Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF ENDOTHELIAL DYSFUNCTION IN DIABETIC PATIENTS
FIELD OF INVENTION
This invention relates to a method for the treatment of endothelial
dysfunction in diabetic
patients. In particular, this invention relates to a method for improving
endothelial
function in treatment of disorders which are related to endothelial
dysfunction, both
macrovascular and microvascular, in diabetic patients.
BACKGROUND OF THE INVENTION
In the chronic disease diabetes mellitus (diabetes), the body loses the
ability to properly
produce or respond to the hormone insulin so that cells of the peripheral
tissues fail to
actively take up glucose from the blood for use or storage. In the diabetic
individual, the
level of glucose in the peripheral blood can become elevated (hyperglycaemia)
and
typically remains so unless some form of intervention is employed (e.g.,
administration of
exogenous insulin) to return glucose in the blood to normal levels. Left
unchecked, the
hyperglycaemia of diabetic individuals can result in shock, organ degeneration
or failure
(e.g., kidney failure, blindness, nerve disease, cardiovascular disease),
tissue necrosis (e.g.,
requiring foot amputation), and even death.
Two major forms of diabetes are type 1 and type 2 diabetes. Type 1 diabetes,
which was
previously known as insulin-dependent diabetes mellitus (1DDM) or juvenile
onset
diabetes, is an autoimmune disease in which the body destroys the insulin-
producing 13
cells (islet cells) of the pancreas resulting in an absolute requirement for
daily
administration of exogenous insulin to maintain normal blood glucose levels.
Type 1
diabetes usually is diagnosed in children and young adults, but can occur at
any age. Type
1 diabetes accounts for 5-10% of diagnosed cases of diabetes.
By far the more prevalent form of diabetes is type 2 diabetes, which was
previously known
as non-insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes was also
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previously known as adult-onset diabetes, however, this form of diabetes is
becoming
increasingly prevalent in the growing population of overweight and clinically
obese
children and young adults. Type 2 diabetes accounts for approximately 90-95%
of all
diagnosed cases of diabetes. Type 2 diabetes typically begins with insulin
resistance, a
disorder in which the body's cells do not respond to insulin properly,
followed by a gradual
loss on part of the pancreas to produce and secrete insulin. Type 2 diabetes
is associated
with a variety of factors including older age, obesity, family history of
diabetes, history of
gestational diabetes, impaired glucose metabolism, physical inactivity, and
various races or
ethnicities. Individuals with type 2 diabetes must attempt to control their
blood glucose
level with careful diet, exercise and weight reduction, and additional
medications.
Major factors contributing to the pro-atherogenic state in diabetes,
particularly type 2
diabetes mellitus (DM2), include dyslipidemia, hyperglycemia, hypertension,
visceral
obesity and insulin resistance (1,2). Observational studies have clearly
demonstrated the
importance of diabetic dyslipidemia in contributing to atherogenesis in
diabetes, illustrated
by the fact that the correlation between low density lipoproteins (LDL) as
well as high
density lipoproteins (HDL) versus cardiovascular events outweighs that of
fasting plasma
glucose (3). Statin intervention studies have revealed a clear benefit of
statin treatment on
reduction of cardiovascular events in DM2 (4, 5); however, in spite of this
impressive
achievement, the majority of DM2 patients will still suffer from
cardiovascular events even
when using statins (6).
During the last two decades, endothelial dysfunction has emerged as one of the
earliest
stages of atherogenesis. Endothelial dysfunction, which is a hallmark in all
diabetic
patients (ie both type 1 and 2) has been shown to have predictive value for
future
cardiovascular events (7-9). In line with the multifactorial pathogenesis of
diabetes-
induced vascular disease (10, 11), numerous therapeutic interventions have
been evaluated
for their potential to improve endothelial function in DM2 patients (9, 12).
Surprisingly,
whereas endothelial function could be fully restored by statin therapy in
dyslipidemic
patients (13), several studies have demonstrated that even intensive statin
treatment cannot
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normalize vascular dysfunction in DM2 (14, 15). The latter emphasizes
possibilities for
additional therapeutic modalities in this high risk group.
High-density lipoproteins (HDLs) represent a broad group of mostly spheroidal
plasma
lipoproteins, which exhibit considerable diversity in their size,
apolipoprotein (apo) and
lipid composition. HDL particles fall into the density range of 1.063-1.21
g/ml (16) and as
they are smaller than other lipoproteins, HDLs can penetrate between
endothelial cells
more readily allowing relatively high concentrations to accumulate in tissue
fluids (17).
The major apolipoprotein of almost all plasma HDLs is apo A-I, which in
association with
phospholipids and cholesterol, encloses a core of cholesteryl esters (16).
Nascent (i.e.
newly synthesised) HDLs secreted by the liver and intestine contain no
cholesteryl esters
and are discoidal in shape (16). The negative association of plasma HDL
concentration
with coronary artery disease has been well documented in epidemiological
studies (18).
Although experiments in animals have demonstrated an anti-atherogenic activity
of HDLs
(19), it is not yet known whether this protective effect is related to the
role of the
lipoprotein in reverse cholesterol transport or to a different mechanism. The
mechanism/mechanisms via which HDLs provide these cardioprotective actions are
not
clearly understood, but may include a role for HDLs in reverse transport of
cholesterol
from peripheral tissues to the liver, inhibition of the oxidation of low-
density lipoproteins,
or modulation of vasodilatation and platelet activation mediated by changes in
the
production of prostacyclin (20). HDLs can also activate endothelial nitric
oxide (NO)
synthase subsequent to its interaction with scavenger receptor-B1 (SR-B1).
In view of the emerging data on the NO promoting effects of HDL, compounds
with HDL-
increasing capacity are of particular interest (21-24). Indeed, in DM2
patients HDL is
positively associated with endothelium-dependent vasomotor responses (8). In
work
leading to the present invention, the inventors have evaluated whether and to
what extent
HDL increase upon infusion of exogenous reconstituted HDL (rHDL) would
translate into
an improvement of vascular function. ApoA-I levels and endothelial function
were
assessed both acutely (4 hours after infusion) as well as 7 days after
infusion of rHDL in
DM2 and matched controls.
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In accordance with an aspect of the present invention, there is provided use
of an effective
amount of high density lipoprotein (HDL) for the treatment of endothelial
dysfunction in a
diabetic patient.
In accordance with a further aspect of the present invention, there is
provided the use of high
density lipoprotein (HDL) in the manufacture of a medicament for
administration to a diabetic
patient for the treatment of endothelial dysfunction in the patient.
In accordance with a further aspect of the present invention, there is
provided an agent for use
in the treatment of endothelial dysfunction in a diabetic patient, said agent
comprising high
density lipoprotein (HDL).
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Bibliographic details of the publications referred to in this specification
are referenced at
the end of the description. The reference to any prior art document in the
specification is
not, and should not be taken as, an acknowledgment or any form of suggestion
that the
document forms part of the common general knowledge.
SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context
requires
otherwise, the word "comprise", and or variations such as "comprises" or
"comprising",
will be understood to imply the inclusion of a stated integer or step or group
of integers or
steps but not the exclusion of any other integer or step or group of integers
or steps.
In one aspect, the present invention provides a method for the treatment of
endothelial
dysfunction in a diabetic patient, which comprises administration (preferably
parenteral
administration) to the patient of an effective amount of high density
lipoprotein (HDL).
In another aspect, the present invention provides the use of high density
lipoprotein (HDL)
in the manufacture of a medicament for administration (preferably parenteral
administration) to a diabetic patient for the treatment of endothelial
dysfunction in the
patient.
In yet another aspect, the present invention provides an agent for
administration
(preferably parenteral administration) in the treatment of endothelial
dysfunction in a
diabetic patient, which comprises high density lipoprotein (HDL).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram which shows that intra-arterial infusion of the
endothelium-
dependent vasodilator serotonin increased forearm blood flow (FBF) in a dose-
dependent
manner in healthy volunteers and patients with DM2. At baseline, the FBF
response to
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serotonin was attenuated in DM2 compared to controls (p< 0.001). After rHDL
infusion,
FBF response to serotonin increased significantly, but did not reach levels
comparable to
controls (p<0.01). Improvement in endothelium-dependent vasodilation persisted
7 days
after rHDL infusion (p<0.01). rHDL infusion did not affect serotonin response
in controls.
Figure 2 is a diagram which shows that at baseline the vasoconstrictor
response to L-
NMMA, reflecting basal nitric oxide (NO) activity, was blunted in DM2 patients
compared
to controls (p<0.001). After rHDL infusion, the L-NMMA constrictor response
was
improved, again still present 7 days after infusion (p<0.01). In line with the
serotonin data,
rHDL infusion had no effect on L-NMMA response in control subjects.
Figure 3 is a diagram which shows that endothelium-independent vasodilation in
response
to sodium nitroprusside was lower in DM2 versus controls (p<0.01) and rHDL
infusion did
not show effect on SNP vasodilator response both in patients and controls
DETAILED DESCRIPTION OF THE INVENTION
Patients with diabetes, particularly type 2 diabetes mellitus (DM2), are
characterized by a
marked increase in cardiovascular risk. Systemic endothelial dysfunction, a
hallmark in
DM2, predicts future risk for cardiovascular events. In view of the relation
between HDL
and the NO pathway, the present inventors have evaluated the effect of rHDL
infusion on
endothelial function in DM2. Specifically, in 7 DM2 patients and 7
normolipidemic
controls, endothelial function was assessed using venous occlusion
plethysmography.
Forearm blood flow (FBF) responses to intra-arterial infusion of the
endothelium-
dependent and independent vasodilators serotonin (5HT) and sodium
nitroprusside,
respectively, and the inhibitor of nitric oxide synthase NG-monomethyl-l-
arginine (L-
NMMA) were measured, both before, 4 hours after and 1 week after infusion of
rHDL
(80mg/kg based on protein).
At baseline HDL was similar in DM2 versus controls (1.1 0.2 vs. 1.2 0.3
mmol/L, ns).
5HT-induced vasodilation (max 17 10%) and L-NMMA induced vasoconstriction (max
-
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17115%) were reduced in DM2 versus controls (5-HT 114122 and L-NMMA -4815%,
both p<0.05). rHDL infusion raised apoA-I levels (1.2 0.2 to 2.8 0.4 vs.
1.2 0.2 to 2.7
0.4 g/L, p<0.01) in DM2 and controls, respectively and restored FBF responses
to 5HT
(86122%, p<0.05) and L-NMMA (-4519%, p<0.01) in DM2. This effect persisted 7
days
after infusion (5HT; 80125%, p<0.05 and L-NMMA -3717%, p<0.01 compared to
baseline). rHDL infusion had no effect in controls. Accordingly, this work
demonstrates
that acute HDL increase improves endothelial function in DM2 and that the
improvement
persists for at least 7 days in spite of return-to-baseline of HDL
concentration.
In one aspect, the present invention provides a method for the treatment of
endothelial
dysfunction in a diabetic patient, which comprises administration to the
patient of an
effective amount of high density lipoprotein (HDL).
Preferably, the administration is parenteral administration.
Reference herein to "treatment" is to be considered in its broadest context.
The term
"treatment" does not necessarily imply that a subject is treated until total
recovery.
Accordingly, treatment includes amelioration of the symptoms of a particular
condition or
disorder as well as reducing the severity of, or eliminating a particular
condition or
disorder.
As used herein, references to "treatment of endothelial dysfunction" are to be
considered as
references to improvement of endothelial function in treatment of disorders
which are
related to endothelial dysfunction. Such disorders include both macrovascular
disorders
(relating to the large blood vessels) such as transient ischaemic attack,
stroke, angina,
myocardial infarction, cardiac failure, and peripheral vascular disease, as
well as
microvascular disorders (relating to the small blood vessels) such as diabetic
retinopathy
(non-proliferative, proliferative, macular oedema), microalbuminuria,
macroalbuminuria,
end stage renal disease, erectile dysfunction, autonomic neuropathy,
peripheral neuropathy,
osteomyelitis and lower limb ischaemia.
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References herein to a "diabetic" patient are to be understood as a reference
to a patient
suffering from either type I diabetes (DM1) or type 2 diabetes (DM2).
In accordance with the present invention, HDL is administered to a diabetic
patient. The
term "HDL" as used herein relates to all forms of high density lipoproteins
and includes
mature HDL, nascent HDL or reconstituted HDL (rHDL) or any mixture thereof, as
well
as rHDL produced from recombinant apolipoprotein or an analogue thereof with
functional
relationship to nascent or reconstituted HDL. Such analogues include
functional peptides
derived from the apolipoprotein (Apo) structure such as those described in
International
Patent Publications Nos. WO 99/16459 and WO 99/16408.
The high density lipoproteins comprise a protein component, and lipid. The
proteins are
preferably apolipoproteins, e.g. human apolipoproteins such as apolipoprotein
A-I (apoA-I),
apolipoprotein A-II (apoA-II) or apolipoprotein A-IV (apoA-IV) or recombinant
apolipoproteins, or functionally homologous peptides with similar properties.
Suitable
lipids are phospholipids, preferably phosphatidyl choline, optionally mixed
with other
lipids (cholesterol, cholesterol esters, triglycerides, sphingolipids, or
other lipids). The
lipids may be synthetic lipids, naturally occurring lipids or combinations
thereof
Preferably, the HDL is reconstituted HDL.
Production of reconstituted HDL is described, by way of example, in US Patent
No.
5652339 and by Matz and Jonas (25) and Lerch et al. (26). Production of rHDL
with
recombinant apolipoproteins is described, by way of example, in European
Patent No. EP
469017 (in yeast), US Patent No. 6559284 (in E. coli), and International
Patent
Publications Nos. WO 87/02062 (in E. coli, yeast and Cho cells) and WO
88/03166 (in E.
coli).
The HDL is administered in an effective amount. An "effective amount" means an
amount
necessary at least partly to attain the desired response, or to delay the
onset or inhibit
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progression or halt altogether, the onset or progression of the particular
condition or
disorder being treated. The amount varies depending upon the health and
physical
condition of the individual to be treated, the racial background of the
individual to be
treated, the degree of protection desired, the formulation of the composition,
the
assessment of the medical situation, and other relevant factors. It is
expected that the
amount will fall in a relatively broad range that can be determined through
routine trials.
Preferred HDL dosage ranges are from 0.1-200 mg, more preferably 10-80 mg, HDL
(weight based on apolipoprotein) per kg body weight per treatment. For
example, the
dosage of HDL which is administered may be about 0.2-100 mg HDL per kg body
weight
(weight based on apolipoprotein) given as an intravenous injection and/or as
an infusion
for a clinically necessary period of time, e.g. for a period ranging from a
few minutes to
several hours, e.g. up to 24 hours. If necessary, the HDL administration may
be repeated
one or several times. The actual amount administered will be determined both
by the
nature of the condition or disorder which is being treated and by the rate at
which the HDL
is being administered.
Preferably, the patient is a human, however the present invention extends to
treatment
and/or prophylaxis of other mammalian patients including primates, livestock
animals (e.g.
sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice,
rabbits, rats, guinea
pigs), companion animals (e.g. dogs, cats) and captive wild animals.
In accordance with the present invention, the HDL is preferably administered
to a patient
by a parenteral route of administration. Parenteral administration includes
any route of
administration that is not through the alimentary canal (that is, not
enteral), including
administration by injection, infusion and the like. Administration by
injection includes, by
way of example, into a vein (intravenous), an artery (intraarterial), a muscle
(intramuscular) and under the skin (subcutaneous). The HDL may also be
administered in
a depot or slow release formulation, for example, subcutaneously,
intradermally or
intramuscularly, in a dosage which is sufficient to obtain the desired
pharmacological
effect.
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Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of the active component which is preferably isotonic with
the blood of
the recipient. This aqueous preparation may be formulated according to known
methods
using suitable dispersing or wetting agents and suspending agents. The sterile
injectable
preparation may also be a sterile injectable solution or suspension in a non-
toxic
parenterally-acceptable diluent or solvent, for example as a solution in a
polyethylene
glycol and lactic acid. Among the acceptable vehicles and solvents that may be
employed
are water, Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose,
trehalose,
glucose) and isotonic sodium chloride solution. In addition, sterile, fixed
oils are
conveniently employed as a solvent or suspending medium. For this purpose, any
bland
fixed oil may be employed including synthetic mono- or di-glycerides. In
addition, fatty
acids such as oleic acid find use in the preparation of injectables.
The formulation of such therapeutic compositions is well known to persons
skilled in this
field. Suitable pharmaceutically acceptable carriers and/or diluents include
any and all
conventional solvents, dispersion media, fillers, solid carriers, aqueous
solutions, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like.
The use of such media and agents for pharmaceutically active substances is
well known in
the art, and it is described, by way of example, in Remington's Pharmaceutical
Sciences,
18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, use
thereof in the
pharmaceutical compositions of the present invention is contemplated.
Supplementary
active ingredients can also be incorporated into the compositions.
Other delivery systems can include sustained release delivery systems.
Preferred sustained
release delivery systems are those which can provide for release of the active
component
of the invention in sustained release pellets or capsules. Many types of
sustained release
delivery systems are available. These include, but are not limited to: (a)
erosional systems
in which the active component is contained within a matrix, and (b)
diffusional systems in
which the active component permeates at a controlled rate through a polymer.
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The present invention also provides the use of high density lipoprotein (HDL)
in the
manufacture of a medicament for administration, preferably parenteral
administration, to a
diabetic patient for the treatment of endothelial dysfunction in the patient.
In yet another aspect, the invention provides an agent for administration,
preferably
parenteral administration, in the treatment of endothelial dysfunction in a
diabetic patient,
which comprises high density lipoprotein (HDL).
The present invention is further illustrated by the following non-limiting
Example.
EXAMPLE
I. Methods
Seven non smoking patients with uncomplicated DM2 (4 men and 3 women) and 7
non
smoking age- and sex matched normolipidemic control subjects (4 men and 3
women)
were enrolled. Inclusion criteria for patients with DM2 were as follows: (1)
fasting plasma
glucose > 7.0 mmol/L, (2) under treatment with diet and metformine; (3) not
using
exogenous insulin; (4) mild dyslipidemia with plasma triglycerides and LDL
cholesterol
levels of less than 2.0 and 3.5 mmo1/1, respectively. The presence of
macrovascular
disease, defined as ECG abnormalities, abnormal ankle-brachial index or a
history of
cardiac, cerebral of peripheral vascular events and autonomic neuropathy were
exclusion
criteria for either patients or control subjects. All female patients were
post-menopausal
and were not on hormone replacement therapy. The median duration of diabetes
was 5.2
1.2 [mean SID] years. Assessments were performed at least 4 weeks after the
cessation of
vasoactive medication, such as ACE inhibitors, angiotensin receptor blockers,
calcium
channel blockers, aspirin, NSAIDs, and vitamin supplementation. None of the
patients
used statin therapy. Alcohol, caffeine and metformin were withheld within 12
hours before
the study. All subjects gave written informed consent and approval was
obtained from the
internal review board of the Academic Medical Center (AMC), University of
Amsterdam,
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Amsterdam, The Netherlands. The study was carried out in accordance with the
principles
of the declaration of Helsinki.
Study Protocol
Vascular function was assessed at baseline and after rHDL infusion using
venous
occlusion strain-gauge plethysmography as previously published (EC-4; Hokanson
Inc,
Bellevue, USA) (27). Measurements were performed in a quiet room with a
constant
temperature (22 C to 24 C) and started at 08:00 a.m. Subjects remained in
supine position
throughout the study. The brachial artery of the nondominant arm was
cannulated with a -
20-gauge, flexible, polyurethane catheter (Arrow Inc, Reading, USA). Insertion
was
followed by a 30 minute interval of saline infusion to allow for re-
establishing baseline
conditions. Thereafter, forearm blood flow (FBF), expressed as millilitres per
minutes per
100 mL of forearm tissue volume (FAY), and was measured simultaneously in both
arms.
A microcomputer-based R-wave--triggered system for online monitoring was used.
During
each measurement, blood pressure cuffs around both upper arms were inflated
(40 mm Hg)
by use of a rapid cuff inflator. Simultaneously, bilateral wrists cuffs were
inflated to
above-systolic blood pressure to exclude hand circulation (200 mmHg). Intra-
arterial blood
pressure and heart rate were monitored continuously. Next, FBF response to
cumulative
doses of the endothelium-dependent vasodilator serotonin (5HT, Sigma; 0.6,
1.8, and 6 ng =
100 mL FAV-1 = min'), the endothelium-independent vasodilator sodium
nitroprusside
(SNP, Spruyt Hillen; 6, 60, 180, and 600 ng = 100 mL FAV'l = midi), and the
competitive
inhibitor of endothelial NO synthase (eNOS) NG-monomethyl-L-arginine (L-NMMA,
Kordia; 50, 100, 200, and 400 lig = 100 mL FAV-1 = min') was measured.
Infusion blocks
of serotonin and sodium nitroprusside were administered in randomized order,
followed by
L-NMMA infusion. All infusates were prepared in the pharmacy of the AMC in
accordance with good manufacturing practice (GMP) guidelines. Agents were
administered
intra-arterially for 6, 4, and 8 minutes at each dose, respectively, with a
constant-rate
infusion pump. Six measurements during the last 2 minutes of each infusion
block were
averaged to determine mean FBF. The 3 different infusion blocks proceeded
after a 15-
minute rest period or until FBF had returned to baseline. Subsequently, a
venous catheter
was inserted in the contralateral arm for administration of rHDL at a dose of
80 mg/kg
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body weight over a period of 4 hours (CSL Behring, Bern, Switzerland) (26,
27).
Thereafter, the infusion blocks were repeated. Patients were then asked not
restart their
medication (besides metformin) and had to return 7 days after rHDL infusion to
repeat
endothelial function measurements.
Laboratory Assessments
Blood samples were drawn from the subjects after a 12-hour overnight fast,
immediately, 4
hours and 7 days after rHDL infusion. After centrifugation within 1 hour after
collection,
aliquots were snap-frozen in liquid nitrogen and stored at -80 C until the
assays were
performed. All measurements were performed at the Vascular and Clinical
laboratory of
the Academic Medical Center, University Hospital of Amsterdam. ALAT and ASAT
were
measured by pyridoxalphosphate activation assay (Roche Diagnostics, Basel,
Switzerland).
HbA lc was measured by HPLC (Reagens Bio-Rad Laboratories B.V., the
Netherlands) on
a Variant II (Bio-Rad Laboratories). Plasma glucose was assessed in duplicate
using the
hexokinase method (Gluco-quant on Hitachi 917; Hitachi). Plasma triglycerides,
total
cholesterol, LDL and HDL levels were determined by standard enzymatic methods
(Roche
Diagnostics, Basel, Switzerland). Plasma levels of apoA-I and apoB were
assessed on
stored plasma by rate nephelometry.
Statistical Analysis
All results of clinical parameters, including plethysmographic data, are
expressed as mean
SD. Descriptive statistics between the 2 groups were compared by means of 2-
tailed
independent Student's t test. FBF was averaged over 6 consecutive recordings
during the
last 2 minutes of each infusion step. FBF recordings made in the first 30
seconds after
wrist-cuff inflation were not used for analysis. Statistical analysis of FBF
measurements,
HDL quality and inflammatory markers for individual subjects between the 2
groups was
performed by 2-way ANOVA for repeated measures. A probability value of P<0.05
was
considered significant and a value of P<0.01 as highly significant. -
Results
Subject characteristics are listed in Table 1. Baseline FBFs were not
significantly different
between patients and control subjects (Table 1). As expected, plasma levels of
fasting
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plasma glucose (P<0.01), hemoglobin Alc (P<0.01), triglycerides (P<0.01) and
apoB
(P<0.05) were higher in DM2 patients. HDL-C and apoA-I levels were comparable
between DM2 patients and controls. After rHDL infusion, plasma apoA-I
increased in
DM2 and control after 4 hours (1.2 0.2 to 2.8 0.4 versus 1.2 0.2 to 2.7
0.4 g/L,
p<0.01 respectively), whereas a slight increase remained in DM2 patients 7
days after
infusion (1.5 0.3 g/L, p<0.05 compared to baseline). Plasma apoB levels were
higher in
DM2 compared to controls and were not affected by rHDL infusion (1.0 0.3 to
0.9 0.3
versus 0.7 0.2 to 0.7 0.2 g/L, ns, respectively).
Acute and long term effects of rHDL infusion on NO bio-availability
Intra-arterial infusion of the endothelium-dependent vasodilator serotonin
increased FBF in
a dose-dependent manner in both groups. At baseline, the FBF response to
serotonin was
attenuated in DM2 compared to controls (p< 0.001, Figure 1). After rHDL
infusion, FBF
response to serotonin increased significantly, but did not reach levels
comparable to
controls (p<0.01, Figure 1). Interestingly, improvement in endothelium-
dependent
vasodilation persisted 7 days after rHDL infusion (p<0.01, Figure 1). rHDL
infusion did
not affect serotonin response in controls (Figure 1).
At baseline the vasoconstrictor response to L-NMMA, reflecting basal NO
activity, was
blunted in DM2 patients compared to controls (p<0.001, Figure 2). After rHDL
infusion,
the L-NMMA constrictor response was improved, again still present 7 days after
infusion
(p<0.01, Figure 2). In line with the serotonin data, rHDL infusion had no
effect on L-
NMMA response in control subjects (Figure 2).
Finally, endothelium-independent vasodilation in response to SNP was lower in
DM2
versus controls (p<0.01, Figure 3) and rHDL infusion did not show effect on
SNP
vasodilator response both in patients and controls.
III Discussion
The present study shows that both basal and stimulated NO activity are
severely
compromised in DM2 patients compared to age and sex-matched controls.
Interestingly,
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despite near normal HDL levels in DM2 patients, infusion of rHDL significantly
improved
endothelial function. This improvement persisted up to 7 days after rHDL
infusion, at
which time HDL levels had already returned towards baseline values. These data
imply
that HDL increasing strategies may offer therapeutic benefit in DM2, even if
HDL-C
levels are not clearly decreased in these patients.
Vascular function at baseline
In line with previous studies (8, 9, 12), the presence of both impaired basal
NO-activity as
well as attenuated NO-release in DM2 upon receptor-mediated stimulation is
confirmed.
Several mechanisms have been shown to contribute to endothelial dysfunction in
diabetes.
Decreased bio-availability of the essential cofactor tetrahydrobiopterin (BH4)
is associated
with uncoupling of endothelial NO-synthase leading to direct production of
oxygen
radicals instead of NO by eNOS (28-30). Other sources may also contribute to
increased
radical production, including NADPH-oxidase as well as mitochondrial
uncoupling (10).
The pivotal role of ROS in diabetic vascular dysfunction has been underscored
by
interventions studies, reporting full restoration of endothelial function upon
intra-arterial
infusion of high concentration of anti-oxidants (9, 12).
Effect of rHDL infusion on vascular function
Infusion of rHDL was associated with a rapid improvement of both basal NO
activity as
well as receptor-stimulated NO-activity, within a few hours after infusion. A
first
explanation would be that rHDL increases NO production. Nitric oxide (NO) is
synthesized by eNOS through the conversion of L-arginine to L-citrulline. Its
activity is
regulated by complex signalling transduction pathways including activation of
the kinases
that alter the phosphorylation of eNOS, i.e. MAP kinase and akt-kinase
signalling, or
increasing intracellular Ca2+ content followed by calcium-calmodulin dependent
activation
of eNOS (33). Yuhanna showed that binding of apoA-I to the endothelial
scavenger
receptor B-1 was accompanied by enhanced endothelium-dependent relaxation
responses
in aortas (24), largely due to akt and MAP-kinase activation (32). In
addition, HDL also
has the capacity to upregulate membrane content of eNOS within endothelial
cells by
preserving eNOS protein stability as well as by preventing eNOS translocation
from the
cell membrane to intracellular organelles (23). All these effects may have
contributed to
CA 02580100 2007-03-01
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the increase in basal NO-availability, assessed as increased vasoconstrictor
responses to
the competitive NO-inhibitor L-NMMA, after rHDL infusion. In contrast, the
afore-
mentioned mechanisms cannot fully explain the increase in serotonin-dependent,
receptor-
stimulated NO availability, which is dependent upon calcium-calmodulin
activation of
eNOS (31). Since serotonin binding to the endothelial 5HT-2A receptor (33) is
unlikely to
change upon rHDL infusion, decreased degradation of NO by oxygen radicals
provides a
second major pathway which can contribute to increased NO-bioavailability.
Indeed, HDL
has potent anti-oxidative properties, not in the least due to the presence of
enzymes such as
paraoxonase and platelet-activating factor hydrolase on the HDL particle (23).
Long-term effects of rHDL on vascular function
Strikingly, endothelium dependent vasodilation was still significantly
improved 1 week
after rHDL infusion. In contrast, both apoAI as well as HDL levels had almost
returned
towards pre-infusion levels. Noticeably, at baseline HDL levels in DM2 were
also not
significantly different from those in control subjects. In contrast, rHDL
infusion had no
effect whatsoever on vascular function in controls. These data imply that, in
spite of
normal HDL concentration, HDL quality may be impaired in DM2. In fact, loss of
HDL
protective effects in DM2 has been partly attributed to non-enzymatic
glycation of
predominantly leucine chains in HDL. Glycation of apoAI-HDL compromises the
ability
of HDL to protect LDL from oxidative damage, amongst other by loss of PON-1
activity
(34). In addition, glycated HDL reduces eNOS expression within the
endothelium, leading
to impaired NO-producing capacity (35). Indeed, antioxidative activity level
of HDL in
DM2 patients are intimately linked to oxidative stress levels and glycemic
control (36).
Study Limitations
Since an effect of rHDL was not seen 4 hours after infusion in the control
group, the
vascular function studies were not repeated after 7 days in the control group.
Consequently, the vascular function data on day 7 in DM2 patients were
compared to
baseline and day 1 observations in controls. However, since the major
conclusion on
vascular function relates to the persistent improvement compared to DM2
patients at
CA 02580100 2007-03-01
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baseline, lack of day 7 study in controls has no impact on the conclusions
drawn in the
present study. Second, although only a relatively small group of DM2 patients
was studied,
the fact that a significant improvement was found already in a limited group
of patients and
reproducibly after 4 hours and 1 week, is supportive of a clear conclusion on
the effect of
HDL on vascular function in DM2 patients, in spite of the small sample size.
Clinical implications for DM2 patients
Statins are the central paradigm for cardiovascular preventive strategies.
However, in view
of the large number of events not prevented during statin therapy, the search
for optimal
combination therapies is in full progress. The promise of HDL increasing
strategies is
expanding rapidly. The strong inverse relationship between HDL-C and
cardiovascular
events is a consistent finding in both non-diabetic as well as diabetic
patients.
Unfortunately, solid data providing evidence for reduction of cardiovascular
risk following
HDL-increasing interventions are scarce, predominantly due to lack of
selective and potent
HDL increasing compounds (37).
Recent data have shown that 5 weekly infusions of rHDL produced with a variant
apolipoprotein (apoA-I Milano) were able to slow progression or even induce
regression of
coronary atheroma volume in patient with recent myocardial infarction (38). In
line with
such a rapid effect, both experimental as well as in vivo studies have shown
that the anti-
atherogenic capacity of HDL is not merely restricted to its role in reverse
cholesterol
transport. The present observation of acute and persistent restoration of
endothelial
dysfunction in DM2 lends further support to effects of HDL beyond its role in
reverse
cholesterol transport. This supports a role for HDL increase in DM2 even if
HDL levels
are not clearly decreased.
30
CA 02580100 2007-03-01
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Table 1.
DM2 CON
(n=7) (n=7)
Age, years 53.6 3.0 48.6 15.1
Sex (female/male) 3/4
BMI, kg/m2 28.9 2.4 25.6 3.6
Smoking (y/n) 0/7 0/7
Systolic blood pressure, mmHg 148 12 135 16
Diastolic blood pressure, mmHg 78 13 83 9
Heart Rate, bpm 65 5 61 4
Fasting plasma glucose, (mmol/L) 8.3 1.2 5.2 0.4 #
HbAlc, % 7.1 0.3 5.4 0.3 #
Total Cholesterol, mmol/L 5.6 0.4 5.3 0.4
LDL-C, mmol/L 2.9 0.6 3.0 0.7
HDL-C, mmol/L 1.1 0.2 1.2 0.3
ApoA-I, g/L 1.2 0.1 1.2 0.2
ApoB, g/L 1.1 0.3 0.8 0.2 *
Triglycerides, mmol/L 1.5 0.4 0.8 0.3 #
Basal FBF, ml = 100 mL FM/4 = min 4.1 2.0 2.6 0.9
* p <0.05, # p <0.01
CA 02580100 2007-03-01
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