Note: Descriptions are shown in the official language in which they were submitted.
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IRAK-RELATED INTERVENTIONS AND DIAGNOSIS
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates generally to a new cluster of molecules
that affects oxidative stress, inflammation, and/or insulin signaling in
white blood cells, particularly nnonocytes, and to identifying the
optimal method or system to modulate the activity of said molecules.
Thereby reducing the risk of diseases associated with activated
nnonocytes such as obesity and obesity-related metabolic syndrome
disorder phenotype characterized by dyslipidennia, hypertension,
glucose intolerance, insulin resistance and diabetes, lipid homeostasis
disorders and/or cardiovascular diseases. More in particular these
molecules are nnicroRNAs (nniRNAs or nniRs) that can be present in the
cell, in cell-derived vesicles that are secreted in blood, and can be
detected in plasma or serum. In addition, we present a method, for
instance a diagnostics method or system, for instance a diagnostic,
which provides information on how to modulate the molecules to treat
or prevent obesity, to separate responders from non-responders, and
to treat or prevent the obesity-related metabolic syndrome disorders.
Several documents are cited throughout the text of this specification.
Each of the documents herein (including any manufacturer's
specifications, instructions etc.) are hereby incorporated by reference.
However, there is no admission that any document cited is indeed
prior art of the present invention.
B. Description of the Related Art
Despite considerable advances in the comprehension of the
pathogenesis of atherosclerosis, cardiovascular diseases remain the
leading cause of mortality and morbidity 1. This can be explained by
the increasing prevalence of obesity and type-2 diabetes mellitus
(T2DM) 2'3. The epidemic of obesity is a global health issue across all
age groups, especially in industrialized countries (American Obesity
Association, 2006). According to WHO's estimate there are more than
300 million obese people (BMI>30) world-wide. Today, for example
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almost 65% of adult Americans (about 127 million) are categorized as
being overweight or obese. There is also evidence that obesity is
increasing problem among children, for example in the USA, the
percentage of overweight children (aged 5-14 years) has doubled in
the last 30 years, from 15% to 32%. The degree of health impairment
of obesity is determined by three factors: 1) the amount of fat 2) the
distribution of fat and 3) the presence of other risk factors. It is the
second leading cause of preventable death in the Western society and
an increasing cause on modernizing societies. Obesity affects all major
bodily systems -heart, lung, muscle and bones-and is considered as a
major risk factor for several chronic disease conditions, including
coronary heart disease, type 2 diabetes mellitus, hypertension, stroke,
and cancers of the breast, endonnetriunn, prostate and colon 4.
A large number of medical conditions have been associated with
obesity. Health consequences are categorized as being the result of
either increased fat mass (osteoarthritis, obstructive sleep apnea,
social stigma) or increased number of fat cells (diabetes, cancer,
cardiovascular disease, non-alcoholic fatty liver disease) 5. Mortality is
increased in obesity, with a BMI of over 32 being associated with a
doubled risk of death 6. There are alterations in the body's response to
insulin (insulin resistance), a pro-inflammatory state and an increased
tendency to thrombosis (pro-thrombotic state) 5. Disease associations
may be dependent or independent of the distribution of adipose tissue.
Central obesity (male-type or waist-predominant obesity,
characterized by a high waist-hip ratio), is an important risk factor for
the metabolic syndrome, the clustering of a number of diseases and
risk factors that heavily predispose for cardiovascular disease. These
are diabetes mellitus type 2, high blood pressure, high blood
cholesterol, and triglyceride levels (combined hyperlipidennia) 7'8. Apart
from the metabolic syndrome, obesity is also correlated with a variety
of other complications. For some of these complaints, it has not been
clearly established to what extent they are caused directly by obesity
itself, or have some other cause (such as limited exercise) that causes
obesity as well. Cardiovascular: congestive heart failure, enlarged
heart and its associated arrhythnnias and dizziness, varicose veins, and
pulmonary embolism. Endocrine: polycystic ovarian syndrome (PCOS),
menstrual disorders, and infertility 9
Gastrointestinal:
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gastroesophageal reflux disease (GERD), fatty liver disease,
cholelithiasis (gallstones), hernia, and colorectal cancer. Renal and
genitourinary: erectile dysfunction 10, urinary incontinence, chronic
renal failure 11, hypogonadisnn (male), breast cancer (female), uterine
cancer (female), stillbirth. Integument (skin and appendages): stretch
marks, acanthosis nigricans, lynnphedenna, cellulitis, carbuncles,
intertrigo. Musculoskeletal: hyperuricennia (which predisposes to gout),
immobility, osteoarthritis, low back pain. Neurologic: stroke, nneralgia
paresthetica, headache, carpal tunnel syndrome, dementia 121
idiopathic intracranial hypertension. Respiratory: obstructive sleep
apnea, obesity hypoventilation syndrome, asthma. Psychological:
Depression, low self esteem, body dimorphic disorder, social
stigmatization.
The economic cost attributable to obesity is substantial and is close to
$100 billion/yr (Wolf & Colditz 1998). Obesity accounts for 2-6% of
total health care costs in several developed countries; some estimates
put the figure as high as 7%. The true costs are undoubtedly much
greater as not all obesity-related conditions are included in the
calculations.
One of the emerging cardiovascular risk factors is subclinical chronic
low-grade inflammation 13. Population studies showed a strong
correlation between pro-inflammatory bionnarkers (such as high
sensitive C-reactive protein (hc-CRP), interleukin-6 (IL-6), and tumor
necrosis factor-a (TNF-a)) and perturbations in glucose homeostasis,
obesity, and atherosclerosis14. Another emerging risk factor is oxidized
LDL (ox-LDL) that activates circulating nnonocytes, thereby increasing
their ability to infiltrate the vascular wall 15. This increased infiltration
is a key event in atherogenesis. The metabolic syndrome clusters
several cardiovascular risk factors including obesity, dyslipidennia,
hypertension, and insulin resistance (IR). Increased inflammation 16'17
and oxidative stress 18-21 were found to be associated with the
metabolic syndrome. It is a primary risk factor for diabetes and
cardiovascular diseases. Recent data suggest that increased oxidative
stress in adipose tissue is an early instigator of the metabolic
syndrome and that the redox state in adipose tissue is a potentially
useful therapeutic target for the obesity-associated metabolic
syndrome 22. Oxidative damage of adipose tissues is associated with
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impaired adipocyte maturation, production of pro-inflammatory
adipocytokines by dysfunctional adipocytes, and increased infiltration
of activated macrophages into the adipose tissues of obese persons
where they produce inflammatory chennokines 23. This enhanced
infiltration is causatively related to the loss of insulin signaling.
There is thus a clear need in the art to have accurate molecules for a
proper treatment of obesity associated disorders such oxidative stress
and inflammation, metabolic syndrome, insulin resistance and type 2
diabetes and related cardiovascular disorders for persons in need
thereto, an to separate responders from non-responders to such a
treatment.
A number of nniRNAs of the present invention have previously been
identified. For example, W02010133970 discloses that miR-103 is
upregulated in liver cells of obese mice and that inhibition of nniR-103
leads to an improvement of several obesitas/insulin resistance
parameters. Surprisingly, as also described in more detail herein
below, the inventors of the current application have found that the
opposite is true in nnonocytes.
Iliopoulos et al. (2010) disclose that miR-181b-1 is pro-inflammatory
in endothelial or cancer cells. They also show that nniR-181b-1 directly
inhibits expression of CYLD, which in its turn is known to inhibit NF-KB
activity 24. Surprisingly, as also described in more detail herein below,
the inventors of the current application have found that nniR-181b is
anti-inflammatory in nnonocytes.
W02010129919 focuses on the influence of let-7 (including let-7a -
let-7i) on asthma and lung inflammation. They show that let-7a, and
likely the other let-7 nniRNAs, directly targets IL-13 expression.
Furthermore, in vivo experiments show that inhibition of nniR-155 (a
let-7 family member) reduces inflammation in lungs. Surprisingly, as
also described in more detail herein below, the inventors of the current
application have found that let-7c and let-7g are anti-inflammatory in
nnonocytes.
Lee et al. (2011) show that miR-130 potently suppresses PPARy in
adipocytes 25. Since PPARy is known to inhibit inflammation in
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nnonocytes 26, one would thus expect nniR-130 to stimulate
inflammation in nnonocytes. However, as described in more detail
herein below, the inventors of the current application have found that
nniR-130 is associated with decreased inflammation in nnonocytes.
The above examples clearly show that the same nniRNA can have very
different, even opposite, effects in different tissues and diseases 27.
Present invention provides such solution to these problems in the art.
SUMMARY OF THE INVENTION
In accordance with the purpose of the invention, as embodied and
broadly described herein, the invention is broadly drawn to molecules
for treatment of the condition and for testing and predicting the
efficacy of his or her treatment.
The present invention solves the problems of the related art by
providing a combination of molecules for the treatment of an activated
nnonocyte, which is characterized by an increased inflammatory state
and/or an increased oxidative stress state and/or a deregulated insulin
signaling. Treatment of said nnonocyte is particularly useful in the
treatment of related diseases such as metabolic syndrome disorder, an
inflammatory disorder, an oxidative stress disorder, an impaired
glucose tolerance, an insulin resistance condition, the progression of
an adipocyte tissue disorder, such as an impaired adipose tissue
accumulation or adipocyte function; metabolic syndrome, and related
cardiovascular diseases. In addition, the diagnostic tools for testing
and predicting the efficacy of the optimal combination of molecules are
provided.
The bionnarkers of present invention in white blood cells (WBCs), or
leukocytes (also spelled "Ieucocytes") for instance T lymphocytes,
nnonocytes and neutrophils and most preferably the nnonocyte type of
white blood cell can be analysed using high speed nnicrofluidic single
cell impedance cytonnetry as for instance been described by David
Holmes and Hywel Morgan 28.
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In particular this invention identifies nniRNAs which regulate nnonocyte
activation and inflammation, oxidative stress and/or insulin signaling in
tissues infiltrated by these nnonocytes (e.g. adipose tissues, vascular
tissues). Said nniRNAs are relevant for treatment of an oxidative stress
state and/or inflammatory state and/or insulin signaling deregulation
in nnonocytes, which leads to the prevention and/or treatment of
obesity and obesity-related metabolic syndrome disorders, and
cardiovascular diseases.
Adiponectin is an antidiabetic adipokine, which enhances insulin action
and inhibits the oxidative stress state and inflammatory state in
nnonocytes, thereby inhibiting causes of among others metabolic
syndrome and cardiovascular diseases. However, resistance to
adiponectin has been shown in obesity and following chronic high fat
feeding and adiponectin treatment may even contribute to lipid
accumulation observed in these conditions. The present invention
identifies nniRNAs that regulate the inhibitory effects of adiponectin or
adiponectin nninnetics on oxidative stress and inflammation in
nnonocytes. The present invention thus provides means to differentiate
reponders from non-responders to adiponectin treatment.
Furthermore, the present invention provides a medicinal solution for
adiponectin resistance. In particular, the modulators of the nniRNAs of
the present invention can be used as a conjunctive therapy to a
treatment with adiponectin or adiponectin nninnetics.
In addition, nniR-146b-5p, IRAK3, 50D2, TNFAIP6, TNFAIP3, TLR2, and
TNFa are known to play an important role in inflammation, oxidative
stress and/or insulin signaling in nnonocytes and are therefore a known
target for the treatment of associated diseases such as obesity and
insulin resistance. Therefore, the modulators of the nniRNAs of the
present invention can be used in a combination therapy with agents
that modulate one or more targets known to play an important role in
inflammation, oxidative stress and/or insulin signaling in nnonocytes,
such as for example selected from nniR-146b-5p, IRAK3, 50D2,
TNFAIP6, TNFAIP3, TLR2, and TNFa. In a particular embodiment, the
modulators of the nniRNAs of the present invention can be used in a
combination therapy with an IRAK3 modulator; more in particular with
a modulator that increases the expression and/or activity of IRAK3.
Said combination therapy can optionally further include agents that
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modulate one or more targets known to play an important role in
inflammation, oxidative stress and/or insulin signaling in nnonocytes,
such as for example selected from nniR-146b-5p, SOD2, TNFAIP6,
TNFAIP3, TLR2, and TNFa.
This method of IRAK3 activation is also particularly suitable for
treating adiponectin resistance disorders or adiponectin deficiency
pathological processes such as endoplasnnatic reticulunn stress-induced
adiponectin downregulation -induced adiponectin -resistance and to
decrease increased risk of cancer due to induced adiponectin
downregulation and leptin upregulation for instance by obesity.
This method of IRAK3 activation is also particularly suitable for use in
a conjunctive therapy with adiponectin or adiponectin nninnetics to
prevent stress-induced damage in the heart; to enhance adiponectin
treatment of hyperproliferation or to enhance adiponectin inhibited
angiogenesis in for instance an anti-cancer or anti-tumor therapy.
Furthermore, the present invention provides a means, for instance
diagnostic tool or a diagnostic method to identify persons who have
WBCs with an increased inflammatory state, an increased oxidative
stress state and/or deregulated insulin signaling, in particular activated
nnonocytes. The nniRNAs of the present invention can be quantified or
qualified in a sample obtained from said person. In a particular
embodiment, said sample is a blood-derived sample. The nniRNAs of
present invention can for example be quantified or qualified on isolated
WBCs, or leukocytes (also spelled "Ieucocytes") for instance T
lymphocytes, nnonocytes and neutrophils and most preferably the
nnonocyte type of white blood cell. A lab on chip nnicrofluidic set-up to
remove red blood cells from the sample and isolate WBCs uses
electrodes to measure each blood cell's electrical properties and
identify said cells as blood flows through the device's channels,
suitable for distinguishing and counting the different types of cell,
providing information used in the diagnosis, and in monitoring the
treatment, of numerous diseases as for instance been described by
David Holmes and Hywel Morgan 28 whereby the blood cells are
identified as they flow through a nnicrofluidic device. Alternative
methods are power-free nnicrofluidics using capillary forces to pull the
blood or other samples. The currently routine blood analysis uses flow
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cytonnetry. In another particular embodiment, the nniRNAs of the
present invention can be quantified or qualified on isolated
nnicrovesicles, particularly on nnonocyte-derived nnicrovesicles.
The above means also allows to identifying persons in which treatment
with adiponectin or adiponectin nninnetics can decrease the risk of
nnonocyte activation in inflammation and/or oxidative stress-related
and/or insulin resistance-related diseases such as obesity, type 2
diabetes, and the metabolic syndrome, atherosclerosis and/or
cardiovascular diseases. The quantification or qualification of the
nniRNAs of the present invention in WBCs, particularly nnonocytes, can
be further complemented with the quantification or qualification of
other markers, such as on or more targets known to play an important
role in inflammation, oxidative stress and/or insulin signaling in
nnonocytes, such as for example selected from nniR-146b-5p, IRAK3,
SOD2, TNFAIP6, TNFAIP3, TLR2, and TNFa.
Such method is particularly useful to identify the responder patients to
a treatment of adiponectin or adiponectin nninnetics. In particular in
patients suffering from obesity, type 2 diabetes, and the metabolic
syndrome, and atherosclerosis or cardiovascular diseases.
The present invention provides a method to identify nniRNA to which
mimics will improve and/or restore the anti-inflammatory and/or
antioxidative and/or the insulin sensitizing response in blood
nnonocytes and infiltrated tissues (e.g. adipose and vascular tissues),
in particular in persons with obesity and obesity-related disorders as
disclosed above. In addition, the present invention provides a method
to identify nniRNA to which mimics will improve and/or restore the anti-
inflammatory and/or antioxidative and/or the insulin sensitizing actions
of adiponectin.
Illustrative embodiments of the invention
DEFINITIONS
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Myeloid refers to the nonlynnphocytic groups of white blood cells,
including the granulocytes, nnonocytes and platelets.
Dyslipidemia (From dys- + lipid (fat) + -ennia (in the blood) =
essentially, disordered lipids in the blood) is a disorder of lipoprotein
metabolism. Dyslipidennias may be manifested by elevation of the
triglyceride concentrations, and a decrease in the "good" high-density
lipoprotein (HDL) cholesterol concentration in the blood. Dyslipidennia
comes under consideration in many situations including diabetes, a
common cause of lipidennia. For adults with diabetes, it has been
recommended that the levels HDL-cholesterol, and triglyceride be
measured every year. Optimal HDL-cholesterol levels are equal to or
greater than 40 nng/dL (1.02 nnnnol/L), and desirable triglyceride levels
are less than 150 nng/dL (1.7 nnnnol/L).
Insulinemia concerns an abnormally large concentration of insulin in
the blood.
Glycemia concerns the presence of glucose in the blood. It is a
medical term meaning that the blood glucose is elevated, typically
above 100 mg/dl.
Hypercholesterolemia is manifested by elevation of the total
cholesterol due to elevation of the "bad" low-density lipoprotein (LDL)
cholesterol in the blood. Optimal LDL-cholesterol levels for adults with
diabetes are less than 100 nng/dL (2.60 nnnnol/L).
Triglycerides are the major form of fat. A triglyceride consists of
three molecules of fatty acid combined with a molecule of the alcohol
glycerol. Triglycerides serve as the backbone of many types of lipids
(fats). Triglycerides come from the food we eat as well as from being
produced by the body. Triglyceride levels are influenced by recent fat
and alcohol intake, and should be measured after fasting for at least
12 hours. A period of abstinence from alcohol is advised before testing
for triglycerides. Markedly high triglyceride levels (greater than
500nng/d1) can cause inflammation of the pancreas (pancreatitis).
Therefore, these high levels should be treated aggressively with low
fat diets and medications, if needed. The word "triglyceride" reflects
the fact that a triglyceride consists of three ("tri-") molecules of fatty
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acid combined with a molecule of the alcohol glycerol ("-glyceride")
that serves as the backbone in many types of lipids (fats).
HDL-cholesterol concerns lipoproteins, which are combinations of
lipids (fats) and proteins, are the form in which lipids are transported
in the blood. The high-density lipoproteins transport cholesterol from
the tissues of the body to the liver so it can be gotten rid of (in the
bile). HDL-cholesterol is therefore considered the "good" cholesterol.
The higher the HDL-cholesterol level, the lower the risk of coronary
artery disease. Even small increases in HDL-cholesterol reduce the
frequency of heart attacks. For each 1 mg/dl increase in HDL-
cholesterol there is a 2 to 4% reduction in the risk of coronary heart
disease. Although there are no formal guidelines, proposed treatment
goals for patients with low HDL-cholesterol are to increase HDL-
cholesterol to above 35 mg/dl in men and 45 mg/dl in women with a
family history of coronary heart disease; and to increase HDL-
cholesterol to approach 45 mg/dl in men and 55 mg/dl in women with
known coronary heart disease. The first step in increasing HDL-
cholesterol levels is life style modification. Regular aerobic exercise,
loss of excess weight (fat), and cessation of cigarette smoking
cigarettes will increase HDL-cholesterol levels. Moderate alcohol
consumption (such as one drink a day) also raises HDL-cholesterol.
When life style modifications are insufficient, medications are used.
Medications that are effective in increasing HDL-cholesterol include
nicotinic acid (niacin), gennfibrozil (Lopid), estrogen, and to a lesser
extent, the statin drugs.
Hypertension or High blood pressure is defined as a repeatedly
elevated blood pressure exceeding 140 over 90 mmHg -- a systolic
pressure above 140 with a diastolic pressure above 90. Chronic
hypertension is a "silent" condition. Stealthy as a cat, it can cause
blood vessel changes in the back of the eye (retina), abnormal
thickening of the heart muscle, kidney failure, and brain damage. For
diagnosis, there is no substitute for measurement of blood pressure.
Not having your blood pressure checked (or checking it yourself) is an
invitation to hypertension. No specific cause for hypertension is found
in 95% of cases. Hypertension is treated with regular aerobic exercise,
weight reduction (if overweight), salt restriction, and medications.
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Diabetes, type 2 is one of the two major types of diabetes, the type
in which the beta cells of the pancreas produce insulin but the body is
unable to use it effectively because the cells of the body are resistant
to the action of insulin. Although this type of diabetes may not carry
the same risk of death from ketoacidosis, it otherwise involves many
of the same risks of complications as type 1 diabetes (in which there is
a lack of insulin). The aim of treatment is to normalize the blood
glucose in an attempt to prevent or minimize complications. People
with type 2 diabetes may experience marked hyperglycemia, but most
do not require insulin injections. In fact, 80% of all people with type 2
diabetes can be treated with diet, exercise, and, if needed be, oral
hypoglycemic agents (drugs taken by mouth to lower the blood sugar).
Type 2 diabetes requires good dietary control including the restriction
of calories, lowered consumption of simple carbohydrates and fat with
increased consumption of complex carbohydrates and fiber. Regular
aerobic exercise is also an important method for treating both type 2
diabetes since it decreases insulin resistance and helps burn excessive
glucose. Regular exercise also may help lower blood lipids and reduce
some effects of stress, both important factors in treating diabetes and
preventing complications. Type 2 diabetes is also known as insulin-
resistant diabetes, non-insulin dependent diabetes, and adult-onset
diabetes.
Systolic: The blood pressure when the heart is contracting. It is
specifically the maximum arterial pressure during contraction of the
left ventricle of the heart. The time at which ventricular contraction
occurs is called systole. In a blood pressure reading, the systolic
pressure is typically the first number recorded. For example, with a
blood pressure of 120/80 ("120 over 80"), the systolic pressure is 120.
By "120" is meant 120 mm Hg (millimeters of mercury). A systolic
murmur is a heart murmur heard during systole, the time the heart
contracts, between the normal first and second heart sounds.
"Systolic" comes from the Greek systole, meaning "a drawing together
or a contraction." The term has been in use since the 16th century to
denote the contraction of the heart muscle.
Osteoarthritis is a type of arthritis caused by inflammation,
breakdown, and eventual loss of cartilage in the joints. It is also
known as degenerative arthritis.
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An ischemic stroke is death of an area of brain tissue (cerebral
infarction) resulting from an inadequate supply of blood and oxygen to
the brain due to blockage of an artery. Ischennic stroke usually results
when an artery to the brain is blocked, often by a blood clot or a fatty
deposit due to atherosclerosis. Symptoms occur suddenly and may
include muscle weakness, paralysis, lost or abnormal sensation on one
side of the body, difficulty speaking, confusion, problems with vision,
dizziness, and loss of balance and coordination. Diagnosis is usually
based on symptoms and results of a physical examination, imaging
tests, and blood tests. Treatment may include drugs to break up blood
clots or to make blood less likely to clot and surgery, followed by
rehabilitation. About one third of people recover all or most of normal
function after an ischennic stroke. Ischennic stroke occurs when local
blood flow is suddenly limited by vessel occlusion. The rate of neuronal
death varies with blood flow. If blood flow falls to less than 15 nnL/100
g/nnin, energy failure and subsequent cell death occur within minutes.
Even suboptimal flow for longer periods may cause the cells to die by
an apoptotic mechanism over days to weeks. Rapid restoration of
blood flow is essential to save brain tissue. The mechanism of stroke
involving the PCA territory is variable. It is commonly due to
ennbolization from the heart, the aortic arch, the vertebral artery, or
the basilar artery. Other mechanisms include intrinsic atherosclerotic
disease and vasospasnn. Migrainous strokes tend to involve PCAs
preferentially. Less commonly, the anterior circulation is to blame
(e.g., internal carotid stenosis), when a fetal PCA is present. Rare
causes of stroke may be considered when usual culprits such as
coagulation abnormalities, vasculitis, synnpathonninnetic drugs, and
metabolic disorders are not present.
Insulin resistance is the diminished ability of cells to respond to the
action of insulin in transporting glucose (sugar) from the bloodstream
into muscle and other tissues. Insulin resistance typically develops
with obesity and heralds the onset of type 2 diabetes. It is as if insulin
is "knocking" on the door of muscle. The muscle hears the knock,
opens up, and lets glucose in. But with insulin resistance, the muscle
cannot hear the knocking of the insulin (the muscle is "resistant"). The
pancreas makes more insulin, which increases insulin levels in the
blood and causes a louder "knock." Eventually, the pancreas produces
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far more insulin than normal and the muscles continue to be resistant
to the knock. As long as one can produce enough insulin to overcome
this resistance, blood glucose levels remain normal. Once the pancreas
is no longer able to keep up, blood glucose starts to rise, initially after
meals, eventually even in the fasting state. Insulin resistance is an
early feature and finding in the pathogenesis of type 2 diabetes
associated with obesity is the development is insulin resistance,
defined as impaired insulin-mediated glucose clearance in insulin-
sensitive tissues (skeletal muscle, liver and adipose tissue). Insulin
resistance is the condition in which normal amounts of insulin are
inadequate to produce a normal insulin response from fat, muscle and
liver cells. Insulin resistance in fat cells reduces the effects of insulin
and results in elevated hydrolysis of stored triglycerides in the absence
of measures which either increase insulin sensitivity or which provide
additional insulin. Increased mobilization of stored lipids in these cells
elevates free fatty acids in the blood plasma. Insulin resistance in
muscle cells reduces glucose uptake (and so local storage of glucose
as (glycogen), whereas insulin resistance in liver cells reduces storage
of glycogen, making it unavailable for release into the blood when
blood insulin levels fall (normally only when blood glucose levels are at
low storage: Both lead to elevated blood glucose levels. High plasma
levels of insulin and glucose due to insulin resistance often lead to
metabolic syndrome and type 2 diabetes, including its complications.
In 2000, there were approximately 171 million people, worldwide, with
diabetes. The numbers of diabetes patients will expectedly more than
double over the next 25 years, to reach a total of 366 million by 2030
(WHO/IDF, 2004). The two main contributors to the worldwide
increase in prevalence of diabetes are population ageing and
urbanization, especially in developing countries, with the consequent
increase in the prevalence of obesity (WHO/IDF, 2004).
Cardiovascular diseases refer to the class of diseases that involve
the heart or blood vessels (arteries and veins). While the term
technically refers to any disease that affects the cardiovascular
system, it is usually used to refer to those related to atherosclerosis
(arterial disease). The circulatory system (or cardiovascular system) is
an organ system that moves nutrients, gases, and wastes to and from
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cells, helps fight diseases and helps stabilize body temperature and pH
to maintain homeostasis. While humans, as well as other vertebrates,
have a closed circulatory system (meaning that the blood never leaves
the network of arteries, veins and capillaries), some invertebrate
groups have open circulatory system. The present diagnostic invention
is particularly suitable for a cardiovascular disease of the group
consisting of hypertension, coronary heart disease, heart failure,
congestive heart failure, atherosclerosis, arteriosclerosis, stroke,
cerebrovascular disease, myocardial infarction and peripheral vascular
disease.
The following terms are similar, yet distinct, in both spelling and
meaning, and can be easily confused: arteriosclerosis,
arteriolosclerosis and atherosclerosis.
Arteriosclerosis also called hardening of the arteries chronic disease
is characterized by abnormal thickening and hardening of the walls of
arteries, with a resulting loss of elasticity. The major form of
arteriosclerosis is atherosclerosis, in which plaques of consisting of
macrophages, fatty deposits in foam cells, or atheronnas, form on the
inner walls of the arteries. These fatty acids are largely due to the
uptake of oxidized LDL by macrophages. Arteriosclerosis is a general
term describing any hardening (and loss of elasticity) of medium or
large arteries (in Greek, "Arterio" meaning artery and "sclerosis"
meaning hardening); arteriolosclerosis is arteriosclerosis mainly
affecting the arterioles (small arteries); atherosclerosis is a hardening
of an artery specifically due to an atheronnatous plaque. Therefore,
atherosclerosis is a form of arteriosclerosis. Arteriosclerosis
("hardening of the artery") results from a deposition of tough, rigid
collagen inside the vessel wall and around the atheronna. This
increases the stiffness, decreases the elasticity of the artery wall.
Arteriolosclerosis (hardening of small arteries, the arterioles) is the
result of collagen deposition, but also muscle wall thickening and
deposition of protein ("hyaline").Calcification, sometimes even
ossification (formation of complete bone tissue) occurs within the
deepest and oldest layers of the sclerosed vessel wall.
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Atherosclerosis causes two main problems. First, the atheronnatous
plaques, though long compensated for by artery enlargement,
eventually lead to plaque ruptures and stenosis (narrowing) of the
artery and, therefore, an insufficient blood supply to the organ it
feeds. If the compensating artery enlargement is excessive, a net
aneurysm occurs. Atherosclerosis chronic disease is caused by the
deposition of fats, cholesterol, calcium, and other substances in the
innermost layer (endothelium) of the large and medium-sized arteries.
Atherosclerosis is a disease affecting the arterial blood vessel. It is
commonly referred to as a "hardening" or "furring" of the arteries. It is
caused by the formation of multiple plaques within the arteries.
These complications are chronic, slowly progressing and cumulative.
Most commonly, soft plaque suddenly ruptures (see vulnerable
plaque), causing the formation of a thrombus that will rapidly slow or
stop blood flow, e.g. 5 minutes, leading to death of the tissues fed by
the artery. This catastrophic event is called an infarction. One of the
most common recognized scenarios is called coronary thrombosis of a
coronary artery causing myocardial infarction (a heart attack). Another
common scenario in very advanced disease is claudication from
insufficient blood supply to the legs, typically due to a combination of
both stenosis and aneurisnnal segments narrowed with clots. Since
atherosclerosis is a body wide process, similar events also occur in the
arteries to the brain, intestines, kidneys, legs, etc.
Pathologically, the atheronnatous plaque is divided into three distinct
components: the nodular accumulation of a soft, flaky, yellowish
material at the centre of large plaques composed of macrophages
nearest the lumen of the artery; sometimes with underlying areas of
cholesterol crystals; and possibly also calcification at the outer base of
older/more advanced lesions.
Thrombogenicity refers to the tendency of a material in contact with
the blood to produce a thrombus, or clot. It not only refers to fixed
thrombi but also to emboli, thrombi which have become detached and
travel through the bloodstream. Thronnbogenicity can also encompass
events such as the activation of immune pathways and the
complement system. All materials are considered to be thronnbogenic
with the exception of the endothelial cells which line the vasculature.
Certain medical implants appear non-thronnbogenic due to high flow
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rates of blood past the implant, but in reality, all are thronnbogenic to
a degree. A thronnbogenic implant will eventually be covered by a
fibrous capsule, the thickness of this capsule can be considered one
measure of thronnbogenicity, and if extreme can lead to the failure of
the implant.
Low-density lipoprotein (LDL) belongs to the lipoprotein particle
family. Its size is approx. 22 nnn and its mass is about 3 million
Da!tons; but, since LDL particles contain a changing number of fatty
acids, they actually have a mass and size distribution. Each native LDL
particle contains a single apolipoprotein B-100 molecule (Apo B-100, a
protein with 4536 amino acid residues) that circles the fatty acids,
keeping them soluble in the aqueous environment. In addition, LDL
has a highly-hydrophobic core consisting of polyunsaturated fatty acid
known as linoleate and about 1500 esterified cholesterol molecules.
This core is surrounded by a shell of phospholipids and unesterified
cholesterol as well as a single copy of B-100 large protein (514 kD).
Cholesterol is an animal sterol that is normally synthesized by the
liver. The main types, low-density lipoprotein (LDL) and high-density
lipoprotein (HDL) carry cholesterol from and to the liver, respectively.
LDL-cholesterol concerns thus the cholesterol in low-density
lipoproteins. Cholesterol is required in the membrane of mammalian
cells for normal cellular function, and is either synthesized in the
endoplasnnic reticulunn, or derived from the diet, in which case it is
delivered by the bloodstream in low-density lipoproteins. These are
taken into the cell by LDL receptor-mediated endocytosis in clathrin-
coated pits, and then hydrolyzed in lysosonnes. Oxidized LDL-
cholesterol concerns a LDL-cholesterol that has been bombarded by
free radicals; it is thought to cause atherosclerosis; the 'bad'
cholesterol; a high level in the blood is thought to be related to various
pathogenic conditions
Metabolic syndrome is a combination of medical disorders that
increase the risk of developing cardiovascular disease and type 2
diabetes. It affects a large number of people, and prevalence increases
with age. Some studies estimate the prevalence in the USA to be up to
25% of the population. Metabolic syndrome is also known as metabolic
syndrome X, syndrome X, insulin resistance syndrome, Reaven's
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syndrome or CHAOS. Metabolic syndrome components were defined as
detailed in the Third Report of the National Cholesterol Education
Program Expert Panel on Detection, Evaluation, and Treatment of High
Blood Cholesterol in adults (ATPIII) report: 1) waist circumference
1.02 cm in men and 88 cm in women; 2) fasting triglycerides
150 mg/dl (1.70 ninno1/1); 3) HDL-cholesterol <40 mg/dl (1.03
ninno1/1) in men and < 50 mg/dl (1.29 ninno1/1) in women; 4) blood
pressure
130/85 mmHg or on anti-hypertensive medication; 5)
fasting-glucose
100 mg/dl (5.55 ninno1/1) or on anti-diabetic
medication.
Activated monocytes are nnonocytes that are associated with
increased inflammation, often due to activation of the toll-like receptor
(TLR)-2 (and/or -4), an increase in interleukin-1 receptor-associated
kinase (IRAK)-1 and 4, and a decrease in the interleukin-1 receptor-
associated kinase (IRAK)-3 (sometimes called IRAKM) and an increase
in NFKB activity 29,301 and/or an increased production of reactive
oxygen species (ROS) and oxidative stress, often due to loss of
antioxidant enzymes like superoxide disnnutase (SODs) 21'31, and/or a
loss of insulin signaling and insulin resistance, for example by loss of
expression of the insulin receptor substrate (IRS)-1 and -2 32.
Activation of nnonocytes renders them more prone to infiltration of in
tissues (e.g. adipose, vascular, pancreas, liver) often due to increased
expression of the nnonocyte chennotactic protein 1 (MCP1 or otherwise
called chennokine CC motif ligand or CCL2) 33. Once infiltrated, these
activated nnonocytes are more prone to give rise to inflammatory M1
macrophages instead of anti-inflammatory M2 macrophages 34-37. In
addition, they lost their capacity to activate their anti-inflammatory
(e.g. increase in IRAK-3) and antioxidative (e.g. increase in
antioxidant SODs and decreased ROS) mechanisms, and thus their
capacity to switch their polarization from M1 to M2 in response to
adiponectin 38'39. These activation mechanisms are illustrated in Figure
2.
"microRNA", also written as nniRNA or nniR, refers to any type of
interfering RNAs, including but not limited to, endogenous nnicroRNAs
and artificial nnicroRNAs. Endogenous nnicroRNAs are small RNAs
naturally present in the genonne which are capable of modulation the
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productive utilization of nnRNA. An artificial nnicroRNA can be any type
of RNA sequence, other than endogeneous nnicroRNA, which is capable
of modulation the productive utilization of nnRNA. For instance, it
includes sequences previously identified as siRNA, regardless of the
mechanism of down-stream processing of the RNA. A nnicroRNA
sequence can be an RNA molecule composed of any one or more of
these sequences. Several types of agents are known that modulate
nnicroRNAs. These include, but are not limited to nnicroRNA mimics and
nnicroRNA inhibitors.
A "miRNA mimic" is an agent used to increase the expression and/or
function of a miRNA. The miRNA mimic can also increase, supplement,
or replace the function of a natural miRNA. In one embodiment, the
miRNA mimic may be a polynucleotide comprising the mature miRNA
sequence. In another embodiment, the miRNA mimic may be a
polynucleotide comprising the pri-miRNA or pre-miRNA sequence. The
miRNA mimic may contain chemical modifications, such as locked
nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-0-
alkyl (e.g. 2f-0-methyl, 2'-13-nnethoxyethyl), 2'-fluoro, and 4' thio
modifications, and backbone modifications, such as one or more
phosphorothioate, nnorpholino, or phosphonocarboxylate linkages.
A "miRNA inhibitor" is an agent that inhibits miRNA function in a
sequence-specific manner. In one embodiment, the miRNA inhibitor is
an antagonnir. "Antagonnirs" are single-stranded, chemically-modified
ribonucleotides that are at least partially complementary to the miRNA
sequence. Antagonnirs may comprise one or more modified
nucleotides, such as 2f-0-methyl-sugar modifications. In some
embodiments, antagonnirs comprise only modified nucleotides.
Antagonnirs may also comprise one or more phosphorothioate linkages
resulting in a partial or full phosphorothioate backbone. To facilitate in
vivo delivery and stability, the antagonnir may be linked to a
cholesterol moiety at its 3' end. Antagonnirs suitable for inhibiting
nniRNAs may be about 15 to about 50 nucleotides in length, more
preferably about 18 to about 30 nucleotides in length, and most
preferably about 20 to about 25 nucleotides in length. "Partially
complementary" refers to a sequence that is at least about 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target
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polynucleotide sequence. The antagonnirs may be at least about 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a
mature nniRNA sequence. In some embodiments, the antagonnirs are
100% complementary to the mature nniRNA sequence.
"Sample" or "biological sample" as used herein can be any organ,
tissue, cell, or cell extract isolated from a subject, a cell-derived
vesicle, such as a sample isolated from a mammal having a metabolic
syndrome disorder or at risk for a metabolic syndrome disorder (e.g.,
based on family history or personal history). For example, a sample
can include, without limitation, cells or tissue (e.g., from a biopsy or
autopsy), peripheral blood, whole blood, red cell concentrates, platelet
concentrates, leukocyte concentrates, blood cell proteins, blood
plasma, platelet-rich plasma, a plasma concentrate, a precipitate from
any fractionation of the plasma, a supernatant from any fractionation
of the plasma, blood plasma protein fractions, purified or partially
purified blood proteins or other components, serum, tissue or fine
needle biopsy samples, or any other specimen, or any extract thereof,
obtained from a patient (human or animal), test subject, healthy
volunteer, or experimental animal. A subject can be a human, rat,
mouse, non-human primate, etc. A sample may also include sections
of tissues such as frozen sections taken for histological purposes. A
"sample" may also be a cell or cell line created under experimental
conditions, that is not directly isolated from a subject.
In a particular embodiment the sample is selected from the group
consisting of (a) a liquid containing cells; (b) a tissue-sample; (c) a
cell-sample; (d) a cell-derived vesicle; (e) a cell biopsy; more in
particular the sample comprises hennatopoietic cells or blood cells;
even more in particular the sample comprises at least one myeloid cell
or debris thereof. In an even further embodiment the sample
comprises at least one of nnonocytes or peripheral blood mononuclear
cells or debris thereof.
In addition, a sample can also be a blood-derived sample, like plasma
or serum. In another particular embodiment, the nniRNAs of the
present invention can be quantified or qualified on isolated
nnicrovesicles, particularly on nnonocyte-derived nnicrovesicles.
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A "control" or "reference" includes a sample obtained for use in
determining base-line expression or activity. Accordingly, a control
sample may be obtained by a number of means including from
subjects not having a metabolic syndrome disorder; from subjects not
suspected of being at risk for developing a metabolic syndrome
disorder; or from cells or cell lines derived from such subjects. A
control also includes a previously established standard, such as a
previously characterized pool of RNA or protein extracts from
nnonocytes of at least 20 subjects without any of the metabolic
syndrome components as defined above. Accordingly, any test or
assay conducted according to the invention may be compared with the
established standard and it may not be necessary to obtain a control
sample for comparison each time.
The inflammatory state of a cell can be measured by determining
well-known inflammatory parameters associated with said cell. These
parameters include certain chennokines and cytokines, including but
not limited to IFN-y, IL-1, IL-6, IL-8, and TNF-a. An increased
inflammatory state of a cell refers to an increased amount of
inflammatory parameters associated with said cell compared to a
control cell. Similarly a normal or decreased inflammatory state of a
cell refers to a similar or decreased amount, respectively, of
inflammatory parameters associated with said cell compared to a
control cell.
Similarly, the oxidative stress state of a cell can be nneausured by
determining well-known oxidative stress parameters, such as e.g. the
amount of reactive oxygen species (ROS). An increased, normal or
decreased oxidative stress state of a cell refers, respectively, to an
increased, similar or decreased amount of oxidative stress parameters
associated with said cell compared to a control cell.
Insulin signaling
Special transporter proteins in cell membranes allow glucose from the
blood to enter a cell. These transporters are, indirectly, under blood
insulin's control in certain body cell types (e.g., muscle cells). Low
levels of circulating insulin, or its absence, will prevent glucose from
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entering those cells (e.g., in type 1 diabetes). More commonly,
however, there is a decrease in the sensitivity of cells to insulin (e.g.,
the reduced insulin sensitivity characteristic of type 2 diabetes),
resulting in decreased glucose absorption. In either case, there is 'cell
starvation' and weight loss, sometimes extreme. In a few cases, there
is a defect in the release of insulin from the pancreas. Either way, the
effect is the same: elevated blood glucose levels.
Activation of insulin receptors leads to internal cellular mechanisms
that directly affect glucose uptake by regulating the number and
operation of protein molecules in the cell membrane that transport
glucose into the cell. The genes that specify the proteins that make up
the insulin receptor in cell membranes have been identified, and the
structures of the interior, transnnennbrane section, and the extra-
membrane section of receptor have been solved.
Two types of tissues are most strongly influenced by insulin, as far as
the stimulation of glucose uptake is concerned: muscle cells
(nnyocytes) and fat cells (adipocytes). The former are important
because of their central role in movement, breathing, circulation, etc.,
and the latter because they accumulate excess food energy against
future needs. Together, they account for about two-thirds of all cells in
a typical human body.
Insulin binds to the extracellular portion of the alpha subunits of the
insulin receptor. This, in turn, causes a conformational change in the
insulin receptor that activates the kinase domain residing on the
intracellular portion of the beta subunits. The activated kinase domain
autophosphorylates tyrosine residues on the C-terminus of the
receptor as well as tyrosine residues in the IRS-1 protein.
1. phosphorylated IRS-1, in turn, binds to and activates phosphoinositol 3
kinase (PI3K)
302. PI3K catalyzes the reaction PIP2 + ATP , PIP3
3. PIP3 activates protein kinase B (PKB)
4. PKB phosphorylates glycogen synthase kinase (GSK) and thereby
inactivates GSK
5. GSK can no longer phosphorylate glycogen synthase (GS)
356. unphosphorylated GS makes more glycogen
7. PKB also facilitates vesicle fusion, resulting in an increase in GLUT4
transporters in the plasma membrane. 40,41
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Unless defined otherwise, technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Singleton et al.,
Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &
Sons (New York, N.Y. 1994), Microarrays in Clinical Diagnostics (C)
2005 Humana Press Inc.) provide one skilled in the art with a general
guide to many of the terms used in the present application.
For purposes of the present invention, the following terms are defined
below.
The term "array" or "microarray" in general refers to an ordered
arrangement of hybridizable array elements such as polynucleotide
probes on a substrate. An "array" is typically a spatially or logically
organized collection, e.g., of oligonucleotide sequences or nucleotide
sequence products such as RNA or proteins encoded by an
oligonucleotide sequence. In some embodiments, an array includes
antibodies or other binding reagents specific for products of a
candidate library. The array element may be an oligonucleotide, DNA
fragment, polynucleotide, or the like, as defined below. The array
element may include any element immobilized on a solid support that
is capable of binding with specificity to a target sequence such that
gene expression may be determined, either qualitatively or
quantitatively.
When referring to a pattern of expression, a "qualitative" difference
in gene expression refers to a difference that is not assigned a relative
value. That is, such a difference is designated by an "all or nothing"
valuation. Such an all or nothing variation can be, for example,
expression above or below a threshold of detection (an on/off pattern
of expression). Alternatively, a qualitative difference can refer to
expression of different types of expression products, e.g., different
alleles (e.g., a mutant or polymorphic allele), variants (including
sequence variants as well as post-translationally modified variants),
etc. In contrast, a "quantitative" difference, when referring to a
pattern of gene expression, refers to a difference in expression that
can be assigned a value on a graduated scale, (e.g., a 0-5 or 1-10
scale, a + +++ scale, a grade 1 grade 5 scale, or the like; it will be
understood that the numbers selected for illustration are entirely
arbitrary and in no-way are meant to be interpreted to limit the
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invention). Microarrays are useful in carrying out the methods
disclosed herein because of the reproducibility between different
experiments. DNA nnicroarrays provide one method for the
simultaneous measurement of the expression levels of large numbers
of genes. Each array consists of a reproducible pattern of capture
probes attached to a solid support. Labeled RNA or DNA is hybridized
to complementary probes on the array and then detected for instance
by laser scanning. Hybridization intensities for each probe on the array
are determined and converted to a quantitative value representing
relative gene expression levels. See the patent publications Nos.
U56040138, U55800992 and U56020135, U56033860, US 6344316,
U57439346, U57371516, U57353116, U57348181, U57347921,
U57335762 , U57335470, U57323308, U57321829, U57302348,
U57276592, U57264929, U57244559, U57221785, U57211390,
U57189509, U57138506, U57052842, U57047141 and U57031845
which are incorporated herein by reference. High-density
oligonucleotide arrays are particularly useful for determining the gene
expression profile for a large number of RNA's in a sample.
A "DNA fragment" includes polynucleotides and/or oligonucleotides
and refers to a plurality of joined nucleotide units formed from
naturally-occurring bases and cyclofuranosyl groups joined by native
phosphodiester bonds. This term effectively refers to naturally-
occurring species or synthetic species formed from naturally-occurring
subunits. "DNA fragment" also refers to purine and pyrinnidine groups
and moieties which function similarly but which have no naturally-
occurring portions. Thus, DNA fragments may have altered sugar
moieties or inter-sugar linkages. Exemplary among these are the
phosphorothioate and other sulfur containing species. They may also
contain altered base units or other modifications, provided that
biological activity is retained. DNA fragments may also include species
that include at least some modified base forms. Thus, purines and
pyrinnidines other than those normally found in nature may be so
employed. Similarly, modifications on the cyclofuranose portions of the
nucleotide subunits may also occur as long as biological function is not
eliminated by such modifications.
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The term "polynucleotide," when used in singular or plural generally
refers to any polyribonucleotide or polydeoxribonucleotide, which may
be unmodified RNA or DNA or modified RNA or DNA. Thus, for
instance, polynucleotides as defined herein include, without limitation,
single- and double-stranded DNA, DNA including single- and double-
stranded regions, single- and double-stranded RNA, and RNA including
single- and double-stranded regions, hybrid molecules comprising DNA
and RNA that may be single-stranded or, more typically, double-
stranded or include single- and double-stranded regions. In addition,
the term "polynucleotide" as used herein refers to triple-stranded
regions comprising RNA or DNA or both RNA and DNA. The strands in
such regions may be from the same molecule or from different
molecules. The regions may include all of one or more of the
molecules, but more typically involve only a region of some of the
molecules. One of the molecules of a triple-helical region often is an
oligonucleotide. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term is
intended herein. Moreover, DNAs or RNAs comprising unusual bases,
such as inosine, or modified bases, such as tritiated bases, are
included within the term "polynucleotides" as defined herein. In
general, the term "polynucleotide" embraces all chemically,
enzymatically and/or metabolically modified forms of unmodified
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of cells, including simple and complex cells.
The term "oligonucleotide" refers to a relatively short polynucleotide,
including, without limitation, single-stranded deoxyribonucleotides,
single- or double-stranded ribonucleotides, RNA: DNA hybrids and
double-stranded DNAs. Oligonucleotides, such as single-stranded DNA
oligonucleotides, are often synthesized by chemical methods, for
example using automated oligonucleotide synthesizers that are
commercially available. However, oligonucleotides can be made by a
variety of other methods, including in vitro recombinant DNA-
mediated techniques and by expression of DNAs in cells.
The terms "differentially expressed gene," "differential gene
expression" and their synonyms, which are used interchangeably, refer
to a gene whose expression is activated to a higher or lower level in a
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subject, relative to its expression in a normal or control subject. A
differentially expressed gene may be either activated or inhibited at
the nucleic acid level or protein level, or may be subject to alternative
splicing to result in a different polypeptide product. Such differences
may be evidenced by a change in nnRNA levels, surface expression,
secretion or other partitioning of a polypeptide, for example.
Differential gene expression may include a comparison of expression
between two or more genes, or a comparison of the ratios of the
expression between two or more genes, or even a comparison of two
differently processed products of the same gene, which differ between
normal subjects and subjects suffering from a disease, or between
various stages of the same disease. Differential expression includes
both quantitative, as well as qualitative, differences in the temporal or
cellular expression pattern in a gene or its expression products. As
used herein, "differential gene expression" can be present when there
is, for example, at least an about a one to about two-fold, or about
two to about four-fold, or about four to about six-fold, or about six to
about eight-fold, or about eight to about ten-fold, or greater than
about 11-fold difference between the expression of a given gene in a
patient of interest compared to a suitable control. However, folds
change less than one is not intended to be excluded and to the extent
such change can be accurately measured, a fold change less than one
may be reasonably relied upon in carrying out the methods disclosed
herein.
In some embodiments, the fold change may be greater than about five
or about 10 or about 20 or about 30 or about 40.
The phrase "gene expression profile" as used herein, is intended to
encompass the general usage of the term as used in the art, and
generally means the collective data representing gene expression with
respect to a selected group of two or more genes, wherein the gene
expression may be upregulated, downregulated, or unchanged as
compared to a reference standard A gene expression profile is
obtained via measurement of the expression level of many individual
genes. The expression profiles can be prepared using different
methods. Suitable methods for preparing a gene expression profile
include, but are not limited to reverse transcription loop-mediated
amplification (RT-LAMP), for instance one-step RT-LAMP, quantitative
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RT-PCR, Northern Blot, in situ hybridization, slot-blotting, nuclease
protection assay, nucleic acid arrays, and immunoassays. The gene
expression profile may also be determined indirectly via measurement
of one or more gene products (whether a full or partial gene product)
for a given gene sequence, where that gene product is known or
determined to correlate with gene expression.
The phrase "gene product" is intended to have the meaning as
generally understood in the art and is intended to generally encompass
the product(s) of RNA translation resulting in a protein and/or a
protein fragment. The gene products of the genes identified herein
may also be used for the purposes of diagnosis or treatment in
accordance with the methods described herein.
A "reference gene expression profile" as used herein, is intended
to indicate the gene expression profile, as defined above, for a pre
selected group which is useful for comparison to the gene expression
profile of a subject of interest. For example, the reference gene
expression profile may be the gene expression profile of a single
individual known to not have an metabolic syndrome disorder
phenotype or a propensity thereto (i.e. a "normal" subject) or the gene
expression profile represented by a collection of RNA samples from
"normal" individuals that has been processed as a single sample. The
"reference gene expression profile" may vary and such variance will
be readily appreciated by one of ordinary skill in the art.
The phrase "reference standard" as used herein may refer to the
phrase "reference gene expression profile" or may more broadly
encompass any suitable reference standard which may be used as a
basis of comparison with respect to the measured variable. For
example, a reference standard may be an internal control, the gene
expression or a gene product of a "healthy" or "normal" subject, a
housekeeping gene, or any unregulated gene or gene product. The
phrase is intended to be generally non-limiting in that the choice of a
reference standard is well within the level of skill in the art and is
understood to vary based on the assay conditions and reagents
available to one using the methods disclosed herein.
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"Gene expression profiling" as used herein, refers to any method that
can analyze the expression of selected genes in selected samples.
The phrase "gene expression system" as used herein, refers to any
system, device or means to detect gene expression and includes
diagnostic agents, candidate libraries, oligonucleotide sets or probe
sets.
The terms "diagnostic oligonucleotide" or "diagnostic
oligonucleotide set" generally refers to an oligonucleotide or to a set
of two or more oligonucleotides that, when evaluated for differential
expression their corresponding diagnostic genes, collectively yields
predictive data.
Such predictive data typically relates to diagnosis, prognosis, selection
of therapeutic agents, monitoring of therapeutic outcomes, and the
like. In general, the components of a diagnostic oligonucleotide or a
diagnostic oligonucleotide set are distinguished from oligonucleotide
sequences that are evaluated by analysis of the DNA to directly
determine the genotype of an individual as it correlates with a
specified trait or phenotype, such as a disease, in that it is the pattern
of expression of the components of the diagnostic oligonucleotide set,
rather than mutation or polymorphism of the DNA sequence that
provides predictive value. It will be understood that a particular
component (or member) of a diagnostic oligonucleotide set can, in
some cases, also present one or more mutations, or polynnorphisnns
that are amenable to direct genotyping by any of a variety of well
known analysis methods, e.g., Southern blotting, RFLP, AFLP, SSCP,
SNP, and the like.
The phrase "gene amplification" refers to a process by which multiple
copies of a gene or gene fragment are formed in a particular cell or cell
line. The duplicated region (a stretch of amplified DNA) is often
referred to as "annplicon." Usually, the amount of the messenger RNA
(nnRNA) produced, i.e., the level of gene expression, also increases in
the proportion of the number of copies made of the particular gene
expressed.
A "gene expression system" refers to any system, device or means
to detect gene expression and includes diagnostic agents, candidate
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libraries oligonucleotide, diagnostic gene sets, oligonucleotide sets,
array sets, or probe sets.
As used herein, a "gene probe" refers to the gene sequence arrayed
on a substrate.
As used herein, a "nucleotide probe" refers to the oligonucleotide,
DNA fragment, polynucleotide sequence arrayed on a substrate.
The terms "splicing" and "RNA splicing" are used interchangeably and
refer to RNA processing that removes introns and joins exons to
produce mature nnRNA with continuous coding sequence that moves
into the cytoplasm of a eukaryotic cell.
"Stringency" of hybridization reactions is readily determinable by one
of ordinary skill in the art, and generally is an empirical calculation
dependent upon probe length, washing temperature, and salt
concentration. In general, longer probes require higher temperatures
for proper annealing, while shorter probes need lower temperatures.
Hybridization generally depends on the ability of denatured DNA to re-
anneal when complementary strands are present in an environment
below their melting temperature. The higher the degree of desired
homology between the probe and hybridizable sequence the higher is
the relative temperature which can be used. As a result, it follows that
higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so. For
additional details and explanation of stringency of hybridization
reactions, see Ausubel et al., Current Protocols in Molecular Biology,
Wiley Interscience Publishers, (1995) and in Current Protocols in
Molecular Biology Copyright 2007 by John Wiley and Sons, Inc.,
2008.
As used herein, a "gene target" refers to the sequence derived from a
biological sample that is labeled and suitable for hybridization to a
gene probe affixed on a substrate and a "nucleotide target" refers to
the sequence derived from a biological sample that is labeled and
suitable for hybridization to a nucleotide probe affixed on a substrate.
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The term "treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in whom
the disorder is to be prevented. The practice of the present invention
will employ, unless otherwise indicated, conventional techniques of
molecular biology (including recombinant techniques), microbiology,
cell biology and biochemistry, which are within the skill of the art.
"Adiponectin" is a protein hormone that modulates a number of
metabolic processes, including glucose regulation and fatty acid
catabolism 42. Adiponectin is exclusively secreted from adipose tissue
into the bloodstream and is very abundant in plasma relative to many
hormones. Levels of the hormone are inversely correlated with body
fat percentage in adults, 43 while the association in infants and young
children is less clear. The hormone plays a role in the suppression of
the metabolic derangements that may result in type 2 diabetes, 42,43
non-alcoholic fatty liver disease (NAFLD) and an independent risk
factor for metabolic syndrome 44. Adiponectin is secreted into the
bloodsteann where it accounts for approximately 0.01% of all plasma
protein at around 5-10 pg/nnL. Plasma concentrations reveal a sexual
dimorphism, with females having higher levels than males. Levels of
adiponectin are reduced in diabetics compared to non-diabetics.
Weight reduction significantly increases circulating levels 45.
Adiponectin automatically self-associates into larger structures.
Initially, three adiponectin molecules form together a honnotrinner. The
trinners continue to self-associate and form hexanners or dodecanners.
Like the plasma concentration, the relative levels of the higher-order
structures are sexually dimorphic, where females have increased
proportions of the high-molecular weight forms. Adiponectin exerts
some of its weight reduction effects via the brain. This is similar to the
action of leptin, 46 but the two hormones perform complementary
actions, and can have additive effects.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Monocytes/macrophages in obesity and obesity-
associated metabolic disorders. Our target cells are nnonocytes
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since they are readily accessible (blood) and their activation
constitutes a reservoir of inflammatory cells that infiltrate in tissues
(adipose, aortic and cardiac tissues) where they actively induce
oxidative stress, inflammation, and cell death and thereby induce
insulin resistance, atherosclerosis, and heart failure.
Figure 2: An overview of miRNAs deregulated in monocytes of
obese subjects and predicted to be involved in regulating key
molecules in the IRAK3-related pathway associated with
increased inflammation and oxidative stress and impaired
insulin signaling and sensitivity. nniR-30a, -101, -103, -126, -
130b, -146b-5p, -151-5p, -181a, -181b, -181d, and -335 are all
decreased in circulating monocytes of obese subjects. Flow of the
pathways at the protein interaction level is indicated by black arrows.
Blunted arrows indicate inhibition and dashed arrows indicate
translocation of transcription factors NFKB and FOX03A to the nucleus.
Phosphorylation is indicated by a circled P. Abbreviations: nniR,
nnicroRNA; ROS, reactive oxygen species.
Figure 3: Expression profiles of 31 candidate miRNAs in
circulating monocytes of obese and lean subjects. Left 2 bars of
each panel show nniRNA levels determined by nniRNA nnicroarray in 6
lean controls and 10 obese subjects. Right 2 bars show levels of the
same nniRNA now validated by qRT-PCR in an extended population (14
lean controls and 21 obese subjects). Data shown are means SEM.
*P < 0.05, **P < 0.01 and ***P < 0.001 obese compared with lean
controls.
Figure 4: miRNAs differentially expressed in monocytes of
obese persons after short-term weight loss. nniRNA levels as
determined by qRT-PCR in 14 lean controls and 21 obese subjects
before and after weight loss. Data shown are means SEM. **P < 0.01
obese compared with lean controls; $P < 0.05 and $$P < 0.01 obese
after weight loss compared with before.
Figure 5: miRNAs differentially expressed in inflammation
associated cell experiments. nniRNA levels as determined by qRT-
PCR in (A) /RAK3-depleted THP-1 cells (n = 4), (B) THP-1 cells
exposed to 10 ng/nnl IL-6 (n = 6) and (C) THP-1 cells exposed to 1
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[tg/nnl gAcrp30 (n = 6). Data shown are means SEM. *P < 0.05, **P
< 0.01 and ***P < 0.001 compared with THP-1 cells transfected with
negative control siRNA, THP-1 control cells or THP-1 cells exposed to
[tg/nnl gAcrp30. Abbreviation: gAcrp30, globular adiponectin.
5 Figure 6: miRNAs differentially expressed in oxidative stress
associated cell experiments. nniRNA levels as determined by qRT-
PCR in THP-1 cells exposed to 10 [tg/nnl ox-LDL (n = 6). Data shown
are means SEM. *P < 0.05 and **ID < 0.01 compared with THP-1
control cells. Abbreviation: ox-LDL, oxidized LDL.
10
Figure 7: miRNAs differentially expressed in insulin resistance
associated cell experiments. nniRNA levels as determined by qRT-
PCR in THP-1 exposed to 10-7 M insulin and 15 nnM glucose (n = 6).
Data shown are means SEM. *P < 0.05 and **P < 0.01 compared
with THP-1 control cells.
Figure 8: miRNA decision tree. Microarray analysis identified a total
of 133 miRNAs that were differentially expressed in circulating
nnonocytes of obese patients compared with lean controls. To gain
insight into this nniRNA expression profile, a bioinfornnatic analysis was
performed. The in silico analysis identified 31 miRNAs with potential
targets in the /RAK3-related gene cluster. The expressions of 18
miRNAs were validated by qRT-PCR in an extended population. The
expression profiles of the 18 miRNAs-of-interest were determined after
short-term weight loss and in inflammation, oxidative stress and
insulin resistance associated cell experiments. We selected 11 miRNAs
based on these expression profiles (depicted in bold). Two of the 11
miRNAs were associated with the occurrence of the metabolic
syndrome, one nniRNA was associated with the occurrence of
cardiovascular risk equivalents (being a Framingham cardiovascular
risk score above 10% per 10 years or type 2 daibetes) and 3 miRNAs
were associated with angiographically documented coronary artery
disease.
*miRNAs are still associated with occurrence of metabolic syndrome,
cardiovascular risk equivalents or coronary artery disease even after
adjusted for other risk factors
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Figure 9: Effect of miRNA inhibitors on inflammation, oxidative
stress and insulin resistance in vitro. Gene expression was
analyzed using qRT-PCR and nnROS production was determined by flow
cytonnetry in THP-1 cells (n = 6) transfected with miRNA inhibitors
against (A) nniR-130b, (B) nniR-151-5p, (C) nniR-103, (D) nniR-156b-
5p, (E) nniR-335, (F) nniR-181d, (G) nniR-126 and (H) nniR-181b.
Data shown are means SEM. **p < 0.01 and ***P < 0.001 compared
with THP-1 cells transfected with negative control miRNA.
Abbreviation: nnROS, mitochondrial reactive oxygen species.
Figure 10: Effect of miRNA mimics on inflammation, oxidative
stress and insulin resistance in vitro. (A) IRAK3, (B) TNFa and
(C) IRSI expression was analyzed using qRT-PCR and (D) iROS
production was determined by flow cytonnetry in THP-1 cells (n = 6)
transfected with nniR-30a, nniR-151-5p, nniR-181d and nniR-335
mimics. Data shown are means SEM. *p < 0.05 and **p < 0.01
compared with THP-1 cells transfected with negative control miRNA.
Abbreviation: iROS, intracellular reactive oxygen species.
Figure 11: miR-146b-5p is an essential mediator of the anti-
inflammatory, antioxidative stress and insulin-sensitizing
actions of globular adiponectin. THP-1 cells were exposed to 10
[tg/nnl gAcrp30 with (n = 4) and without (n = 6) inhibition of nniR-
146b-5p. Gene expression was analyzed by measuring relative RNA
levels using qRT-PCR and ROS production was determined by flow
cytonnetry. Data shown are means SEM. **p < 0.01 and ***p < 0.001
compared with THP-1 cells exposed to 10 [tg/nnl gAcrp30.
Abbreviations: gAcrp30, globular adiponectin, iROS, intracellular ROS;
nnROS, mitochondria! ROS; ROS, reactive oxygen species.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
The following detailed description of the invention refers to the
accompanying drawings. Also, the following detailed description does
not limit the invention. Instead, the scope of the invention is defined
by the appended claims and equivalents thereof.
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The risk for developing heart disease is directly related to the
concomitant burden of obesity-related cardiovascular risk factors
clustered in the metabolic syndrome (MetSyn) 8: dyslipidennia (i.e.
high triglycerides and low HDL-cholesterol), hypertension, and type 2
diabetes.
Persons with the metabolic syndrome (MetSyn) are at increased risk of
developing coronary heart diseases (CHD) as well as increased
mortality from CHD and any other cause 47'48.
The Third Report of the National Cholesterol Education Program Expert
Panel on Detection, Evaluation, and Treatment of High Blood
Cholesterol in adults (ATPIII), draws attention to the importance of
MetSyn and provides a working definition of this syndronne49.
Findings from the Third National Health and Nutrition Examination
Survey showed that MetSyn is highly prevalent in the United States.
Its prevalence has increased from 6.7% among participants aged 20 to
29 years, to 43.5% and 42.0% for participants aged 60 to 69 years
and aged at least 70 years, respectively 3.
Over 75% of hypertension cases are reported to be directly
attributable to obesity, and the risk of developing hypertension is five
to six times greater in obese adult Americans age 20 to 45 compared
to non¨obese individuals of the same age. Obesity and insulin
resistance, and the interaction between these two components, are
associated with a high cardiovascular risk 8851. As many as 90% of
individuals with type 2 diabetes are overweight or obese. Obesity-
related type 2 diabetes is a leading cause of morbidity and mortality in
western societies, and is quickly approaching pandemic proportions 82.
In addition to heart disease, obesity is reported to increase the risk of
ischennic stroke independent of other risk factors, including age and
systolic blood pressure. The incidence of osteoarthritis increases with
BMI and is associated with arthritis of the hand, hip, back and, in
particular, the knee. Increased weight adds stress to bones and joints
due to increased load. Lastly, there is evidence that some cancers
(endonnetrial, breast and colon) are associated with obesity.
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Although obesity and insulin resistance, and the interaction between
these two components, are associated with a high cardiovascular risk
5051, the severity of insulinennia and glycaennia during the diabetic
phase can only to a minor extent explain this increased cardiovascular
risk.
A possible pathogenic mechanism which links obesity with type-2
diabetes and with cardiovascular risk is nnonocyte activation. Indeed,
obesity is associated with increased infiltration in the adipose tissue of
activated nnonocytes/nnacrophages that also produce inflammatory
chennokines 23.
Increased oxidative stress causes increased activated nnonocyte
infiltration and is an early instigator of MetSyn. Several findings
support this hypothesis. As an example, we demonstrated that MetSyn
is associated with elevated levels of circulating oxidized LDL (oxLDL), a
marker of oxidative stress. High triglycerides, low HDL-cholesterol, and
high glucose and insulin predicted elevated levels of oxLDL
independent of LDL-cholesterol levels. The association between
MetSyn and elevated levels of oxLDL has been confirmed in European
and Japanese cohorts 53-55. Persons with high oxLDL levels showed a
greater disposition to myocardial infarction, adjusting for all
established cardiovascular risk factors 18-21,56 Two other studies
confirmed that elevated levels of circulating oxLDL predict future
cardiovascular events even after adjustment for traditional
cardiovascular risk factors and C-reactive protein 5758. Recently, we
have shown that persons with high oxLDL showed a 4.5-fold greater
disposition to future MetSyn after 5 years follow-up, adjusted for age,
gender, race, study centre, cigarette smoking, BMI, physical activity,
and LDL-cholesterol, little changed by further adjustment for C-
reactive protein, and adiponectin. In particular, oxLDL predicted the
development of obesity, dyslipidennia and pre-diabetes. Several studies
showed that oxLDL can induce the activation of nnonocytes as
evidenced by increased capacity of nnonocytes to infiltrate vascular
tissues in response to oxLDL-induced nnonocyte chennoattractant
protein-1 by endothelial cells, by the oxLDL-induced activation of toll-
like repceptor (TLR)-2 and 4-mediated pro-inflammatory response
resulting in production of inflammatory cytokines, by the 0xLDL-
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induced NF-KB activation and by the oxLDL-induced mitochondrial
dysfunction resulting in a further enehancennent of ROS production 59.
We hypothesized that the identification of a cluster of genes and
associated proteins which are associated with nnonocyte/nnacrophage
activation, as evidenced by their inflammation and oxidative stress
state, and of which the expression pattern is improved by weight loss
that significantly reduces cardiovascular risk could lead to a better
estimate of the risk for cardiovascular disease for obese persons. We
started from the observation in obese miniature pigs on an atherogenic
diet that toll-like receptor 2 (TLR2) was over expressed in plaque
macrophages isolated by laser capture micro dissection and correlated
with atherosclerotic plaque complexity 60. Then, we performed micro
array analysis of RNA extracted from nnonocytes of obese women.
Because we found that TLR2 was over expressed, we searched for
genes that correlated with TLR2. Structural modeling predicted a
cluster of genes that besides TLR2 contains the following genes and
associated proteins: IL1 receptor-associated kinase 3 (IRAK3), Tumor
Necrosis Factor (TNF)-Associated Factor 6 (TRAF6), the myeloid
differentiation marker MYD88, TNF-alpha-induced protein 3 and 6
(TNFAIP3; TNFAIP6), the Insulin Receptor Substrate 2 (IR52),
nnitogen-activated protein kinase 13 (MAPK13), the Forkhead Box 03A
(FOX03A), and superoxide disnnutase 2 (50D2). These genes and
associated proteins form the backbone of a pathway that links the toll-
like receptor-mediated inflammation with the protection against
oxidative stress by means of 50D2. Earlier (W02009121152) we
presented evidence that some of these predicted molecules indeed are
novel (bio)nnarkers of cardiovascular risk in association with obesity,
lipid homeostasis disorder related cardiovascular disease and/or an
impaired glucose tolerance condition and that some are even causal
bionnarkers. Especially, low expression was found to be associated with
high prevalence of metabolic syndrome and high cardiovascular risk.
Description of the molecules
Toll-like receptors, nuclear factor NF-kappa-B, tumor necrosis factor
alpha, and al receptor-associated kinases
The Toll like /Interleukin 1 receptor family consists of a large number
of transnnennbrane proteins which are involved in host defense and
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have conserved intracellular domains. This superfannily is divided into
2 subgroups based on the components of the extracellular domains:
the Toll like receptors (TLRs) with leucine-rich repeats, and the
Interleukin-1 receptors (IL1R5) with innnnunoglobulin-like motifs.
TOLL-LIKE RECEPTOR 2; TLR2
Aliprantis et al. (1999) found that bacterial lipoproteins induced
apoptosis in THP-1 nnonocytic cells through human TLR2 61. In
addition, bacterial lipoproteins also initiated apoptosis in an epithelial
cell line transfected with TLR2. Bacterial lipoproteins stimulated NF-
kappa-B, a transcriptional activator of multiple host defense genes,
and activated the respiratory burst through TLR2. Thus, TLR2 is a
molecular link between microbial products, apoptosis, and host
defense mechanisms. Aderenn and Ulevitch (2000) reviewed the role of
TLRs in innate immunity 62. Whereas lipopolysaccharide (LPS) activates
cells through TLR4, gram-positive cell-wall components, including
peptidoglycan and lipoteichoic acid, as well as nnycobacterial cell-wall
components such as lipoarabinonnannan and nnycolylarabinogalactan,
and yeast cell-wall zynnosan, activate cells via TLR2. Takeuchi et al.
(2000) showed that TIr2- and, particularly, Myd88-deficient mice are
highly susceptible, in terms of growth in blood and kidney and
decreased survival, to infection with Staphylococcus aureus compared
to wildtype mice 63. In vitro, TIr2-deficient macrophages produced
reduced TNF and IL6 in response to S. aureus compared to wildtype or
TIr4-deficient macrophages, whereas Myd88-deficient macrophages
produced no detectable TNF or IL6. The authors concluded that TLR2
and MYD88 are critical in the defense against gram-positive bacteria.
Shishido et al. (2003) found that TIr2-deficient mice survived longer
than wildtype mice after induced myocardial infarction 64. There was
no difference in inflammation or infarct size between knockout mice
and wildtype mice. They concluded that TLR2 plays an important role
in ventricular remodeling after myocardial infarction. In
atherosclerosis-susceptible Ldlr (606945)-null mice, Mu!lick et al.
(2005) demonstrated that complete deficiency of TIr2 led to a
reduction in atherosclerosis whereas intraperitoneal injection of a
synthetic TLR2/TLR1 agonist dramatically increased atherosclerosis 65.
In Ldlr-null mice, transplantation of TIr2 -/- bone marrow (BM) cells
had no effect on atherosclerosis, suggesting the presence of an
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endogenous TLR2 agonist activating TLR2 in cells that were not of BM
cell origin. In Ldlr-null mice, complete deficiency of TIr2 as well as a
deficiency of TIr2 only in BM-derived cells led to striking protection
against agonist-mediated atherosclerosis, suggesting a role for BM-
derived cell expression of TLR2 in transducing the effects of an
exogenous TLR2 agonist. They stated that these findings support the
concept that chronic or recurrent microbial infections contribute to
atherosclerotic disease and also suggest the presence of host-derived
endogenous TLR2 agonists.
The Homo sapiens toll-like receptor (TLR2) nnRNA has been deposited
in the NCBI database under the accession number NG_016229 (28803
bp nnRNA linear PRI 05-NOV-2010) with the nucleotide sequence as in
sequence ID 1.
The Homo sapiens toll-like receptor 2 (TLR2) protein has been
deposited in the NCBI database under the accession number
AAH33756 VERSION AAH33756.1 (784 aa PRI 07-OCT-2003) with the
amino acid sequence depicted in sequence ID 2.
Nuclear factor of kappa light polypeptide gene enhancer in B-
cells 1 (NFKB1)
NFKB has been detected in numerous cell types that express
cytokines, chennokines, growth factors, cell adhesion molecules, and
some acute phase proteins in health and in various disease states.
NFKB is activated by a wide variety of stimuli such as cytokines,
oxidant-free radicals, inhaled particles, ultraviolet irradiation, and
bacterial or viral products. Inappropriate activation of NFKB has been
linked to inflammatory events associated with autoinnnnune arthritis,
asthma, septic shock, lung fibrosis,
glonnerulonephritis,
atherosclerosis, and AIDS. In contrast, complete and persistent
inhibition of NFKB has been linked directly to apoptosis, inappropriate
immune cell development, and delayed cell growth. For reviews, see
Chen et al. (1999) 66 and Baldwin (1996) 67. Yang et al. (2004)
observed that after hyperoxic exposure, neonatal mice showed
increased Nfkb binding, whereas adult mice did not 68. Neonatal
Nfkb/luciferase transgenic mice demonstrated enhanced in vivo Nfkb
activation after hyperoxia. Inhibition of Nfkbia resulted in decreased
BcI2 protein levels in neonatal lung homogenates and decreased cell
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viability in lung primary cultures after hyperoxic exposure. In addition,
neonatal Nfkb1-null mice showed increased lung DNA degradation and
decreased survival in hyperoxia compared with wildtype mice. They
concluded that there are maturational differences in lung NFKB
activation and that enhanced NFKB may serve to protect the neonatal
lung from acute hyperoxic injury via inhibition of apoptosis. In
transgenic mice deficient for the LDL receptor (LdIr) and with a
macrophage-restricted deletion of Ikbkb, an activator of NFKB, Kanters
et al. (2003) found an increase in atherosclerosis, as characterized by
increased lesion size, more lesions, and necrosis 69. In vitro studies
showed that Ikbkb deletion in macrophages resulted in a reduction of
TNF and the antiinflannnnatory cytokine 1110. The findings suggested
that inhibition of the NFKB pathway affects the pro- and
antiinflannnnatory balance that controls the development of
atherosclerosis.
The nnRNA of Homo sapiens nuclear factor NF-kappa-B p105 subunit
(NFKB1), isofornn 1has been deposited in the NCBI database under the
accession number NM_003998 4093 bp nnRNA linear (PRI 23-JAN-
2011) with the nnRNA nucleotide sequence as in sequence ID 3. The
protein of NFKB1, isofornn 1 has been deposited in the NCBI database
under the accession number NP_003989 969 aa linear (PRI 23-JAN-
2011) with the amino acid sequence depicted in sequence ID 4. The
nnRNA of Homo sapiens nuclear factor NF-kappa-B p105 subunit
(NFKB1), isofornn 2 has been deposited in the NCBI database under
the accession number NM_001165412 4090 bp nnRNA linear (PRI 22-
JAN-2011) with the nnRNA nucleotide sequence as in sequence ID 5.
The protein of NFKB1, isofornn 2 has been deposited in the NCBI
database under the accession number NP_001158884 968 aa linear
(PRI 22-JAN-2011) as depicted in sequence ID 6.
Nuclear factor of kappa light polypeptide gene enhancer in B-
cells inhibitor alpha (NFKBIA or IKBa)
The NFKB complex, a master regulator of proinflannnnatory responses,
is inhibited by NFKBIA proteins, which inactivate NFKB by trapping it in
the cytoplasm. Phosphorylation of serine residues on the NFKBIA
proteins by kinases (IKK1 or IKK2) marks them for destruction via the
ubiquitination pathway, thereby allowing activation of the NFKB
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complex. Nenci et al. (2007) demonstrated that the transcription factor
NFKB functions in gut epithelial cells to control epithelial integrity and
the interaction between the mucosal immune system and gut
nnicroflora 7 . Intestinal epithelial-specific inhibition of NFKB through
conditional ablation of NEMO or both IKK1 and IKK2, IKK subunits
essential for NFKB activation, spontaneously caused severe chronic
intestinal inflammation in mice. NFKB deficiency led to apoptosis of
colonic epithelial cells, impaired expression of antimicrobial peptides,
and translocation of bacteria into the mucosa. Concurrently, this
epithelial defect triggered a chronic inflammatory response in the
colon, initially dominated by innate immune cells but later also
involving T lymphocytes. Deficiency of the gene encoding the adaptor
protein MyD88 prevented the development of intestinal inflammation,
demonstrating that Toll-like receptor activation by intestinal bacteria is
essential for disease pathogenesis in this mouse model. Furthermore,
NEMO deficiency sensitized epithelial cells to TNF-induced apoptosis,
whereas TNF receptor-1 inactivation inhibited intestinal inflammation,
demonstrating that TNFR1 signaling is crucial for disease induction.
They concluded that a primary NFKB signaling defect in intestinal
epithelial cells disrupts immune homeostasis in the gastrointestinal
tract, causing an inflammatory bowel disease-like phenotype. Their
results further identified NFKB signaling in the gut epithelium as a
critical regulator of epithelial integrity and intestinal immune
homeostasis and have important implications for understanding the
mechanisms controlling the pathogenesis of human inflammatory
bowel disease. Cytokine signaling is thought to require assembly of
nnulticonnponent signaling complexes at cytoplasmic segments of
membrane-embedded receptors, in which receptor-proximal protein
kinases are activated. Matsuzawa et al. (2008) reported that, upon
ligation, CD40 formed a complex containing adaptor molecules TRAF2
and TRAF3, ubiquitin-conjugating enzyme UBC13, cellular inhibitor of
apoptosis protein-1 and -2, IKK-gamma, and MEKK1. TRAF2, UBC13,
and IKK-gamma were required for complex assembly and activation of
MEKK1 and MAP kinase cascades 71. However, the kinases were not
activated unless the complex was translocated from the membrane to
the cytosol upon CIAP1/CIAP2-induced degradation of TRAF3. They
proposed that this 2-stage signaling mechanism may apply to other
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innate immune receptors and may account for spatial and temporal
separation of MAPK and IKK signaling
The nnRNA of Homo sapiens NF-kappa-B inhibitor alpha (NFKBIA) has
been deposited in the NCBI database under the accession number
NG_007571.1 1579 bp nnRNA linear (PRI 25-DEC-201) with the nnRNA
nucleotide sequence as in sequence ID 7. The protein of NFKBIA has
been deposited in the NCBI database under the accession number
NP_065390 317 aa linear (PRI 25-DEC-2010) with the amino acid
sequence depicted in sequence ID 8.
Tumor necrosis factor alpha (TNF-a)
Tumor necrosis factor (TNF) is a multifunctional proinflannnnatory
cytokine secreted predominantly by nnonocytes/nnacrophages that has
effects on lipid metabolism, coagulation, insulin resistance, and
endothelial function. TNF was originally identified in mouse serum after
injection with Mycobacterium bovis strain bacillus Calnnette-Guerin
(BCG) and endotoxin. Serum from such animals was cytotoxic or
cytostatic to a number of mouse and human transformed cell lines and
produced hemorrhagic necrosis and in some instances complete
regression of certain transplanted tumors in mice 72'73. Kamata et al.
(2005) found that TNF-a-induced reactive oxygen species (ROS),
whose accumulation could be suppressed by mitochondrial superoxide
disnnutase (50D2; 147460), caused oxidation and inhibition of JNK
(see 601158)-inactivating phosphatases by converting their catalytic
cysteine to sulfenic acid 74. This resulted in sustained INK activation,
which is required for cytochronne c release and caspase-3 cleavage, as
well as necrotic cell death. Treatment of cells or experimental animals
with an antioxidant prevented H202 accumulation, INK phosphatase
oxidation, sustained INK activity, and both forms of cell death.
Antioxidant treatment also prevented TNF-a-mediated fulnninant liver
failure without affecting liver regeneration. Zinman et al. (1999)
studied the relationship between TNF-a and anthroponnetric and
physiologic variables associated with insulin resistance and diabetes in
an isolated Native Canadian population with very high rates of NIDDM
(125853) 75. Using the homeostasis assessment (HOMA) model to
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estimate insulin resistance, they found moderate, but statistically
significant, correlations between TNF-a and fasting insulin, HOMA
insulin resistance, waist circumference, fasting triglycerides, and
systolic blood pressure; in all cases, coefficients for females were
stronger than those for males. The authors concluded that in this
homogeneous Native Canadian population, circulating TNF-a
concentrations are positively correlated with insulin resistance across a
spectrum of glucose tolerance. The data suggested a possible role for
TNF-a in the pathophysiology of insulin resistance.
The Homo sapiens tumor necrosis factor alpha (TNF-a) nnRNA has
been deposited in the NCBI database under the accession number _
NG_007462 (9763 bp bp nnRNA linear PRI 13-NOV-2010) with the
nucleotide sequence as in sequence ID 9. The Homo sapiens tumor
necrosis factor alpha (TNF-a) protein has been deposited in the NCBI
database under the accession number AC037640 VERSION
AC037640.1 (232 aa PRI 06-APR-2009) with the amino acid sequence
depicted in sequence ID 10.
IL1 Receptor-Associated Kinase 1 (IRAK1)
Signal transduction pathways in these receptor families ultimately lead
to activation of members of the Rel and AP1fannily of transcription
factors. An important mediator in this pathway is IL1 Receptor-
Associated Kinase 1 (IRAK1). Using a nnurine EST sequence encoding
a polypeptide with significant homology to IRAK1 to screen a human
phytohennagglutinin-activated peripheral blood leukocyte (PBL) cDNA
library, Wesche et al. 76 isolated a full-length cDNA clone encoding a
596-amino acid protein. Sequence analysis revealed an overall
sequence similarity of 30 to 40% with IRAK1 and IRAK2 as well as
structural similarity in an N-terminal death domain and a central
kinase domain. Kanakaraj et al. 77 determined that Irak-deficient mice
had impaired responses to interleukin-18 (IL18, as measured by JNK
and NFKB activation. They also noted a severe impairment in gamma-
interferon (IFNG) production and the induction of natural killer cell
cytotoxicity by IL18. Infection with nnurine cytonnegalovirus showed
that IRAK is essential for IFNG production but not for IL18 expression
or NK cell cytotoxicity, which may be compensated for by IFNA/IFNB.
Thomas et al. 78 noted that Irak-deficient mice were viable and fertile.
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They observed diminished NFKB activation in fibroblasts from Irak
knockout mice when stimulated with IL1. NFKB activation in response
to TNF was unimpaired. Treatment of splenocytes with IL12 alone or in
combination with IL18, but not with IL18 alone, resulted in the
production of normal amounts of IFNG. On the other hand, Irak
deletion did not impair delayed-type hypersensitivity responses or cell-
mediated immunity to infection with the intracellular bacterium Listeria
nnonocytogenes. Jacob et al. 79 found that absence of Irak1 in mice
significantly attenuated the serologic and cellular immunologic
phenotypes independently attributed to the Sle1 and S1e3
susceptibility loci for systemic lupus erythennatosus (SLE) in mice.
The Homo sapiens interleukin-1 receptor-associated kinase 1 (IRAK1)
nnRNA has been deposited in the NCBI database as under the
accession number ACCESSION NM_001025242.1 (LOCUS:
NM_001025242 3499bp nnRNA linear PRI 04-AUG-2010) with the
nucleotide sequence as in sequence ID 11. The Homo sapiens
interleukin-1 receptor-associated kinase 1 (IRAK1) protein has been
deposited in the NCBI database as under the accession number
ACCESSION AAH54000 VERSION AAH54000.1 (LOCUS AAH54000 693
aa linear PRI 07-OCT-2003) with the amino acid sequence depicted in
sequence ID 12.
IL1 Receptor-Associated Kinase 3 (IRAK3)
Another IRAK was found to be predominantly expressed in PBL and the
nnonocytic cell lines U937 and THP-1, in contrast to the other IRAKs
that are expressed in most cell types. Because of the restriction of
expression of this IRAK to nnonocytic cells, the authors termed the
protein IRAKM, now called IRAK3. The IRAK3 (or IRAKM or
Interleukin-1 Receptor-Associated Kinase 3 or Interleukin-1 Receptor-
Associated Kinase M) gene consists of 12 exons spanning a region of
approximately 60 kb in chromosome 12q14.3 80. Like IRAK2, the
expression of IRAK3 in THP-1 cells is upregulated in the presence of
phorbol ester and iononnycin, which also induce differentiation of these
cells into more mature macrophages 76. IRAK-3 (IRAK-M) is a member
of the interleukine-1 receptor-associated kinase (IRAK) family. The
IRAK family is implicated in the Toll-like receptor (TLR) and II-1R
signaling pathway. IRAK3 interacts with the myeloid differentiation
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(MYD) marker MYD88 and TRAF6 signaling proteins in a manner
similar to the other IRAKs. However, Kobayashi et al. 81 showed that
IRAK3, in contrast to other IRAKs, is induced upon TLR stimulation but
negatively regulates TLR signaling. IRAK3 -/- cells exhibited increased
cytokine production upon TLR/IL1 stimulation and bacterial challenge,
and Iraknn -/- mice showed increased inflammatory responses to
bacterial infection. Endotoxin tolerance, a protection mechanism
against endotoxin shock, was significantly reduced in IRAKM -/- cells.
Thus, the authors concluded that IRAK3 regulates TLR signaling and
innate immune homeostasis. Data with IRAK-M knockout mice have
revealed that IRAK-M serves as a negative regulator of IL-1R/TLR
signaling. Moreover IRAK-M expression is mainly restricted to cells of a
myeloid origin.
The Homo sapiens interleukin-1 receptor-associated kinase 3 (IRAK3)
nnRNA has been deposited in the NCBI database as under the
accession number ACCESSION NM_007199, VERSION NM_007199.2
(LOCUS: NM_007199 8351 bp nnRNA linear PRI 03-AUG-2010) with
the nucleotide sequence as in sequence ID 13. The Homo sapiens
interleukin-1 receptor-associated kinase 3 (IRAK3) protein has been
deposited in the NCBI database as under the accession number
NP_009130 ACCESSION VERSION NP_009130.2 GI:216547519
(LOCUS NP_009130 596 aa linear PRI 03-AUG-2010) with the amino
acid sequence as in sequence ID 14.
ILI. Receptor-Associated Kinase 4 (IRAK4)
IRAK4 is another kinase that activates NF-kappaB in both the Toll-like
receptor (TLR) and T-cell receptor (TCR) signaling pathways 82-85. The
protein is essential for most innate immune responses. Mutations in
this gene result in IRAK4 deficiency and recurrent invasive
pneunnococcal disease. Multiple transcript variants encoding different
isofornns have been found for this gene. The transcript variant 1
represents the longest transcript. Variants 1 and 2 both encode the
same isofornn A. Variants 3, 4, and 5 all encode the same isofornn B.
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This variant (4) lacks two alternate exons and uses a downstream start
codon, compared to variant 1. The resulting isofornn B, also known as
the short form, has a shorter N-terminus, compared to isofornn A.
The Homo sapiens interleukin-1 receptor-associated kinase 4 (IRAK4)
nnRNA has been deposited in the NCBI database as under the
accession number ACCESSION M_001114182, VERSION
NM_001114182.2 (LOCUS: NM_001114182 4351 bp nnRNA linear PRI
05-AUG-2010) with the nucleotide sequence as in sequence ID 15. The
Homo sapiens interleukin-1 receptor-associated kinase 4 (IRAK4)
protein has two isofornns A and B. Isofornn A been deposited in the
NCBI database as under the accession number NP_057207
ACCESSION VERSION
NP_057207.2 GI: 166795293 (LOCUS
NP_057207 460 aa linear PRI 01-AUG-2010) with the amino acid
sequence as in sequence ID 16. Isofornn B been deposited in the NCBI
database as under the accession number NP_001138729 ACCESSION
VERSION NP_001138729.1 (336 aa linear PRI 05-AUG-2010) with the
amino acid sequence as in sequence ID 17.
TNF-alpha-induced protein 6 (TNFAIP6)
Lee et al. 86 described a gene, which they designated TSG6 (current
name TNFAIP6), that is transcribed in normal fibroblasts and activated
by binding of TNF-alpha and IL1 at AP-1 and NF-1L6 sites in its
promoter. The cDNA was isolated from a library made from TNF-
treated human fibroblasts. TNFAIP6 is a member of the hyaluronan-
binding protein family, which includes cartilage link protein,
proteoglycan core protein, and the adhesion receptor CD44. The
predicted polypeptide is 277 amino acids long and includes a typical
cleavage signal peptide. TNFAIP6 is highly homologous to CD44,
particularly in the hyaluronic acid-binding domain. Western blots with
antibodies made to a TNFAIP6 fusion protein detected a 39-kD
glycoprotein in TNF-treated cells, and hyaluronate binding was shown
by co-precipitation. TNFAIP6 expression is rapidly activated by TNF-
alpha, IL1, and lipopolysaccharide in normal fibroblasts, peripheral
blood mononuclear cells, synovial cells, and chondrocytes.
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The nnRNA of Homo sapiens tumour necrosis factor, alpha-induced
protein 6 (TNFAIP6) has been deposited in the NCBI database under
the accession number NM 007115 VERSION NM_007115.3 (1439 bp
bp PRI 27-DEC-2010) with the nnRNA nucleotide sequence as in
sequence ID 18. The protein of Homo sapiens tumour necrosis factor,
alpha-induced protein 6 (TNFAIP6) has been deposited in the NCBI
database under the accession number CAD13434 VERSION
CAD13434.1 (277 bp PRI 07-OCT-2008) as depicted in Sequence ID
19.
Insulin Receptor Substrate 1 and 2 (IRS1 and IR52)
The Insulin Receptor Substrate 1 (IRS1) acts as an interface between
signalling proteins with Src homology-2 domains (5H2 proteins) and
the receptors for insulin, IGF2, growth hormone, several interleukins,
and other cytokines. It regulates gene expression and stimulates
nnitogenesis and appears to mediate insulin/IGF1-stimulated glucose
transport. Thus, the finding that the homozygous Irs1 KO mouse
survives with only mild resistance to hypertension was surprising. This
dilemma was provisionally resolved by the discovery by Sun et al. 87 of
a second IRS signalling protein in mouse. They purified and cloned a
likely candidate from mouse myeloid progenitor cells and, because of
its resemblance to IRS1, they designated it IR52. Withers et al. 88
demonstrated that homozygous absence of the Irs2 gene results in
type II diabetes in mice. Heterozygous and wild type animals were
unaffected. The authors demonstrated profound insulin resistance in
both skeletal muscle and liver in the homozygous Irs2 -/- mice. Male
mice lacking the Irs2 locus showed polydypsia and polyuria without
ketosis and died from dehydration and hyperosnnolar coma. A similar
disease progression was observed in female mice, with the exception
that the females rarely died. The authors concluded that dysfunction of
IR52 may contribute to the pathophysiology of human type II
diabetes. Tobe et al. 89 observed that Irs2-deficient mice showed
increased adiposity with increased serum leptin level, suggesting leptin
resistance before the mice developed diabetes. Using oligonucleotide
nnicroarray and Northern blot analyses to analyze gene expression
they detected increased expression of SREBP1, a downstream target of
insulin, in Irs2-deficient mouse liver. Using high dose leptin
administration, they provided evidence that leptin resistance in Irs2-
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deficient mice is causally related to SREBP1 gene induction. The
authors concluded that Irs2 gene disruption results in leptin resistance,
causing SREBP1 gene induction, obesity, fatty liver, and diabetes.
Taguchi et al. 90 showed that, in mice, less Irs2 signalling throughout
the body or only in brain extended life span up to 18%. At 22 months
of age, brain-specific Irs2 knockout mice were overweight,
hyperinsulinennic, and glucose intolerant; however, compared with
control mice, they were more active and displayed greater glucose
oxidation, and during meals they displayed stable SOD2 concentrations
in the hypothalamus. Thus, they concluded that less Irs2 signalling in
aging brains can promote healthy metabolism, attenuate meal-induced
oxidative stress, and extend the life span of overweight and insulin-
resistant mice.
The nnRNA of Homo sapiens insulin receptor substrate 1 (IRS1) has
been deposited in the NCBI database under the accession number
ACCESSION NG_015830 VERSION NG_015830.1 (74474 bp nnRNA
linear PRI 04-NOV-2010) with the nnRNA nucleotide sequence as in
sequence ID 20. The protein of IRS1 has been deposited in the NCBI
database under the accession number AAH53895 VERSION
AAH53895.1 (1242bp PRI 15-JUL-2006) with the amino acid sequence
depicted in sequence ID 21. The nnRNA of Homo sapiens insulin
receptor substrate 1 (IR52) has been deposited in the NCBI database
under the accession number NG_008154 VERSION NM_ NG_008154.1
(39731 bpbp nnRNA PRI 10-NOV-2010) with the nnRNA nucleotide
sequence as in sequence ID 22. The protein of IR52 has been
deposited in the NCBI database under the accession number Q9Y4H2
VERSION Q9Y4H2.2 (1338 bp PRI11-JAN-2011) as depicted in
sequence ID 23.
FOX03A
Survival factors can suppress apoptosis in a transcription-independent
manner by activating the serine/threonine kinase AKT1, which then
phosphorylates and inactivates components of the apoptotic
machinery, including BAD and caspase-9. Brunet et al. 91
demonstrated that AKT1 also regulates the activity of FKHRL1 (current
name FOX03A). In the presence of survival factors, AKT1
phosphorylates FKHRL1, leading to the association of FKHRL1 with 14-
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3-3 proteins and its retention in the cytoplasm. Survival factor
withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation,
and target gene activation. Within the nucleus, FKHRL1 most likely
triggers apoptosis by inducing the expression of genes that are critical
for cell death, such the TNF ligand superfannily 6 (TNFSF6). Nennoto
and Finkel 92 observed that exposure to intracellular ROS induced an
increase in phosphorylated Fkhr11 and a shift from a nuclear to a
cytosolic localization. They found that serum starvation, a stimulus
that increases oxidative stress, resulted in lower levels of hydrogen
peroxide in Shc1 -/- cells or in cells expressing a ser36-to-ala (536A)
Shc1 mutant compared with wild type cells. Serum starvation also
increased Fkhr11-dependent transcriptional activity, which was further
augmented in the Shc1-deficient cells. Increased ROS exposure failed
to induce increased Fkhr11 phosphorylation in the mutant cells. Essers
et al. 93 reported an evolutionarily conserved interaction of beta-
catenin with FOX() transcription factors, which are regulated by insulin
and oxidative stress signalling. In mammalian cells, beta-catenin binds
directly to FOX() and enhances FOX() transcriptional activity. In C.
elegans, loss of the beta-catenin BAR1 reduces the activity of the
FOX() ortholog DAF16 in dauer formation and life span. Association of
beta-catenin with FOX() was enhanced in cells exposed to oxidative
stress. Furthermore, BAR1 was required for the oxidative stress-
induced expression of the DAF16 target gene sod3 and for resistance
to oxidative damage. They concluded that their results demonstrated a
role for beta-catenin in regulating FOX() function that is particularly
important under conditions of oxidative stress.
The nnRNA of Homo sapiens forkhead box 03 (FOX03), transcript
variant 1,
has been deposited in the NCBI database under the
accession number NM_001455 VERSION NM_001455.3 (7341 bp PRI
14-JAN-2011) with the nnRNA nucleotide sequence as in sequence ID
24. The nnRNA of Homo sapiens forkhead box 03 (FOX03), transcript
variant 2, has been deposited in the NCBI database as under the
accession number NM_201559 VERSION
NM_201559.2 (7314 bp
PRI 16-JAN-2011) with the nnRNA nucleotide sequence as in sequence
ID 25. The protein of Homo sapiens forkhead box 03 (FOX03),
transcript 1, as presented in CD5344..2365 is depicted in sequence ID
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26. The protein of Homo sapiens forkhead box 03 (FOX03), transcript
2, as presented in CDS317..2338 is depicted in sequence 27.
Adiponectin
Adiponectin (ADPN or ACRP30) is a hormone secreted by adipocytes
that regulates energy homeostasis and glucose and lipid metabolism.
Adipocytes also produce and secrete proteins such as leptin (LEP),
adipsin (factor D), various other complement components (e.g.,
properdin) and C3a), and TNF), suggesting a possible link to the
immune system. Adiponectin, an adipose tissue-specific plasma
protein, has antiinflannnnatory effects on the cellular components of the
vascular wall 94'98.
By constructing and screening an adipose tissue cDNA library for novel
genes, Maeda et al. 96 isolated a cDNA encoding APM1, an adipose
tissue-specific collagen-like factor. Sequence analysis predicted that
the 244-amino acid secretory protein has a signal peptide but no
transnnennbrane hydrophobic stretch, and a short N-terminal
noncollagenous sequence followed by a short collagen-like motif of G-
X-Y repeats. APM1 shares significant similarity to collagen X), collagen
VIII, and complement protein C1q within the C terminus. Northern blot
analysis detected a 4.5-kb APM1 transcript in adipose tissue but not in
muscle, intestine, placenta, uterus, ovary, kidney, liver, lung, brain, or
heart.
Saito et al. 97 cloned an adipose tissue-specific gene they termed
GBP28. They stated that the GBP28 protein is encoded by the APM1
nnRNA identified by 96. By genonnic sequence analysis, Saito et al. 97
and Schaffler et al. 98 determined that the GBP28 gene spans 16 kb
and contains 3 exons, and that the promoter lacks a TATA box. By
Southern blot and genonnic sequence analyses, Das et al. 99
determined that the mouse gene, which they termed Acrp30
(adipocyte complement-related protein, 30-kD), contains 3 exons and
spans 20 kb. Using FISH, Saito et al. 97 mapped the APM1 gene to
chromosome 3q27. However, also by FISH, Schaffler et al. 98 mapped
the APM1 gene to 1q21.3-q23. By radiation hybrid analysis, Takahashi
et al. 100 confirmed that the APM1 gene maps to 3q27. Using FISH, Das
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et al. 99 mapped the mouse Acrp30 gene to chromosome 16 in a region
showing homology of synteny with human 3q27. By RNase protection
and Western blot analysis, Schaffler et al. 98 showed that APM1 is
expressed by differentiated adipocytes as a 33-kD protein that is also
detectable in serum. By sequence comparisons, they found links
between APM1 and TNF family ligands as well as to cytokines
expressed by T cells.Using cell [LISA analysis, Ouchi et al. 101
determined that the APM1 gene product, which they termed
adiponectin, suppressed TNF-induced nnonocyte adhesion to aortic
endothelial cells (HAECs), as well as expression of vascular cell
adhesion molecule-1 (VCAM1), selectin E (SELE), and intercellular
adhesion molecule-1 (ICAM1) on HAECs, in a dose-dependent manner.
These results indicated that adiponectin may attenuate the
inflammatory response associated with atherogenesis. In addition,
Ouchi et al. 95 found that plasma adiponectin values were significantly
lower in patients with coronary artery disease compared with those of
subjects matched for age and body mass index. By innnnunoblot
analysis, Ouchi et al. 102 extended these studies to show that
adiponectin suppresses TNF-induced I-kappa-B-alpha (IKBA)
phosphorylation and nuclear factor kappa-B (NFKB) activation without
affecting the interaction of TNF and its receptors or other TNF-
mediated phosphorylation signals. The inhibitory effect was
accompanied by cAMP accumulation, which could be blocked by
adenylate cyclase or protein kinase A (PKA) inhibitors. These results,
together with a finding by Arita et al. 103 that plasma adiponectin
values are low in obese subjects, suggested that adiponectin levels
may be helpful in assessing the risk for coronary artery disease.Using
hennatopoietic colony formation assays, Yokota et al. 1" showed that
adiponectin inhibited nnyelonnonocytic progenitor cell proliferation, at
least in part due to apoptotic mechanisms, at physiologic
concentrations of the protein (approximately 2.0 to 17 micrograms/I-in!
in plasma). Analysis of colony formation from CD34-positive stem cells
in the presence of a combination of growth factors showed that CFU-
GM (nnyelonnonocytic) but not BFU-E (erythrocytic) colony formation
was inhibited by adiponectin and by complement factor C1q.
Proliferation of lymphoid cell lines was not inhibited by adiponectin.
Northern blot analysis revealed that adiponectin-treated cells had
reduced expression of the antiapoptotic BCL2 gene but not of
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apoptosis-inducing factors such as BAX. Analysis of macrophage
function established that adiponectin suppresses phagocytic activity as
well as lipopolysaccharide (LPS)-induced TNF, but not interleukin-1B
(IL1B) or interleukin-6 (IL6), production and expression. Blockade of
C1QRP, a C1q receptor on macrophages, abrogated the suppression of
phagocytic function but not the inhibition of TNF production or
nnyelonnonocytic cell proliferation mediated by adiponectin. Yokota et
al. {Yokota, 2000 623 /id} suggested that adiponectin is an important
regulator of hennatopoiesis and inflammatory responses that acts
through C1QRP and other receptors. Yannauchi et al. 105 demonstrated
that phosphorylation and activation of the 5-prime-AMP-activated
protein kinase (AMPK) are stimulated with globular and full-length
adiponectin in skeletal muscle and only with full-length adiponectin in
the liver. In parallel with its activation of AMPK, adiponectin stimulates
phosphorylation of acetyl coenzyme A carboxylase (ACC1), fatty acid
oxidation, glucose uptake and lactate production in nnyocytes,
phosphorylation of ACC and reduction of molecules involved in
gluconeogenesis in the liver, and reduction of glucose levels in vivo.
Blocking AMPK activation by a dominant-negative mutant inhibits each
of these effects, indicating that stimulation of glucose utilization and
fatty acid oxidation by adiponectin occurs through activation of AMPK.
Yannauchi et al. 105 concluded that their data provided a novel
paradigm, that an adipocyte-derived antidiabetic hormone,
adiponectin, activates AMPK, thereby directly regulating glucose
metabolism and insulin sensitivity in vitro and in vivo. Yokota et al. 106
found that brown fat in normal human bone marrow contains
adiponectin and used marrow-derived preadipocyte lines and long-
term cultures to explore potential roles of adiponectin in
hennatopoiesis. Recombinant adiponectin blocked fat cell formation in
long-term bone marrow cultures and inhibited the differentiation of
cloned stronnal preadipocytes. Adiponectin also caused elevated
expression of COX2 by these stronnal cells and induced release of
prostaglandin E2. A COX2 inhibitor prevented the inhibitory action of
adiponectin on preadipocyte differentiation, suggesting involvement of
stronnal cell-derived prostanoids. Furthermore, adiponectin failed to
block fat cell generation when bone marrow cells were derived from
COX2 heterozygous mice. Yokota et al. 106 concluded that
preadipocytes represent direct targets for adiponectin action,
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establishing a paracrine negative feedback loop for fat regulation. They
also linked adiponectin to the COX2-dependent prostaglandins that are
critical in this process.
Using SDS-PAGE to analyze human and mouse adiponectin from serum
or adipocytes and recombinant adiponectin expressed in mammalian
cells, Waki et al. 107,108 detected 3 different molecular mass species
and characterized them as low-molecular weight (LMW) trinners (67
kD), middle-molecular weight (MMW) hexanners (136 kD), and high-
molecular weight 12- to 18-nners (greater than 300 kD). A disulfide
bond through an N-terminal cysteine was required for the formation of
nnultinners larger than a trinner. Noting that Arita et al. 103 had found
total adiponectin concentrations to be higher in females than in males,
Waki et al. 107,108 analyzed serum samples from healthy young
Japanese volunteers and found that HMW nnultinners, but not MMW or
LMW nnultinners, were significantly less abundant in males than
females. Sivan et al. 109 sought to determine if adiponectin is present
in human fetal blood, to define its association with fetal birth weight,
and to evaluate whether dynamic changes in adiponectin levels occur
during the early neonatal period. Cord blood adiponectin levels were
extremely high compared with serum levels in children and adults and
were positively correlated with fetal birth weights. No significant
differences in adiponectin levels were found between female and male
neonates. Cord adiponectin levels were significantly higher compared
with maternal levels at birth, and no correlation was found between
cord and maternal adiponectin levels. Sivan et al. 109 concluded that
adiponectin in cord blood is derived from fetal and not from placental
or maternal tissues. Kunnada et al. 110 incubated human nnonocyte-
derived macrophages with physiologic concentrations of recombinant
human adiponectin to determine the effect of adiponectin on matrix
nnetalloproteinases (MMPs) and tissue inhibitors of nnetalloproteinases
(TIMPs). Adiponectin treatment increased TIMP1nnRNA levels in a
dose-dependent manner without affecting MMP9 nnRNA. Adiponectin
also augmented TIMP1 secretion into the media. Adiponectin
significantly increased IL10 nnRNA expression and protein secretion.
Cotreatnnent of cells with adiponectin and anti-IL10 monoclonal
antibodies abolished adiponectin-induced TIMP1 nnRNA expression.
Kunnada et al. 110 concluded that adiponectin acts as an
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antiinflannnnatory signal by selectively increasing TIMP1 expression
through IL10 induction. Biochemical, genetic, and animal studies
established a critical role for Acrp30/adiponectin in controlling whole-
body metabolism, particularly by enhancing insulin sensitivity in
muscle and liver, and by increasing fatty acid oxidation in muscle.
Wong et al. 111 described a widely expressed and highly conserved
family of adiponectin paralogs. They focused particularly on the mouse
paralog most similar to adiponectin, CTRP2. At nanonnolar
concentrations, bacterially produced CTRP2 rapidly induced
phosphorylation of AMP-activated protein kinase, acetyl-coA
carboxylase, and nnitogen-activated protein kinase in cultured
nnyotubes, which resulted in increased glycogen accumulation and
fatty acid oxidation. The authors suggested that the discovery of the
family of adiponectin paralogs has implications for understanding the
control of energy homeostasis and could provide new targets for
pharnnacologic intervention in metabolic diseases such as diabetes and
obesity. To study how the biologic activities of adiponectin are
transmitted, Hug et al. 112 performed a series of expression cloning
studies to identify cell surface molecules capable of binding
adiponectin, using a magnetic-bead panning method that may present
higher-valency forms of the adiponectin ligand. Specifically, they
transduced a C2C12 nnyoblast cDNA retroviral expression library into
Ba/F3 cells and panned infected cells on recombinant adiponectin
linked to magnetic beads. They identified T-cadherin (see 601364) as
a receptor for the hexanneric and high molecular weight species of
adiponectin but not for the trinneric or globular species. Only
eukaryotically expressed adiponectin bound to T-cadherin, implying
that posttranslational modifications of adiponectin are critical for
binding. T-cadherin is expressed in endothelial and smooth muscle
cells, where it is positioned to interact with adiponectin. Because T-
cadherin is a glycosylphosphatidylinositol-anchored extracellular
protein, it may act as a coreceptor for a signaling receptor through
which adiponectin transmits metabolic signals. Iwabu et al. 113
provided evidence that adiponectin induces extracellular calcium influx
by adiponectin receptor-1 (ADIPOR1), which was necessary for
subsequent activation of calciunn/calnnodulin-dependent protein kinase
kinase-beta (CaMKK-beta; CAMKK2), AMPK, and SIRT1, increased
expression and decreased acetylation of PGC1-alpha (604517), and
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increased mitochondria in nnyocytes. Moreover, muscle-specific
disruption of AdipoR1 suppressed the adiponectin-mediated increase in
intracellular calcium concentration, and decreased the activation of
CaMkk, AMPK, and SIRT1 by adiponectin. Suppression of AdipoR1 also
resulted in decreased PGC1-alpha expression and deacetylation,
decreased mitochondria! content and enzymes, decreased oxidative
type I nnyofibers, and decreased oxidative stress-detoxifying enzymes
in skeletal muscle, which were associated with insulin resistance and
decreased exercise endurance.
Yang et al. 114 studied the changes of plasma adiponectin levels with
body weight reduction among 22 obese patients who received gastric
partition surgery. A 46% increase of mean plasma adiponectin level
was accompanied by a 21% reduction in mean BMI. The authors
concluded that body weight reduction increased the plasma levels of a
protective adipocytokine, adiponectin. In addition, they inferred that
the increase in plasma adiponectin despite the reduction of the only
tissue of its own synthesis suggests that the expression of adiponectin
is under feedback inhibition in obesity. Lindsay et al. 115 found that 70
Pima Indian patients who later developed type II diabetes had, at
baseline, lower concentrations of adiponectin than did controls. Those
individuals with high concentrations of the protein were less likely to
develop type II diabetes than those with low concentrations. Stefan et
al. 116 measured fasting plasma adiponectin and insulin concentrations
and body composition in 30 5-year-old and 53 10-year-old Pima Indian
children. Cross-sectionally, plasma adiponectin concentrations were
negatively correlated with percentage body fat and fasting plasma
insulin concentrations at both 5 and 10 years of age. At age 10 years,
percentage body fat (p = 0.03), but not fasting plasma insulin, was
independently associated with fasting plasma adiponectin
concentrations. Longitudinally, plasma adiponectin concentrations
decreased with increasing adiposity. Longitudinal analyses indicated
that hypoadiponectinennia is a consequence of the development of
obesity in childhood. Taganni et al 117 studied adiponectin levels in 31
female patients with anorexia nervosa and in 11 with bulimia nervosa.
Serum adiponectin concentrations in anorexia nervosa and bulimia
nervosa were significantly lower than those in normal-weight controls.
These results were unexpected in light of reports that circulating
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adiponectin levels are downregulated in obesity 103 and that weight
reduction increases plasma adiponectin levels 114 levels were high in
constitutionally thin subjects and low in obese subjects, which
provided a negative correlation with body mass index (BMI) and body
fat mass. In contrast, serum leptin levels correlated very well with BMI
and fat mass among all the patients and controls. The concentrations
of adiponectin after weight recovery increased to the normal level
despite a relatively small increase in BMI. The authors suggested that
abnormal feeding behavior in patients with eating disorders may
reduce circulating adiponectin levels, and that weight recovery can
restore it. Williams et al. 118 determined the extent to which low
maternal plasma adiponectin is predictive of gestational diabetes
mellitus (GDM), a condition that is biochemically and epidemiologically
similar to type II diabetes, using a prospective, nested case-control
study design to compare maternal plasma adiponectin concentrations
in 41 cases with 70 controls. Adiponectin concentrations were
statistically significantly lower in women with GDM than controls (4.4
vs 8.1 nnicroginnl, P less than 0.001). Approximately 73% of women
with GDM, compared with 33% of controls, had adiponectin
concentrations less than 6.4 nnicroginnl. After adjusting for
confounding, women with adiponectin concentrations less than
6.4nnicroginnl experienced a 4.6-fold increased risk of GDM, as
compared with those with higher concentrations (95% confidence
interval, 1.8-11.6). The authors concluded that their findings were
consistent with other reports suggesting an association between
hypoadiponectinennia and risk of type II diabetes.Using Spearman
univariate analysis, Liu et al. 119 demonstrated that both total and high
molecular weight adiponectin levels were inversely associated with
body mass index (BMI), fasting glucose, homeostasis model of
assessment of insulin resistance, triglycerides, and alanine
anninotransferase (ALT), with the high molecular weight isofornn also
positively correlated with high-density lipoprotein cholesterol (r =
0.19; p = 0.036). They concluded that high molecular weight
adiponectin, but not hexanneric or trinneric, tracks with the metabolic
correlates of total adiponectin and that an independent inverse
association exists between ALT and high molecular weight adiponectin.
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The Homo sapiens adiponectin (ADPQ or ACRP30) has to transcript
variants. The longer one (variant 1) nnRNA has been deposited in the
NCBI database as under the accession number NM_001177800
VERSION NM_001177800.1 (4629 bp nnRNA linear PRI 01-AUG-2010)
with the nucleotide sequence as in sequence ID 28. The second variant
differs in the 5' UTR compared to variant 1. Both variants 1 and 2
encode the same protein. It has been deposited in the NCBI database
as under the accession NM_004797 VERSION NM_004797.3 (LOCUS:
NM_004797 4578 bp nnRNA linear PRI 02-AUG-2010) with the
nucleotide sequence as in sequence ID 29.
The Homo sapiens adiponectin (ADPQ or ACRP30) protein has been
deposited in the NCBI database as under the accession number
ABZ10942 ACCESSION VERSION ABZ10942.1 GI:167077467 (LOCUS
ABZ10942.1 GI:167077467 244 aa linear PRI 11-FEBR-2008) with the
amino acid sequence as in sequence ID 30.
Isolation of monocyte-derived microvesicles from plasma
samples
Plasma samples from patients are easy to collect and contain nniRNAs
120-1231 which have diagnostic potential in metabolic syndrome and
cardiovascular disease 124'125. The main physiological carrier of plasma
nniRNAs are nnicrovesicles (MVs) which are small vesicles shed from
almost all cell types under both normal and pathological conditions
126,127. Interestingly, MVs bear surface receptors/ligands of the original
cells and have the potential to selectively interact with specific target
cells. They are involved in cell-to-cell communication including the
communication between adipocytes and macrophages and between
circulating nnonocytes and vascular endothelial cells 123. Due to the
presence of specific surface receptors/ligands, peripheral blood MVs
can be divided in origin-based subpopulations which can be used to
determine nniRNA expression profiles in MVs derived from one specific
cell type. In detail, peripheral blood MVs derived from mononuclear
phagocyte cell lineage can be detected with anti-CD14, anti-CD16,
anti-CD206, anti-CCR2, anti-CCR3 and anti-CCR5 antibodies 122. By
labeling the antibodies with a fluorescent group or magnetic particles,
these cell-specific MVs can be isolated using FACS or magnetic cell
separation technology.
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Methods to determine the gene expression / activity
Preparation of Reagents Using Biomarkers
The bionnarkers described herein may be used to prepare
oligonucleotide probes and antibodies that hybridize to or specifically
bind the bionnarkers mentioned herein, and homologues and variants
thereof.
Probes and Primers
A "probe" or "primer" is a single-stranded DNA or RNA molecule of
defined sequence that can base pair to a second DNA or RNA molecule
that contains a complementary sequence (the target). The stability of
the resulting hybrid molecule depends upon the extent of the base
pairing that occurs, and is affected by parameters such as the degree
of connplennentarity between the probe and target molecule, and the
degree of stringency of the hybridization conditions. The degree of
hybridization stringency is affected by parameters such as the
temperature, salt concentration, and concentration of organic
molecules, such as fornnannide, and is determined by methods that are
known to those skilled in the art. Probes or primers specific for the
nucleic acid bionnarkers described herein, or portions thereof, may
vary in length by any integer from at least 8 nucleotides to over 500
nucleotides, including any value in between, depending on the purpose
for which, and conditions under which, the probe or primer is used. For
example, a probe or primer may be 8, 10, 15, 20, or 25 nucleotides in
length, or may be at least 30, 40, 50, or 60 nucleotides in length, or
may be over 100, 200, 500, or 1000 nucleotides in length. Probes or
primers specific for the nucleic acid bionnarkers described herein may
have greater than 20-30% sequence identity, or at least 55-75%
sequence identity, or at least 75-85% sequence identity, or at least
85-99% sequence identity, or 100% sequence identity to the nucleic
acid bionnarkers described herein. Probes or primers may be derived
from genonnic DNA or cDNA, for example, by amplification, or from
cloned DNA segments, and may contain either genonnic DNA or cDNA
sequences representing all or a portion of a single gene from a single
individual. A probe may have a unique sequence (e.g., 100% identity
to a nucleic acid bionnarker) and/or have a known sequence. Probes or
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primers may be chemically synthesized. A probe or primer may
hybridize to a nucleic acid bionnarker under high stringency conditions
as described herein.
Diagnostic Use
In a preferred embodiment, the invention involves methods to
assess quantitative and qualitative aspects of the bionnarker gene
expression(s), e.g. nniRNAs. of which the increased or decreased
expression as provided by the present invention is indicative for the
combination of oxidative stress and inflammation and insulin
resistance related to the progression of a metabolic syndrome disorder
in a subject or the increased risk to develop related cardiovascular
diseases in said subject. Techniques well known in the art, e.g.,
quantitative or semi-quantitative RT PCR for instance real time RT
PCR, for instance nnRNA analysis by the fluorescence-based real-time
reverse transcription polynnerase chain reaction (qRT-PCR or RT-qPCR)
or reverse transcription loop-mediated amplification (RT-LAMP), for
instance one-step RT-LAMP, or real-time NASBA for detection,
quantification and differentiation of the RNA and DNA targets 1281 or
Northern blot, can be used.
In a particular embodiment, the analyzing techniques include the
application of detectably-labeled probes or primers. The probes or
primers can be detectably-labeled, either radioactively or non-
radioactively, by methods that are known to those skilled in the art,
and their use in the methods according to the invention, involves
nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid
amplification by the polynnerase chain reaction (e.g., RT-PCR), single
stranded conformational polymorphism (SSCP) analysis, restriction
fragment polymorphism (RFLP) analysis, Southern hybridization,
northern hybridization, in situ hybridization, electrophoretic mobility
shift assay (EMSA), fluorescent in situ hybridization (FISH), and other
methods that are known to those skilled in the art.
By "detectably labeled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide probe
or primer, a gene or fragment thereof, or a cDNA molecule. Methods
for detectably-labeling a molecule are well known in the art and
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include, without limitation, radioactive labeling (e.g., with an isotope
such as 32P or 35S) and nonradioactive labeling such as, enzymatic
labeling (for example, using horseradish peroxidase or alkaline
phosphatase), chennilunninescent labeling, fluorescent labeling (for
example, using fluorescein), bioluminescent labeling, or antibody
detection of a ligand attached to the probe. Also included in this
definition is a molecule that is detectably labeled by an indirect means,
for example, a molecule that is bound with a first moiety (such as
biotin) that is, in turn, bound to a second moiety that may be observed
or assayed (such as fluorescein-labeled streptavidin). Labels also
include digoxigenin, luciferases, and aequorin.
Therefore, in a first aspect, the present invention provides an in vitro
method to determine activation of a nnonocyte in a sample, said
method comprising measuring the expression level of one or more
nnicroRNAs selected from the group consisting of let-7c, let-7g, nniR-
18a, nniR-27b, nniR-30a, nniR-30b, nniR-30d, nniR-101, nniR-103, nniR-
107, nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b, nniR-
181d, and nniR-335 in said sample.
In another embodiment, the present invention provides the in vitro
method of the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-101, nniR-103,
nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b, nniR-181d,
and nniR-335; in particular wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-103, nniR-151-5p, nniR-
181a, nniR-181b, nniR-181d, and nniR-335.
In yet another embodiment, the present invention provides the in vitro
method of the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-130b, nniR-151-
5p, nniR-181d, and nniR-335.
In a further embodiment, the invention provides the in vitro method of
the invention wherein the activation of the nnonocyte is indicative for
the inflammatory state of said nnonocyte and comprises measuring the
expression level of one or more nnicroRNAs selected from the group
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consisting of let-7c, let-7g, nniR-18a, nniR-30a, nniR-30b, nniR-101,
nniR-103, nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b,
nniR-181d, and nniR-335 in said sample.
In another embodiment, the invention provides the in vitro method of
the invention, wherein the activation of the nnonocyte is indicative for
the inflammatory state of said nnonocyte and comprises measuring the
expression level of one or more nnicroRNAs selected from the group
consisting of let-7c, let-7g, nniR-18a, nniR-30a, nniR-30b, nniR-101,
nniR-103, nniR-126, nniR-130b, nniR-151-5p, nniR-181b, and nniR-335 in
said sample.
In yet another embodiment, the invention provides the in vitro method
of the invention, wherein the one or more nnicroRNAs are selected
from the group consisting of nniR-30a, nniR-101, nniR-103, nniR-126,
nniR130b, nniR-151-5p, nniR-181b, and nniR-335; in particular, wherein
the one or more nnicroRNAs are selected from the group consisting of
nniR-30a, nniR-130b, nniR-151-5p, and nniR-335.
In an even further embodiment, the invention provides the in vitro
method of the invention, wherein the activation of the nnonocyte is
indicative for the oxidative stress state of said nnonocyte and
comprises measuring the expression level of one or more nnicroRNAs
selected from the group consisting of let-7c, let-7g, nniR-27b, nniR-30a,
nniR-30b, nniR-30d, nniR-101, nniR-103, nniR-107, nniR-151-5p, nniR-
181a, and nniR-181b in said sample; in particular wherein the one or
more nnicroRNAs are selected from the group consisting of nniR-30a,
nniR-101, nniR-103, nniR-151-5p, nniR-181a, and nniR-181b; more in
particular wherein the one or more nnicroRNAs are selected from the
group consisting of nniR-30a, and nniR-151-5p.
In a still further embodiment, the invention provides the in vitro
method of the invention, wherein the activation of the nnonocyte is
indicative for insulin signalling deregulation of said nnonocyte and
comprises measuring the expression level of one or more nnicroRNAs
selected from the group consisting of nniR-30a, nniR-103, nniR-126,
nniR-130b, nniR-151-5p, nniR-181a, nniR181b, nniR-181d, and nniR-335
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in said sample; in particular wherein the activation of the nnonocyte is
indicative for insulin signalling deregulation of said nnonocyte and
comprises measuring the expression level of one or more nnicroRNAs
selected from the group consisting of nniR-30a, nniR-103, nniR-126,
nniR-130b, nniR-151-5p, nniR-181a, and nniR181b in said sample; more
in particular wherein the one or more nnicroRNAs are selected from the
group consisting of nniR-30a, nniR-130b, and nniR-151-5p.
It is also an object of the invention to provide an in vitro method to
predict if a subject will respond to adiponectin or adiponectin mimetic
treatment, said method comprising determining the activation of
nnonocytes in a sample by measuring the expression level of one or
more nnicroRNAs selected from the group consisting of let-7c, let-7g,
nniR-18a, nniR-27b, nniR-30a, nniR-30b, nniR-30d, nniR-101, nniR-103,
nniR-107, nniR-126, nniR-130b, nniR-146b-5p, nniR-151-5p, nniR-181a,
nniR-181b, nniR-181d, and nniR-335; in particular nniR-30a and nniR-
146b-5p; in said sample,
whereby
a) the absence of activated nnonocytes is an indication that said
subject will respond to said adiponectin or adiponectin mimetic
treatment, and
b) the presence of activated nnonocytes is an indication that said
subject will not respond to said adiponectin or adiponectin mimetic
treatment.
The present invention also provides an in vitro method to predict if a
subject will respond to adiponectin or adiponectin mimetic treatment,
said method comprising determining the activation of nnonocytes
according to the invention,
whereby
a) the absence of activated nnonocytes is an indication that said
subject will respond to said adiponectin or adiponectin mimetic
treatment, and
b) the presence of activated nnonocytes is an indication that said
subject will not respond to said adiponectin or adiponectin mimetic
treatment.
In a particular embodiment, the present invention provides diagnosis,
treatment and/or monitoring methods for a subject that suffers from
or is at risk of suffering from at least one disease or disorder selected
from the group comprising obesity, metabolic syndrome, type 2
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diabetes mellitus, hyperglycemia, low glucose tolerance, insulin
resistance, a lipid disorder, dyslipidennia,
hyperlipidennia,
hypertriglyceridennia, hypercholesterolennia, low HDL levels, high LDL
levels, atherosclerosis, and a cardiovascular disease.
It is a further object of the invention to provide an in vitro method of
the invention wherein the activation of the nnonocyte is indicative for a
cardiovascular disease, in particular a coronary artery disease, in a
person, said method comprising measuring the expression level of one
or more nnicroRNAs selected from the group consisting of nniR-30a,
nniR-101, nniR-130b, and nniR-181a in said sample; in particular one or
more nnicroRNAs selected from the group consisting of nniR-30a, nniR-
101, and nniR-181a.
In another embodiment, the invention provides an in vitro method of
the invention wherein the activation of the nnonocyte is indicative for a
cardiovascular disease, in particular a coronary artery disease, in a
person, said method comprising measuring the expression level of one
or more nnicroRNAs selected from nniR-30a and nniR-130b.
In another further embodiment, the invention provides an in vitro
method of the invention wherein the activation of the nnonocyte is
indicative for the cardiovascular risk of a person, said method
comprising measuring the expression level of one or more nnicroRNAs
selected from the group consisting of nniR-101, nniR-130b, nniR-181a,
nniR-181b, nniR-181d, and nniR-335 in said sample; in particular one or
more nnicroRNAs selected from nniR-130b and nniR-181b.
In yet another embodiment, the invention provides an in vitro method
of diagnosing the cardiovascular risk of a person, said method
comprising measuring the expression level of one or more nnicroRNAs
selected from the group consisting of nniR-130b, nniR-181d, and nniR-
335 in a nnonocyte obtained from said person.
It is also a further object of the invention to provide an in vitro method
of the invention wherein the activation of the nnonocyte is indicative
for metabolic syndrome in a person, said method comprising
measuring the expression level of one or more nnicroRNAs selected
from the group consisting of nniR-30a, nniR-130b, and nniR-181a in said
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sample; in particular one or more nnicroRNAs selected from nniR-130b
and nniR-181a.
In a different embodiment, the present invention provides an in vitro
method of the invention wherein the activation of the nnonocyte is
indicative for metabolic syndrome in a person, said method comprising
measuring the expression level of one or more nnicroRNAs selected
from nniR-30a and nniR-130b.
The in vitro methods of the invention may further comprise analysing
the expression level and/or activity of one or more members selected
from the group consisting of nniR-146b-5p, IRAK3, SOD2, TNFAIP6,
TNFAIP3, TLR2, and TNFa in said sample; in particular the expression
level and/or activity of IRAK3 and optionally one or more members
selected from the group consisting of SOD2, TNFAIP6, TNFAIP3, TLR2,
and TNFa in said sample.
In a particular embodiment, a sample consists of one or more cells,
tissues, or parts thereof. For example, in another particular
embodiment, said sample is a blood-derived sample; more in
particular plasma, serum, or a fraction thereof. The samples used in
the present invention can also comprise tissues containing activated
nnonocytes and/or macrophages that originate from activated
nnonocytes; examples of such tissues can include artheroslerotic
plaques, cardiac tissues, liver tissues, and pancreatic tissues.
In another particular embodiment, said sample consists essentially of
nnonocytes or nnonocyte-derived material, in particular nnonocytes or
nnonocyte-derived nnicrovesicles.
The methods of the present invention to determine activation of at
least one nnonocyte can also be used in a method of monitoring the
progression of the treatment of a disease associated with activated
nnonocytes. In particular of at least one disease or disorder selected
from the group comprising obesity, metabolic syndrome, type 2
diabetes mellitus, hyperglycemia, low glucose tolerance, insulin
resistance, a lipid disorder, dyslipidennia,
hyperlipidennia,
hypertriglyceridennia, hypercholesterolennia, low HDL levels, high LDL
levels, atherosclerosis, and a cardiovascular disease in a person. In
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different particular embodiments, the methods of monitoring the
progression of treatment comprise determining nnonocyte activation,
the inflammatory state of a nnonocyte, the oxidative stress state of a
nnonocyte, insuling signalling deregulation of a nnonocyte, or
determining the cardiovascular risk of a subject according to any one
of the different embodiments of the present invention.
Methods of treatment
Detection of the bionnarkers described herein may enable a
medical practitioner to determine the appropriate course of action for a
subject (e.g., further testing, drug or dietary therapy, surgery, no
action, etc.) based on the diagnosis. Detection of the bionnarkers
described herein may also help determine the presence or absence of
a syndrome or disorder associated with activated nnonocytes, early
diagnosis of such a syndrome or disorder, prognosis of such a
syndrome or disorder, or efficacy of a therapy for such a syndrome or
disorder. In alternative aspects, the bionnarkers and reagents prepared
using the bionnarkers may be used to identify therapeutics for such a
syndrome or disorder. The methods according to the invention allow a
medical practitioner to monitor a therapy for a syndrome or disorder
associated with activated nnonocytes in a subject, enabling the medical
practitioner to modify the treatment based upon the results of the test.
In said aspect of the present invention, it has for example been
found that a syndrome or disorder associated with activated
nnonocytes can be treated by administering to a subject in need
thereof an effective amount of a therapeutic or a combination of
therapeutics that increase(s) or decrease(s) the expression of nniRNAs
in the nnonocytes or macrophages or any white blood cell. Said
therapeutic may include an agent that increases the expression of
IRAK3.
Syndromes or disorders associated with activated nnonocytes
include (1) non-insulin dependent Type 2 diabetes mellitus (NIDDM),
(2) hyperglycemia, (3) low glucose tolerance, (4) insulin resistance,
(6) a lipid disorder, (7) dyslipidennia, (8) hyperlipidennia, (9)
hypertriglyceridennia, (10) hypercholesterolennia, (11) low HDL levels,
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(12) high LDL levels, (13) atherosclerosis, and (14) metabolic
syndrome.
The effective amount of a compound, which is required to
achieve a therapeutic effect will be, of course, vary with the type of
therapeutic component, such as small molecules, peptides, etc ; the
route of administration; the age and condition of the recipient; and the
particular disorder or disease being treated. In all aspects of the
invention, the daily maintenance dose can be given for a period
clinically desirable in the patient, for example from 1 day up to several
years (e.g. for the mammal's entire remaining life); for example from
about (2 or 3 or 5 days, 1 or 2 weeks, or 1 month) upwards and/or for
example up to about (5 years, 1 year, 6 months, 1 month, 1 week, or
3 or 5 days). Administration of the daily maintenance dose for about 3
to about 5 days or for about 1 week to about 1 year is typical.
Nevertheless, unit doses should preferably be administered from twice
daily to once every two weeks until a therapeutic effect is observed.
Adiponectin is an adipocytokine, which is mainly produced by the
adipose tissue. Although it is the most abundantly produced protein of
the fat cell, plasma levels are reduced in obese patients. There is
growing evidence that reduced adiponectin concentrations indicate an
increased cardiovascular risk because hypoadiponectinennia is
associated with the components of the metabolic syndrome, in
particular with insulin resistance, elevated triglycerides, and low HDL
Apart from this, adiponectin possesses anti-inflammatory properties
and exerts direct antiatherosclerotic and cardioprotective effects 129'130.
Therefore, it was suggested that low adiponectin concentrations are a
cardiovascular risk factor and that therapeutic strategies that enhance
the secretion or action or mimetic the action of this adipocytokine
might reduce the incidence of cardiovascular diseases (CVDs).
However, several recently published studies on the prospective
association between adiponectin and CVD events/mortality showed
inconsistent results. Five studies reported that adiponectin was not
independently associated with future CVD 131-135. Low adiponectin
concentrations turned out as a risk factor for future CVD in some
1
studies 136-142 whereas others showed that high adiponectin levels
were associated with an increased risk of CVD and/or mortality 143-149
The underlying mechanisms for these contradictory results are still
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unclear but may be due to differences in the study populations. Toward
this, it was speculated that low adiponectin predicts cardiovascular
events in low-risk populations for CVD, whereas in high-risk
populations, a counter-regulatory increase of adiponectin occurs that is
responsible for the elevated cardiovascular risk associated with high
adiponectin levels. In this application, we present a method to identify
persons that will be vulnerable to adiponectin or adiponectin nninnetics
and ways to overcome this vulnerability.
Therefore, in a second aspect, the invention provides an agent that
modulates one or more nnicroRNAs selected from the group consisting
of let-7c, let-7g, nniR-18a, nniR-27b, nniR-30a, nniR-30b, nniR-30d, nniR-
101, nniR-103, nniR-107, nniR-126, nniR-130b, nniR-151-5p, nniR-181a,
nniR-181b, nniR-181d, and nniR-335; in particular from the group
consisting of nniR-30a, nniR-103, nniR-126, nniR-130b, nniR-151-5p,
nniR-181b, nniR-181d, and nniR-335; for use in the treatment of at
least one activated nnonocyte or the prevention of activation of at least
one nnonocyte in a subject.
In a particular embodiment, the present invention provides agents
according to the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-101, nniR-103,
nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b, nniR-181d,
and nniR-335; in particular wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-130b, nniR-151-
5p, nniR-181d, and nniR-335.
In another particular embodiment, the present invention provides
agents of the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-103, nniR-151-5p, nniR-
181a, nniR-181b, nniR-181d, and nniR-335.
In a further embodiment, the present invention provides agents of the
invention, wherein the one or more nnicroRNAs are selected from the
group consisting of let-7c, let-7g, nniR-18a, nniR-30a, nniR-30b, nniR-
101, nniR-103, nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b,
nniR-181d and nniR-335; in particular let-7c, let-7g, nniR-18a, nniR-30a,
nniR-30b, nniR-101, nniR-103, nniR-126, nniR-130b, nniR-151-5p, nniR-
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181b, and nniR-335; and wherein the activated nnonocyte is
characterized by an increased inflammatory state.
In another further embodiment, the invention provides agents
according to the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-103, nniR-130b,
nniR-151-5p, nniR-181d, and nniR-335; and wherein the activated
nnonocyte is characterized by an increased inflammatory state.
In another further embodiment, the invention provides agents
according to the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-101, nniR-103,
nniR-126, nniR130b, nniR-151-5p, nniR-181b, and nniR-335; in particular
nniR-30a, nniR-130b, nniR-151-5p, and nniR-335; and wherein the
activated nnonocyte is characterized by an increased inflammatory
state.
In another embodiment, the present invention provides agents
according to the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of let-7c, let-7g, nniR-27b, nniR-30a,
nniR-30b, nniR-30d, nniR-101, nniR-103, nniR-107, nniR-151-5p, nniR-
181a, and nniR-181b; in particular from nniR-30a, nniR-101, nniR-103,
nniR-151-5p, nniR-181a, and nniR-181b; more in particular from nniR-
30a, and nniR-151-5p; and wherein the activated nnonocyte is
characterized by an increased oxidative stress state.
In yet another embodiment, the present invention provides agents
according to the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-126, nniR-130b, nniR-151-
5p, and nniR-181b and wherein the activated nnonocyte is
characterized by an increased oxidative stress state.
In a particular embodiment, the present invention provides agents
according to the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-103, nniR-126,
nniR-130b, nniR-151-5p, nniR-181a, nniR-181b, nniR-181d, and nniR-
335; in particular from nniR-30a, nniR-103, nniR-126, nniR-130b, nniR-
151-5p, nniR-181a, and nniR181b; more in particular from nniR-30a,
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nniR-130b, and nniR-151-5p; and wherein the activated nnonocyte is
characterized by insulin signalling deregulation.
In another particular embodiment, the present invention provides
agents according to the invention, wherein the one or more nnicroRNAs
are selected from the group consisting of nniR-103, nniR-130b, nniR-
151-5p, nniR-181d, and nniR-335 and wherein the activated nnonocyte
is characterized by insulin signalling deregulation.
In a particular embodiment, the agents according to the different
embodiments of the present invention can be used for the treatment of
activated nnonocytes or the prevention of nnonocyte activation in a
subject that suffers from at least one disease or disorder selected from
the group comprising obesity, metabolic syndrome, type 2 diabetes
mellitus, hyperglycemia, low glucose tolerance, insulin resistance, a
lipid disorder, dyslipidennia, hyperlipidennia, hypertriglyceridennia,
hypercholesterolennia, low HDL levels, high LDL levels, atherosclerosis,
and a cardiovascular disease.
It is furthermore an aim of the invention to provide agents according
to the invention, wherein the one or more nnicroRNAs are selected
from the group consisting of nniR-101, nniR-130b, nniR-181a, nniR-
181b, nniR-181d, and nniR-335; in particular one or more nnicroRNAs
selected from nniR-130b and nniR-181b; and wherein the treatment of
said activated nnonocyte or the prevention of activation of said
nnonocyte in said subject leads to a decreased cardiovascular risk of
said subject.
In a different embodiment, the one or more nnicroRNAs are selected
from the group consisting of nniR-130b, nniR-181d, and nniR-335; and
the treatment of said activated nnonocyte or the prevention of
activation of said nnonocyte in said subject leads to a decreased
cardiovascular risk of said subject.
In yet another embodiment, the present invention provides agents of
the invention, wherein the one or more nnicroRNAs are selected from
the group consisting of nniR-30a, nniR-101, nniR130b, and nniR-181a; in
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particular one or more nnicroRNAs selected from the group consisting
of nniR-30a, nniR-101, and nniR-181a;
and wherein the treatment of said activated nnonocyte or the
prevention of activation of said nnonocyte treats and/or prevents a
cardiovascular disease, in particular a coronary artery disease, in said
person.
In a particular embodiment, the present invention provides agents of
the invention, wherein the one or more nnicroRNAs are selected from
nniR-30a and nniR-130b; and wherein the treatment of said activated
nnonocyte or the prevention of activation of said nnonocyte treats
and/or prevents a cardiovascular disease, in particular a coronary
artery disease, in said person.
In another particular embodiment, the present invention pvoides
agents of the invention, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-130b, and nniR-
181a in said sample; in particular one or more nnicroRNAs selected
from nniR-130b and nniR-181a, or from nniR-30a and nniR-130b; and
wherein the treatment of said activated nnonocyte or the prevention of
activation of said nnonocyte treats and/or prevents metabolic
syndrome in said person.
In a further embodiment, the agent of the invention that modulates
one or more nnicroRNAs is a nnicroRNA mimic, such as for example an
antagomir.
As also described elsewhere in the present application, the agents of
the present invention can also be used in combination with other
agents that are known to reduce, prevent or treat nnonocyte activation
or that are known to reduce, prevent or treat diseases associated with
nnonocyte activation. It is therefore also an object of the present
invention to provide a combination comprising
= an agent according to the invention, and
= adiponectin or an adiponectin mimetic,
for use in the treatment of at least one activated nnonocyte or the
prevention of activation of at least one nnonocyte in a subject.
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In a different embodiment, the inventions provides a combination
comprising
= an agent according to the invention, and
= a modulator of one or more members selected from the group
consisting of IRAK3, SOD2, TNFAIP6, TNFAIP3, TLR2, and TNFa
for use in the treatment of at least one activated nnonocyte or the
prevention of activation of at least one nnonocyte in a subject.
In particular, such a combination comprises
= an agent according to the invention, and
= a modulator of IRAK3, and
= optionally a modulator of one or more members selected from
the group consisting of SOD2, TNFAIP6, TNFAIP3, TLR2, and
TNFa
for use in the treatment of at least one activated nnonocyte or the
prevention of activation of at least one nnonocyte in a subject.
In a futher embodiment, the present inventions provides a
combination comprising
= an agent according to the invention and/or an agent that
modulates nniR-146b-5p, and
= adiponectin or an adiponectin mimetic,
for use in the treatment of at least one activated nnonocyte or the
prevention of activation of at least one nnonocyte in a subject.
In another embodiment, the present invention provides a combination
comprising
= an agent according to the invention and/or an agent that
modulates nniR-146b-5p, and
= a modulator of one or more members selected from the group
consisting of IRAK3, SOD2, TNFAIP6, TNFAIP3, TLR2, and TNFa
for use in the treatment of at least one activated nnonocyte or the
prevention of activation of at least one nnonocyte in a subject; in
particular a combination comprising
= an agent according to the invention and/or an agent that
modulates nniR-146b-5p, and
= a modulator of IRAK3, and
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= optionally a modulator of one or more members selected from
the group consisting of SOD2, TNFAIP6, TNFAIP3, TLR2, and
TN Fa
for use in the treatment of at least one activated nnonocyte or the
prevention of activation of at least one nnonocyte in a subject.
In a particular embodiment, the invention provides a combination
according to the invention further comprising adiponectin or an
adiponectin mimetic, for use in the treatment of at least one activated
nnonocyte or the prevention of activation of at least one nnonocyte in a
subject.
Furthermore, the present invention provides the use of an agent
according to the different embodiments of the invention to treat and/or
prevent one or more elements selected from the group comprising
nnonocyte activation, the oxidative stress state of a nnonocyte, the
inflammatory state of a nnonocyte, and insulin signaling deregulation in
a nnonocyte.
In addition, it is the object of the present invention to provide a
method of treating and/or prevention a disease or disorder associated
with activated nnonocytes in a subject. It is therefore another aspect of
the invention to provide a method of treating and/or preventing a
disease or disorder associated with activated nnonocytes in a subject in
need thereof, said method comprising modulating one or more
nnicroRNAs selected from the group consisting of let-7c, let-7g, nniR-
18a, nniR-27b, nniR-30a, nniR-30b, nniR-30d, nniR-101, nniR-103, nniR-
107, nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b, nniR-
181d, and nniR-335 in a nnonocyte cell in said subject. In particular,
the method wherein the one or more nnicroRNAs are selected from the
group consisting of nniR-30a, nniR-101, nniR-103, nniR-126, nniR-130b,
nniR-151-5p, nniR-181a, nniR-181b, nniR-181d, and nniR-335, in
particular from the group consisting of nniR-103, nniR-151-5p, nniR-
181a, nniR-181b, nniR-181d, and nniR-335; more in particular from
nniR-30a, nniR-130b, nniR-151-5p, nniR-181d, and nniR-335.
In another embodiment, the activated nnonocytes in the method
according to the invention have an increased inflammatory state and
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the one or more nnicroRNAs in the method of treating and/or
preventing are selected from the group consisting of let-7c, let-7g,
nniR-18a, nniR-30a, nniR-30b, nniR-101, nniR-103, nniR-126, nniR-130b,
nniR-151-5p, nniR-181a, nniR-181b, nniR-181d, and nniR-335; in
particular from let-7c, let-7g, nniR-18a, nniR-30a, nniR-30b, nniR-101,
nniR-103, nniR-126, nniR-130b, nniR-151-5p, nniR-181b, and nniR-335;
more in particular from nniR-30a, nniR-103, nniR-130b, nniR-151-5p,
nniR-181d, and nniR-335.
In yet another embodiment, the present invention provides a method
of treating and/or preventing as hereinbefore, wherein the one or
more nnicroRNAs are selected from the group consisting of nniR-30a,
nniR-101, nniR-103, nniR-126, nniR130b, nniR-151-5p, nniR-181b, and
nniR-335; in particular wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-130b, nniR-151-
5p, and nniR-335.
The invention further provides a method of treating and/or prevention
as hereinbefore, wherein the activated nnonocytes have an increased
oxidative stress state and wherein the one or more nnicroRNAs are
selected from the group consisting of let-7c, let-7g, nniR-27b, nniR-30a,
nniR-30b, nniR-30d, nniR-101, nniR-103, nniR-107, nniR-151-5p, nniR-
181a, and nniR-181b; in particular from nniR-126, nniR-130b, nniR-151-
5p, and nniR-181b. In a different embodiment, the present invention
provides said method, wherein the one or more nnicroRNAs are
selected from the group consisting of nniR-30a, nniR-101, nniR-103,
nniR-151-5p, nniR-181a, and nniR-181b; in particular from nniR-30a,
and nniR-151-5p.
In a further embodiment, the present invention provides a method of
treating and/or preventing as described hereinbefore, wherein the
activated nnonocytes have deregulated insulin signalling and wherein
the one or more nnicroRNAs selected from the group consisting of nniR-
30a, nniR-103, nniR-126, nniR-130b, nniR-151-5p, nniR-181a, nniR-181b,
nniR-181d, and nniR-335; in particular from nniR-30a, nniR-103, nniR-
126, nniR-130b, nniR-151-5p, nniR-181a, and nniR181b. In a different
embodiment, the present invention provides said method, wherein the
one or more nnicroRNAs are selected from the group consisting of nniR-
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103, nniR-130b, nniR-151-5p, nniR-181d, and nniR-335; in particular
from nniR-30a, nniR-130b, and nniR-151-5p.
In another particular embodiment, the disease or disorder associated
with activated nnonocytes that have deregulated insulin signalling is
selected from the group comprising obesity, metabolic syndrome, type
2 diabetes mellitus, hyperglycemia, low glucose tolerance, insulin
resistance, a lipid disorder, dyslipidennia,
hyperlipidennia,
hypertriglyceridennia, hypercholesterolennia, low HDL levels, high LDL
levels, atherosclerosis, and a cardiovascular disease.
In a particular embodiment, the method of the invention is
characterized in that the disease or disorder associated with activated
nnonocytes is a cardiovascular disease, in particular a coronary artery
disease, and that the one or more nnicroRNAs are selected from the
group consisting of nniR-30a, nniR-101, nniR130b, and nniR-181a; in
particular one or more nnicroRNAs selected from the group consisting
of nniR-30a, nniR-101, and nniR-181a; more in particular nniR-30a
and/or nniR-130b.
In another particular embodiment, the method of the invention is
characterized in that the disease or disorder associated with activated
nnonocytes is metabolic syndrome, and that the one or more
nnicroRNAs are selected from the group consisting of nniR-30a, nniR-
130b, and nniR-181a; in particular one or more nnicroRNAs selected
from nniR-130b and nniR-181a; more in particular nniR-30a and/or nniR-
130b.
In a particular embodiment, the downregulation of a nnicroRNA of the
invention is indicative for an activated nnonocyte. In another particular
embodiment, the downregulation of a nnicroRNA of the invention is
indicative for a nnonocyte with an increased inflammatory state. In yet
another particular embodiment, the downregulation of a nnicroRNA is
indicative for a nnonocyte with an increased oxidative stress state, with
the exception for nniR-151-5p, for which an upregulation is indicative
for a nnonocyte with an increased oxidative stress state. In another
particular embodiment, the downregulation of a nnicroRNA of the
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invention is indicative for a nnonocyte with deregulated insulin
signalling.
It is furthermore an object of the present invention to provide
methods, agents, us of agents, and compositions according to the
different embodiments of the invention wherein one or more of the
following restrictions apply:
= let-7c can not be selected
= let-7g can not be selected
= nniR-18a can not be selected
= nniR-27b can not be selected
= nniR-30a can not be selected
= nniR-30b can not be selected
= nniR-30d can not be selected
= nniR-101 can not be selected
= nniR-103 can not be selected
= nniR-107 can not be selected
= nniR-126 can not be selected
= nniR-130b can not be selected
= nniR-151-5p can not be selected
= nniR-181a can not be selected
= nniR-181b can not be selected
= nniR-181d can not be selected
= nniR-335 can not be selected
Compositions
It is also an object of the present invention to provide a composition
comprising the above mentioned components. In particular, suitable
for use in treating and/or preventing activation of a nnonocyte and
accordingly useful in a metabolic syndrome or a disease associated
with the activation of said nnonocytes, condition or disorder selected
from the group consisting of (1) non-insulin dependent Type 2
diabetes mellitus (NIDDM), (2) hyperglycemia, (3) low glucose
tolerance, (4) insulin resistance, (6) a lipid disorder, (7) dyslipidennia,
(8) hyperlipidennia, (9) hypertrig lyceridennia,
(10)
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hypercholesterolennia, (11) low HDL levels, (12) high LDL levels, (13)
atherosclerosis, in a subject in need thereof.
Therefore, it is an object of the present invention to provide a
pharmaceutical composition comprising an agent or a combination
according to the invention, for use in the treatment of at least one
activated nnonocyte or the prevention of activation of at least one
nnonocyte in a subject.
In addition, the present invention provides the use of an agent
according to any one of the different embodiments of the invention in
the preparation of a pharmaceutical composition.
The compositions of the present invention, for use in the methods of
the present invention, can be prepared in any known or otherwise
effective dosage or product form suitable for use in providing topical or
systemic delivery of the therapeutic compounds, which would include
both pharmaceutical dosage forms as well as nutritional product forms
suitable for use in the methods described herein.
The above mentioned components may be administrated to induce an
increase or a decrease of nnicroRNAs in myeloid cells in particular in
blood nnonocytes. Such administration can be in any form by any
effective route, including, for example, oral, parenteral, enteral,
intraperitoneal, topical, transdernnal (e.g., using any standard patch),
ophthalmic, nasally, local, non-oral, such as aerosal, spray, inhalation,
subcutaneous, intravenous, intramuscular, buccal, sublingual, rectal,
vaginal, intra-arterial, and intrathecal, etc. Oral administration is
prefered. Such dosage forms can be prepared by conventional
methods well known in the art, and would include both pharmaceutical
dosage forms as well as nutritional products.
Pharmaceutical compositions
The pharmaceutical compositions of the present invention can be
prepared by any known or otherwise effective method for formulating
or manufacturing the selected product form. For example, the above
mentioned components can be formulated along with common
excipients, diluents, or carriers, and formed into oral tablets, capsules,
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sprays, mouth washes, lozenges, treated substrates (e. g. oral or
topical swabs, pads, or disposable, non-digestible substrate treated
with the compositions of the present invention); oral liquids (e. g.
suspensions, solutions, emulsions), powders, or any other suitable
dosage form.
Non-limiting examples of suitable excipients, diluents, and carriers
include: fillers and extenders such as starch, sugars, nnannitol, and
silicic derivatives; binding agents such as carboxynnethyl cellulose and
other cellulose derivatives, alginates, gelatin, and polyvinyl pyrolidone;
moisturizing agents such as glycerol; disintegrating agents such as
calcium carbonate and sodium bicarbonate; agents for retarding
dissolution such as paraffin; resorption accelerators such as
quaternary ammonium compounds; surface active agents such as
acetyl alcohol, glycerol nnonostearate; adsorptive carriers such as
kaolin and bentonite; carriers such as propylene glycol and ethyl
alcohol, and lubricants such as talc, calcium and magnesium stearate,
and solid polyethyl glycols.
Antagomirs are one of a novel class of chemically engineered
oligonucleotides. Antagonnirs are used to silence endogenous nniRNA.
An antagonnir is a small synthetic RNA that is perfectly complementary
to the specific nniRNA target with either nnispairing at the cleavage site
of Ago2 or some sort of base modification to inhibit Ago2 cleavage.
Usually, antagonnirs have some sort of modification, such as 2'
nnethoxi groups and phosphothioates, to make it more resistant to
degradation. It is unclear how antagonnirization (the process by which
an antagonnir inhibits nniRNA activity) operates, but it is believed to
inhibit by irreversibly binding the nniRNA. Antagonnirs are now used as
a method to constitutively inhibit the activity of specific nniRNAs 15"51.
Understanding the nniRNA signature in susceptible individuals may
facilitate the partitioning of patients into distinct subpopulations for
targeted therapy with antagonnirs 152.
In addition to the antinniRs, there is also the opportunity to mimic or
reexpress miRNAs by using synthetic RNA duplexes designed to
mimic the endogenous functions of the nniRNA of interest, with
modifications for stability and cellular uptake. The "guide strand" is
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identical to the nniRNA of interest, whereas the "passenger strand" is
modified and typically linked to a molecule such as cholesterol for
enhanced cellular uptake. Another way to increase the level of a
nniRNA is by the use of adeno-associated viruses (AAVs). Delivered in
viral vectors, the nniRNA of interest can be continually expressed,
resulting in robust replacement expression of nniRNAs downregulated
during disease. Additionally, the availability of a number of different
AAV serotypes allows for the potential of tissue-specific expression
because of the natural tropism toward different organs of each
individual AAV serotype, as well as the different cellular receptors with
which each AAV serotype interacts. The use of tissue-specific
promoters for expression allows for further specificity in addition to the
AAV serotype. Furthermore, AAV is currently in use in a number of
clinical trials for gene therapy, of which the safety profiles have looked
quite positive. In line with this, Kota et al recently showed AAV-
mediated delivery of nniR-26a blunts tumor genesis in a mouse model
of liver cancer 153. Although systemic viral delivery of nniRNAs to the
heart during disease has not been performed yet, there have been a
number of studies using AAV9 to successfully deliver RNA interference
to cardiac tissue and effectively restore cardiac function during disease
in rodents 154.
To deliver miRNA mimics in vivo effectively and into specific cells
and organs, one can explore encapsulation technologies. In this
approach, inhibitors/mimics are sequestered in various kinds of
liposonnes/nanoparticles to further protect them from degradation and
to direct them to the appropriate tissues 155'156. Another technique
involves conjugating a cationic protein (carrier) with a monoclonal
antibody targeting a specific cell surface receptor. The antibody only
binds to the cells expressing the surface antigen that it recognizes.
Oftentimes, the antigen is a receptor. Prior to administration to the
animal, the "conjugate" is loaded with nniRNA inhibitors/mimics. The
carrier binds the nniRNA through electrostatic forces. The
inhibitor/mimic is negatively charged and the carrier is positively
charged. Upon binding to the cell receptor, the conjugate complex is
internalized and the inhibitor/mimic released into the cytoplasm to
silence or mimic the desired nniRNA 157'158.
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Cell surface markers have been used for targeting cargo to mouse
nnonocytes. Sialoadhesin (CD169 or Siglec-1) is an endocytic receptor
expressed on nnonocytes and macrophages 159. Sialoadhesin-specific
innnnunoconjugates have been used for targeting toxin and antigens to
these specific cell types 160. Dectin-1, a major receptor that recognized
b-glucans, is highly expressed on nnonocytes and macrophages.
Complexes of antisense ODN with b-1,3-glucans, have been shown to
be incorporated into macrophages via dectin-1 161. Recently a rabies
virus glycoprotein (RVG) peptide was used for delivery of siRNA
payload to macrophages and nnicroglial cells in the brain. Here the 29-
nner RVG peptide that binds specifically to acetylcholine receptor
expressed on myeloid cells are linked to a positively-charged nona-D-
arginine (9R) residues for binding of siRNA for efficient delivery in vitro
and in vivo 162. Dendrinners are branched, synthetic polymers with
layered architectures that have recently shown considerable promise in
several therapeutic applications. In a recent report by Hayder M et al.
163, a phosphorus-containing dendrinner with an N3P3
(cyclotriphosphazene) core and phenoxynnethyl-nnethylhydrazone
(PMMH) branches, and capped with anionic azabisphosphonate (ABP)-
end groups was chemically synthesized and shown to be able to
selectively target nnonocytes. Rapid internalization of nnonocytes with
the dendrinner is followed by anti-inflammatory activation in
nnonocytes as has been shown in mouse model of arthritis (also see
review 164).
The zebrafish has proven to be a powerful vertebrate model in
genetics and developmental biology. Nowadays it has been emerging
as a model for human disease and therapy. The relatively low cost,
availability of transgenic lines, external and rapid development of the
embryo and transparency during development makes the zebrafish a
popular and attractive model. The zebrafish is an excellent model for
the functional validation of nniRNAs in vivo 165. It can also be used as
screening model to identify therapeutic agents that influence the
nniRNA expression during inflammatory diseases. The myeloid lineage-
which shows close homology structurally, biochemically and
functionally to their mammalian counterparts- appears already after
12-16 hours post fertilization (hpf) and by 24 hpf a functional
cardiovascular system has been formed 166'167. Until now 415 zebrafish
nniRNAs have been identified demonstrating the conservation of this
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important mechanism in vertebrates 168 Gain- and loss of function
experiments can be performed to unravel the nniRNA biology in relation
to inflammation, oxidative stress and insulin resistance. Fertilized eggs
of the zebrafish are easily manipulated by nnicroinjections of small
oligonucleotide fragments inhibiting or mimicking a specific nniRNA
169,170. The nnonocyte/nnacrophage population can be tracked in the
transparent embryos by using the Tg(fms:GAL4.VP16)i186 transgenic
line in which the nnonocytes/nnacrophages are fluorescently labeled 171.
Crossing this line with the Flil-eGFP line makes it possible to evaluate
the interaction of the nnonocytes/nnacrophages with the endothelial
layer of the blood vessels. The behavior, number and interaction of the
nnonocytes/nnacrophages with blood vessels and surrounding tissue
can be monitored by simple in vivo time lapse microscopy 165,171-174
Besides the zebrafish screening model, mice can be applied for long
term in vivo research. Several mouse models of obesity, metabolic
syndrome and atherosclerosis are available in which the therapeutic
-.
potential of nniRNAs can be evaluated 175182 RNA-analogs have been
developed to achieve silencing or mimicking of endogenous nniRNAs
and can be systemically administered in mice to study the nniRNAs of
interest 158'183'184.
Drawing and Table Description
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
The present invention will become more fully understood from the
detailed description given herein below and the accompanying
drawings which are given by way of illustration only, and thus are not
!imitative of the present invention, and wherein:
Figure 1: Monocytes/nnacrophages in obesity and obesity-associated
metabolic disorders.
Figure 2: An overview of nniRNAs deregulated in nnonocytes of obese
subjects and predicted to be involved in regulating key molecules in
the IRAK3-related pathway associated with increased inflammation
and oxidative stress and impaired insulin signaling and sensitivity.
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Figure 3: Expression profiles of 31 candidate nniRNAs in circulating
nnonocytes of obese and lean subjects.
Figure 4: nniRNAs differentially expressed in nnonocytes of obese
persons after short-term weight loss.
Figure 5: nniRNAs differentially expressed in inflammation associated
cell experiments.
Figure 6: nniRNAs differentially expressed in oxidative stress associated
cell experiments.
Figure 7: nniRNAs differentially expressed in insulin resistance
associated cell experiments.
Figure 8: nniRNA decision tree
Figure 9: Effect of nniRNA inhibitors on inflammation, oxidative stress
and insulin resistance in vitro
Figure 10: Effect of nniRNA mimics on inflammation, oxidative stress
and insulin resistance in vitro.
Figure 11: nniR-146b-5p is an essential mediator of the anti-
inflammatory, antioxidative stress and insulin-sensitizing actions of
globular adiponectin.
Table 1: Characteristics and gene expressions before and after weight
loss in obese patients
Table 2: List of differentially expressed nniRNAs in obese patients (P <
0.05)
Table 3: List of differentially expressed nnicroRNAs-of-interest in obese
patients with their theoretical targets as determined by in silico target
prediction (P < 0.05)
Table 4: Characteristics of patients in the second cohort
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Table 5: AUC of ROC curves regarding diagnostic power to distinguish
CAD patients from healthy controls
Table 6: Association of nniRNA expressions in nnonocytes with
occurrence of angiographically documented CAD
Table 7: Primers used in qRT-PCR
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Examples
Study design, materials and methods
Materials
All chemicals were obtained from Sigma-Aldrich unless stated
otherwise. Human THP-1 nnonocytic cells (TIB-202) were obtained
from ATCC.
Study cohort
This study complies with the Declaration of Helsinki, and the locally
appointed Ethics Committee approved the study protocol. All human
participants gave written informed consent and underwent coronary
angiography for (suspected) stable or unstable coronary artery disease
(CAD). For practical purposes, the presence of significant CAD was
defined in this study as at least one stenosis of 50% in a major
coronary artery (left main, LAD, Cx or RCA), while absence of CAD was
defined as having no detectable lesions on angiography, including
lumina! irregularities. Patients with a history of cardiovascular disease,
including coronary, peripheral artery or cerebrovascular disease, where
excluded from the CAD negative group.
The first cohort comprised 14 lean control (27% male; BMI < 30
kg/m2) and 21 obese individuals (33% male; BMI > 30 kg/m2). These
21 morbidly obese subjects were referred to our hospital for bariatric
surgery. Before they were included, patients were evaluated by an
endocrinologist, an abdominal surgeon, a psychologist and a dietician.
Only after multidisciplinary deliberation the selected patients received
a laparoscopic Roux-en-Y gastric bypass. A 30 ml fully divided gastric
pouch is created and the jejunum, 30 cm distal of the ligament of
Treitz, is anastonnosed to it with a circular stapler of 25 mm. To
restore intestinal transit, a fully stapled entero-entero anastonnose is
constructed 120 cm distal on the alimentary limb. In this way the food
passage is derived away from almost the whole stomach, the
duodenum and the proximal jejunum 185-187. All participants in the first
cohort were without symptoms of clinical cardiovascular disease. The
samples were collected between March 29th, 2005 and May 30th, 2006.
The second cohort comprised 126 subjects (83% male, BMI = 28 1
kg/m2, nnean SEM) of which 39% had the metabolic syndrome and
65% had a positive angiogrann. This population was used for ROC and
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regression analysis. The samples of the second cohort were collected
between June 8th, 2010 and January 31st, 2011.
Monocyte isolation
Blood samples were collected, and PBMCs were prepared from the
anti-coagulated blood using gradient separation on Histopaque-1077
after removal of the plasma fraction. Cells were washed three times in
Ca2+- and Mg2+-free Dulbecco's (D)-PBS. PBMCs were incubated for 15
min at 4 C with CD14 nnicrobeads at 20 p1/1 x 107 cells. The cells were
washed once, re-suspended in 500 pl Ca2+- and Mg2+-free DPBS
containing 0.5% BSA/1 x 108 cells. The suspension was then applied
to an LS column in a MidiMACS Separator (Miltenyi) 188'189. We selected
CD14 + nnonocytes because CD14 intensity expression on circulating
nnonocytes was found to be associated with increased inflammation in
patients with diabetes 190.
Blood analysis
Blood samples were centrifuged to prepare plasma samples for
analysis. Total and HDL-cholesterol and triglyceride levels were
determined with enzymatic methods (Boehringer Mannheim). LDL-
cholesterol levels were calculated with the Friedewald formula. Plasma
glucose was measured with the glucose oxidase method (on Vitros
750XRC, Johnson & Johnson), and insulin with an immunoassay
(Biosource Technologies). Ox-LDL 191 and IL-6 were measured with
[LISA (Mercodia and R&D Systems). Hs-CRP (Beckman Coulter) was
measured on an Innnnage 800 Innnnunochennistry System. Blood
pressure was taken three times with the participant in a seated
position after 5 minutes quiet rest. The average of the last two
measurements was used for systolic and diastolic blood pressure.
Micro vesicle isolation, total RNA isolation and quantitative RT-PCR
analysis
Microvesicles (MV) were isolated from cell culture medium by
differential centrifugation according to previous publications 120,121.
Briefly, after removing cells and other debris by centrifugation at 300g,
and 16,500g, the supernatant was centrifuged at 100,000g for 70 min
(all steps were performed at 4 C). MVs were collected from the pellet
and resuspended in RNase-free water. The presence of MVs after
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ultracentrifugation was determined with flow cytonnetry. To confirm
that nnicrovesicles were the correct size, flow cytonnetry gates were set
using 1 micron beads (Invitrogen).
Total RNA was extracted with the TRIzol reagent (Invitrogen) and
purified on a nniRNeasy Mini Kit column (Qiagen). The RNA quality was
assessed with the RNA 6000 Nano assay kit using the Agilent 2100
Bioanalyzer.
For nnRNA expression analysis, first-strand cDNA was generated from
total RNA by reverse transcription using the VILO cDNA synthesis kit
(Invitrogen). Quantitative (q)RT-PCR was performed on a 7500 Fast
Real-Time PCR system using the Fast SYBR Green Master mix (Applied
Biosystenns) according to the manufacturer's instructions. Table 7
summarizes forward and reverse primers used in qRT-PCR analysis.
RNA expression levels were expressed as the ratio compared to
controls as previously described 60,192 To make sure that primer
sequences, used in qRT-PCR, target the right gene, their specificity
was validated by Basic Local Alignment Search Tool (BLAST) 193.
Furthermore, cDNA clones (OriGene) for IRAK3 (and TNFAIP3 and -6,
and 50D2) were used to double check the primer specificity. In
addition, PCR fragments were validated for GC/AT ratio, length, and
amplification specificity with dissociation curve analysis and agarose
gel electrophoresis 194.
For nniRNA expression analysis, first-strand cDNA was generated from
total RNA by reverse transcription using the Universal cDNA synthesis
kit (Exiqon). Quantitative (q)RT-PCR was performed on a 7500 Fast
Real-Time PCR system using the Universal SYBR Green master mix
and fully validated and optimized LNA PCR primer sets according to the
manufacturer's instructions (Exiqon). We used RNU5G as
housekeeping gene for normalization of the nniRNA content in
nnonocytes. However, no housekeeping nniRNA has been established
and validated to normalize for the nniRNA content in plasma and MV
samples. Therefore, we supplemented the plasma samples (after
addition of TRIzol) with 10 fnnol Caenorhabditis elegans nniR-39 (cel-
nniR-39) as described previously 195. The expression levels of nniRNAs
in MVs were directly normalized to the total protein content of MVs.
MiRNA Array Profiling and Target Prediction
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The quality of the total RNA was verified by an Agilent 2100
Bioanalyzer profile. 600 ng total RNA from sample and reference was
labelled with Hy3TM and Hy5TM fluorescent label, respectively, using the
miRCURYTM LNA Array power labelling kit (Exiqon, Denmark) following
the procedure described by the manufacturer. The Hy3Tm-labeled
samples and a Hy5Tm-labeled reference RNA sample were mixed pair-
wise and hybridized to the miRCURYTM LNA Array version 5th
Generation (Exiqon), which contains capture probes targeting all
nniRNAs for human, mouse or rat registered in the nniRBASE version
15.0 at the Sanger Institute. The hybridization was performed
according to the miRCURYTM LNA array manual using a Tecan HS4800
hybridization station (Tecan). After hybridization the nnicroarray slides
were scanned and stored in an ozone free environment (ozone level
below 2.0 ppb) in order to prevent potential bleaching of the
fluorescent dyes. The miRCURYTM LNA array nnicroarray slides were
scanned using the Agilent G2565BA Microarray Scanner System
(Agilent Technologies) and the image analysis was carried out using
the InnaGene 8.0 software (BioDiscovery). The quantified signals were
background corrected (Nornnexp with offset value)196 and normalized
using the global Lowess (LOcally WEighted Scatterplot Smoothing)
regression algorithm. nniRNAs, differentially expressed in nnonocytes of
obese women compared to lean controls at a P-value < 0.05, were
considered for further analysis. To gain insight into the functional
significance of differential nniRNA expression in nnonocytes of obese
women, a bioinfornnatic analysis was performed, which determined
predicted nniRNAs for each of the members of the gene cluster using
the DIANA-nnicroT, nniRanda, PicTar and TargetScan target prediction
algorithms 197 and compared them with the list of differentially
expressed nniRNAs.
Cell culture
THP-1 nnonocytic cells were subcultured in RPMI 1640 as described
previously in detail 60,192 For globular adiponectin incubation
experiments, cells were cultured at a density of 1 x 106 cells/ml in
RPMI 1640 supplemented with 10% FBS and 5 pg/nnl gentannicin. After
24 h, 1 or 10 pg/nnl globular adiponectin (PeproTech) was added and
the cells were incubated for 6 to 24 h. Globular adiponectin is a
recombinant protein derived from human globular domain adiponectin
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cDNA expressed in Escherichia co/i. This protein was endotoxin free
(<2 EU/pg) according to the manufacturer. The ox-LDL incubation
experiments were performed like previously described 6 . For
insulin/glucose incubation experiments, cells were cultured at a density
of 1 x 106 cells/ml in glucose-free RPMI 1640 supplemented with 10%
FBS, 5 pg/nnl Gentannicin, and 5.5 nnnno1/1 D-glucose in a 5% CO2
incubator at 37 C. After 24 h, 10-7 nno1/1 insulin and 9.5 nnnno1/1 D-
glucose or 9.5 nnnno1/1 D-nnannitol (osmotic control) was added and
incubated for 24 h under normal growth conditions. For IL-6
experiments, THP-1 cells were stimulated with 10 ng/nnl recombinant
IL-6 (PeproTech) for 24 h. Cell viability, as determined by trypan blue
exclusion, was > 80%. nnROS and iROS formation were measured with
MitoSOX and CelIROX (Invitrogen). Cells were incubated with PBS
containing 5 pM MitoSOX or 2.5 pM CelIROX for 10 or 30 minutes at
37 C and 5% CO2. The labeled cells were washed twice with PBS and
then suspended in warm PBS for analysis by flow cytonnetry (Becton,
Dickinson and Company).
RNA interference
To deplete IRAK3 nnRNA, THP-1 cells were transiently transfected with
chemical synthesized HP GenonneWide siRNAs (Qiagen; target
sequence: 5'-CACATTCGAATCGGTATATTA-3' (Hs_IRAK3_5) and 5'-
CTGGATGTTCGTCATATTGAA-3' (Hs_IRAK3_6)). To inhibit nniR-30a, -
103, -126, -130b, -146b-5p, -151-5p, 181b, -181d or -335 THP-1 cells
were transiently transfected with nnIRCURY LNA nniRNA Power
Inhibitors (Exiqon):
nniR-30a: 5'-TTCCAGTCGAGGATGTTTAC-3',
nniR-103: 5'-CATAGCCCTGTACAATGCTGC-3',
nniR-126: 5'-GCATTATTACTCACGGTACG-3',
nn i R-130 b : 5'-TG CCCTTTCATCATTG CACT- 3',
nn i R-146 b- 5 p : 5'-GCCTATGGAATTCAGTTCTC-3',
nn i R-151 -5 p : 5'-CTAGACTGTGAG CTCCTCG -3',
nn i R-181 b: 5'-ACCCACCGACAGCAATGAATGT-3',
miR-181d: 5'-CCACCGACAACAATGAATGT-3',
nniR-335: 5'-CAIiiiiCGTTATTGCTCTTG-3'.
To overexpress nniR-30a, -103, -130b, -146b-5p, -151-5p, 181b, -
181d or -335 THP-1 cells were transiently transfected with synthetic
nniScript nniRNA mimics (Qiagen):
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nniR-30a: 5'-UGUAAACAUCCUCGACUGGAAG-3',
nniR-103: 5'-AGCAGCAUUGUACAGGGCUAUGA-3',
nniR-130b: 5'-CAGUGCAAUGAUGAAAGGGCAU-3',
nniR-146b-5p: 5'-UGAGAACUGAAUUCCAUAGGCU-3',
nniR-151-5p: 5'-UCGAGGAGCUCACAGUCUAGU-3',
nniR-181b: 5'-AACAUUCAUUGCUGUCGGUGGGU-3',
nniR-181d: 5'-AACAUUCAUUGUUGUCGGUGGGU-3',
nniR-335: 5'-UUUUUCAUUAUUGCUCCUGACC-3'.
As a negative control, we used AllStars Negative Control siRNA
(Qiagen) or nniRCURY LNA nniRNA Power Inhibitor Control (Exiqon; 5'-
GTGTAACACGTCTATACGCCCA-3'); as a positive control, we used
Mrn/Hs_MAPK1 control siRNA (Qiagen; target sequence: 5'-
AATGCTGACTCCAAAGCTCTG-3'). Cells were transfected with 50 nM of
siRNA using HiPerfect reagent (Qiagen) according to the
manufacturer's instructions with modifications as follows. THP-1 cells
were seeded at density 1.5 x 105/well (24 well-plate) in 100 pl of
culture medium. Next, complexes (3 pnnol of siRNA/nniRNA plus 6 pl of
HiPerfect reagent) were formed in 0.1 ml of serum-free RPMI-1640 for
10 min at room temperature and then added to each well. After 6
hours of incubation under normal growth conditions, 400 pl of growth
medium was added to each well and the cells were incubated for 42
hours. Gene silencing was monitored at the nnRNA level by means of
qRT-PCR.
Statistical analysis
Patient groups were compared with an unpaired t-test with Welch's
correction; in vitro data were compared with the Mann-Whitney U test.
Spearman rank correlation was determined with GraphPad Prism 5.
Receiver operating characteristic curve (ROC) analysis was performed
with MedCalc statistical software for biomedical research. Odds ratios
were determined by Chi-square test with Yates' correction (GraphPad
Prism 5). Regression analysis was performed with the Statistical
Package for the Social Sciences (SPSS for Windows; release 16). A P-
value of less than 0.05 was considered statistically significant.
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Results
miRNAs differentially expressed in circulating monocytes of obese
patients and involved in regulating the IRAK3-related gene cluster
Our target cells for the identification of novel bionnarkers and/or
therapeutic targets are nnonocytes since they are readily accessible
(blood) and constitute a reservoir of inflammatory cells that infiltrate
in tissues (adipose, aortic and cardiac tissues) during obesity where
they actively induce oxidative stress, inflammation, and cell death and
thereby induce insulin resistance, atherosclerosis, and heart failure 198
(Figure 1).
The first patient cohort used in this study comprised 14 lean controls
(29% male; age: 33 3 years, nnean SEM) and 21 morbidly obese
individuals (33% male; age: 39 3 years), without clinical symptoms
of cardiovascular disease. Obese subjects in the first cohort had higher
IL-6, hs-CRP, leptin and glucose levels, and lower adiponectin levels,
indicating the presence of systemic inflammation. The higher levels of
circulating ox-LDL indicated systemic oxidative stress. Furthermore,
insulin and triglyceride concentrations were higher; HDL-cholesterol
was lower. Obese individuals had higher systolic and diastolic blood
pressure. Insulin resistance, calculated by a homeostasis model
assessment (HOMA-IR) index, was 86% higher in obese subjects
(Table 1A). A cluster of risk factors for cardiovascular disease and
T2DM including raised blood pressure, dyslipidennia (elevated
triglycerides and/or decreased HDL-cholesterol), raised fasting
glucose, and central obesity have become known as the metabolic
syndrome. A person qualifies for the metabolic syndrome with three
abnormal findings out of five 199. Four controls used in this study had 1
metabolic syndrome component; 1 had 2. Two obese patients had 1, 7
had 2, 5 had 3, and 7 had 4 metabolic syndrome components. Thus,
57% of the obese individuals had the metabolic syndrome. Finally, we
also collected blood of the obese subjects three months after bariatric
surgery. The blood characteristics after short-term weight loss are
depicted in Table 1A. In aggregate, there was less systemic
inflammation but no reduction in circulating ox-LDL. Triglycerides,
HOMA-IR and adiponectin concentrations were restored to levels of
lean persons (Table 1A). The numbers of leukocytes (3.70 1.68 x 106
vs. 3.56 0.80 x 106 per ml blood) and CD14+ nnonocytes (3.24 1.32
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X 105 vs. 3.30 1.56 x 105 per ml blood) were similar in lean and obese
subjects.
Our lab has previously identified a theoretical model (also referred to
as the IRAK3-related pathway) in circulating nnonocytes of obese
patients that links IRAK-mediated inflammation with oxidative stress
and impaired insulin signaling and sensitivity. In detail, this model
contains TLR2 as cell surface marker, NFKB and FOX03A as
transcription factors, TNFa as inflammatory output, SOD1, SOD2 and
CAT as oxidative stress markers, IRAK3 and TNFAIP3 and -6 as
putative inhibitors of the TLR2/NFKB inflammatory pathway and IRS1
and IRS2 as markers of insulin signaling (Figure 2 and Table 1B).
IRAK3, predominantly expressed in nnonocytes/nnacrophages 81, was
the only inhibitor of which the expression was decreased in obese
patients compared to lean controls and was associated with increased
inflammation, evidenced by increased expression of TLR2 and TNFa
(Table 1B). Low IRAK3 and high SOD2 was associated with a high
prevalence of metabolic syndrome (odds ratio: 9.3; sensitivity: 91%;
specificity: 77%). By comparison, the odds ratio of hs-CRP, a widely
used marker of systemic inflammation, was 4.0 (sensitivity: 69%;
specificity: 65%). Weight loss was associated with an increase in
IRAK3 and a decrease in SOD2, in association with a lowering of
systemic inflammation and a decreasing number of metabolic
syndrome components (Table 1). We identified the increase in reactive
oxygen species in combination with obesity-associated low adiponectin
and high glucose and IL-6 as cause of the decrease in IRAK3 in human
nnonocytic THP-1 cells in vitro.
In this study, we identified nniRNAs differentially expressed in
circulating nnonocytes of obese patients and involved in modulating the
IRAK3-related pathway. nniRNA nnicroarrays (Exiqon, nniRBASE version
15.0) were performed on total RNA from isolated nnonocytes of 6 lean
and 10 obese individuals. A total of 133 nniRNAs were differentially
expressed between obese and lean controls (Table 2). To gain insight
into this nniRNA expression profile, a bioinfornnatic analysis was
performed, which determined predicted nniRNAs for each of the
members of the IRAK3-related gene cluster using the DIANA-nnicroT,
nniRanda, PicTar and TargetScan target prediction algorithms 197 and
compared them with the list of differentially expressed nniRNAs (Table
3). The in silico analysis identified 31 nniRNAs with potential targets in
the presented gene cluster. Figure 2 illustrates the interactions
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between the selected nniRNAs and their potential targets in the IRAK3-
related pathway associated with increased inflammation and oxidative
stress and impaired insulin signaling and sensitivity.
Next, we validated the expressions of the 31 candidate nniRNAs in 14
lean and 21 obese subjects. The expression levels of the nniRNAs were
assessed by qRT-PCR, and normalized by expression levels of RNU5G,
identified as most stable reference gene (GeNornn 200) .
The fold
changes of nniRNA levels for the obese individuals vs. lean controls are
shown in Figure 3. Of the 31 nniRNAs that were significantly
deregulated in the nnicroarray analysis, 18 nniRNAs were validated with
qRT-PCR in the extended population. For mature nniRNA sequences,
see sequence IDs 31-74.
Effect of short-term weight loss (in vivo intervention) that is associated
with an increase in insulin sensitivity and a reduction of inflammation
The expression profile of the 18 selected nniRNAs was also determined
three months after bariatric surgery. The expressions of nniR-103,
nniR-151-5p, nniR-181a, nniR-181b, nniR-181d and nniR-335, all
decreased in obese subjects, were significantly increased after short-
term weight loss (Figure 4). Interestingly, the expression profile of
nniR-103, nniR-151-5p, nniR-181a, nniR-181b and nniR-335 correlated
negatively with BMI (r, = -0.34, r, = -0.44, r, = -0.34, r, = -0.34 and
r, = -0.44 respectively, all P < 0.05) and systemic markers of
inflammation (hs-CRP, leptin and glucose). The expressions of nniR-
151-5p, nniR-181a, nniR-181b and nniR-335 were also associated with
the number of metabolic syndrome components (r, = -0.42, r, = -
0.34, r, = -0.31 and R, = -0.36 respectively, all P < 0.01). In these
obese persons (before and after weight loss), nniR-181a, nniR-181b
and nniR-335 correlated positively with the IRAK3 expression (r, =
0.40, r, = 0.41 and r, = 0.50 respectively, all P < 0.01); and nniR-126,
nniR-151-5p and nniR-335 correlated negatively with the SOD2
expression (r, = -0.35, r, = -0.47 and r, = -0.34 respectively, all P <
0.05).
miRNAs associated with inflammation (in vitro) and related oxidative
stress
In order to establish the role of nniRNAs in inflammation we followed
three approaches: 1) measuring their expressions in response to
silencing of IRAK3, an inhibitor of the TLR/TNFa-mediated
inflammation; 2) measuring their expressions in the antioxidative and
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anti-inflammatory response of cells exposed to the inflammatory
cytokine IL-6; and 3) establishing their role in the maintenance of the
anti-inflammatory (and antioxidative) response to adiponectin.
First, we depleted the expression of IRAK3 in THP-1 cells by means of
siRNAs. Knockdown of IRAK3 (-71%, n = 10, P < 0.001) resulted in an
increased inflammatory state of the cells as can be seen from an
increased expression of TNFa (+38%, P < 0.001), and an increased
oxidative stress state, evidenced by a higher production of
mitochondria! ROS (nnROS, +33%) and intracellular ROS (iROS,
+62%, both P < 0.05). To prove that these effects were not caused by
off-target effects, THP-1 cells were transfected with a second siRNA
targeting IRAK3 at a different location. The gene expressions were not
different when we compared the effect of the two siRNAs (data not
shown). IRAK3-depletion decreased the expressions of let-7c, let-7g,
nniR-18a, nniR-30b, nniR-101, nniR-130b, nniR-146b-5p, nniR-181b and
nniR-335 (Figure 5A).
Next, we incubated THP-1 cells with high levels of IL-6 characteristic
for obese patients (Table 1A). Exposure to IL-6 decreased the
expression of TNFa (-29%%, P < 0.01). There was no effect on m-
and iROS production. These protective effects of THP-1 cells were
associated with an increased expression of nniR-103, nniR-126, nniR-
130b, nniR-146b-5p and nniR-151-5p indicating that these nniRNAs can
protect against inflammation and oxidative stress caused by IL-6
(Figure 58).
Furthermore, we investigated the effect of high (as in lean controls
and obese persons after weight loss) and low (as in obese persons)
levels of globular adiponectin (gAcrp30) because this domain of the
adiponectin protein appears to be responsible for the anti-
108,201 .
inflammatory effects of adiponectin
Exposure of THP-1 cells to
low levels of adiponectin resulted in a decreased expression of IRAK3
(RNA and protein) compared to cells exposed to high levels of
adiponectin. This decrease was associated with more TNFa (+75%, P
< 0.001) and nnROS production (+20%, P < 0.01), and less nniR-30a
and nniR-146b-5p (Figure 5C).
miRNAs associated with response to oxidative stress (in vitro) and
related inflammation
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We measured the protective effects of THP-1 cells exposed to ox-LDL,
evidenced by a decrease in TNFa expression (-88%; P<0.001); this
resulted in a 13% (P<0.01) reduction of ROS. The increased
expressions of let-7c, let-7g, nniR-27b, nniR-30a, nniR-30b, nniR-30d,
nniR-101, nniR-103, nniR-107, nniR-146b-5p, nniR-181a and nniR-181b
indicate that these nniRNAs can protect against oxidative stress and
inflammation induced by ox-LDL. In contrast, the expression of nniR-
151-5p was decreased in THP-1 cells exposed to ox-LDL (Figure 6).
miRNAs associated with insulin resistance (in vitro)
Insulin resistance in obese patients is characterized by high levels of
insulin and glucose and thus an increased HOMA-IR index (Table 1A).
Incubation of THP-1 cells with 10-7 M insulin and 15 nnM glucose
resulted in a decreased expression of IRAK3 (-13%, P < 0.01), IRS1 (-
29%, P < 0.05) and IRS2 (-29%, P < 0.01), and more TNFa (+18%, P
< 0.05) and iROS (+5%, P < 0.01). Induction of insulin resistance in
those cells, associated with increased inflammation and oxidative
stress was associated with less nniR-30a, nniR-103, nniR-126, nniR-
130b, nniR-146b-5p, nniR-151-5p, nniR-181a and nniR-181b (Figure 7).
Association of miRNA expressions in monocytes with occurrence of
metabolic syndrome and angiographically documented CAD
Next, we determined the association of the nniRNA expressions with
the occurrence of the metabolic syndrome and CAD in the second
independent cohort.
The characteristics of the 126 subjects are summarized in Table 4. In
aggregate, 25% of the patients were obese and 65% were diagnosed
with CAD. Moreover, 16 patients had 0; 24 had 1; 37 had 2; 28 had 3;
17 had 4; and 4 patients had 5 metabolic syndrome components.
Thus, 39% of these individuals had the metabolic syndrome. MiR-130b
and nniR-181a predicted a higher number of metabolic syndrome
components even after adjusting for smoking, insulin, adiponectin and
IL-6. The R2-value of the model with nniR-130b was 0.277 (P < 0.001);
that with nniR-181a was 0.262 (P < 0.001). MiR-30a also correlated
with a higher number of metabolic syndrome components, but its
association was lost after adjusting for smoking, insulin, adiponectin
and IL-6.
We used binary logistic regression analysis to determine the
association of nniRNAs with occurring cardiovascular risk equivalents,
being a Framingham cardiovascular risk score above 10% per 10 years
202 or type 2 diabetes. MiR-101, nniR-130b, nniR-181a, nniR-181b, nniR-
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181d, and nniR-335 were all significantly (P < 0.05) associated with
cardiovascular risk equivalents. Their respective B-values were -
1.92 0.89, nnean SEM), -1.30 0.52, -1.49 0.75, -1.66 0.77, -
1.28 0.56, and -1.90 0.84. When adjusting for age, gender, smoking
and BMI, nniR-130b and nniR-181b remained significant predictors of
cardiovascular risk equivalents. When adjusting for age, gender,
smoking, BMI, HDL, triglycerides, glucose, insulin, and systolic blood
pressure, nniR-130b remained a significant predictor of occurring
cardiovascular risk equivalents. As indicated above BMI (obesity), HDL
and triglycerides (dyslipidennia), glucose (diabetes) and blood pressure
(hypertension) are measures of the metabolic syndrome 199.
ROC analysis revealed that nniR-30a, nniR-101, nniR-130b and nniR-
181a were associated with CAD (Table 5). Table 6 shows the odds
ratios for CAD in relation to nniRNA expressions in nnonocytes
determined by Chi-square test with Yates' correction. Odd ratios of low
nniR-30a, low nniR-101, low nniR-130b and low nniR-181a varied
between 2 and 12. Binary logistic regression analysis showed that
nniR-30a, nniR-101 and nniR-181a, but not nniR-130b were associated
with CAD after adjusting for age gender, smoking and even BMI,
suggesting that these nniRNAs are associated with CAD even in the
absence of obesity. Their respective B- and P-values were -3.42 1.57
(P < 0.05), -2.32 1.16 (P < 0.05), and -3.04 1.47 (P < 0.05). All 3
nniRNAs remained associated with CAD even after adjustment for age,
gender, smoking, BMI, HDL, triglycerides, glucose, insulin, and systolic
blood pressure.
Overview of the followed selection procedure to identify miRNAs-of-
interest
Figure 8 depicts an overview of the followed selection procedure to
identify nniRNAs-of-interest in activated nnonocytes. It summarizes the
involvement of the selected nniRNAs in processes related to
inflammation, oxidative stress and insulin resistance, in said
nnonocytes. Furthermore, this figure also illustrates nniRNAs associated
with the occurrence of the metabolic syndrome, cardiovascular risk
equivalents and angiographically documented CAD. The selected
nniRNAs are depicted in bold.
miRNAs detectable in plasma samples and monocyte-derived
micro vesicles
Because circulating plasma nniRNAs secreted by all kind of cell types
-
(including nnonocytes) 120123 may have diagnostic potential in
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1
metabolic syndrome and cardiovascular disease 124,125 we measured
the selected nniRNAs (Figure 8) in plasma samples of obese patients
and patients with angiographically documented CAD. All nniRNAs,
except nniR-30a and nniR-181d, were easily detectable in plasma
samples of obese patients and patients with CAD: nniR-30a and nniR-
181d had low expression levels.
The main physiological carrier of plasma nniRNAs are nnicrovesicles
(MVs) which are derived from circulating blood cells and other tissues
directly affected by disease 126. To determine the presence of the
selected nniRNAs in nnonocyte-derived MVs, we isolated MVs from THP-
1 culture medium by ultracentrifugation and measured the miRNA
expressions by qRT-PCR. All nniRNAs, discussed in Figure 8, were
detectable in MVs secreted by THP-1 cells.
Association between miRNA expressions and expressions of IRAK3-
related targets (illustrated in Figure 2)
MiR-30a (R = 0.20; P < 0.05), nniR-151 (R = 0.22; P < 0.01), nniR-
181b (R = 0.27; P < 0.001), nniR-181d (R = 0.17; P < 0.05) and nniR-
335 (R = 0.26; P < 0.01) correlated with the expression of IRAK3 in
nnonocytes of 126 lean and obese patients, characterized in Table 4.
Mir-30a (R = 0.22; P < 0.05), nniR-101 (R = 0.27; P < 0.01), nniR-103
(R = 0.26; P < 0.01), nniR-126 (R = 0.22; P < 0.01), nniR-151-5p (R =
0.24; P < 0.01), nniR-181b (R = 0.25; P < 0.01), nniR-181d (R = 0.26;
P < 0.01), and nniR-335 (R = 0.25; P < 0.05) correlated with the
expression of TNFAIP3. MiR-30a (R = 0.30; P < 0.001), nniR-101 (R =
0.35; P < 0.001), nniR-103 (R = 0.44; P < 0.001), nniR-126 (R = 0.29;
P < 0.001), nniR-130b (R = 0.30; P < 0.001), nniR-151-5p (R = 0.32;
P < 0.001), nniR-181a (R = 0.30; P < 0.001), nniR-181b R = 0.39; P <
0.001), nniR-181d (R = 0.36; P < 0.001), and nniR-335 (R = 0.37; P <
0.001) correlated with the expression of SODI. MiR-130b correlated
with the expression of SOD2. In multiple regression analysis (using a
model containing all nniRNAs), nniR-181d was the best predictor of
SODI, nniR-103 was the best predictor of TNFAIP3, nniR-181d was the
best predictor of SODI, and nniR-335 was the best predictor of SOD2.
Effect of miRNA inhibition on monocyte activation as evidenced by
inflammation, oxidative stress and insulin signaling in vitro
To confirm the functional role of selected nniRNAs in regulating
inflammation (evidenced by TNFa upregulation), oxidative stress
(evidenced by nnROS production) and insulin resistance (evidenced by
IRSI downregulation) in nnonocytes, we transiently transfected THP-1
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cells with a LNA-modified nniRNA inhibitors targeting nniR-103, nniR-
130b, nniR-146b-5p, nniR-151-5p, nniR-181d and nniR-335.
Sequestration of endogenous nniR-130b (possible target: TNFa) and
nniR-151-5p (possible target: NFKB1) levels in THP-1 cells resulted in
more TNFa and nnROS production and less IRSI suggesting the
involvement of these nniRNAs in the protection against inflammation
and oxidative stress (ROS production) and loss of insulin signaling
(Figure 9A-B). Furthermore, depletion of nniR-103 (possible target:
TNFa), nniR-146b-5p (targets: IRAK1 and TRAF6 203) and nniR-335
(possible target: NFKBIa) increased the TNFa expression and
decreased the IRSI expression suggesting the involvement of these
nniRNAs in the regulation of inflammation and subsequently the insulin
signaling (Figure 9C-E). MiR-181d depletion (possible targets: TNFAIP6
and IRS2) also resulted in more TNFa and less IRSI. However, nniR-
181d in contrast to nniR-103, nniR-146b-5p and nniR-335 can target the
inflammatory as well as the insulin signaling pathway directly (Figure
9F). Sequestration of nniR-126 (possible target: SOD2) and nniR-181b
(possible targets: TNFAIP6 and IRS2) levels in THP-1 cells resulted in
more nnROS production without increasing the TNFa expression or
reducing the insulin sensitivity (Figure 9G-H).
In conclusion, several inhibitors of nniRNAs of the invention increased
inflammation, oxidative stress, and/or insulin resistance, which again
underscores the importance of these nniRNAs in nnonocyte activation.
Effect of miRNA mimics on inflammation, oxidative stress and insulin
signaling in vitro
To determine wether mimics of the nniRNAs of the invention are able to
reduce nnonocyte activation parameters, we transiently transfected
THP-1 cells with synthetic nniRNA mimics and determined the effect of
nniRNA overexpression in nnonocytes.
Overexpression of nniR-30a, nniR-181d and nniR-335 decreased
inflammation in THP-1 cells: nniR-30a by increasing IRAK3, nniR-181d
by decreasing TNFa and nniR-335 by both increasing IRAK3 and
decreasing TNFa (Figure 10A-B). Furthermore, transfection of nniR-
130b increased the IRSI expression and nniR-151-5p overexpression
decreased the iROS production (Figure 10C-D).
In conclusion, several mimics of nniRNAs of the invention indeed
reduced one or more of the important nnonocyte activation
parameters.
miR-146b-5p is required for the protective actions of adiponectin
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The causal relation between decreased IRAK3 and nniR-146b-5p and
nniR-30a levels in THP-1 cells (Figure 5C) suggest that these nniRNAs
are a mediator of the protective actions of adiponectin. To determine
whether nniR-146b-5p is an essential mediator of adiponectin-related
anti-inflammatory and antioxidative stress properties, THP-1 cells were
transfected with nniR-146b-5p inhibitor and exposed to high levels of
gAcrp30. The sequestration of nniR-146b-5p and subsequent exposure
to high gAcrp30 resulted in an increased expression of TNFa, more
iROS production and decreased IRSI expression compared to cells
exposed to high levels of gAcrp30 (Figure 11). Interestingly, there was
no significantly decrease in IRAK3 expression detectable suggesting
that nniR-146b-5p is an essential mediator of the anti-inflammatory,
antioxidative stress and insulin-sensitizing actions of gAcrp30.
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Tables
Table 1: Characteristics and gene expressions before and after weight loss in
obese
patients.
Lean controls (n = 14) Obese patients (n = 21)
Before weight loss After weight loss
A. Characteristics
Age (years) 33+3 39+3 39+3
BMI (kg/m2) 21+1 44+1*** 36+1***/ $$$
Leptin (ng/ml) 8.7+1.4 65.6+8.0*** 21.0+3.5**/$$$
Adiponectin (pg/ml) 10.9+1.8 3.9+0.6** 7.0+1.0$$$
Glucose (mg/di) 83+2 111+7*** 89+4$$$
Insulin (mU/I) 10.3+1.8 16.5+2.1* 6.3+0.8$$$
HOMA-IR 2.1+0.4 3.9+0.5** 1.8+0.2$$$
Triglycerides (mg/di) 80+7 132+11*** 99+8$$
LDL-C (mg/di) 110+9 85+6* 92+3
HDL-C (mg/di) 64+4 49+3** 47+2**
SBP (mmHg) 120+3 136+3** 118+1$$$
DBP (mmHg) 75+3 86+2** 62+1***/$$$
IL-6 (pg/ml) 1.8+0.2 4.8+0.4*** 3.4+0.4***/$
Hs-CRP (mg/I) 0.49+0.10 5.65+1.12*** 3.45+0.82**/$
Ox-LDL (IU/I) 50+5 71+4** 69+4**
B. Gene expressions
CAT 1.03+0.03 0.90+0.03** 0.99+0.04
FOX03A 1.06+0.10 1.65+0.18** 1.00+0.06$$
INSR 0.99+0.04 0.65+0.02*** 0.75+0.04*** /$
IRAKI 0.99+0.04 1.21+0.04*** 1.07+0.04$$
IRAK3 0.98+0.04 0.49+0.03*** 0.79+0.05**/$$$
IRAK4 1.03+0.07 0.86+0.03* 0.90+0.04
IRS2 1.05+0.16 1.01+0.06 0.96+0.08
MyD88 0.98+0.04 1.24+0.03*** 1.12+0.03*! $$
NFKBI 1.01+0.05 0.92+0.03 1.12+0.07$$
NFKBIa 1.23+0.20 3.34+0.53** 2.67+0.47** /$
SODI 1.01+0.04 0.81+0.02*** 0.89+0.04*/$
SOD2 1.00+0.05 2.65+0.28*** 1.91+0.19 ***/$
TLR2 0.99+0.08 1.54+0.06*** 1.02+0.09$$$
TNFAIP3 1.05+0.13 1.54+0.11** 1.03+0.11$$
TNFAIP6 1.05+0.16 4.43+0.69*** 2.32+0.34** $$$
TNFa 1.05+0.09 2.18+0.31** 1.06+0.12$$
TRAF6 0.98+0.32 2.26+0.25*** 2.18+0.24***
Data shown are means + SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 obese
compared with
lean controls; $P < 0.05, $$P < 0.01 and $$$P < 0.001 compared with before
weight loss.
Abbreviations: BMI, body mass index; C, cholesterol; DBP, diastolic blood
pressure; HOMA-IR,
homeostasis model assessment of insulin resistance; hs-CRP, high sensitivity C-
reactive
protein; ox-LDL, oxidized LDL; SBP, systolic blood pressure.
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Table 2: List of differentially expressed microRNAs in
obese patients (P < 0.05)
Obese (n = 10) vs. Lean (n = 6)
miRNA p-value Fold change
hsa-miRPlus-F1026 0,0001 1,2
hsa-miR-1255a 0,0002 1,2
hsa-let-7c 0,0002 0,8
hsa-miRPlus-E1077 0,0003 1,4
hsa-miR-513a-5p 0,0003 1,8
hsa-miR-1275 0,0003 2,3
hsa-miR-361-5p 0,0004 0,8
hsa-miR-652 0,0005 0,8
hsa-miRPlus-E1151 0,0005 1,3
hsa-miR-30e* 0,0007 0,9
hsa-miR-1201 0,0007 0,9
hsa-miR-494 0,0008 2,3
hsa-miRPlus-E1097 0,0009 1,2
hsa-miR-18b 0,0009 0,8
hsa-miR-126 0,0010 0,8
hsa-miR-151-5p 0,0011 0,8
hsa-miRPlus-F1037 0,0012 0,9
hsa-miRPlus-E1101 0,0012 1,4
hsa-miR-3202 0,0014 1,3
hsa-miR-374a 0,0015 0,7
hsa-miR-765 0,0017 1,4
hsa-miR-374b 0,0017 0,8
hsa-miR-2116 0,0017 1,2
hsa-miR-30a 0,0017 0,8
hsa-miR-583 0,0018 1,3
hsa-miR-30b 0,0019 0,9
hsa-miR-602 0,0019 1,3
hsa-miRPlus-F1066 0,0019 1,2
hsa-miR-300 0,0019 1,9
hsa-miR-148b 0,0019 0,8
hsa-miRPlus-E1238 0,0019 1,3
hsa-miRPlus-E1209 0,0020 1,9
hsa-miR-186 0,0022 0,8
hsa-miR-101 0,0023 0,7
hsa-miR-30d 0,0027 0,9
hsa-miR-532-5p 0,0028 0,9
hsa-miR-32* 0,0029 1,3
hsa-miR-625* 0,0031 0,8
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hsa-miR-199b-5p 0,0033 0,9
hsa-miR-195 0,0033 0,9
hsa-miR-146b-5p 0,0036 0,8
hsa-miR-32 0,0038 0,7
hsa-miR-184 0,0038 1,2
hsa-miR-1827 0,0041 1,2
hsa-let-7g 0,0044 0,9
hsa-miRPlus-E1200 0,0045 1,1
hsa-miR-17* 0,0047 0,9
hsa-miR-335* 0,0047 1,2
hsa-miRPlus-E1110 0,0049 1,4
hsa-miR-331-3p 0,0049 0,9
hsa-miRPlus-A1015 0,0050 0,9
hsa-miR-642 0,0052 1,3
hsa-miR-29c 0,0055 0,8
hsa-miR-320a 0,0055 0,8
hsa-miRPlus-A1072 0,0055 1,3
hsa-miR-146b-3p 0,0056 1,2
hsa-miR-27b 0,0056 0,9
hsa-miRPlus-E1232 0,0058 1,3
hsa-miR-140-5p 0,0061 0,9
hsa-miRPlus-F1225 0,0062 1,2
hsa-miR-18a 0,0062 0,8
hsa-miR-181a 0,0063 0,8
hsa-miRPlus-E1212 0,0064 1,2
hsa-miR-181b 0,0065 0,9
hsa-miR-107 0,0069 0,8
hsa-miRPlus-E1285 0,0071 1,1
hsa-miR-214 0,0071 1,2
hsa-miR-17 0,0072 0,9
hsa-miRPlus-F1147 0,0078 0,9
hsa-miR-340 0,0081 0,8
hsa-miR-106a 0,0081 0,9
hsa-miR-1299 0,0081 1,1
hsa-miR-27a 0,0083 0,9
hsa-miR-938 0,0091 1,2
hsa-miR-744 0,0093 0,9
hsa-miR-525-5p 0,0095 1,2
hsa-miR-103 0,0096 0,9
hsa-miR-19a 0,0096 0,8
hsa-miRPlus-F1086 0,0099 1,1
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hsa-miR-196a* 0,0101 0,9
hsa-miR-574-5p 0,0103 1,1
hsa-miRPlus-F1074 0,0103 0,9
hsa-miR-660 0,0107 0,9
hsa-miR-920 0,0110 1,2
hsa-miR-500* 0,0111 0,9
hsa-miR-665 0,0111 1,3
hsa-miR-181d 0,0111 0,9
hsa-miR-505 0,0113 0,9
hsa-miRPlus-F1155 0,0123 1,1
hsa-miR-19b 0,0126 0,8
hsa-miR-485-3p 0,0126 1,2
hsa-miR-551b 0,0132 1,4
hsa-miR-142-5p 0,0134 0,8
hsa-miR-335 0,0138 0,8
hsa-miRPlus-F1166 0,0145 1,2
hsa-miR-92b 0,0146 0,9
hsa-miRPlus-E1098 0,0149 1,2
hsa-miR-34b 0,0152 0,9
hsa-miRPlus-E1072 0,0156 1,1
hsa-miR-221 0,0162 0,9
hsa-miR-425* 0,0164 0,9
hsa-let-71 0,0165 0,9
hsa-miR-30e 0,0168 0,9
hsa-miRPlus-E1146 0,0168 1,2
hsa-miR-362-3p 0,0170 0,8
hsa-miR-620 0,0182 1,1
hsa-miR-1297 0,0194 0,9
hsa-let-7e 0,0198 1,2
hsa-miR-197 0,0211 0,9
hsa-miR-423-3p 0,0214 0,9
hsa-miR-340* 0,0216 1,3
hsa-miR-146a 0,0219 0,8
hsa-miR-1304 0,0222 1,1
hsa-miRPlus-E1175 0,0223 1,1
hsa-miRPlus-E1093 0,0226 1,1
hsa-miRPlus-E1090 0,0231 1,2
hsa-miR-342-3p 0,0237 0,8
hsa-miRPlus-F1064 0,0244 1,1
hsa-miR-502-3p 0,0252 0,9
hsa-miR-339-5p 0,0252 0,8
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hsa-let-7f 0,0285 0,9
hsa-miR-2113 0,0294 1,4
hsa-miR-484 0,0329 0,9
hsa-miR-1285 0,0338 1,1
hsa-miR-143 0,0355 1,5
hsa-miRPlus-F1180 0,0357 0,9
hsa-miR-130a 0,0370 0,8
hsa-miR-223* 0,0396 0,9
hsa-miR-130b 0,0414 0,9
hsa-miR-320d 0,0421 0,8
hsa-let-7a 0,0440 0,9
hsa-miR-25 0,0469 0,9
hsa-miR-574-3p 0,0486 1,1
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Table 3: List of differentially expressed microRNAs-of-interest in obese
patients with
their theoretical targets as determined by in silico target prediction (P <
0.05)
Obese (n = 10) vs. Lean (n = 6)
miRNA p-value Fold change Targets
hsa-miR-25 0,0469 0,9 ABCA1, PAFAH1B1, IRS2
CAT, FOX03A, ZNF217, KLF9, IRS1,
hsa-miR-30a 0,0017 0,8 IRS2
hsa-miR-101 0,0023 0,7 TLR2, ZNF217, PAFAH1B1
hsa-miR-30d 0,0027 0,9 CAT, FOX03A, INSR, IRS1, IRS2
hsa-miR-19a 0,0096 0,8 TLR2, TNFAIP3, TNFAIP6, TNF,
ZNF217
hsa-miR-30b 0,0019 0,9 CAT, FOX03A, IRS1, IRS2
hsa-miR-19b 0,0126 0,8 TLR2, TNFAIP3, TNFAIP6, ZNF217
hsa-miR-30e-5p 0,0168 0,9 CAT, FOX03A, IRS1, IRS2
hsa-miR-126 0,0010 0,8 NFKBIa, SOD2, FOX03A, IRS1
hsa-miR-151-5p 0,0011 0,8 TLR2, TLR4, NFKB1
hsa-let-7g 0,0044 0,9 TNFAIP3, MyD88, IRS1
hsa-miR-29c 0,0055 0,8 PAFAH1B1, NFKB1, IL13
hsa-miR-27b 0,0056 0,9 IRAK4, INSR, IRS1
hsa-miR-106b 0,0081 0,9 IRAK1, IKKy, ZNF217
hsa-miR-142-3p 0,0134 0,8 TLR2, IRAK1, ZNF217
hsa-miR-425-3p 0,0164 0,9 NFKB1, CAT, IRS1
hsa-let-7f 0,0285 0,9 TNFAIP3, MyD88, IRS1
hsa-miR-130a 0,0370 0,8 TNFAIP6, TNF, ZNF217
TNFAIP6, TNF, ZNF217, ABCA1, ESR1,
hsa-miR-130b 0,0414 0,9 IRF1, PAFAH1B1
hsa-let-7c 0,0002 0,8 TNFAIP3, IRS1
hsa-miR-30e-3p 0,0007 0,9 NFKB1, SOD2
hsa-miR-374a 0,0015 0,7 TNFAIP6, ZNF217
hsa-miR-374b 0,0017 0,8 TNFAIP6, ZNF217
hsa-miR-146b-5p 0,0036 0,8 IRAK1, TRAF6$
hsa-miR-18a 0,0062 0,8 TNFAIP3, IRS2
hsa-miR-181a 0,0063 0,8 TNFAIP6, IRS2
hsa-miR-181a* 0,0063 0,8 TLR4, IKKy
hsa-miR-181b 0,0065 0,9 TNFAIP6, IRS2
hsa-miR-107 0,0069 0,8 IKKy, TNF
hsa-miR-17 0,0072 0,9 IRAK1, ZNF217
hsa-miR-27a 0,0083 0,9 INSR, IRS1
hsa-miR-103 0,0096 0,9 IKKy, TNF
hsa-miR-181d 0,0111 0,9 TNFAIP6, IRS2
hsa-let-71 0,0165 0,9 TNFAIP3, IRS1
hsa-miR-620 0,0182 1,1 TLR2, TNFAIP6
hsa-let-7e 0,0198 1,2 TNFAIP3, IRS1
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hsa-miR-146a 0,0219 0,8 IRAK1, TRAF6$
hsa-miR-143 0,0355 1,5 TLR2, IRS1
hsa-let-7a 0,0440 0,9 TNFAIP3, IRS1
hsa-let-7d 0,078 0,9 TNFAIP3, MyD88
hsa-miR-18b 0,0009 0,8 TNFAIP3
hsa-miR-765 0,0017 1,4 IKKy
hsa-miR-602 0,0019 1,3 CAT
hsa-miR-532-5p 0,0028 0,9 NFKB1, IRS1
hsa-miR-195 0,0033 0,9 SOCS, PAFAH1B1
hsa-miR-32 0,0038 0,7 IRS2
hsa-miR-340 0,0081 0,8 IRS1
hsa-miR-27a 0,0083 0,9 IRAK4
hsa-miR-525-5p 0,0095 1,2 IRAK3
hsa-miR-660 0,0107 0,9 IRAK4
hsa-miR-551b 0,0132 1,4 IRAK4
hsa-miR-142-5p 0,0134 0,8 TNFAIP6
hsa-miR-335 0,0138 0,8 FOX03A, NFKBIa
hsa-miR-92b 0,0146 0,9 IKKy
hsa-miR-34b 0,0152 0,9 IKKy
hsa-miR-197 0,0211 0,9 FOX03A
hsa-miR-342-3p 0,0237 0,8 IRAK3
hsa-miR-502-3p 0,0252 0,9 IRAK1
hsa-miR-339-5p 0,0252 0,8 IRAK1
stargets are functionally validated 203
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Table 4: Characteristics of patients in the second cohort.
Patients
(n = 126)
Age (years) 51+1
Gender (% male) 66
Smoking (%) 25
Obesity (%) 25
T2DM (%) 10
MetS (%) 39
CAD (%) 65
BMI (kg/m2) 28+1
Leptin (ng/ml) 11.1+1.0
Adiponectin (pg/ml) 9.4+0.5
Glucose (mg/di) 110+3
Insulin (mU/1) 18.6+1.7
HOMA-IR 6.2+0.7
Triglycerides (mg/di) 116+4
LDL-C (mg/di) 98+3
HDL-C (mg/di) 50+1
SBP (mmHg) 138+2
DBP (mmHg) 79+1
IL-6 (pg/ml) 3.7+0.2
Hs-CRP (mg/1) 2.98+0.32
Ox-LDL (IU/1) 51+2
Data shown are means + SEM. Abbreviations: BMI, body mass index; C,
cholesterol; CAD,
coronary artery disease; DBP, diastolic blood pressure; HOMA-IR, homeostasis
model
assessment of insulin resistance; hs-CRP, high sensitivity C-reactive protein;
MetS, metabolic
syndrome; ox-LDL, oxidized LDL; SBP, systolic blood pressure; T2DM, type 2
diabetes
mellitus.
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Table 5: AUC of ROC curves regarding diagnostic power to distinguish CAD
patients
from healthy controls.
microRNA AUC 9513/0CI P-value
miR-30a 0.64 0.54-0.74 0.011
miR-101 0.74 0.65-0.82 < 0.0001
miR-130b 0.72 0.63-0.80 0.0001
miR-181a 0.82 0.74-0.89 < 0.0001
Abbreviations: AUC, area under the curve; CI, confidence intervals.
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Table 6: Association of miRNA expressions in monocytes with occurrence of
angiographically documented CAD.
microRNA Cut point* OR Sensitivity Specificity PPV
NPV
(%) (%) (%) (%)
Low miR-30a 0.195 9.6 (2.1-44.3) 35 95 91 48
Low miR-101 0.633 7.6 (2.1-44.3) 57 85 90 45
Low miR-130b 0.925 10.0 (4.1-24.8) 86 62 45
69
Low miR-181a 0.521 11.8 (4.5-31.0) 73 81 89
59
*Cut points were determined by ROC curve analysis. Data are means (and 95%
confidence
intervals). Abbreviations: OR, odds ratio; NPV, negative predictive value;
PPV, positive
predictive value.
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Table 7: Primers used in qRT-PCR
Forward primer Reverse primer
ACTB 5'-GGACCTGACCGACTACCTCATG-3 5'-CGACGTAGCAGAGCTTCTCCTT-3'
CAT 5'-CATCCAGAAGAAAGCGGTCAA-3' 5'-TCAGCATTGTACTTGTCCAGAAGAG-
3'
FOX03A 5'-CAACAAAATGAAATCCATAGAAGCA-3' 5'-
AGTGTATGAGTGAGAGGCAATAGCA-3'
IN SR 5'-TGTGTACCTCTTGTGGCGTTTC-3' 5'-CTCAGTGCACCTCTCTCTTACATTG-
3'
IRAK1 5'-TCAGTCCTAGCAAGAAGCGAGAA-3' 5'-ACTGGCCCGAGGTTGGA-3'
IRAK3 5'-TGCAACGCGGGCAAA-3' 5'-TTTAGTGATGTGGGAGGATCTTCA-
3'
IRAK4 5'-AGTGATGGAGATGACCTCTGCTTAG-3' 5'-TGAGCAATCTTGCATCTCATGTG-
3'
IRS1 5'-CATCCATTTCAGTTTGTTTACTTTATCC-3' 5'-
TTATTCTGGTGTCACAGTGCATTTT-3'
IR52 5'-GCTTCCCCAGTGCCTATCTTC-3' 5'-
AAACCAACAACTTACATCTCCAATGA-3'
MyD88 5'-TGCATATCTTTGCTCCACTTTCA-3' 5'-ATTCCCTCCCAAGATCCTAAGAA-
3'
NFKB1 5'-CCCTGACCTTGCCTATTTGC-3' 5'-CGGAAGAAAAGCTGTAAACATGAG-
3'
NFKBIA 5'-TGGCCACACGTGTCTACACTTAG-3' 5'-CAGCACCCAAGGACACCAA-3'
SOD1 5'-TTGGGCAAAGGTGGAAATGA-3' 5'-CACCACAAGCCAAACGACTTC-3'
SOD2 5'-TGGAAGCCATCAAACGTGACT-3' 5'-TTTGTAAGTGTCCCCGTTCCTT-3'
TLR2 5'-TGCAAGTACGAGCTGGACTTCTC-3' 5'-GTGTTCATTATCTTCCGCAGCTT-
3'
TNFAIP3 5'-TCCCTGCTCCTTCCCTATCTC-3' 5'-ATGTTTCGTGCTTCTCCTTATGAA-
3'
TNFAIP6 5'-GGCCATCTCGCAACTTACAAG-3' 5'-GCAGCACAGACATGAAATCCA-3'
TN Fa 5'-CAAGCCTGTAGCCCATGTTGTA-3' 5'-TTGGCCAGGAGGGCATT-3'
TRAF6 5'-CATGAAAAGATGCAGAGGAATCAC-3' 5'-GAACAGCCTGGGCCAACAT-3'
125
