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(Circulation Research. 1999;85:1118.)
© 1999 American Heart Association, Inc.

Molecular Medicine

Transcriptional Profile of Mechanically Induced Genes in Human Vascular Smooth Muscle Cells

Yajun Feng, Jeong-Hee Yang, Hayden Huang, Scott P. Kennedy, Thomas G. Turi, John F. Thompson, Peter Libby, Richard T. Lee

From the Vascular Medicine and Atherosclerosis Unit (Y.F., J.-H.Y., H.H., P.L., R.T.L.), Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; Pfizer Central Research (S.P.K., T.G.T., J.F.T.), Groton, Conn.

Correspondence to Richard T. Lee, MD, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail rtlee{at}bics.bwh.harvard.edu

	Abstract

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Abstract—Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.

Key Words: vascular smooth muscle • genomics • strain

	Introduction

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In tissues subjected to deformations, cells monitor and respond to their mechanical environment. In the artery wall, the vascular smooth muscle cell (VSMC) contributes prominently to regulating tissue strength.¹ VSMCs synthesize and organize extracellular matrix,² regulate extracellular matrix degradation,³ and regulate vascular tone. These cells may therefore play a pivotal role in arterial structure in pathophysiological settings that change arterial mechanics such as hypertension and atherosclerotic plaque stability.⁴

Mechanical forces directly regulate VSMC functions in vitro.^{5 6 7 8 9} Several mechanisms may mediate the responses of human VSMCs to deformation. Large deformations in vitro (>10%) lead to transient cell injury, release of fibroblast growth factor-2 (FGF-2), and cell proliferation,¹⁰ similar to observations after experimental balloon injury in vivo.¹¹ In contrast, we have previously demonstrated that human VSMCs regulate immediate-early gene expression at very small strains (1%), well below the amplitudes that cause cell injury and FGF-2 release.¹² Thus, it is possible that the responses of VSMCs to smaller deformations that do not injure cells may mediate changes in the vascular system.

Although VSMCs appear to respond exquisitely to small deformations, the profile of mechanically induced genes at small strains remains incompletely defined. Recently developed molecular techniques can demonstrate differentially expressed genes. Differential display, a polymerase chain reaction–based technique, has been applied to mechanically induced genes in endothelial cells exposed to fluid shear stress.^{13 14} This approach has revealed previously unsuspected biomechanical regulation of several genes, including cyclooxygenase-2 and superoxide dismutase. One disadvantage of a polymerase chain reaction–based technique such as differential display is that it does not reveal relative quantitative changes. A second disadvantage is that differential display does not provide the approximate number of genes explored (the denominator in the fraction of genes induced).

In contrast to differential display, DNA microarray technology allows expression monitoring of thousands of genes on the same chip. Using two-color fluorescence patterns, the transcriptional profile of a stimulus may be described.¹⁵ For example, a stimulus such as serum treatment of fibroblasts induced hundreds of genes when an array of >8600 genes was explored, indicating clusters of genes potentially involved in wound repair.¹⁶ Because relatively little is known about mechanically induced genes in human VSMCs, and because these genes may be relevant to hypertension, atherosclerosis, aortic aneurysm formation, and other diseases, we studied the transcriptional profile of mechanically induced genes in human VSMCs. Unlike the diverse response to serum stimulation of fibroblasts, only a few genes are induced by deformation of human VSMCs; the functions of these genes indicate a defense against excessive deformation. These data suggest that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a few genes that comprise a highly specific molecular response, such as the response to mechanical stimuli.

	Materials and Methods

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Materials
DMEM and Ham’s F-12 were obtained from Biowhittaker. Dulbecco’s PBS solution, Hanks’ balanced salt solution, fibronectin, and other materials required for tissue culture were purchased from Gibco-BRL. [-³²P] dCTP (3000 Ci/mmol) was purchased from Dupont NEN. The cDNA for the human vascular endothelial growth factor (VEGF) gene was the generous gift of Dr Judith Abraham, Scios (Sunnyvale, Calif).

Culture of VSMCs
Cells were prepared from explants from excess aortic tissue from the donor at the time of organ harvest for orthotopic cardiac transplantation at Brigham and Women’s Hospital. VSMCs were maintained in DMEM, 10% FCS, and 1% penicillin/streptomycin sulfate. These conditions are selective for growth of VSMCs over endothelial cells. VSMCs were maintained at 37°C, 5% CO₂ up to passage 6 to 7 for experiments. Under these conditions, 50% to 60% of human VSMCs routinely stain positive for smooth muscle actin. The protocol was approved by the Brigham and Women’s Hospital Committee for Human Research.

Mechanical Strain Device
Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured, an approach that produces controlled cellular strain and allows visualization of cells. This device provides a nearly homogeneous biaxial strain profile; that is, strains that are equal at all locations on the membrane and in all directions.¹⁰ An advantage of this device over some commonly used systems is that it eliminates locations on the substrate that have very high strains (20% to 30%) in one direction. The membrane undergoes cyclic deformation as the platen assembly moves sinusoidally with a frequency and amplitude derived by the motor speed and the cam size, respectively. We have previously measured membrane strains with a high-resolution video device¹⁰ ; the cams used for this study gave strains of 1%, 4%, and 9%.

The cell culture silicone membrane itself supports negligible adhesion of VSMCs. For the preparation of VSMCs to be subjected to mechanical strain, autoclaved membrane dishes were coated with 2 µg/mL of fibronectin in Hanks’ solution for 6 to 12 hours at 4°C and then washed twice with 10 mL PBS. VSMCs were plated on the coated membrane dish at a density of 700 000 cells/dish in 13 mL of DMEM containing 10% FBS and incubated 16 to 24 hours. Cells were then washed with 10 mL of Hanks’ solution four times to remove residual serum and incubated with 10 mL of serum-free IT medium (equal volumes of DMEM and Ham’s F-12 supplemented with 1 µmol/L insulin and 5 µg/mL transferrin) for 48 hours. Before mechanical strain, 10 mL of fresh IT medium was exchanged. Mechanical strain was then applied at a specified constant frequency and amplitude, and control dishes received no mechanical strain.

Transcriptional Profiling
The DNA microarray experiment was performed with human aortic smooth muscle cells cultured on fibronectin-coated membranes with serum-free medium for 48 hours. Cells from a single patient donor were then exposed to 12 hours or 24 hours of cyclic deformation (1 Hz, 4% amplitude) or no deformation, and RNA was prepared. The choice of these time points was based on previous observations indicating that small strains regulated gene induction at these time points.¹² The DNA microarray hybridization experiment was performed using the public domain UniGem 1.0 array (Incyte Inc) using methods previously described.^{15 16} The UniGem 1.0 array has 5000 well-characterized genes with putative functions; this study reports findings from analyses of these genes. A complete listing of genes contained within UniGem 1.0 can be found at http://www.incyte. com/products/arrays/genelists/index.html. Data were analyzed using the GemTools software package (Incyte Inc). The sensitivity of the assay was detection of one transcript in 75 000.

Microarray reproducibility was determined using two independent assays. First, 200 ng of human RNA was labeled with either Cy3 or Cy5dCTP, mixed, and hybridized to an array. Fluorescent ratios were calculated for all called elements. These data demonstrated that when the same RNA is used for both fluorescent channels, 99% of elements of the UniGem 1.0 microarray give differential expression values within 2-fold. A second series of experiments used RNA isolated from two unrelated cell lines. Comparison of these two RNAs over three separate hybridizations yielded an average correlation coefficient of r=0.97. Additional reproducibility data using these methods are available at http://www.incyte.com/science/gem/thp-1.html. For the present study, we used a threshold value of 2.5-fold to define differential gene induction to minimize false-positive elements. Furthermore, we compared the results for >1000 genes to hybridizations with the Affymetrix GeneChip HU6800, using a different cell donor and methods previously described.¹⁶

Northern Analyses
The cDNA clones for differentially expressed genes were ordered from the IMAGE consortium. Each clone was sequenced from both 5' and 3' ends to confirm identity. Positive elements in the DNA microarray were confirmed by Northern analysis in at least three independent experiments using three different patient sources of human aortic smooth muscle cells. Total RNA was isolated by the guanidium thiocyanate and phenol chloroform method.¹⁷ For Northern blotting, 15 µg RNA was loaded on a 1.0% agarose-formaldehyde gel (2.0 mol/L), transferred to a nylon membrane (Amersham Life Science), and UV cross-linked with a UV Stratalinker (Stratagene). The probe was hybridized with ExpressHyb solution (Clontech) at 68°C for 1 hour. The membrane was washed with 2x SSC, 0.05% SDS solution for 30 to 40 minutes and three times at room temperature and 0.1x SSC, 0.1% SDS solution with continuous shaking at 50°C for 40 minutes. The membrane was exposed to film at -80°C, and radiographs were scanned and analyzed with Optimas 5.0 software (Optimas Co). Densitometric units were normalized to the ethidium-stained 28S ribosomal subunit on the membrane.

Protein Assays
To measure plasminogen activator inhibitor-1 (PAI-1) protein secreted into the medium, an enzyme-linked immunoassay was performed with a PAI-1 ELISA kit (Biopool). For Western analyses, cell lysates (50 µg) were loaded on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane in 25 mmol/L Tris base (pH 8.5), 0.2 mol/L glycine, and 20% methanol. The nitrocellulose membrane was blocked by 5% nonfat dried milk in TBS washing buffer containing 20 mmol/L Tris base (pH 7.6), 137 mmol/L NaCl, and 0.1% Tween 20 for 2 hours. For the detection of cyclooxygenase-1 and tenascin-C, the membrane was incubated with 1:2000 diluted goat anti-human Cox-1 polyclonal antibody (Santa Cruz Biotechnology, Inc) and mouse anti–rat tenascin-C antibody (Genex), respectively, for 1 hour at room temperature and washed with TBS washing buffer. The secondary antibody coupled to peroxidase was diluted 1:5000 and incubated with the membrane for 30 minutes. After washing with TBS washing buffer, the membrane was developed with the enhanced chemiluminescent (ECL) method (Amersham Life Science).

All data shown are representative of at least 3 independent experiments using different cell sources. A value of P<0.05 was considered statistically significant.

	Results

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Induction of VEGF and Transcript Imaging
We first sought to identify a positive control gene that could be used to confirm the effect of deformation in the samples used for transcriptional profiling. Based on previous reports of mechanically induced genes in cultured smooth muscle cells or endothelial cells, we studied platelet-derived growth factor (PDGF)-A, PDGF-B, superoxide dismutase, and transforming growth factor-ß.^{13 18 19 20} When using 4% cyclic biaxial deformation (an amplitude that does not cause cell injury or FGF-2 release in these cells), Northern analyses of these genes failed to demonstrate induction by strain in human aortic smooth muscle cells in repeated experiments (data not shown).

Mechanically induced VEGF gene expression has been reported in kidney mesangial cells²¹ and in heart tissue²² ; therefore, we hypothesized that VEGF may be mechanically responsive in human aortic smooth muscle cells. VEGF was modestly but reproducibly induced by 4% deformation at 12 and 24 hours. Induction of VEGF at 24 hours was 1.9±0.5-fold (n=3, P<0.05) compared with control. Although this average value of induction was below the threshold for detection by the DNA microarray, it was sufficient to confirm that the cells used for the transcript imaging responded to 4% cyclic deformation (Figure 1).

View larger version (80K): [in this window] [in a new window]   Figure 1. Mechanical strain induces VEGF expression. Human aortic smooth muscle cells were subjected to 4% cyclic strain for 12 and 24 hours. Deformation induced the two mRNAs of VEGF by Northern analysis. These data are representative of 3 independent experiments.

Transcriptional profiles of mechanically induced genes at 12 and 24 hours were remarkably similar and restricted to only a few genes (Table). Among the 5000 genes with putative functions, only three genes were induced >2.5-fold: cyclooxygenase-1 (3.5-fold at 12 hours, 2.7-fold at 24 hours); PAI-1 (5.5-fold at 12 hours, 3.4-fold at 24 hours); and tenascin-C (2.6-fold at 12 hours). Thus, although the 12- and 24-hour microarray hybridizations were performed only once, the results of these two hybridizations regarding induced genes were nearly identical. Among the 5000 genes, 3160 elements were called positive at 24 hours; therefore, 3157 genes were called positive but did not change >2-fold. In addition, we used aortic smooth muscle cells from another patient source and otherwise identical experimental conditions (4% strain, 1 Hz, 24 hours) in an Affymetrix GeneChip hybridization to evaluate reproducibility. Among 1000 genes that were hybridized to the UniGem chip but did not have differential regulation, all 1000 genes were not significantly changed in the GeneChip experiment. Furthermore, the positive control of PAI-1 was upregulated 6-fold, and the negative control of thrombomodulin (see below) was downregulated 7.1-fold in the GeneChip experiment, indicating that results were consistent using different cell sources. Further studies confirmed and extended the results of the DNA microarray-based profiling for the three induced genes.

View this table: [in this window] [in a new window]   Table 1. Differentially Expressed Genes in Human Aortic Smooth Muscle Cells

Cyclooxygenase-1
Cyclooxygenases participate in synthesis of prostaglandins G/H2 from arachidonic acid. The functional differences between cyclooxygenase-1 and cyclooxygenase-2 remain incompletely defined, although the tissue expression of these two enzymes is distinct.^{23 24} In contrast to cyclooxygenase-2 ("inducible Cox"), which is highly expressed in many cell types in response to numerous cytokines as well as shear stress in endothelial cells, cyclooxygenase-1 has been called the "constitutive Cox," because it generally does not respond to cytokine stimuli. However, recent studies have documented inducibility of cyclooxygenase-1, including by shear stress in endothelial cells.^{25 26 27} Northern analyses of separate cell lines confirmed that both alternatively spliced mRNAs of cyclooxygenase-1 are mechanically induced in human aortic smooth muscle cells in a time-dependent manner (Figure 2A). At 24 hours, cells subjected to 4% cyclic deformation had 3.4±0.8-fold increase in mRNA levels in cyclooxygenase-1 expression compared with controls (n=4, P<0.05). In contrast, cyclooxygenase-2 levels did not increase greater than 2-fold in repeated experiments.

View larger version (46K): [in this window] [in a new window]   Figure 2. Mechanical strain induces cyclooxygenase-1 expression. A, Human aortic smooth muscle cells were subjected to 4% cyclic strain for 0, 6, 12, or 24 hours. Total RNA was harvested and analyzed by Northern analysis for the two mRNAs of cyclooxygenase-1 as well as cyclooxygenase-2. B, Aortic smooth muscle cells were subjected to 0%, 1%, 4%, or 9% strain for 24 hours. Deformation of 1% and above induced expression of the two mRNAs of cyclooxygenase-1 by Northern analysis (arrows). These data are representative of 4 independent experiments. C, Western analysis demonstrating an increase in cyclooxygenase-1 protein after 24 hours of strain. Duplicate control (0% strain) and strain (4% strain) lanes are shown.

Because our cell deformation method provides uniform biaxial strain, strain amplitude thresholds for cellular responses may be established; for example, cyclic strains as small as 1% suppress matrix metalloproteinase-1 expression by aortic smooth muscle cells.¹² Amplitude-dependence studies determined that cyclooxygenase-1 was induced by strains as small as 1% (Figure 2B), and Western analyses indicated that deformation also increases cyclooxygenase-1 protein synthesis (4.0±1.5-fold, n=3, Figure 2C). In contrast to the induction of cyclooxygenase-1, cyclooxgenase-2 was highly induced by interleukin-1ß (10 ng/mL) but not by deformation (Figure 3).

View larger version (99K): [in this window] [in a new window]   Figure 3. Interleukin-1 induces cyclooxygenase-2 but not cyclooxygenase-1 expression. Human aortic smooth muscle cells were cultured with interleukin-1ß (10 ng/mL). Cyclooxgenase-2, which was not induced by strain, was inducible with cytokine stimulation. The two mRNAs of cyclooxygenase-1 are designated by arrows. These data are representative of 3 independent experiments.

PAI-1
Small strain amplitudes induced both isoforms of PAI-1 mRNA (Figure 4). At 4% strain, PAI-1 mRNA expression was induced 8.3±2.2-fold (n=5) at 24 hours. Cyclic strains as small as 1% increased PAI-1 mRNA levels at 24 hours (data not shown). In contrast to cyclooxgenase-1, PAI-1 induction increased in response to human recombinant tumor necrosis factor- (10 ng/mL) and phorbol 12-myristate 13-acetate (100 ng/mL). Secreted PAI-1 in the medium was analyzed by ELISA assay in independent experiments, demonstrating a 62±25% (n=3, P<0.05) increase in PAI-1 protein secretion after 24 hours of cyclic deformation.

View larger version (55K): [in this window] [in a new window]   Figure 4. Mechanical strain induces PAI-1 expression. Human aortic smooth muscle cells were subjected to 4% cyclic strain for 12 and 24 hours. Deformation, as well as phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) and tumor necrosis factor (TNF)- (10 ng/mL), induced both PAI-1 mRNAs (arrows). These data are representative of 5 independent experiments.

Tenascin-C
Tenascin-C is a large extracellular matrix protein that can form hexamers. Tenascin-C expression is regulated by growth factors and cytokines and is temporally regulated in development.^{28 29} Tenascin-C has antiadhesive properties in vitro and is prominent in remodeling tissues.³⁰ Tenascin-C transcripts were induced by cyclic deformation 3.0±1.0-fold in VSMCs at 12 hours (Figure 5; n=3, P<0.05), with less induction apparent at 24 hours. Amplitude-response experiments indicated that tenascin-C induction is amplitude dependent and apparent at 1% biaxial strains. Furthermore, Western analysis demonstrated a 2.5-fold increase in tenascin-C protein after 24 hours of strain (Figure 5C).

View larger version (44K): [in this window] [in a new window]   Figure 5. Mechanical strain induces tenascin-C. A, Human aortic smooth muscle cells were subjected to 4% cyclic strain for 0, 6, 12, or 24 hours. Total RNA was analyzed for tenascin-C expression by Northern analysis. B, Aortic smooth muscle cells were subjected to 0%, 1%, 4%, or 9% strain for 24 hours. Deformation of 1% and above induced tenascin-C expression by Northern analysis. These data are representative of 3 independent experiments. C, Western analysis demonstrating induction of tenascin-C protein after 24 hours of interleukin-1 (IL-1, 10 ng/mL) or strain (4% strain). Duplicate lanes are shown; the arrows designate two protein isoforms of tenascin-C.

Downregulated Genes
DNA microarray technology has the capability of identifying decreases in mRNA levels (Table). The expression of only 13 genes decreased >2.5-fold at either 12 or 24 hours. Among the downregulated genes was matrix metalloproteinase-1 (-3.9-fold at 12 hours); we had reported this phenomenon before these transcriptional profiling experiments.¹² Another downregulated gene was thrombomodulin (-2.6-fold at 24 hours), an integral membrane glycoprotein that binds thrombin, the final enzyme of the procoagulant pathway³¹ ; we confirmed by Northern analysis that thrombomodulin was downregulated 3.3±1.0-fold by cyclic deformation (Figure 6). The thrombin-thrombomodulin complex is the primary physiological activator of protein C. However, thrombomodulin is expressed on many nonendothelial cells, and recent data suggest that thrombomodulin may regulate cell proliferation independent of thrombin.³²

View larger version (47K): [in this window] [in a new window]   Figure 6. Mechanical strain downregulated thrombomodulin expression. Human aortic smooth muscle cells were subjected to 4% cyclic strain for 24 hours. Northern analyses demonstrated reduction of thrombomodulin expression. These data are representative of 3 independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arterial smooth muscle cells reside in a mechanically active environment subjected to variable mechanical loads. Because smooth muscle cells comprise the primary structural cells of the artery, the responses of these cells to deformation may represent an important defense against excess mechanical load. This study identified mechanically induced genes in human aortic smooth muscle cells by transcriptional profiling; interestingly, these genes have potential extracellular matrix or vasomotion roles. Compared with stimuli such as interferons, which may induce dozens or hundreds of genes,³³ cellular deformation appears to be a highly specific stimulus with a response restricted to a small number of genes. Preliminary data from our laboratories indicate that cytokines also induce many more genes in VSMCs than deformation (unpublished data, 1999).

Cells may use multiple mechanisms for controlling extracellular matrix degradation.³⁴ Plasmin can directly digest extracellular matrix components in addition to activating matrix metalloproteinases.¹⁴ Many cell types including endothelial cells and VSMCs synthesize the endogenous inhibitor PAI-1.^{35 36} Although PAI-1 synthesized by endothelial cells may play a critical role in fibrinolytic balance, PAI-1 within the artery also regulates extracellular matrix proteolysis and vascular repair.^{37 38} PAI-1, as an inhibitor of plasminogen activators, can function as a direct inhibitor of matrix degradation or indirectly by preventing activation of matrix metalloproteinases.³⁹ Increased levels of PAI-1 could tip the balance of matrix synthesis and degradation, promoting extracellular matrix accumulation. This hypothesis agrees with the observation that mice lacking PAI-1 have increased pulmonary fibrosis when exposed to bleomycin.⁴⁰

In addition to genes relevant to extracellular matrix remodeling such as PAI-1 and tenascin-C, we describe in the present study the mechanical induction of VEGF and cyclooxygenase-1. VEGF, an angiogenic and growth factor, also has vasodilatory properties through a nitric oxide–dependent mechanism.^{41 42} Cyclooxgenase-1 is generally considered the "constitutive Cox" when compared with the highly "inducible Cox," cyclooxygenase-2. In endothelial cells, both of these enzymes may be inducible by shear stress,²⁷ although mechanical deformation predominantly induces cyclooxygenase-1 in VSMCs. It is possible that this apparent differential regulation is related to different functions of the cyclooxgenases in these cells. In vascular smooth muscle, induction of cyclooxygenases could potentially increase synthesis of prostacyclin, a vasodilator, or prostaglandin E2, an inhibitor of smooth muscle cell proliferation.⁴³

This transcriptional profile of VSMCs may underestimate the number of mechanically induced genes. First, we only studied two time points, and some changes may occur at different time points. Second, we used a threshold of 2.5-fold change in steady-state mRNA levels, and expression of some proteins may be importantly changed with smaller changes in steady-state mRNA. Because the 5000 genes explored in this DNA microarray represent <10% of expressed human genes, further study will be needed to define more completely the totality of mechanically regulated genes in the genome. It should also be noted that the artery is a mechanically anisotropic 3D structure, and in vitro cell monolayer deformation methods cannot completely simulate in vivo mechanical stimuli. Finally, VSMCs are heterogeneous both in vivo and in vitro,⁴⁴ and we cannot exclude the hypothesis that a specific subpopulation of cells accounts for most of the molecular responses reported in the present study.

VSMC responses to deformation may be relevant to atherosclerotic plaque stability and the development of hypertensive vascular disease. For example, PAI-1 expression may—like downregulation of matrix metalloproteinase 1—represent a response by VSMCs to strengthen the surrounding matrix. On one hand, expression of PAI-1 could strengthen vascular tissue and render a fibrous cap more resistant to rupture. On the other hand, the long-term effects could be detrimental, leading to excess matrix accumulation and vascular sclerosis. Further identification of other genes—particularly those without currently known functions—expressed in VSMCs subjected to mechanical deformation may provide new therapeutic targets for vascular disease.

	Acknowledgments

 
This work was supported in part by a grant-in-aid from the American Heart Association and a grant from the National Heart, Lung, and Blood Institute (HL-54759).

	Footnotes

 
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received August 2, 1999; accepted August 31, 1999.

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