© 1999 American Heart Association, Inc.
Original Contribution |
Exposure of Human Vascular Endothelial Cells to Sustained Hydrostatic Pressure Stimulates Proliferation
Involvement of the 
V Integrins
From the Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY (E.A.S., R.B.); the Department of Pediatrics, New York Medical College, Valhalla, NY (M.S.M.); and the Department of Cardiovascular Research, Genentech, Inc, South San Francisco, Calif (M.E.G.).
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Abstract—The present study investigated the effects of sustained hydrostatic pressure (SHP; up to 4 cm H2O) on human umbilical vein endothelial cell (HUVEC) proliferation, focal adhesion plaque (FAP) organization, and integrin expression. Exposure of HUVECs to SHP stimulated cell proliferation and a selective increase in the expression of integrin subunit
V. The increase in
V was observed as early as 4 hours after exposure to
pressure and preceded detectable increases in the bromodeoxyuridine
labeling index. Laser confocal microscopy studies demonstrated
colocalization of the V integrin to FAPs. The individual
FAPs in pressure-treated cells demonstrated a reduced area and
increased aspect ratio and were localized to both
peripheral and more central regions of the cells, in
contrast to the predilection for the cell periphery in cells maintained
under control pressure conditions. The pressure-induced changes in
V distribution had functional consequences on the cells:
adhesivity of the cells to vitronectin was increased, and
V antagonists blocked the pressure-induced
proliferative response. Thus, the present study suggests a role for
V integrins in the mechanotransduction of pressure by
endothelial cells.
Key Words: mechanotransduction • hydrostatic pressure • integrin • human umbilical vein endothelial cell • cell proliferation
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Introduction |
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Anumber of disease processes (such as tumor angiogenesis and glaucoma-induced retinal microangiopathy) and normal physiological events (such as developmental growth, wound healing, and ovulation) are characterized by endothelial cell proliferation. Endothelial cell proliferation is stimulated by a variety of growth factors, such as basic fibroblast growth factor (bFGF)1 and vascular endothelial growth factor (VEGF),2 as well as by mechanical forces such as cyclic stretch (as reviewed in Reference 33 ), fluid shear,4 and sustained hydrostatic pressure [SHP]).5 6
The mechanisms by which endothelial cells distinguish SHP and convert this mechanical force to a biochemical response (ie, cell division) remain unclear. Among the candidate mechanotransducers are integrins, a family of transmembrane glycoproteins, which have been shown to initiate a biochemical cascade of responses leading to activation of cytosolic transcription factors and/or to regulation of gene expression (as reviewed in Reference 77 ). Direct mechanical linkage of cytoskeletal elements to adherent integrins may provide another mechanotransduction pathway.8
Recent research has associated intracellular signals from the
vitronectin receptors (VnRs; integrins
Vß3 and
Vß5) with
endothelial cell proliferation.2 9
Functional blocking antibodies to
Vß3 integrins
selectively inhibit endothelial cell proliferation and
angiogenesis in vivo in response to bFGF but not to VEGF.9
Conversely, blocking monoclonal antibodies specific for the
Vß5 VnR selectively
inhibit the in vivo proliferative effects of VEGF but not of
bFGF.2
The effects of SHP on normal, human endothelial cells
are poorly understood; for this reason, the present study used
human umbilical vein endothelial cells (HUVECs) as a
model system. In addition, the use of human cells permitted
investigation of the SHP signal transduction pathways of
endothelial cells, because suitable immunological
reagents specific for human integrins and other intracellular proteins
were available. In the present study, we demonstrated that exposure
of human endothelial cells to SHP stimulated cell
proliferation and V integrin expression and
altered the distribution of focal adhesion plaques (FAPs).
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Materials and Methods |
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The supplies used in the present study were purchased commercially as follows: HUVECs, EGM-UV medium, and bovine brain extract (BBE) were from Clonetics; FBS was from HyClone; medium 199 (M199) was from Gibco BRL; glass coverslips were from VWR; Thermanox coverslips were from Nunc, Inc; human fibronectin and laminin were from Collaborative Biomedical Products; monoclonal antibodies to vinculin (clone hVIN-1), fluorescein-conjugated streptavidin, 4-(2-aminoethyl)benzenesulfonyl fluoride, Hoechst 33258 dye, salmon sperm DNA, 3,3'-diaminobenzidine tablets (FastDAB), human vitronectin, and bovine gelatin were all from Sigma Chemical Co (St Louis, Mo); BSA was obtained from Bayer; monoclonal antibodies to integrin subunit
V (clone
LM142), integrin Vß3
(clone LM609), and integrin
Vß5 (clone P1F6) and
polyclonal antisera to integrin subunits V,
ß1, ß3, and
ß5 were from Chemicon International (Temecula,
Calif); normal sera (horse, goat, rabbit, and mouse), biotinylated
secondary antisera, Vectashield mounting medium, and the Vectastain
Elite ABC reagents were from Vector Laboratories;
4',6-diamidino-2-phenylindole (DAPI) was from Calbiochem; BrdU,
FixDenat, and anti-BrdU monoclonal antibody were from
Boehringer Mannheim (Indianapolis, Ind); AquaMount was from
Lerner Laboratories; protein A–agarose beads and the bicinchoninic
acid protein assay kit were from Pierce; and the Enhanced
ChemiLuminescence detection kit was from Amersham.
Pressure Experiments
HUVECs (passage numbers 2 to 9) were cultured in EGM-UV medium
supplemented with 10% FBS and 0.4% BBE. For pressure experiments,
HUVECs were trypsinized and seeded at 1/3 confluent density
either to borosilicate glass coverslips (for immunocytochemical
experiments), Thermanox coverslips (for BrdU labeling experiments), or
plastic tissue-culture flasks (for other experiments); all substrates
had been precoated with adsorbed human fibronectin (1 µg/mL). Cells
were cultured for an additional 16 hours to 3 days before use in
experiments. Unless otherwise noted, the cells were cultured in M199
supplemented with 10% FBS and 0.4% BBE for the duration of the
experiments. Pressure experiments were conducted in pressure chambers
that consisted of sterile, polypropylene cylinders (35-mm diameter;
10-cm height) with a removable, watertight cap on the bottom end.
Coverslips (on which HUVECs had been cultured) were secured to the
inside of the removable caps before the latter were attached to the
pressure chambers. The chambers were then filled with M199 to attain
the desired hydrostatic pressure head; the medium was added slowly and
gently to minimize shear stress on the cells. Filled chambers were
capped loosely with sterile, 35-mm lids and were maintained immobile
under standard cell-culture conditions for the duration of the
experiments. Previous studies by our group determined the pH,
pO2, and
pCO2 characteristics of this
model.5 Unless otherwise noted, controls were HUVECs
maintained under 0.2 cm H2O pressure, and cells
were exposed to 4 cm H2O SHP for various periods.
Because a large quantity (25 mL) of medium was required to generate the
4 cm H2O pressure heads in this model, some
controls consisted of HUVECs maintained under 0.2 cm
H2O pressure in 25 mL of medium in 100-mm Petri
dishes; these experiments were conducted at 37°C in a humidified, 5%
CO2–95% air atmosphere for periods of 4 hours
to 4 days. HUVECs grown on tissue-culture plasticware were either
maintained under control conditions or exposed to SHP in M199 at
37°C; these cells were exposed to pressure for various times ranging
from 15 minutes to 4 days.
Immunocytochemistry
HUVECs on glass coverslips were fixed in 3.7% formalin in PBS
for 15 minutes and then permeabilized in 0.1% Triton
X-100 in PBS for 5 minutes. Preliminary studies indicated that the
monoclonal antibodies to
Vß3 (LM609) and
Vß5 (P1F6) and the
polyclonal antisera to ß3 and
ß5 integrins used in the present study did
not have adequate sensitivity and/or specificity for their antigens in
the formalin-fixed endothelial cells used in the
present study; however, the monoclonal antibody to
V (LM142) and the monoclonal antibody to
vinculin (hVIN-1) stained single bands on Western blots and recognized
antigens in formalin-fixed cells (data not shown). The fixed,
permeabilized cells were therefore probed using these
antibodies as previously described.10
Microscopy and Image Analysis
The endothelial cells used in the
immunocytochemical experiments were examined with a Zeiss LSM 410
laser-scanning confocal microscope (Carl Zeiss) at the University of
Connecticut Center for BioImaging Technology, Farmington, Conn.
Microscopy was performed with a 63x objective and
fluorescein optics (490-nm excitation/520-nm emission).
Interference-reflectance microscopy (IRM) was performed with the same
microscope and lens. Images were stored digitally as TIFF files and
were subsequently analyzed using PhotoShop (Adobe) and ImagePro
Plus (Media Cybernetics) software.
Protein Extraction
Cells in situ were first rinsed in 4°C PBS (containing 1
mmol/L CaCl2 and MgCl2) and
then in serum-free M199. Proteins and DNA were extracted by incubating
the cells in 4°C extraction buffer [10 mmol/L Tris-HCl,
150 mmol/L NaCl, 2 mmol/L CaCl2, 1
mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 U/mL
aprotinin, 15 µg/mL leupeptin, 0.36 mmol/L 1,10-phenanthroline,
1% (vol/vol) Nonidet NP-40, and 1% (vol/vol) Triton X-100; pH 7.5].
Protein extracts were precleared by using protein A–agarose beads, and
the supernatant was reserved; extracts that were not used immediately
after extraction were snap-frozen and stored at -80°C. The quantity
of protein in each extract sample was determined using a Pierce
bicinchoninic acid protein detection kit according to manufacturer's
directions. Total DNA was eluted from the protein extracts by ethanol
precipitation, and DNA was quantified by techniques previously
described in the literature.11
Proteins used for Western blot experiments were extracted as follows. Cells in situ were rinsed twice in 4°C PBS, removed with plastic cell scrapers, and pelleted by centrifugation. The supernatant was discarded, and 1 mL of immunoprecipitation buffer (0.625 mmol/L HEPES-KOH, pH 7.5; 22.5 mmol/L potassium acetate; 8 µmol/L EDTA; 10 mmol/L DTT; 40 U/mL aprotinin; 15 µg/mL leupeptin; 15 µg/mL pepstatin; 1 mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride; 1 mmol/L NaF; and 1 mmol/L NaVO4) was added. The cell pellet was sheared in this buffer by aspiration (10 times) through a 23-gauge needle, precleared with protein A–agarose beads at 4°C for 30 minutes, and cleared of debris and beads by centrifugation (15 000g, 5 minutes). Glycerol was added to 30% final concentration, and extracts that were not used immediately were snap-frozen and stored at -80°C.
Gel Electrophoresis
Aliquots of protein samples were separated by
SDS–polyacrylamide gel electrophoresis. Proteins were
transferred from the gels to nitrocellulose membranes by electroelution
using a GENIE transfer apparatus (Idea Scientific) for 40
minutes. Proteins bound to the membranes were detected by Western blot
with polyclonal antisera to integrin subunits
V, ß1,
ß3, and ß5, and p65, as
well as with monoclonal antibodies to vinculin, and were visualized
with an Enhanced ChemiLuminescence detection kit according to
manufacturer's directions. Membranes were stripped of bound antibodies
and reprobed several times with different primary antibodies. Stripping
was accomplished by soaking the membranes in a solution of 100
mmol/L ß-mercaptoethanol, 62.5 mmol/L Tris-HCl, and 2% SDS at
50°C for 30 minutes. Band densities were quantified using PhotoShop
software (Adobe).
BrdU and DAPI Labeling
HUVECs that had been seeded to Thermanox coverslips were either
maintained under control pressure conditions or exposed to SHP in M199
for periods of 30 minutes to 4 days. In some experiments, the medium
contained 100 nmol/L SB223245 (a selective V
integrin antagonist12 ) to determine the
requirement of V integrins for
endothelial cell proliferation. BrdU was added to the
medium to a final concentration of 10 µmol/L either 24 hours (to
assay for inhibition of proliferation using integrin
antagonists) or 4 hours (to assay for the time at which
proliferation was upregulated in response to pressure) before the end
of the experiment. At the end of the experiment, the cells were fixed
and permeabilized in FixDenat solution at 37°C for 30
minutes. The fixed, permeabilized cells were rinsed in
PBS and subsequently incubated in anti-BrdU monoclonal antibody
solution (1:1000 in PBS) in a 4°C, humidified chamber overnight.
Cells were rinsed and incubated in biotin-conjugated anti-mouse
antisera at 37°C for 2 hours and subsequently incubated in Vector
Elite ABC reagent at 37°C for 1 hour. The cells were rinsed 3 times
in PBS and incubated in Sigma FastDAB solution that had been prepared
according to manufacturer's instructions immediately before use; cells
were incubated in this solution until a visible, brown precipitate had
formed (
2 minutes), at which time the reaction was quenched with a
large volume of distilled water.
Cell proliferation was determined after HUVECs were either maintained under control conditions, exposed to SHP, or maintained under control pressure conditions in 25 mL of medium for 24 hours. These cells were subsequently fixed (as described in the Immunocytochemistry section) and incubated in 5 µg/mL DAPI to stain the nuclei, which were then counted.
The BrdU- or DAPI-labeled cells on coverslips were rinsed in PBS, mounted in AquaMount, and examined with a 40x objective either by transmitted-light or UV fluorescence microscopy. Visual fields thus imaged were stored digitally and analyzed with ImagePro Plus software to count the stained nuclei; counts were averaged over 4 to 6 fields per coverslip and 3 to 6 coverslips per data point.
Cell Cycle Analysis
HUVECs were seeded to fibronectin-coated tissue-culture
plasticware at 1/3 confluent density and cultured for 3 days
under control pressure conditions. Cells were serum deprived by
incubation in M199 containing 1% BSA and no serum for 24 hours. Cells
were subsequently incubated in fresh M199 containing either 1% BSA or
both 10% serum and 0.4% BBE under control pressure conditions and
under 4 cm H2O SHP for 24 hours. HUVECs were
subsequently detached from their substrates with 5 mmol/L EDTA in
Ca2+/Mg2+-free PBS and then
quenched in an equal volume of PBS with Ca2+ and
Mg2+. Cells were pelleted by
centrifugation and the supernatant was discarded. The
cells were resuspended by slow addition of 1 mL 70% ethanol to the
pellet under continuous vortexing; these cells were fixed in 70%
ethanol at 4°C for 24 hours. Fixed cells were repelleted and
incubated in 100 U/mL RNase A (in
Ca2+/Mg2+-free PBS) at
37°C for 2 hours, after which time propidium iodide (final
concentration 50 µg/mL) was added and the cells incubated for 10
minutes. Fluorescence was analyzed with a FACScan flow
cytometer (Becton Dickinson).
Cell Adhesion Experiments
ELISA (96-well, not tissue-culture treated) plates were coated
with 1 µg/mL vitronectin, fibronectin, gelatin, or
laminin in 0.1 mol/L NaCO3 (pH 9.5) at 37°C for
18 hours and washed with M199 (containing 1% BSA).
HUVECs were either maintained under control conditions or exposed to 4 cm H2O pressure for 4 days. After this time, the cells were detached (as described in the Cell Cycle Analysis section), pelleted by centrifugation, and resuspended in M199 containing 1% BSA; aliquots of these cell suspensions (3x105 cells/mL) were seeded to the protein-coated plates and allowed to adhere for 1 hour under standard cell-culture conditions. At the end of that time, the medium was removed from each plate by flicking, followed by gentle centrifugation (<500 rpm) of the plate, upside-down, for 3 minutes. Cells in each well were counted manually by phase-contrast microscopy and a 10x objective.
Data Analysis
Numerical data consisting of multiple comparisons were first
analyzed by 1-way ANOVA; if the ANOVA indicated a significant
difference between groups, the difference between each control and
experimental group was then evaluated with a post hoc Bonferroni's
t test. Numerical data comparing a control versus a single
experimental group were evaluated with an unpaired, 2-tailed Student's
t test. Numerical data comparing each control versus a
matched experimental group were evaluated with a paired, 1-tailed
Student's t test. For all of these tests, a value of
P<0.05 was considered statistically significant.
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Results |
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SHP Stimulated HUVEC Proliferation
Several methods were used to assess the effects of SHP on HUVEC proliferation, including determination of cell numbers, total protein and DNA contents, cell cycle analyses, and BrdU labeling index. Compared with HUVECs maintained under control pressure conditions, exposure of HUVECs to SHP for 4 days resulted in a significant (P<0.05) increase in cell number (from 1.91±0.08x107 to 3.21±0.05x107 cells/175-cm2; mean±SEM, n=4) as well as an increase in total protein and total DNA content (data not shown). Independent experiments also demonstrated that pressure-treated cells showed a significant increase in the number of BrdU-labeled nuclei, observable as early as 24 hours after exposure to elevated pressure (Figure 1A
); exposure of HUVECs to large
volumes of medium did not affect the number of labeled nuclei (Figure 1B). Cell cycle analysis demonstrated that exposure of
HUVECs to SHP for 1 day resulted in an increased percentage of cells in
the S/G2/M phases of the cell cycle, with a
concomitant decrease in the percentage of cells in the
G0/G1 phases (Figure 2). It should be noted that these
increases in cell proliferation occurred above a high background
proliferation due to the use of culture medium supplemented with serum
and BBE.
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SHP Altered the Distribution and Concentration of VnRs
The Vß3 and
Vß5 VnRs play
important roles in endothelial cell
proliferation2 9 and were investigated in the present
study by Western blot, immunocytochemistry, and adhesion assays. The
amount of V, ß1,
ß3, and ß5 integrin
subunits in HUVEC lysates prepared from cells exposed to SHP for
various periods was compared by Western blot analysis (Figure 3A and 3B). Equal amounts of protein were
loaded onto each lane; blots were probed with an antibody to the
transcription factor p65; similar band density in each lane
demonstrated equal loading of protein across the blots (Figure 3B). Compared with controls, pressure-treated cells exhibited
increased total V, reproducibly detectable
after as little as 4 hours of exposure to SHP. Exposure of HUVECs to
SHP for 3 days resulted in increased levels of
ß3 (Figure 3B); this effect, however,
was not reproduced consistently in all runs of the experiment.
The levels of ß5 and ß1
were not affected by exposure of HUVECs to SHP at any time examined in
the present study (Figure 3A).
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SHP Altered V and Vinculin
Distribution
Immunocytochemical techniques were used to determine
whether V and vinculin localization
was affected by exposure of HUVECs to SHP. When HUVECs were maintained
under control conditions, integrin subunit V
localized predominantly to large, rounded regions around the cell
border (Figure 4A). In contrast, after
exposure of the cells to SHP for 4 days, V
localized to numerous regions throughout the spread edge of the cells
(Figure 4E); these V-rich regions had
an elongated shape (Table). Similarly,
regions immunoreactive for vinculin were large, rounded, and localized
predominantly to the cell border in HUVECs maintained under control
conditions (Figure 4B); after exposure of these cells to
pressure, regions immunoreactive for vinculin were located throughout
the spread edge of the cells (arrows) and had an elongated shape, as
evidenced by the morphology of individual regions (Figure 4F)
and confirmed by computer-aided morphology analysis of the
aspect ratios of >700 immunoreactive regions (Table). Because
vinculin is characteristically associated with FAPs and the
distributions of V and vinculin were similar,
IRM was used to determine whether V
colocalized with FAPs. Both under control pressure conditions (Figure 4C and 4D) and after exposure of the HUVECs to SHP for 4 days
(Figure 4G and 4H), regions of intense
V staining (Figure 4C and 4G; arrows)
colocalized with areas of cell-substrate contact (Figure 4D and 4H; arrows); the areas of cell-substrate contact were elongated (Figure 4H), identical to the elongated shape of the regions of
V staining in HUVECs that had been exposed to
SHP (Figure 4G). In agreement with many published reports,
parallel experiments demonstrated that vinculin localized to FAPs (data
not shown). Computer-aided morphometric analysis confirmed
that, after exposure of HUVECs to SHP, the areas of the individual
V- and vinculin-immunoreactive FAPs were
smaller than those in controls (specifically, 23.3±0.5 pixels
for V in cells under pressure versus 44.0±1.3
pixels in control cells; the Table) and were more elongated
(2.11±0.03 aspect ratio for V in cells
under pressure versus 1.88±0.02 aspect ratio in control cells; the
Table). Cells that had been cultured under control pressure
conditions in large volumes of medium (25 mL; similar to that of the
hydrostatic pressure heads) had FAP morphology similar to cultures
maintained under control pressure conditions in 2 mL medium (data not
shown).
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Exposure of HUVECs to SHP Selectively Increased Subsequent Cell
Adhesion to Vitronectin
The effect of pressure on endothelial cell
adhesion to vitronectin-coated surfaces was examined to
determine whether the pressure-induced increase in the total pool of
V integrins resulted in a functional change in
V integrin–mediated adhesion. Compared with
controls, HUVECs that had been exposed to SHP for 4 days exhibited
greater cell adhesion on surfaces that had been precoated with adsorbed
vitronectin at concentrations ranging from 0.5 to 10
µg/mL (data not shown). To determine the selectivity of this
response, the adhesion of control and pressure-treated HUVECs to
fibronectin, laminin, and gelatin was also determined. Pressure-treated
cells demonstrated significant increases in adhesion to
vitronectin-coated surfaces; in contrast, cell adhesion to
laminin-coated, gelatin-coated, and fibronectin-coated surfaces did not
change (Figure 5). Furthermore, the
adhesion of these cells to the vitronectin-coated surfaces
could be completely blocked in the presence of 2.5 µg/mL each LM609
and P1F6 (data not shown). These data are consistent with a
pressure-induced, selective increase in functional VnRs.
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Integrin V Inhibitors Blocked
Pressure-Induced Cell Proliferation
Cell proliferation was determined by BrdU uptake assays for HUVECs
that were either maintained under control conditions or exposed to SHP
in the presence and absence of 100 nmol/L SB223245 (a specific
V integrin
antagonist12 ) for 4 days. Coincubation of
HUVECs with 100 nmol/L SB22324512 blocked the
pressure-induced increase in cell proliferation (9.4±0.9 BrdU-labeled
nuclei per high-power field for cells under pressure versus 2.1±0.9
for cells under pressure in the presence of SB223245).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several pathological conditions, including atherosclerosis,13 tumors,14 15 and glaucoma,16 expose endothelial cells to increased pressure and feature enhanced endothelial proliferation/turnover as an etiological component of the disease. In vivo, endothelial cells are exposed to several mechanical forces, namely, fluid shear, substrate tension, and hydrostatic pressure. Exposure of endothelial cells to these mechanical forces in vitro also affects cell morphology and function.3 5 6 17 In particular, SHP has been shown to affect the proliferation of numerous cell types, including endothelial cells,5 6 chondrocytes,18 fibroblasts,18 19 20 and intimal vascular smooth muscle cells.21 22 Although the direction (ie, stimulatory or inhibitory) of the responses of these different cell types to pressure is likely dependent on cell type, culture conditions, and the pressure regimens to which the cells are exposed,19 these results suggest that molecular signal transduction mechanisms can be activated by SHP and that these mechanisms undoubtedly play a role in physiological and pathological processes in vivo.
The present study used a model of SHP to investigate the effects of this mechanical force independent of shear and tensile stresses. The 4 cm H2O pressure used in the present study falls within the low range of physiological hydrostatic pressures to which the endothelial cells may be exposed in vivo. In the body, the total hydrostatic pressure to which vascular endothelial cells are exposed is not the same as the hydrodynamic pressure value, typically measured as "blood pressure" with a sphygmomanometer. The hydrostatic pressure is the net sum of several pressures, including the vascular hydrostatic pressure (ie, the force containing blood within the vessels) and components of the vascular hydrodynamic pressure, as well as the plasma and tissue oncotic pressures and the tissue hydrostatic pressure (as reviewed in Reference 2323 ). Within a given vascular bed, the hydrostatic pressure to which endothelial cells are exposed is directly correlated with, but generally lower than, arterial blood pressure. For example, the normal abdominal aortic pressure of the rat ranges from 132 to 172 cm H2O; however, the measured tissue interstitial pressure (which is a reasonable estimate of the hydrostatic pressure) in the rat kidney ranges from 4 to 29 cm H2O.24 A change in blood pressure (such as transient changes due to alterations in posture and chronic changes due to disease, eg, hypertension) exposes endothelial cells to changes in hydrostatic pressure. Additionally, the parameters that contribute to variations in blood pressure (namely, posture, hypertension, etc) suggest that endothelial cells at various sites of the vascular tree are exposed to different hydrostatic pressures; for example, in the upright human body, the hydrostatic pressure in the veins of the neck is maintained at atmospheric pressure,25 the veins of the skull at subatmospheric pressure,25 and the large vessels of the feet at pressures that may exceed 100 cm H2O above atmospheric.25
The present study provided evidence that HUVECs respond
consistently to a physiological pressure of
4 cm H2O. The findings of this study fall into
several major categories: (1) SHP stimulates the proliferation of human
endothelial cells, even when these cells have a high
baseline proliferation due to the presence of 10% FBS and 0.4% BBE in
the culture medium; (2) SHP upregulates integrin
V expression; and (3) SHP results in
remodeling of FAPs. The stimulatory effects of pressure on
endothelial cell proliferation were confirmed by using
various techniques, specifically, determination of total cell number,
total DNA and protein contents, ratio of cells entering the
proliferative phases of the cell cycle, and BrdU labeling index. These
observations are in agreement with previous studies that demonstrated
increased proliferation of bovine endothelial cells
after exposure to SHP.5 6 The present study, however,
is the first to demonstrate that exposure of normal, human
endothelial cells to SHP resulted in significantly
(P
0.05) increased cell proliferation. Compared with bovine
endothelial cell results,5 6 the
magnitude of the response of HUVECs to pressure was less pronounced. It
is important to note that, in contrast to bovine
endothelial cells, culture of HUVECs requires exogenous
growth factors (specifically, BBE, a crude extract rich in bFGF);
consequently, the observed increase in HUVEC proliferation under SHP
occurred over an artificially high baseline rate of cell proliferation,
which (in comparison) caused the pressure-induced increases in
proliferation to appear smaller.
Exposure of HUVECs to SHP resulted in a 300% increase in the
expression of the integrin subunit V and in a
smaller, more variable increase in the expression of integrin
subunit ß3. This increase was selective for
V expression because expression of integrin
subunits ß1 and ß5 was
not affected (Figure 3
). Immunostaining and IRM
experiments demonstrated that these biochemical changes in integrins
were accompanied by changes in the shape and distribution of FAPs
(Figure 4 and the Table). In agreement with these
biochemical changes, HUVECs exposed to SHP also demonstrated increased
adhesion to vitronectin-coated plasticware; adhesion of
these cells to other ligands (such as fibronectin, laminin, and
gelatin) was not affected (Figure 5). Furthermore, the
pressure-induced increase in cell adhesion to vitronectin
could be blocked by the combination of 2.5 µg/mL each of LM609 and
P1F6 (but not by 5 µg/mL of either antibody alone) in the culture
medium (data not shown); this result provided evidence that the altered
expression of VnRs that occurs after exposure of HUVECs to pressure may
result in changes in the interactions of these cells with proteins of
the extracellular matrix.
The observation that a mechanical force (ie, hydrostatic pressure)
changes human endothelial cell morphology, FAP
composition, and FAP organization is consistent with results of
studies that exposed endothelial cells to cyclic strain
and shear; however, the details of the biochemical responses of
endothelial cells to each of these mechanical stimuli
are unique. For example, exposure of bovine endothelial
cells to SHP results in changes in cell shape (from polygonal to
elongated) over a time course of 4 days.5 In contrast,
exposure of bovine and human endothelial cells to
cyclic strain and laminar shear stress results in similar polygonal to
elongated changes in cell shapes as early as 4 hours after the onset of
stimulation.26 27 Furthermore, exposure of HUVECs to
cyclic strain does not affect the level (either total or surface) of
2, 5,
ß1, and ß3
integrins27 ; under cyclic strain conditions, however,
integrin subunits ß1,
5 (cells seeded on collagen), and
2 (cells seeded on fibronectin) are localized
to long streaks parallel to the long axes of the cells.27
Bovine aortic endothelial cells exposed to shear stress
upregulate 5ß1 but
downregulate Vß3
expression and redistribute
Vß3 to the
"upstream" end of the cells.26 In contrast, the
results of the present study demonstrate that exposure of human
endothelial cells to SHP results both in the
upregulation and redistribution of specific integrins (namely,
V heterodimers) and in a unique reorganization
of FAP morphology and distribution. These differences in integrin
distribution and expression in endothelial cells
exposed to different external mechanical forces are consistent
with the "tensegrity" model proposed by Ingber.28 The
tensegrity model hypothesizes that an external mechanical force acting
on a cell is transmitted by tensile actin filaments to proteins
(specifically, integrins) that anchor the cell to the extracellular
matrix28 ; thus, different mechanical forces that apply
distinct stresses to endothelial cells could induce
specific reorganization of integrins to a pattern that is optimally
suited to resist those stresses.
Through tensegrity and/or other mechanisms, integrins and other
proteins present in the FAPs (eg, focal adhesion kinase and
paxillin) mediate both outside-in and inside-out signaling pathways (as
reviewed in References 7 and 297 29 through 31); activation of these
pathways leads to altered gene expression, cell migration, and cell
proliferation. The V integrins in particular
have been reported to modulate proliferation of
endothelial cells in vivo.2 9 32 Blocking
monoclonal antibodies to
Vß3 (LM609) and to
Vß5 (P1F6) inhibited
angiogenesis in several in vivo models.2 9 In the
present investigation, time-course studies demonstrated that
increases in total immunoreactive V could be
detected after 4 hours of HUVEC exposure to 4 cm
H2O SHP, yet a significant increase in cell
proliferation (determined by BrdU labeling index) was not detected
until 24 hours, suggesting that alteration of
V preceded the proliferative response. Use of
functional blocking antibodies in the present in vitro study,
however, suggests that LM609 and P1F6 (at 5 µg/mL) did not affect
pressure-induced proliferation (data not shown). It is important to
note that a mixture of 2.5 µg/mL each of LM609 and P1F6 antibodies
significantly reduced adhesion of HUVECs to
vitronectin-coated tissue-culture plasticware (data not
shown), suggesting that the concentrations of antibodies used to block
integrin-mediated proliferation were sufficient to inhibit attachment
of the VnRs to their ligands but may not have effected detachment of
adherent VnRs from their ligands. Higher concentrations of these
antibodies were not tested due to the very high cost of such
experiments. However, 100 nmol/L SB223245 did prevent pressure-induced
increases in HUVEC proliferation; SB223245 has been reported in the
literature to specifically inhibit interactions of
Vß3 integrins with
their ligands.12 It should be noted that the observations
with SB223245 must take into account the potential "toxic" effects
of Vß3
inhibitors: detachment of cells from substrates leads to
death in anchorage-dependent cells; furthermore blocking of
V integrins in proliferating cells can lead to
apoptosis in the absence of cell detachment.9
Although no evidence of extensive cell death was observed in the
present study, a reduction in the number of proliferating cells in
the presence of V integrin
inhibitors may be due to cell death rather than exclusively
to an inhibition of integrin-mediated signaling leading to
pressure-induced cell proliferation. Thus, at present, the precise
role that V heterodimers may play in the
pressure-induced proliferative response of endothelial
cells cannot be unequivocally proven.
Reorganization of adhesion plaques in response to SHP is probably just one of the steps in the mechanotransduction/mechanoresponse to this ubiquitous mechanical force. It is clear that signaling events that occur downstream of integrin activation must also be triggered by hydrostatic pressure. Additionally, a role for either soluble or intracellular growth factors cannot be ruled out. The results of Acevedo et al5 provided evidence that exposure of bovine pulmonary artery endothelial cells to SHP resulted in release of bFGF from the cells and in the accumulation of this growth factor, and perhaps others (for example, VEGF), in the supernatant medium. These conditioned media from pressure-treated cells stimulated subsequent proliferation of cells under control pressure conditions, providing evidence that the released factor(s) mediated the cell proliferation response to SHP. Thus, the proliferative response of HUVECs to SHP could also be due to either increased synthesis and/or release of soluble growth factor(s) or to changes in growth factor signaling. It should be noted that, in the present study, it was necessary for the survival of the HUVECs in culture that the experiments in the present study be carried out in the presence of high concentrations of exogenous growth factors (BBE), a situation that complicated attempts to examine growth factor signaling in the current model.
In summary, the results of the present study demonstrated, for the
first time, that a modest (specifically, 4 cm
H2O) level of SHP over a period of 1 to 4 days
stimulates cell proliferation, changes in integrin expression, and
changes in organization of FAPs in human endothelial
cells. The present study also suggests that VnRs play a role (to
date, not specifically determined) in the pressure-induced increase in
endothelial cell proliferation. It is unclear at this
time, however, whether changes in V
expression, distribution, and function mediate or occur in response to
the observed changes in cell proliferation rate. Further research on
the mechanism(s) responsible for regulation of
V synthesis and on the role of various
integrin-associated signaling molecules is needed to elucidate
pertinent details of the intracellular signaling mechanism(s) that
governs pressure-induced increases in endothelial cell
proliferation.
|
Acknowledgments |
|---|
The authors wish to thank the Whitaker Foundation for a graduate fellowship to Eric A. Schwartz.
|
Footnotes |
|---|
Correspondences to Dr Mary E. Gerritsen, Senior Scientist, Cardiovascular Research, Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080-4990.
Received September 10, 1998; accepted November 24, 1998.
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