Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION OF FUNCTIONAL PROTEINS:
BALANCE OF SHEAR STRESS AND GRAVITY
ORIGIN OF THE INVENTION
The jointly made invention described herein was made by an
employee of the United States Govennment and may be manufactured and used by
or
for the Government of the United States of America for governmental purposes
without the payment of any royalties hereon or therefor.
The invention described herein was also made by inventors in the
performance of work under an agreement with Tulane Educational Fund and is
subject to the provisions of Section 305 of the National Aeronautics and Space
Act
of 1958, Public Law 85-568 (72 Stat. 435; 42 U.S.C. 2457).
RELATED APPLICATIONS
The present application claims the benefit of 35 U.S.C. ~111(b)
provisional patent application 60/043205 filed April 8, 1997.
BACKGROUND OF THE INVENTION
Federal Funding Notice
The present invention was funded by NIH Grant DK46117, NIH R21,
and NASA NRA Grant 9-811. Consequently, the United States government has
certain rights in this invention.
Field of the Invention
The present invention relates generally to the fields of protein
chemistry, endocrinology and gene therapy. More specifically, the present
invention
relates to a method for production of functional proteins in culture in
response to
shear stress using a rotating wall vessel.
Description of the Related Art
A successful and documented modality to induce polarization and
differentiation of cells in culture is the rotating wall vessel (1-4). In
rotating wall
vessels gravity is balanced by equal and opposite physical forces including
shear
stresses. In engineering terms this has been claimed to simulated microgravity
at
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boundary conditions [Wolf D.A. and R.P. Schwarz. (1991) NASA Technical Paper
3143].
Rotating wall vessels, including models with perfusion, are a
quantum advance. The rotating wall vessel is a horizontally rotated
cylindrical cell
culture device with a coaxial tubular oxygenator (1, 5-7). The rotating wall
vessel
induces expression of select tissue-specific proteins in diverse cell cultures
(1-2, 8-
9). Examples of expression of tissue-specific proteins include
carcinoembryonic
antigen expression in MIP-101 colon carcinoma cells (2), prostate specific
antigen
induction in human prostate fibroblasts (7), through matrix material induction
during
chondrocyte culture (8). The quiescent cell culture environment of the
rotating wall
vessel balances gravity with shear and other forces without obvious mass
transfer
tradeoff (1-2, 4). The rotating wall vessel provides a culture environment
suitable
for co-cultures of diverse cell types, and three dimensional tissue construct
formation.
i 5 Rotating wall vessel technology is being used in clinical medical
practice recently by facilitating pancreatic islet implantation (4, 10).
Pancreatic
islets are prepared in rotating wall vessels to maintain production and
regulation of
insulin secretion. The islets are alginate encapsulated to create a non-
inflammatory
immune haven, and are implanted into the peritoneal cavity of Type I diabetic
patients. This implantation of pancreatic islets has maintained normoglycemia
for
18 months in diabetic patients, and progressed to Phase III clinical trials
(4, 10).
These vessels have also been applied to, for example, mammalian skeletal
muscle
tissue, cartilage, salivary glands, ovarian tumor cells, and colon crypt cells
(11-13).
Previous studies on shear stress response in endothelial cells, and rotating
wall
vessel culture have been limited to structural genes (14-16). These studies
did not
address the issue of a process for the production of functional molecules,
such as
hormones. Shear stress response elements have not previously been demonstrated
in
epithelial cells, either for structural genes of production of functional
molecules.
Vitamin D dependent rickets has been a disease familiar to family
farms and larger animal husbandry industries for centuries (17-18). The
development of renal replacement therapy by dialysis in humans expanded
vitamin
D deficient bone disease from an occasional human clinical caveat to a common
clinical problem. This led to identification of the active form of vitamin D
as 1,25-
diOH D, and the development of a mufti-billion dollar per year worldwide
market,
predominantly in end-stage renal disease patients, to provide replacement
hormone
clinically (18). The active 1,25-diOH form of vitamin D, is mainly used to
treat
bone disease in dialysis patients but has *also been implicated as a therapy
for
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osteoporosis, and some forms of cancer. Recently, the effects of vitamin D
have
been recognized to play a central role not only in other common bone lesions
such as
osteoporosis due to aging and steroid induced osteoporosis, but in immune
function
and surveillance, growth and development, and cardiac and skeletal muscle
function
( 19-22).
Several active forms of vitamin D have been identified, vitamin D
receptors cloned, and nuclear binding proteins for the hormone identified and
cloned
( 17-22). Studies on the regulation of 1 a-hydroxylase activity are limited by
the
lack of a renal cell line with regulated expression of the enzyme. The only
reports of
1-a-hydroxylase activity in culture utilize freshly isolated chicken renal
cortical
cells in which the activity declines precipitously within 48 hours of plating
in culture
(28).
The importance of the renal 1-a-hydroxylase is best understood by
comparing the kinetics of the renal enzyme to other forms in the body (29-30).
Demonstration that nephrectomy in pregnant rats did not completely abolish
1,25
diOH-D, formation sparked an intensive search for extrarenal sites of 1 a-
hydroxylase activity (29). Although 1 a-hydroxylase activity has been reported
in
monocytes, liver, aortic endothelium and a variety of placental and fetal
tissues, the
enzyme kinetics contrast sharply with the renal 1 a-hydroxylase. Extrarenal 1-
a-
hydroxylase has a much higher Km indicating that much higher substrate levels
are
needed for activity (29). In the uremic patient, extrarenal 1,25-diOH D,
production
is very limited due to a relative lack of substrate. Administrating large
quantities of
25-OH D, substrate to anephric patients modestly boosts plasma 1,25-diOH D,
levels (29).
The lack of a differentiated polarized line of renal tubular epithelial
cells for investigative purposes persists despite extensive searches by
several
laboratories (3 i-38). Renally derived cell lines transformed with viruses or
tumor
cells to produce immortality continue as some of the most popular cell
biological
tools to study polarized delivery (31, 33, 35). But these renally derived
immortal
cell lines such as MDCK or LLP-CK1 retain few if any of the differentiated
features
characteristic of renal epithelial cells. Similarly, primary cultures rapidly
' dedifferentiate and modalities as diverse as basement membrane matrices,
growth
supplements or Millipore inserts achieve only modest degrees of polarity (37-
38).
' The pathognomonic structural features of renal proximal tubular
epithelial cells are the abundance of apically derived microvilli, the
glycoprotein
content of associated intermicrovillar clefts, and the highly distinctive
arrangement
of subapical endosomal elements (39-40). Renal epithelial cells of the
proximal
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tubule are characterized by thousands of long apical microvilli. The apical
endosomal machinery begins in intermicrovillar clefts. The endosomal pathway
is
characterized by clathrin coated vesicles, small spherical endosomal vesicles,
with
deeper larger endosomal vacuoles (33, 39). From the endosomal vacuoles
proteins
and lipids either recycle to apical surface in dense apical tubules or shuttle
to
lysosomes to be degraded.
A cluster of apical proteins with homologous sequence repeats are
especially desirable to express in cultured cells as they are thought to be
molecular
mediators of renal injury (41-43). Two of these proteins megalin (gp330) and
cubulin (gp280) (Moestrup, et al., J. Biol. Chem.13273 (9):5325-5242 (1998)
are
molecular mediators of tubular vacuolation and ensuing secondary damage.
Megalin (gp330) is a receptor found on the luminal surface of the proximal
tubular
cells of the kidney. Megalin binds several proteins and drugs including
aminoglycoside antibiotics and other polybasic drugs. Megalin is expressed in
the
kidney, lung, testes, ear, and placenta. The only cells which express megalin
in
culture are immortalized placental cells. There is no known renal cell culture
which
expresses megalin. Gp280 is a receptor found on the luminal surface of the
proximal tubular cells of the kidney. Gp280 binds several proteins and drugs
including intrinsic factor-cobalamin (vitamin B12 bound to its carrier
protein) and
myeloma light chains. Cubulin (gp280) is expressed in the kidney, ear, and
placenta. The only cells which express cubulin (gp280) in culture are
immortalized
placental cells. There is no known renal cell culture which expresses cubulin
(gp280).
Erythropoietin (EPO) is a hormone produced in the kidney, and
secreted into the blood. Erythropoietin controls the rate of production of red
blood
cells by the bone marrow. Erythropoietin may be produced by the interstitial
cells
between the tubules or the proximal tubular cells or both. Erythropoietin
production
is lost in all known renal cell culture systems. Erythropoietin is mainly used
to treat
anemia in dialysis patients but is also popular to treat the anemia of AIDS
patients
and many forms of cancer.
The prior art is deficient in the lack of effective means of producing
functional proteins including hormones in response to shear stress. Further,
the prior
art is deficient in the identification of shear stress response elements in
epithelial cell
genes. The present invention fulfills this longstanding need and desire in the
art.
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SUMMARY OF THE INVENTION
In one embodiment of the present invention, there is provided a
method of producing a functional protein, comprising the steps of: isolating
. mammalian cells; placing said cells into a rotating wall vessel containing a
cell
culture comprising culture media and culture matrix; producing three-
dimensional
cell aggregates under simulated microgravity conditions; and detecting
expression of
the functional protein in the cell culture.
In another embodiment of the present invention, there is provided a
method of inducing expression of at least one gene in a cell, comprising the
steps of
contacting said cell with an transcription factor decoy oligonucleotide
sequence
directed against a nucleotide sequence encoding a shear stress response
element; and
determining the expression of said gene in said cell.
In yet another embodiment of the present invention, there is provided
a transcription factor decoy, comprising an oligonucleotide sequence directed
against a nucleotide sequence encoding a shear stress response element.
Other and further aspects, features, and advantages of the present
invention will be apparent from the following description of the presently
preferred
embodiments of the invention given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features, advantages and
objects of the invention, as well as others which will become clear, are
attained and
can be understood in detail, more particular descriptions of the invention
briefly
summarized above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings form a part of the
specification. It is to be noted, however, that the appended drawings
illustrate
preferred embodiments of the invention and therefore are not to be considered
limiting in their scope.
Figure 1 shows homogeneity and structure of human renal epithelial
' cells in culture. Flow cytometry frequency histograms demonstrate number of
cells
positive for the proximal tubular marker y-glutamyl transferase. Figure lA
shows
the number of cells with y-glutamyl transferase activity as the frequency of
activity
in 2000 cells compared to an unstained control with trapping agent alone. This
is
the raw digest of human renal cells. Figure 1B shows that following
differential
trypsinization, the percentage of proximal tubular cells present can be
increased to
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99+1%. Figure 1C and 1D show transmission electron micrographs of human
epithelial cells in culture. The intact renal cortex in vivo (far left panel),
is compared
to culture of the natural mixture of human renal cortical cells in
conventional 2-
dimensional culture (middle left panel) which is completely devoid of
microvilli.
Rotating wall vessel culture of pure proximal tubular cells shows some
microvilli
(middle right panel} but there are far more microvilli during rotating wall
vessel
culture of the natural mix of renal cortical cells (far right panel). Compared
to these
representative images, some areas of the natural mixture of cells in the
rotating wall
vessel show much greater abundance of microvilli, and well defined desmosomes
(lower panel) which are lacking in the other cultures.
Figure 2 shows protein expression in the rotating wall vessel. Figure
2A shows analysis of the expression and endosomal compartmentation of megalin,
and cubulin in renal cells following rotating wall vessel culture. The ability
of flow
cytometry to make simultaneous measurements of entrapped fluorescein dextran
as
an endosomal marker and antibody binding allows construction of three
dimensional
frequency histograms displaying entrapped fluorescein dextran fluorescence
against
antibody binding on horizontal axes. A control sample shows vesicles negative
for
fluorescein on the left and fluorescein containing endosomes on the right
(2000
vesicles depicted left panel). A control without fluorescein entrapped shows
only
the left population (not shown). Co localization of anti-cubulin binding
demonstrates that all the fluorescein positive endosomes are positive for
cubulin,
while non-endosomal membranes can be subdivided into cubulin positive and
negative populations (middle panel). This pattern is repeated for anti-megalin
binding in renal cortical cells (right panel).
Figure 2B shows quantitatian of cubulin, and megalin antibody
binding to renal cell membranes under various culture conditions. Analysis of
protein expression in cultured cells by antibody binding used classic serial
log
dilution antibody curves. An increase in binding with a decrease in dilution
is
pathognomonic for specific antibody binding during flow cytometry analysis.
Binding of anti-cubulin antisera to membrane vesicles prepared from renal
cortical
cells after 16 days in culture, detected by the fluorescence of a
phycoerthyrein
tagged secondary antibody, shows an almost two log increase in binding with
antibody dilution (upper left panel below). This increased cubulin antibody
binding
in the cells grown in the rotating wall vessel (STLV) is more than five times
the
expression seen in stirred fermentors. Similarly, there was no detectable
expression
in the conventional cultures resulting in a flat line (not shown). Binding of
normal
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serum and minimal dilution of primary antisera were not detectably different.
Binding curves for anti-megalin antiserum showed a similar pattern (not
shown).
. Figure 2C depicts non-specific (minimum) and peak binding of each
antiserum following rotating wall vessel culture and two-dimensional SDS-PAGE
analysis of protein content of cells following rotating wall vessel culture.
Analysis
of the protein content of cultures of the natural mixture of rat renal
cortical cells
after 16 days culture in gas permeable bags as a control (left panel) or
rotating wall
vessel (right panel) depicts changes in a select set of proteins. Molecular
weight
(14-220 kDa) on the abscissa is displayed against isoelectric point (pH 3-10)
on the
ordinate.
Figure 3 shows gene expression in the rotating wall vessel. Figure
3A and Figure 3B show differential display of genetic expression of rat renal
cortical
cells grown in conventional culture or rotating wall vessels. Differential
display of
expressed genes was compared in aliquots of the same cells grown in a 55 ml
1 S rotating wall vessel (STLV) or conventional gas permeable 2-dimensional
bag
controls. For differential display, copies of expressed genes were generated
by
polymerase chain reaction using random 25mer primers and separated on a 6% DNA
sequencing gel (Figure 3A). Bands of different intensity between control and
STLV, representing differentially expressed genes, were identified by visual
inspection, excised and reamplified using the same primers. Differential
expression
and transcript size were confirmed by Northern hybridization (Figure 3B). PCR
products were then subcloned into the pGEM-T vector and sequenced. Sequences
were compared to the Genebank sequences using the BLAST search engine. One
expressed gene which decreased in the STLV (band D on gelabove) was identified
as rat manganese-containing superoxide dysmutase (98% match 142 of 144
nucleotides). Two genes which increased in the STLV, band A was identified as
the
interleukin-1 beta gene (100% match for 32 of 32 nucleotides) and Band B which
corresponded to a 20 kB transcript on a Northern blot appears to be a
unidentified
gene that has a 76% homology to the mouse GABA transporter gene. Figure 3C and
Figure 3D show RT-PCR of time dependent change in genes during rotating wall
vessel culture. Semi quantitative RT-PCR shows increases in the epithelial
genes
megalin, villin and extra-cellular calcium sensing receptor (ECaR), the shear
stress
response element genes ICAM, VCAM and MnSOD (Figure 3C). There was no
change in b-actin or GADPH. Unlike in endothelial cells many of these changes
are
prolonged as at 16 days megalin, ECaR, ICAM, VCAM and villin changes persist
(Figure 3D).
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Figure 4 shows structure and effects of antisense probe for shear
stress response element on rat renal cortical epithelial cells. Figure 4A
shows the
structure. The probe with sequence CTGAGACCGATATCGGTCTCAG (SEQ ID
No:l) has two possible conformations. As a single strand it would fold back on
itself to form a binding element for the transcription factor. As a double
strand it
would then have two binding sites for the transcription factor, one in the
sense
orientation and one in the antisense orientation.
Figure 4B shows effects of antisense shear stress response element
probe on time dependent gene expression. The antisense probe added to
conventional 2-dimensional cultures of rat renal cortical cells at 80 nm
increases
MnSOD in a time dependent manner. Comparison is made to controls with the
active binding site scrambled. In contrast the probe has no effect on villin
gene
expression.
Figure 5 shows gene expression in the rotating wall vessel: automated
gene analysis. Abundance of the expression of over 18,300 genes was assayed by
annealing poly A RNA from human renal cortical epithelial cells grown in a
rotating
wall vessel for 8 days to a filter robotically loaded with oligonucleotide
primers.
Poly A RNA from a non adherent bag culture serves as a control. The filters
are
shown at the top of the diagram then the analysis of shear stress responsive
genes,
renal epithelium specific genes, and other genes germane to the current
analysis.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a method of producing a
functional protein, comprising the steps of isolating mammalian cells; placing
said
cells into a rotating wall vessel containing a cell culture comprising culture
media
and culture matrix; producing three-dimensional cell aggregates under
simulated
microgravity conditions; and detecting expression of the functional protein in
the
cell culture. Generally, simulated microgravity conditions comprise a balance
between gravity and oppositely directed physical forces. Representative
examples
such physical forces include sedimentational shear stress, centrifugal forces,
viscosity and coriolus forces.
Preferably, the functional protein is selected from the group
consisting of a hormone, a toxin receptor and a shear stress dependent
functional
biomolecule. Representative examples of hormones which can be produced
according to the method of the present invention include 1,25-dihydroxy-
vitamin D3
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and erythropoietin. Representative examples of toxin receptors which can be
produced according to the method of the present invention include megalin and
cubulin. Representative examples of shear stress dependent functional
biomolecule
which can be produced according to the method of the present invention include
is
selected from the group consisting of villin, magnesium dependent superoxide
dismutase, nitric oxide synthase, c-fos, c-jun, platelet derived growth factor-
b,
transforming growth factor-b, tissue-type plasminogen activator and monocyte
chemotactic protein-1, megalin, cubulin, erythropoietin and 1-a-hydroxylase.
Generally, any mammalian cell could be used in the methods of the
present invention. Representative examples of mammalian cells include renal
cortical cells, renal fibroblast cells, hepatocytes, pancreatic islets, renal
interstitial
cells, parathyroid cells, thyroid cells, pituitary cells, ovarian cells and
testicular cells.
Generally, the cell is selected from the group consisting of epithelial cell
and
endothelial cell. Preferably, cell contains shear stress response elements.
1 S Representative examples of shear stress response element include GAGACC
and
GGTCTC.
In the methods of the present invention, the rotating wall vessel is
initiated and maintained from about 6 rotations per minute to about 16
rotations per
minute. Preferably, the sedimentational shear stress is from about 0.2
dynes/cm2 to about 1.0 dynes/cm2. The culture matrix may contain a core
structure
selected from the group consisting of cell aggregates and microcarrier beads,
although other components to such a culture matrix are well known to those
having
ordinary skill in this art.
The present invention is also directed to a method of inducing
expression of at least one gene in a cell, comprising the steps of: contacting
said cell
with an transcription factor decoy oligonucleotide sequence directed against a
nucleotide sequence encoding a shear stress response element; and determining
the
expression of said gene in said cell. Generally, oligonucleotide comprises a
terminal
phosphothiorate moiety and a phosphodiester backbone and a structure which
allows
the oligonucleotide to pass cell membranes and accumulate in the nuclear
compartment of the cell. Generally, the cell is a cultured cell. Preferably,
the cell is
' selected from the group consisting of an epithelial cell and an endothelial
cell.
Representative examples of which can be used in this method include renal
cortical
cell, renal fibroblast cell, hepatocyte, pancreatic islet, renal interstitial
cell,
parathyroid cell, thyroid cell, pituitary cell, ovarian cell and testicular
cell. In one
embodiment, the cell is grown in two dimensional culture. Representative
examples
of shear stress response elements include GAGACC and GGTCTC.
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Preferably, the gene encodes a protein selected from the group consisting of
megalin, cubulin, erythropoietin and 1-a-hydroxylase. The concentration of the
oligonucleotide useful in this method generally ranges from about 10 nm to
about 10
The present invention is also directed to a transcription factor decoy,
comprising an oligonucleotide sequence directed against a nucleotide sequence
encoding a shear stress response element. Preferably, the nucleotide sequence
encoding a shear stress response element has a sequence selected from the
group
consisting of GAGACC and GGTCTC.
In one preferred technique, the rotating wall vessel is generally
initiated and maintained at 10 rotations per minute. Preferably, the rotating
wall
vessel provides a balance of forces comprising gravity and equal and opposite
sedimentational shear stress. Useful sedimentational shear stress rates within
the
context of the claimed methods are from about 0.2 dyneslcm2 to 1.0 dynes/cm2.
As used herein, rotating wall vessels= refers to a cylidrical
horizontal rotating culture vessel with a coaxial oxygenator.
As used herein, shear stress response element= refers to a sequence
of a family of genes in the cell nucleus which binds one or more transcription
factors
in response to shear stress on the cell. A representative example of a shear
stress
response element is GAGACC or its complementary sequence GGTCTC.
As used herein, shear stress conditions= refers to flow of liquid, or
current of liquid over cells which causes genes to turn on or off.
As used herein, slow turning lateral vessel :- refers to one specific
size and shape of a rotating wall vessel.
As used herein, differential display- refers to displaying on a filter,
gel or chip a discrete set of genes turned on or off in a cell under two
different
conditions.
As used herein, simulated microgravity= refers to balance of gravity
by oppositely directed forces including shear stresses during rotational wall
vessel
culture.
As used herein, graded gravitational sedimentation shear-'.-- refers to
the shear imparted to a particle or cell falling through fluid.
As used herein, functional protein= refers to a protein with biological
effects.
As used herein, three-dimensional co-culture process= refers to cells
grown in a matrix or on beads (or other three-dimensional structural suport)
in a
three-dimensional array, rather than on a flat plate.
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As used herein, coriolus force= refers to an incidental flow field
caused by the rotating gravity vector in the rotating wall vessel.
As used herein, shear stress= refers to the force felt at the surface of
the particle as it moves through the fluid.
As used herein, gravity induced sedimentation= refers to the force on
a particle in the rotating wall vessel making it fall through the fluid due to
gravity.
As used herein, centrifugal force= refers to the force on a particle in
the rotating wall vessel which pulls it towards the wall due to rotational
speed.
As used herein, transcription factor decoy- refers to an
oligonucleotide folded to form a double stranded DNA which binds a nuclear
trancription factor. The transcription factor decoy prevents the transcription
factor
from binding promoter regions regulating expression of specific genes.
The following examples are given for the purpose of illustrating
various embodiments of the invention and are not meant to limit the present
invention in any fashion.
EXAMPLE 1
Human Renal Cortical Cells
Human renal cortical cells were isolated by Clonetics Inc. (San
Diego, CA) from kidneys unsuitable for transplantation. Differential
trypsinization
resulted in cell fractions highly purified for proximal tubular cells compared
to the
natural mixture of cells in the renal cortex. The co-culture of the natural
cell mix,
and highly purified proximal tubular cells were cultured separately in a
special
growth medium with 2% fetal calf serum.
EXAMPLE 2
Rat Renal Cortical Cells
Rat renal cells were isolated from renal cortex harvested from
euthenized Sprague Dawley rats (Harlan Sprague-Dawley, Cleveland OH) as
described (44). In brief, renal cortex was dissected out with scissors, minced
finely
in a renal cell buffer 137 mmol NaCI, 5.4 mmol KCI, 2.8 mmol CaCl2, 1.2 mmol
' 30 MgCl2, 10 mmol HEPES-Tris, pH 7.4. The minced tissue was placed in 10 ml
of a
solution of 0.1 % Type IV collagenase and 0.1 % trypsin in normal saline. The
' solution was incubated in a 37oC shaking water bath for 45 minutes with
intermittent titration. The cells were spun gently (800rpm for 5 minutes), the
supernatant aspirated, the cells resuspended in 5 ml renal cell buffer with
0.1
bovine serum, .and passed through a fine (70 mm) mesh. The fraction passing
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through the mesh was layered over a discontinuous gradient of 5% bovine serum
albumin and spun gently. The supernatant was again discarded. The cells were
resuspended in DMEM/F-12 medium (ciprofloxacin and fungizone treated) and
placed into culture in various culture vessels in a 5% C02 95% 02 incubator.
EXAMPLE 3
Culture Techniques: Rotating Wall Vessels
When grown under conventional conditions in DMEM/F I2
supplemented with fetal calf serum and an antibiotic cocktail such as
ciprofloxacin
and fungizone, both the highly purified cells as well as the cell mix form a
monolayer. Fetal calf serum was used at optimal concentration: 2% for human
cells
and 10% for rat cells. In order to increase epithelial cell differentiation
(1, 45), renal
cells were cultured in a rotating wall vessels known as a SS ml slow turning
lateral
vessel (STLV) (I, 45). To initiate cell culture, the slow turning lateral
vessel was
filled with medium, and seeded by addition of cell suspension (2X106
cells/ml).
Residual air was removed through a syringe port and vessel rotation was
initiated at
10 rotations per minute, and maintained for 10-16 days. Medium was changed
every
2 to 3 days depending on glucose utilization. Concomitant with cells,
microcarrier
beads were added an 5 mg/ml to promote aggregate formation in the slow turning
lateral vessel. Without beads the cells became shattered in the vessel in a
few hours.
Beads were cytodex-3 in all protocol except when electron microscopy was
planned
when the much more expensive, but easily sectioned Cultisphere GL cells were
added to the vessels.
EXAMPLE 4
Stirred Controls and Static Controls
To provide a stirred control stirred fermentors which mixed in the
horizontal plane were loaded with identical concentrations of cells and beads
from
the same pool added to the slow turning lateral vessel (1, 31, 46). Gas
permeable
Fluoroseal bags (Fluoroseal Inc, Urbana IL) in 7 or 55 ml size were selected
as
conventional static controls. Culture beads were added to the conventional
controls
at the same density as the slow turning lateral vessel cultures (1, 45).
EXAMPLE 5
Electron Microscopy Quantitation of Number of Microvilli
Transmission electron micrographs were performed on cell
aggregates from the rotating wall vessels and conventional monolayers. Cells
were
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washed with ice cold phosphate buffered saline, then fixed for electron
microscopy
with 2.5% glutaraldehyde in phosphate buffered saline (9, 47). The samples
were
then transferred to 1% osmium tetroxide in 0.05 M sodium phosphate (pH 7.2)
for
several hours, dehydrated in an acetone series followed by embedding in Epon.
Lead-stained thin sections were examined and photographed using a Phillips
EM/200 electron microscope. For electron microscopy the easily sectioned
Cultispere GL beads, replaced Cytodex-3 which is almost impossible to section.
EXAMPLE 6
Analysis of the Proximal Tubule Epithelial Marker, g-glutamyl Transpeptidase
The renal cortical cells were 75+4% (n~) proximal tubules as
determined by flow cytometry analysis of aliquots for the proximal marker g-
glutamyl transferase using Schiff base trapping of cleavage products of L-g-
glu-4-
methoxy-4-b-naphthylamine (44) (Figure 1 ).
EXAMPLE 7
Analysis of the Endosomal Distribution of Megalin and Cubulin by Flow
Cytometry
To quantitate the total and endosomal expression of cubulin, megalin
and aquaporin-2 cells in conventional culture, stirred fermentors and slow
turning
lateral vessel rotating wall vessels, 0.3 mg/ml lOS fluorescein-dextran was to
each
cell culture for 10 minutes at 37oC in the C02 incubator. This loads an
entrapped
fluorescent dye into the early endosomal pathway (9, 47). Cells were then
immediately diluted into ice cold phosphate buffered saline and washed once.
Next,
the cells were homogenized with 6 passes of a tight fitting glass-Teflon motor
driven
homogenizer. A post-nuclear supernatant was formed as the 11,OOOg supernatant,
180,000g pellet of membrane vessels (Figure 2A).
Aliquots of membrane vesicles were labeled with megalin or cubulin
antisera. The megalin and cubulin antisera were rabbit polyclonals raised to
affinity
purified and chromatographically pure receptor (43, 48). Membrane vesicles
were
' first pre-incubated in SO% normal goat serum for 2 hours to reduce non-
specific
binding of secondary antisera raised in goat. After washing aliquots of
membrane
vesicles were stained with serial log dilution of antisera and incubated at
4oC
overnight. After fiu-ther washing 1:40 of goat anti-rabbit affinity purified
rat pre-
absorbed phycoerthyrein conjugated secondary antiserum was added, and
incubated
for 4 hours at room temperature. Prior to flow cytometry the membrane vesicles
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were washed and resuspended in 200 mM mannitol, 100 mM KCI, 10 mM HEPES,
pH 8.0 with Tris to which had been added 10 mM nigericin. In the presence of
potassium, nigericin collapses pH gradients, ensuring optimal fluorescence of
the
highly pH dependent fluorescein-dextran emission. Fluorescein-dextran and
antibody staining tagged by phycoerythrein were now analyzed and co-localized
on
a vesicle-by-vesicle basis by flow cytometry (Figure 2B).
EXAMPLE 8
Differential Display
Differential display of expressed genes was compared in aliquots of
the same cells grown in a 55 ml rotating wall vessel {slow turning lateral
vessel) or
conventional gas permeable 2-dimensional bag controls (Figure 3A and 3B).
Differential display was performed using Delta RNA Fingerprinting system
(Clontech labs, Palo Alto CA}. Copies of expressed genes were generated by
polymerase chain reaction using random 25mer primers and separated on a 6% DNA
sequencing gal. Bands of different intensity between control and slow turning
lateral vessel, representing differentially expressed genes, were identified
by visual
inspection, excised and reamplified using the same primers. Differential
expression
and transcript size were confirmed by Northern hybridization. PCR products
were
then subcloned into the pGEM-T vector (Promega, Madison WI) and sequenced
using fMOL cycle sequencing system (Promega, Madison, WI). Sequences were
compared to the Genebank sequences using the BLAST search engine (National
Center for Biotechnology Information). For genes of interest the bands were
labeled
with 32P for confirmation of the changes by Northern blot analysis.
EXAMPLE 9
Detection of Gene Expression in Cell Cultures by RT-PCR
Cell aggregates from the rotating wall vessel culture were washed
once in ice cold phosphate buffered saline and snap frozen at -70oC until RNA
was
isolated. Total RNA was first isolated, followed by isolation of poly A+ RNA.
Following reverse transcription, 10%-20% of each cDNA was amplified
(Robocycler 40, Stratagene, La Jolla, CA) using 95oC denaturation, 63oC
annealing
and 72oC extension temperatures. Amplification was for a total of 30 cycles
with
the first three cycles having extended denaturation and annealing times.
Positive and
negative strand PCR primers, respectively, were derived from published
sequences
using Generunner software. 20% of the PCR reaction was electrophoresed on
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agarose/ethidium bromide gels and visualized under UV light so that a
comparison
of amplified gene fragments could be made to DNA standards (HaeIII digested
X174 DNA, Promega) electrophoresed on the same gel (Figure 3C and 3D).
Representative fragments amplified for each gene in question were isolated
from
gels and direct sequenced to assure identity of the PCR product. In addition,
5% of
the same cDNA were subjected to PCR for expression of the housekeeping mRNA,
glyceraldehyde 3-phosphate dehydrogenase, and b-actin to assure that similar
amounts of input RNA and that similar efficiencies of reverse transcription
were
being compared. Each cDNA was run in at least three dilutions to ensure that
measurements were made on the initial linear portion of the response curve.
EXAMPLE 10
Genetic Decoys
Double stranded genetic decoys matching the sequence of a known
shear stress response element were synthesized (Chemicon International Inc.,
La
Jolla, CA) (structure and sequence shown at top of Figure 4). These decoys had
a
terminal phosphothiorate moiety to prevent intracellular lysis, and a
phosphodiester
backbone to facilitate passage across cell membranes (49). Passage to and
accumulation in the nuclear compartment of cultured cells was confirmed by
confocal imaging of a fluorescein tagged decoy. Three decoys were synthesized:
the
active decoy, a random sequence control in which the six bases of the shear
stress
response element were scrambled, and a fluorescein conjugated form of the
decoy.
Decoys were placed in the cell culture medium of rat renal cortical cells
grown as
above in conventional two-dimensional culture. Aliquots of cells exposed to
control
or active sequence decoy at 80 nm concentration were harvested at 2, 6 and 24
hours
after exposure.
EXAMPLE 11
Genetic Discovery Array
A sample of human renal cortical cells grown in conventional flask
culture was trypsinized and split into a gas permeable bag control and a
rotating wall
' 30 vessel (55 ml slow turning lateral vessel). After 8 days of culture on 5
mg/ml
cytodex-3 beads, cells were washed once with ice cold phosphate buffered
saline,
the cells were then lysed and mRNA was selected with biotinylated oligo(dT)
then
separated with streptavidin paramagnetic particles (PolyATtract System 1000,
Promega Madison, WI). 32P labeled cDNA probes were then generated by reverse
transcription with 32P dCTP. The cDNA probes were hybridized to identical Gene
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Discovery Array Filters (Genome Systems Inc. St. Louis, MO). The Gene
Discovery Array filters contain 18,394 unique human genes from the LM.A.G.E.
Consortium [LLNL](15) cDNA Libraries which are robotically arrayed on each of
a
pair of filter membranes. Gene expression was then detected by phosphor
imaging
and analyzed using the Gene Discovery Software [Genome Systems] (50).
EXAMPLE 12
Assay of 1-a-hydroxylase Activity
As the 1-a-hydroxylase enzyme has never been isolated or cloned it is
assayed functionally by the production of 1,25-dihydroxy-vitamin D3 from
ultrapure
exogenous 25-hydroxy vitamin D3. For each measurement, the classic Michaelis
Menton kinetics of the enzyme are determined by assaying equal aliquots of
renal
cell aggregates in a curve of 25-OH D3 substrate concentrations from 0.1 to 10
mg/ml in 6 steps. All incubations are performed in the presence of the anti-
oxidant
DPED at 10 mM to ensure no contribution of non-enzymatic oxygenation (23-26).
1 S 1,25-diOH D3 generated in vitrol3was quantitated as described (23-27). In
vitro(3incubations were terminated by adding a volume of acetonitrile equal to
the
incubation volume. Each incubation tube received 1,000 cpm of 3H-1,25
dihydroxy
D3 to estimate recovery losses during the extensive extraction and
purification
scheme. The I,25-dihydroxy D3 is extracted from the incubation medium by C18
solid-phase extraction (24-25). Following extraction, the samples are
evaporated to
dryness under N2 and dissolved in 2 ml of methylene chloride. The samples are
then applied to silica Bond-Elut cartridges and the 1,25-dihydroxy D3-
containing
fraction is isolated and collected (26). The individual fractions containing
1,25-
diOH D3 and then subjected to normal phase HPLC on a Beckman model 344 liquid
chromatography system. Normal-phase HPLC was performed with a Zorbax-Sil
column (26) (4 X 25 cm) developed in and eluted with methylene
chloride/isopropanol (96:4 v/v) with a flow rate of 2 mllmin. The 1,25-
dihydroxy
D3 eluted from this system was dried under N2 resuspended in ethanol and
quantitated by radio receptor assay or radio immunoassay (25-26). Plasma 1-25-
dihydroxy vitamin D3 was assayed in a similar fashion, but as the product is
already
formed, assay begins with extraction into acetonitrile (23-26). Hence, all
measurement of 1-a-hydroxylase activity in cells included determination of the
Michaelis Menton Km and Vmax of the enzyme. The Michaelis Menton parameters
were determined by automated curve fitting.
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EXAMPLE 13
Culturing Renal Fibroblasts and Assay for Production of Erythropoietin
As renal fibroblasts are the source of erythropoietin secreted into the
circulation, renal fibroblasts were cultured. Freshly dissected rat renal
cortex was
minced and collagenase\trypsin digested prior to removal of debris on a single
discontinuous 5% albumin gradient. The mixture of rat renal cortical cells was
placed into culture in DMEM\F 12 with 20% fetal bovine serum. After two wPPk~
r~
encourage fibroblast overgrowth in the rich medium, fibroblast growth factor
was
added. The resultant culture had fibroblastic features in the culture flask
and was
inoculated into a high aspect rotating vessell (HARV) for culture under
increased
shear stress conditions. The cells aggregate on the beads and slowly
increasing their
numbers. After 3 weeks growing the fibroblasts in a HARV, erythropoietin was
assayed in the cell supernatant. The media were concentrated 1 SX and assayed
via
RIA. The media alone was also concentrated 15X as the control.
EXAMPLE 14
Culturing Hapatocytes and Assay for Production of Erythropoietin
As hepatocytes are a source of erythropoietin secreted into the
circulation, immortalized human hepatocyes were cultured under control and
applied
shear stress conditions. The Hep3B cells were placed into culture in DMEM with
10% fetal bovine serum in static flask culture. The resultant culture was
split, one
half remaining in static flask culture and the other half was inoculated into
a HARV
for culture under increased shear stress conditions. The cells aggregated on
the
beads. After 24 hours growing the Hep3B cells in a HARV, erythropoietin was
assayed in the cell supernatant. The media were assayed by RIA. The static
flask
media was also assayed as the control.
EXAMPLE 1 S
Shear Stress Response Elements Mediate Changes in Erythropoietin Gene
Expression
The immortal hepatic cell line, Hep3B, constitutively produces
erythropoietin. The 5' promoter and 3' enhancer regions of the gene contain
putative
shear stress response elements. The role of these elements in the enhancement
of
erythropoietin production in response to shear was tested by using integrated
perfused rotating wall vessel culture to reintroduce graded shear. This
protocol
utilizes a library of promoters driving luciferase reporters genes, with
various
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constructs lacking the putative shear stress response elements. It also allows
DNA
footprinting analysis of the histones which bind the promoter and enhancer
elements.
EXAMPLE 16
Results
The proportion of proximal tubular cells in human renal cell fractions
isolated by differential trypsinization was assayed using an entrapped
fluogenic
substrate for the proximal enzyme marker g-glutamyl-transferase (44). Flow
cytometry analysis on a cell-by-cell basis showed the natural cell mixture in
the
human renal cortex to be 85+4%, n=4 proximal tubular cells (Figure lA, left
panel).
Following differential trypsinization, and selection of the pure fractions,
proximal
tubular enrichments as high as 99+1 % could be achieved (right panel). As
reported
in other systems, rotating wall vessels were conducive to vigorous cell
growth, as
evidenced by the high rates of glucose consumption assayed as 30 mg/dl
glucose/100,000 cells/day. A cell doubling time of 4+3 days was assayed using
Alamar blue in the rotating wall vessel compared to 4+2days in conventional
culture
(n=4).
The ultrastructure of cultures of pure proximal tubular cells or renal
cortical cell mixtures of human kidneys were grown in rotating wall vessels
for 16
days, and were examined by transmission electron microscopy (Figures 1B and
1C).
Quantitation of the number of microvilli present by counting random plates at
the
same magnification demonstrates not only that the rotating wall vessel induces
microvillus formation, but co-culture with the normal mix of renal cortical
cells
increases the effect (Table 1 ). Normal cortical cell mix in conventional two-
dimensional culture has 2 1 microvilli per field; "pure" proximal tubular
culture in
rotating wall vessel has 10 4 microvilli per field; and the normal cortical
cell mix. in
rotating wall vessel has 35 11 microvilli per field.
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TABLE 1
Human proximal tubular cells microvilli counted on transmission electron-
micrographs of cells grown for 16 days under various culture conditions
Culture Conditions % Proximal Microvilli
Tubular MarkersPer
Field
conventional 2-D culture 85 2 1
pure= culture in rotating wall 99 10 4
vessel
normal cortical cell mix in rotating85 35 11
wall vessel
i o examine the expression of megalin and cubulin in renal cells in
culture, there are advantages to using human cells instead of rat cells.
Specifically,
the rat sequences of megalin and cubulin have been cloned, while the human
sequences have not, and the antisera recognizes the rat but not the human
isoforms
of these proteins. Hence, the natural mixture of cells in the rat renal cortex
was
placed into culture in rotating wall vessels, stirred fermentors, and
traditional culture
for analysis of protein expression.
As the endosomal pathway has been implicated to play a central role
in the function and pathophysiology of cubulin and megaiin, entrapped
endosomal
markers were co-localized with receptor antibody binding. The ability of flow
cytometry to make simultaneous measurements of entrapped fluorescein dextran
as
an endosomal marker and antibody binding allows construction of three
dimensional
frequency histograms displaying entrapped fluorescein dextran fluorescence
against
antibody binding on horizontal axes and number of vesicles in each channel up
out
of the page (Figure 2A). A control sample shows vesicles negative for
fluorescein
on the left and fluorescein containing endosomes on the right (200 vesicles
depicted,
left panel). A control without fluorescein entrapped shows only the left
population
(not shown). Co localization of anti-cubulin binding demonstrates that all the
fluorescein positive endosomes were positive for cubulin, while non-endosomal
membranes could be subdivided into cubulin positive and negative populations.
(middle panel). This pattern was repeated for anti-megalin binding in renal
cortical
cells (right panel) in culture.
Next, analysis of protein expression in cultured cells by antibody
binding used classic serial log dilution antibody curves. An increase in
binding with
a decrease in dilution is pathognomonic for specific antibody binding during
flow
cytometry analysis. Binding of anti-cubulin antisera to membrane vesicles
prepared
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from renal cortical cells after 16 days in culture, detected by the
fluorescence of a
phycoerthyrein tagged secondary antibody, shows an almost two log increase in
binding with antibody dilution (Figure 2B). This increase in the cells grown
in the
rotating wall vessel (slow turning lateral vessel) is more than five times the
expression seen in stirred fermentors. Similarly there was no detectable
expression
in the conventional cultures resulting in a flat line (not shown). Comparison
of
maximal binding of the anti-cubulin antibody to minimum taken to be the
antibody
dilution at which there is no further decline in signal with primary antibody
dilution
is shown in Figure 2C. Binding of normal serum and minimal dilution of primary
antisera were not detectably different. Binding curves for anti-megalin
antiserum
showed a similar pattern (not shown) but the peak binding was a little lower
(Figure
2C}. Again stirred fermentor has much less expression than the rotating wall
vessel
(slow turning lateral vessel) and the conventional cell membranes have no
detectable
binding (not shown).
I S To examine the proportion of proteins changing in the rotating wall
vessel, two-dimensional gel SDS-PAGE analysis on cultures grown in the
rotating
wall vessel and bag controls were performed (Figure 2d). The results shown in
Figure 2D demonstrates changes were in a selected group of proteins.
To identify the genes changing during rotating wall vessel culture,
differential display were performed. Differential display of expressed genes
was
compared in aliquots of the same cells grown in a 55 ml rotating wall vessel
(slow
turning lateral vessel) or conventional gas permeable 2-dimensional bag
controls.
Differential display of copies of expressed genes were generated by polymerase
chain reaction using random 25mer primers and separated on a 6% DNA sequencing
gel. Bands of different intensity between control and slow fuming lateral
vessel,
representing differentially expressed genes, were identified by visual
inspection,
excised and reamplified using the same primers. Differential expression and
transcript size were confirmed by Northern hybridization. PCR products were
then
subcloned into the pGEM-T vector and sequenced. Sequences were compared to the
Genebank sequences using the BLAST search engine. One expressed gene which
decreased in the slow turning lateral vessel (band D on gel, Figure 3A) was
identified as rat manganese-containing superoxide dismutase (98% match I42 of
144 nucleotides). Two genes which increased in the slow turning lateral
vessel,
band A was identified as the interleukin-1 beta gene (100% match for 32 of 32
nucleotides) and Band B which corresponded to a 20 kB transcript on a Northern
blot appears to be a unidentified gene that has a 76% homology to the mouse
GABA
transporter gene.
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To examine the genetic changes in specific genes, the expression of
tissue specific epithelial cell markers and classic shear stress response
dependent
genes were examined by RT-PCR (Figure 3c). Several genes specific for renal
proximal tubular epithelial cells, including megalin, cubulin, the
extracellular
S calcium sensing receptor, and the microvillar structural protein villin,
increase early
in rotating wall vessel culture. Similarly there were dynamic time dependent
changes in classic shear stress dependent genes including intercellular
adhesion
molecule 1 (ICAM) and vascular cell adhesion molecule (VCAM) (increased) and
manganese dependent superoxide dismutase (suppressed). Many but not all of
these
changes were prolonged, as after 16 days in culture gene expression of
megalin,
ICAM, VCAM and the extracellular calcium sensing receptor were still elevated,
while villin and manganese dependent superoxide dismutase were at control
levels.
Expression of control GADPH, b-actin and 18S genes did not change throughout
the
time course.
1 S To test for a role of a putative endothelial shear stress response
element in these renal cortical cell changes, an antisense probe for the
sequence was
synthesized (Figure 4A). A control probe had the active motif scrambled.
Confocal
imaging of a fluorescein conjugated form of the probe confirmed nuclear
delivery of
the probe (images not shown). Culture of rat renal cortical cells in 80 nm of
the
probe, resulted in a time dependent increase in magnesium dependent superoxide
dismutase, but no change in villin gene expression (Figures 4B and 4C). The
control
probe had no effect.
In order to confirm the genetic responses to rotating wall vessel
culture and the analysis with human cells, automated gene display analysis of
expressed RNA was performed on human renal cortical cells grown in a control
gas-
permeable bag and the slow turning lateral vessel for 8 days (50). Of the more
than
18,000 genes assayed a select group was again observed to change (Figure 5).
In
particular, vectored changes in all the classic shear stress response genes
assayed by
RT-PCR and differential display in rat cell culture were confirmed. A battery
of
tissue specific genes was increased including villin, angiotensin converting
enzyme,
parathyroid hormone receptor and sodium channels. Other physical force
dependent
genes such as heat shock proteins 27/28 kDa and 70-2 changed, as did focal
adhesion kinase, and a putative transcription factor for shear stress
responses NF-kb
- changed. Fusion proteins such as synabtobrevin 2 mildly decreased gene
expression, and clathrin light chains hugely increased gene expression.
To determine whether renal cells grown in simulated microgravity
have 1-a-hydroxylase activity, the 1-a-hydroxylase activity of cell cultures
were
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compared grown in traditional 2-D culture in gas permeable bags, and NASA
rotating wall vessels. Both rat renal cells (Table 2) and human embryonic
renal cells
were assayed (Table 3).
TABLE 2
The 1 a-hydroxylase activity of the various rat renal cell cultures detected
as
production of 1.25-diOH D3
Cell Sample 1,25-diOH D3 Volume of 1,25-diOH D3
concentration Supernatant Production
(pg/ml) (ml) (pg)
Boiled static <2, not detectable7 ml Not detectable
control I
Boiled static <2, not detectable7 ml Not detectable
control II
Static control <2, not detectable7 ml Not detectable
I
Static control <2, not detectable7 ml Not detectable
II
Boiled rotating<2, not detectableSS ml Not detectable
wall vessel
Rotating wall 14.2 55 ml ~g 1
vessel
The results shown in TABLE 2 indicate that rat renal cells show
increased structural differentiation during culture in simulated microgravity
conditions, and express much greater 1-a-hydroxylase activity than under
conventional culture conditions.
TABLE 3
The 1 a-hydroxylase activity of the various human embryonic renal cell
cultures
detected as production of 1.25-diOH D3
Cell Sample 1,25-diOH D3 Volume of 1,25-diOH
Concentration Supernatant D3
(pg/ml) (ml) Production
(pg)
Boiled static 8.2 10 g2
control
Static control 14.6 10 146
Rotating wall 24.8 55 1364
vessel
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TABLE 3 indicates that human embryonic kidney cells show
increased structural differentiation during culture in simulated microgravity
conditions, and express 10 fold greater 1-a-hydroxylase activity than under
conventional culture conditions.
S TABLE 4
Renal fibroblasts cell sunernatanr Prvthrnnnir~tin acca..
J
Condition Erythropoietin (mu/ml)
Shear stress culture l,g
Control media conc 1 SX 0.23
TABLE S
Hepatocvtes cell sunernatanr et'vthrnnniPtin aecav
Condition . Erythropoietin (mu/ml)
Shear stress culture 141.7 mu/1 x 106 cells
Control static flask undetectable
Results of cell supernatant erythropoietin assay from renal fibroblasts
and hepatocytes culture were shown in Table 4 and Table S, respectively. The
results shown in TABLES 4 and S indicate erythropoietin production was
increased
in both renal and hepatic cells during graded gravitational sedimentation
shear.
Erythropoietin has the classic shear stress response elements in the
promoter and enhancer regions which control expression of its gene. The
results
1 S shown in Tables 4 and S also indicate that the expression of the
erythropoietin gene
was upregulated by those shear stress response elements during graded
gravitational
sedimentation shear in the vessel.
EXAMPLE 17
Discussion
Rotating wall vessels have been used by other investigators as
"simulated microgravity". The present invention contends that gravity is still
active,
and that in a rotating wall vessel gravity is balanced by equal and opposite
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sedimentational shear stress. A centrifugal force due to spinning the cells,
quantitatively much smaller than gravity, is also present and offset by equal
and
opposite sedimentational shear stress. Thus, the present invention presents a
new
concept that rotating wall vessels provide this new balance of forces,
including
application of sedimentational shear, rather than microgravity.
The rotating wall vessel bioreactor provides quiescent co-localization
of dissimilar cell types (1, 46), mass transfer rates that accommodate
molecular
scaffolding and a micro-environment that includes growth factors (l, 46).
Engineering analysis of the forces active in the vessel is complex (l, S-7).
This
study provides the first evidence for the cell biological mechanisms by which
the
vessel induces changes in tissue specific gene and protein expression.
There are two possible explanations why the rotating wall vessel
induces an order of magnitude more expression of the renal toxin receptors
cubulin
and megalin than stirred fermentor culture. First, there are significant
differences in
1 S the degree of shear stress induced. The rotating wall vessel induces 0.5-
1.0
dynes/cm2 shear stress (1), while stirred fermentors induce 2-40 dynes/cm2
depending on design and rotation speed (1, 5, 46). This degree of stress
damages or
kills most epithelial cells (1, 5, 46). Second, impeller trauma in the stirred
fermentor, is absent in the rotating wall vessel. This explains why there was
far
more cubulin and megalin induced in renal cultures in rotating wall vessel
culture
than a stirred fermentor, and both receptors were not detectable in
conventional 2-
dimensional culture.
Rotating wall vessel culture induced changes in a select set of genes,
as evidenced by the genetic differential display gels and 2-dimensional
protein gel
analysis. For example, erythropoietin production is controlled by a shear
stress
element which mediates changes observed during graded gravitation
sedimentation
shear. 1-a-hydroxylase activity is maintained and increased in both renal
cortical
epithelial cells and human embryonic kidney cells, wherein the induction of
the
enzyme (1-a-hydroxylase) converts 25-hydroxy-vitamin D3 to the active 1,25-
dihydroxy-vitamin D3 form. The present invention is the first demonstration of
a
process for production of molecules including hormones and other biomolecules
induced by shear stress and other forces. The mechanistic information can be
interpreted from knowledge of the pattern of response and distribution of
certain
gene products.
Megalin and cubulin represent the first pattern of change, as these
proteins are restricted in distribution to renal cortical tubular epithelial
cells. The
increase in megalin mRNA and protein; and cubulin protein expression is
therefore
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unequivocal evidence for changes in the epithelial cells. This provides an
important
new tool for studies of nephrotoxicity. Long suspected to play a role in renal
toxicity, the tissue restricted giant glycoprotein receptors megalin and
cubulin, have
recently been shown to be receptors for common nephrotoxins.~ Megalin is a
receptor for polybasic drugs such as the aminoglycoside antibiotic gentamicin
(48)
and vitamin D binding protein (51), and cubulin is the receptor for vitamin-
B12
intrinsic factor (52). Although these receptors are expressed by transformed
placental cells in culture (9, 43), there is currently no renal model
expressing these
markers for toxicology investigations (53). Rotating wall culture provides a
fresh
approach to expression of renal specific markers in culture for study on the
pharmacology, biochemistry and toxicology which define the unique properties
and
sensitivities of renal epithelial cells.
The second pattern of change is represented by villin. Message for
the microvilli protein villin increases in the rotating wall vessel in the
first day of
culture, and soon reformation of microvilli was observed. A decoy matching the
nuclear binding motif of a putative shear stress response element failed to
induce
similar changes. Although the promoter for villin has not been cloned, this
suggests
the changes in villin were induced by other transcription factors which may be
due
to shear stress or other stimuli in the bioreactor. Villin is also restricted
to brush
border membranes such as renal proximal tubular cells, or colonic villi (54-
55). The
observed increases in villin message resolved after 16 days of rotating wall
vessel
culture.
Magnesium dependent superoxide dismutase represents a third
pattern of response: a mitochondria) enzyme, ubiquitous is distribution,
modulated
by the classic shear stress response element in endothelial cells (56-57).
Magnesium
dependent superoxide dismutase message decreased early in the first day of
rotating
wall vessel culture, and this was persistent after 16 days in culture. These
changes
were confirmed when magnesium dependent superoxide dismutase was identified as
suppressed in the differential display analysis of gene changes, and Northern
blot
confirmation was performed. A decoy for the classic shear stress response
element
induced an increase in magnesium dependent superoxide dismutase (MnSOD),
which indicates that similar changes to the rotating wall vessel can be
induced by the
use of genetic decoys. Thus, the biological process of genetic induction by
defined
shear stress elements can be produced by multiple means including genetic
decoys
or use of the rotating wall vessel. Other shear stress response element
dependent
genes, specifically, intercellular adhesion molecule 1 {ICAM) and vascular
cell
adhesion molecule (VCAM) had changes in the rotating wall vessel opposite to
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magnesium dependent superoxide dismutase, mirroring observations made during
flow induced stress in endothelial cells (56-57}. This provides three lines of
evidence consistent with a role for shear stress as one mediator of genetic
changes
induced in the rotating wall vessel.
Differential display of the genes activated and deactivated under
rotating wall vessel culture conditions showed rotating wall vessel culture
was
associated with decreased expression of manganese dependent superoxide
dismutase
mRNA and increased expression of interleukin-1 b gene mRNA. This greatly
extends and brings together previous observations on the interactions of
stress,
manganese dependent superoxide dismutase expression and interleukin-1. Topper
et
al. reported an oppositely directed effect, i.e., differential display of
vascular
endothelial cells exposed to high stress demonstrates increased manganese
dependent superoxide dismutase gene expression (57). Other direct evidence
links
superoxide dismutase and interleukin-1 as increases in manganese superoxide
dismutase levels and decreases in interleukin-1 levels in HT-1080 fibrosarcoma
cells
(58). In more indirect evidence overexpression of mitochondrial manganese
superoxide dismutase promotes the survival of tumor cells exposed to
interleukin-1
(59). The present study provides direct evidence that modest shear stress
decreases
magnesium dependent superoxide dismutase in association with an inverse effect
on
interleukin-1.
The data here demonstrates internal consistency. The changes in
magnesium dependent superoxide disrnutase were observed on differential
display,
confirmed by Northern blot analysis, and matched responses were detected by RT-
PCR. Megalin demonstrated matched changes in RT-PCR gene and protein
expression. Changes in villin observed by RT-PCR were associated with dramatic
reformation of microvilli, in which villin is a major structural protein.
Although
semi-quantitative RT-PCR is prone to inherent variation due to the massive
amplification of signals, the use of multiple controls which remain unchanged
(b-
actin, GAPDH and 18S), and experimental confirmation that reactions were
linearly
related to cDNA concentration, minimizes these problems. The internally
consistent
findings by other methods strongly suggests this RT-PCR data is valid.
Study of the mechanisms of action of the rotating wall vessel to
induce gene and protein expression during cell culture has been hampered by
nomenclature. First, the attachment of the moniker "simulated microgravity",
based
on engineering analysis of boundary conditions, clouds intuitive analysis of
the cell
biology as there is no cellular equivalent for this term (1, 6-7). Similarly
the reduced
shear stress in the rotating wall vessel compared to stirred fermentors leads
to the
26
CA 02286349 1999-10-07
WO 98/45468 PCT/US98/06826
term "reduced shear stress culture" ( 1 ), whereas there is increased shear
stress
compared to conventional 2-dimensional culture (1, 5). As cell aggregates
remain
suspended in the rotating wall culture vessels, gravity is balanced by an
equal and
opposite force. Engineering arguments on the relative contributions of fluid
shear,
drag, centrifugal force, coriolus motion, and tangential gravity-induced
sedimentation are themselves tangential to the cell biology. Several lines of
evidence are documented that shear stress responses are one of the component
of the
biological response. This opens the door for analysis of other biological
response
mediators in the vessels, and investigation as to whether unloading of gravity
plays
as big a role as the oppositely directed balancing forces.
Using the rotating wall vessel as a tool, data here provide the first
evidence that shear stress response elements, which modulate gene expression
in
endothelial cells, are also active in epithelial cells, although other
investigators failed
to see an effect of shear stress on epithelial cells. The present invention
demonstrates that epithelial cells have shear stress response elements and
change
gene expression in response to physical forces including but not limited to
sedimentational shear stress. As the rotating wall vessel gains popularity as
a
clinical tool to produce hormonal implants it is desirable to understand
mechanisms
by which it induces genetic changes (10, 60), if necessary to prolong the
useful life
of implants. Several lines of evidence are provided that shear stress response
elements are the first mechanism identified by which the rotating wall vessel
induces
genetic changes. Using a putative endothelial cell shear stress response
element
binding site as a decoy, the role of this sequence in the regulation of
selected genes
in epithelial cells was validated. However, many of the changes observed in
the
rotating wall vessel are independent of this response element. It remains to
define
other genetic response elements modulated during rotating wall vessel culture,
and
whether the induced changes are secondary to the balancing forces, or
primarily
related to unloading gravity.
The following references were cited herein.
1. GOODWIN TJ, et al., J cell Biochem 51:301-311, 1993.
2. JESSUP JM, et al., J Cell Biochem 51:290-300, 1993.
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7. Zhau HE, et al., In Vitro Cell Dev Biol 33:375-380, 19978.
27
CA 02286349 1999-10-07
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8. Baker TL, et al., In Vitro Cell Dev Biol 33:358-365, 1997
9. Hammond TG, et al., J. Mem. Biol. In press
10. Soon-Shiong P., et al., PNAS USA 90:5843-5847, 1993
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21. Holick MF., et al., Bone 17(2S):107S-111S, 1995.
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28. Henry HL. et al., J Biol Chem 254:2722-2729, 1979.
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1995.
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1977
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1989
34. TAUB ML, et al., In Vitro Cell & Dev Biol 25:770-775,
1989
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1989
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40. MAUNSBACH AB., J.Ultrastruct.Res. 16:1-12, 1966.
41. RONCO P, et al., J. Immunol. 136:125-130,1986.
42. SAHALI D, et al., J Exp Med 167:213-218, 1988.
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S 44. Hammond, T.G. et al., Kidney Int 42:997-1005, 1992.
4S. Schwarz, R.P.,et al., J.Tiss.Cult.Meth. 14:51-S8, 1992.
46. Topper JN, et al., J. Vasc Res 33:S100A.
47. Hammond, T. G., et al., Cytometry 14: 411-420, 1993.
48. Moestrup, et al., Journal of Clinical Invest, 96:1404-1413, 1995.
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Schlingensiepen, et al., (Blackwell Science) pp. 1-87.
S0. Lennon, G.G., et al., Genomics 33:1 S 1-1 S2, 1996.
S 1. Christensen EI, et al., JASN 8:S9A, 1997.
S2. Seetharam B. et al., J Clin Invest. 99, 2317-22,1997
1 S S3. Orlando RA, et al., PNAS USA 90:4082-4086, 1993.
S4. Arpin M, et al., J Cell Biol 107:1759-1766, 1988.
SS. Chantret I, et al., Cancer Res. 48:1936-1942, 1988.
S6. Resnick N et al., FASEB J. 9:874-882, 1995.
S7. Tuttle R et al., Curr. Opin. Cell Biol. 3: 70-72, 1993.
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Any patents or publications mentioned in this specification are
indicative of the levels of those skilled in the art to which the invention
pertains.
2S These patents and publications are herein incorporated by reference to the
same
extent as if each individual publication was specifically and individually
indicated to
be incorporated by reference.
One skilled in the art will readily appreciate that the present invention
is well adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those inherent therein. The present examples along with
the
methods, procedures, treatments, molecules, and specific compounds described
herein are presently representative of preferred embodiments, are exemplary,
and are
not intended as limitations on the scope of the invention. Changes therein and
other
uses will occur to those skilled in the art which are encompassed within the
spirit of
3S the invention as defined by the scope of the claims.
29
CA 02286349 1999-10-07
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SEQUENCE LISTING
( 1 ) GENERAL INFORMATION
(i) APPLICANT: Hammond et al.
(ii) TITLE OF INVENTION: Production of Functional Proteins:
Balance
of Shear
Stress
and Gravity.
(iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Benjamin Aaron Adler, Ph.D. J.D.
(B) STREET: 8011 Candle Lane
(C) CITY: Houston
(D) STATE: Texas
(E) ZIP: 77071
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 1.44 Mb floppy disk
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Macintosh
(D) SOFTWARE: Microsoft Word for Macintosh
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION 1VLTMBER:
(B) FILING DATE:
(vii) PRIOR APPLICATION DATE:
(A) APPLICATION NUMBER: 60/043,205
(B) FILING DATE: 04/08/97
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Benjamin Aaron Adler, Ph.D.
(B) REGISTRATION NUMBER: 35,423
(C) REFERENCE/DOCKET N~JMBER: D6081
(ix) TELECOMMUNICATION INFORMATION:
CA 02286349 1999-10-07
WO 98/45468 PCT/CJS98/06826
(A) TELEPHONE: (713) 777-2321
(B) TELEFAX: (713) 777-6908
(2) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 by
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single-stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(A) DESCRIPTION: cDNA to mRNA
(iii) HYPOTHETICAL: no
(iv) ANTISENSE: yes
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(ix) FEATURE:
(x) PUBLICATION INFORMATION:
{xi) SEQUENCE DESCRIPTION: SEQ ID
NO.: 1
CTGAGACCGA TATCGGTCTC
AG 22
31
