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(Circulation Research. 1998;83:1027-1034.)
© 1998 American Heart Association, Inc.

Original Contributions

Differential Regulation of Protease Activated Receptor-1 and Tissue Plasminogen Activator Expression by Shear Stress in Vascular Smooth Muscle Cells

Maria Papadaki¹, Johannes Ruef¹, Kytai Truong Nguyen, Fengzhi Li, Cam Patterson, Suzanne G. Eskin, Larry V. McIntire, , Marschall S. Runge

From the Division of Cardiology, University of Texas Medical Branch (J.R., F.L., C.P., M.S.R.), Galveston; Institute of Biosciences and Bioengineering, Rice University (M.P., K.T.N., L.V.M.); and the Department of Cell Biology, Texas Biotechnology Corporation (S.G.E.), Houston, Tex.

Correspondence to Marschall S. Runge, MD, PhD, University of Texas Medical Branch, Division of Cardiology, 5.106 John Sealy Hospital, 301 University Blvd, Galveston, TX 77555-0553.

	Abstract

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Abstract—Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm²), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm²), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface–associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca²⁺] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Key Words: shear stress • protease activated receptor-1 • tissue plasminogen activator • smooth muscle cell

	Introduction

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The observed predisposition for atherosclerotic lesion formation at sites of disturbed blood flow¹ has led to extensive investigation of the role of hemodynamic forces in this process. Because endothelial cells (ECs) interface directly with fluid shear forces, the effects of shear stress on EC function and gene expression have been investigated extensively. Exposure of EC to shear stress in vitro produces marked alteration in cell morphology, consistent with the proposed in vivo role of shear stress.² Moreover, shear stress–mediated regulation of endothelial gene expression has been demonstrated for proteins, with key roles in maintaining homeostasis,^{3 4} cell migration,⁵ and cell growth.^{6 7}

It has been suggested recently that vascular smooth muscle cells (VSMCs) also may be responsive to shear stress.⁸ In vivo studies of VSMCs growth rates after balloon catheter injury have demonstrated an inverse correlation between growth rates and calculated shear stress forces.^{9 10} In vitro studies have confirmed the in vivo observations¹¹ and demonstrated that the synthesis of transforming growth factor-ß₁ (TGF-ß₁), tissue plasminogen activator (tPA),¹² heme oxygenase-1,¹³ nitric oxide,¹⁴ and prostaglandin¹⁵ increased under shear stress. Consistent with these observations, modeling studies supported the concept that VSMCs in the normal vasculature are exposed to significant shear stresses, on the order of 1 dyn/cm², because of interstitial fluid flow driven by transmural pressure gradients.¹⁶

On the basis of these findings, the present study was designed to determine whether shear stress also mediates gene expression in VSMCs, potentially leading to the pathological proliferation of VSMCs at sites of disturbed blood flow in the vasculature.¹ tPA and the human protease activated receptor-1 (PAR-1), 2 genes likely to be important in the maintenance of vascular integrity, were selected for the study. The PAR-1 gene was chosen because: (1) PAR-1 expression is known to increase dramatically after experimental injury in animal models, after percutaneous transluminal coronary angioplasty in patients, and in human atherosclerosis^{17 18 19} ; and (2) the known roles of thrombin on VSMCs function can be modulated by regulation of PAR-1 expression.^{20 21} tPA was selected for the study because: (1) the fibrinolytic activity of tPA limits thrombus formation in areas of vascular injury²² ; and (2) tPA gene expression in vascular EC is known to be regulated in response to shear stress in vitro.^{3 4} In addition, the effects of shear stress on the expression of urokinase-like plasminogen activator and the urokinase-like plasminogen activator receptor (uPAR) were examined.

PAR-1 is a 7-transmembrane G protein–linked receptor that is proteolytically and irreversibly activated by thrombin.^{23 24} Activation of PAR-1 in VSMCs reportedly mobilizes calcium and induces a cascade of growth-related signaling events.² PAR-1 is rapidly desensitized after activation, followed by internalization and trafficking in the lysosomes. Recovery of thrombin responsiveness is due to replenishment of the cell surface with new receptors.²⁶ The presence of a protected intracellular PAR-1 pool in many cell types (such as ECs, smooth muscle cells, and fibroblasts) with at least as many receptors as are present initially on the cell surface provides a mechanism for quick recovery after activation, independent of protein synthesis.^{17 24 26 27} In the absence of an agonist, PAR-1 cycles at very low rates between the cell surface and the intracellular pool,²⁴ and the time required for new PAR-1 synthesis after cleavage by thrombin is 24 hours.²⁸

tPA forms a ternary complex with fibrin and plasminogen and catalyzes the conversion of inactive plasminogen to plasmin.²⁹ In addition to its role in fibrinolysis, plasmin degrades extracellular matrix components and facilitates cell migration in many cell types.^{30 31}

The data presented here indicate that high shear stress downregulates human PAR-1 expression, whereas low shear stress upregulates it, consistent with the known variation in human PAR-1 expression in vascular injury and atherosclerosis.³² In contrast, tPA expression increases in areas of high shear stress and decreases in areas of low shear stress, where thrombus formation is most likely.

	Materials and Methods

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Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co.

Cell Culture and Shear Stress Experiments
HASMCs were obtained from the abdominal aorta of a 9-year-old kidney transplant donor.¹¹ The culture medium was DMEM supplemented with 20% heat-inactivated FBS (HyClone Laboratories, Logan, Utah), 2 mmol/L L-glutamine, 200 U/mL penicillin, and 100 µg/mL streptomycin (Gibco BRL). HASMCs were used at passages P3 to P10. Rat aortic smooth muscle cells (RASMCs) were isolated as previously described³³ and grown in DMEM supplemented with 10% heat-inactivated FBS, 200 U/mL penicillin, and 100 mg/mL streptomycin. P3-P8 RASMCs were used in the experiments. Cells were seeded on glass slides (75x38 mm; Fisherbrand, Fisher Scientific), coated with 1 µg/cm² human plasma fibronectin (Collaborative Biomedical Products) and placed in complete (serum-containing) medium. On reaching 95% confluence, HASMCs or RASMCs cultures were either maintained in stationary conditions or exposed to different levels of shear stress using parallel plate chambers connected to recirculating flow loops.^{3 11 34} A well-defined shear stress, created by gravity-driven flow through the chamber, was established by choosing a predetermined vertical distance between the upper and lower fluid reservoir. The flow apparatus was assembled in a laminar flow hood, filled with 15 to 20 mL of complete medium, placed in a 37°C humidified room, and gassed with a mixture of 95% air and 5% CO₂. Shear stress experiments were performed for 2, 4, 6, 12, and 24 hours.

RNase Protection Assay
After exposure to shear stress, HASMCs were washed in PBS, and total RNA was isolated using RNAzolB (Tel-Test, Inc). Target human cDNA subclones were prepared by generating PCR fragments from full-length cDNA. In brief, a 223-bp tPA fragment and a 177-bp uPA fragment were cloned into pGEM4Z (Promega) to create plasmids pGTPA and pGUPA. A 510-bp uPAR cDNA fragment was cloned into PCRII (Invitrogen) to create pUPAR. A 222-bp human PAR-1 cDNA fragment was cloned into pAlter-1 (Promega). pTR-1GAPDH (Ambion, Inc) contains a 149-bp fragment of glyceraldehyde phosphate dehydrogenase (GAPDH), which was used as an internal control. To prepare antisense cRNA riboprobe templates, all plasmids were linearized and transcribed by SP6 or T7 RNA polymerase using an in vitro transcription kit, Maxi-script (Ambion), following the manufacturer's guidelines with minor modifications: 100 mCi of [³²P]UTP at 800 Ci/mmol (Amersham Corp) and 0 to 6 mL of 0.05 mmol/L UTP in 20 mL total volume was used. After transcription for 60 minutes at 37°C, the RNA templates were digested with 2 U RNase-free DNase I for 25 minutes at 37°C, and full-length antisense RNA probes were recovered from 8 mol/L urea/5% acrylamide-denaturing PAGE gel with elution buffer (0.5 mol/L ammonium acetate, 1 mmol/L EDTA, 0.2% SDS) at 37°C overnight. RNase protection assays were performed using a RNase protection assay kit, RPAII (Ambion): 3 µg total RNA was incubated with 106 cpm [³²P]-labeled antisense cRNA probes in 20 mL hybridization buffer (80% deionized formamide, 100 mmol/L sodium citrate, pH 6.4, 300 mmol/L sodium acetate, pH 6.4, 1 mmol/L EDTA), heated to 95°C for 5 minutes, and hybridized overnight at 42°C. RNase digestion buffer (200 mL) with 2.5 U/mL of RNase A and 100 U/mL of RNase T were added to the hybridization mixtures and incubated for 20 minutes at 37°C. RNase inactivation/precipitation mixture (300 mL) was added. After precipitation and centrifugation, the protected fragments were resuspended in 6 mL gel-loading buffer (8% sucrose, 0.025% bromophenol blue, 0.025% xylene cyanol) and separated on a 5% nondenaturing PAGE gel. The dried gel was exposed to autoradiography.

mRNA Stability Experiments
Control and shear-stressed (25 dyn/cm² for 12 hours) HASMCs were exposed to 4 µg/mL actinomycin-D for 0, 2, 4, and 6 hours to determine the half-life of human PAR-1 mRNA. Human PAR-1 mRNA levels were quantified by Northern blot analysis and normalized to GAPDH.

Northern Blot Analysis
Total RNA was obtained from sheared or control HASMCs or RASMCs using the fast-RNA isolation kit (BIO101), according to the manufacturer's instructions. Total RNA (5 to 7 µg) was fractionated in a 1.3% formaldehyde agarose gel, transferred to a nitrocellulose membrane, and immobilized by UV cross-linking (Stratagene). cDNA probes for human and rat PAR-1 and for GAPDH were labeled with [-³²P]dCTP (DuPont NEN), using random primers. Blots were prehybridized for 20 minutes and then hybridized with labeled cDNA for 1 hour at 68°C in QuikHyb solution (Stratagene). After hybridization, blots were washed twice in 2xSSC/0.1% SDS at room temperature and then washed twice in 0.2xSSC/0.1% SDS buffer at 55°C. The blots were exposed to Biomax MS film (Kodak) at -80°C for 16 to 20 hours. Appropriate exposures of the resulting autoradiographs were subjected to scanning densitometry, and signals were analyzed using NIH Image software.

Flow Cytometry
For flow cytometry experiments, RASMCs were used on the basis of the previous species-specific characterization of the polyclonal rat PAR-1 antibody, TR-R9.¹⁷ Shear stress experiments were performed for 24 and 48 hours. Because of the presence of intracellular PAR-1 pools in VSMCs, experiments were performed with -thrombin preincubation to stimulate PAR-1 turnover. After preincubation of RASMCs with -thrombin (4 U/mL) for 15 minutes, cells were rinsed twice with serum-free medium and then exposed to 25 dyn/cm² for 24 hours. At the end of each experiment, slides were washed 3 times with PBS without Mg²⁺ and Ca²⁺ (Gibco BRL), whereafter 1.5 mL of 40-mmol/L EDTA was added to each slide for 10 minutes; finally, cells were scraped into microcentrifuge tubes. After centrifugation for 10 minutes at 3000 rpm, cell numbers were determined by Coulter counter, and the pellet was resuspended in 500 µL PBS/10⁶ cells. The polyclonal antibody TR-R9 (20 µg/mL) was added to the counted cells and incubated for 30 minutes at 4°C, thus achieving the same antibody-cell ratios for all samples. Samples without primary antibody served as negative controls. After centrifugation for 4 minutes at 3000 rpm, the pellet was resuspended in 500 µL PBS/10⁶ cells, and 20 µg/mL of FITC-labeled goat anti-rabbit IgG secondary antibody (Vector Laboratories) was added. After incubation for 30 minutes, the cells were centrifuged and resuspended in PBS/1% formaldehyde. Flow cytometry was performed using a fluorescence analyzer and cell sorter (Becton Dickinson). From each sample, 6000 cells were analyzed, and the results were expressed as geometric mean fluorescence.

tPA ELISA
Detection of tPA in the cell culture medium was conducted using an ELISA assay (Diagnostica Stago). In brief, conditioned media from stationary control or shear-stressed cultures were collected, coated on 96-well plates, and incubated for 2 hours at room temperature. After repeated washes, the wells were incubated with a peroxidase-coupled anti-human tPA antibody for 2 hours at room temperature. The color reaction was performed with an ortho-phenylenediamine substrate and measured at 492 nm using a microplate reader (Bio-Rad).

[Ca²⁺] Measurements in HASMCs
HASMCs were seeded at a density of 4x10⁴ cells/cm² on sterile glass coverslips (24x50 mm, No. 1 thickness; Sigma) coated with 2 µg/cm² human plasma fibronectin. Cultures either were exposed to 25 dyn/cm² for 24 hours or maintained under stationary control conditions. After 2 washes with HBSS (Gibco, BRL), cells were loaded with Fura-2-AM (Molecular Probes; 1 µmol/L final concentration in HBSS) and Pluronic (0.1% of final volume). HASMCs were incubated for 30 minutes at 37°C, washed twice in HBSS, and incubated for an additional 30 minutes to allow for the cellular esterases to hydrolyze Fura-2-AM to its nonpermeable fluorescent form, Fura-2. The fluorescence ratio imaging system used for Fura-2 measurements have been described in detail elsewhere.³⁵ Briefly, at each time point (time intervals, 20 to 400 s), 2 images were acquired at the 2 excitation wavelengths (340 and 380 nm), the background was subtracted, and ratio (340 nm/380 nm) was calculated. The ratio values corresponded to intracellular changes in [Ca²⁺]. -Thrombin (5 U/mL) was added to both control and shear-stressed cultures at 100 s after the onset of flow.

Mitogenic Assays
Number of cells from HASMCs exposed to 25 dyn/cm² for 24 hours and matched stationary control cells was measured by Coulter Counter 24 hours after incubation in complete media in the presence or absence of -thrombin. For thymidine incorporation experiments, control and sheared HASMCs treated as above were labeled with 2 µCi/mL [³H]thymidine in the presence or absence of -thrombin (5 U/mL) and incubated at 37°C for 24 hours. At the end of the incubation period, cells were harvested, and the relative [³H]thymidine incorporation was determined using a liquid scintillation counter (United Technologies Packard).

Statistical Analysis
Results are expressed as mean±SEM. When data from >2 groups were compared, 2-way ANOVA was used, followed by Fisher least significance difference post-hoc test. To indicate significance between normalized treatment and control groups, a 2-tailed Student t test for paired samples was used. To determine trends for normalized tPA protein and [Ca²⁺] mobilization over time, a univariate repeated measures ANOVA was used, and when overall significance was indicated, mean/regression coefficient comparisons were performed to test significance in within-subject groups. Two-tailed Student t tests for paired samples were used to compare the geometric means of cell surface rat PAR-1 protein fluorescence units between stationary control and flow groups. Differences were considered significant when P<0.05. All calculations were performed with SuperANOVA 1.11 (Macintosh).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Shear Stress–Induced Changes in PAR-1 and tPA mRNA Expression
As a first step in the systematic examination of effects of shear stress on VSMCs, we examined the role of shear stress on regulation of mRNA species for the following genes: PAR-1, tPA, uPA, and uPAR. Cultured HASMCs were exposed to stationary conditions or to shear stress for defined time intervals (2, 4, 6, 12, and 24 hours), and mRNA expression was measured by RNase protection assays. Levels of shear stress were chosen in the physiological range (5, 15, or 25 dyn/cm²).^{36 37} Figure 1 shows autoradiographs of representative RNase protection assays. At 6 hours, human PAR-1 mRNA expression was decreased under high–shear stress conditions (25 dyn/cm²) in comparison to stationary conditions or low shear (Figure 1A). Similar effects were observed at 4, 12, and 24 hours (Figure 1B). In contrast, after correction with GAPDH, no consistent changes were observed for uPA and uPAR. Regulation of tPA by shear stress was also observed in HASMCs (Figure 2). Under low-shear conditions (5 dyn/cm²) for 24 hours, tPA mRNA expression was decreased in comparison to static conditions or to 15 dyn/cm². Conversely, tPA mRNA expression was upregulated by 25 dyn/cm².

View larger version (88K): [in this window] [in a new window]   Figure 1. Shear stress regulates PAR-1 mRNA expression in HASMCs. Autoradiographs of representative RNase protection assays with mRNA from shear-stressed HASMCs. The protected bands for uPAR, PAR-1 (human thrombin receptor [HTR]), uPA, and GAPDH mRNA levels are indicated. A, Representative stationary control and shear-stressed samples from a 6-hour experiment. B, Experiments in which HASMCs were exposed to 25 dyn/cm2 for different time periods (4, 12, and 24 hours).

View larger version (53K): [in this window] [in a new window]   Figure 2. tPA mRNA is expressed in response to shear stress in a pattern opposite of PAR-1. Autoradiographs of a RNase protection assay for tPA and GAPDH from stationary control HASMCs and from HASMCs exposed to 5, 15, and 25 dyn/cm2 for 24 hours.

To quantify the effects of shear stress more accurately, results from multiple experiments were corrected densitometrically for loading and expressed as a ratio of flow-to-stationary conditions (Figure 3). Because consistent differences were not noted with uPA and uPAR, only the results for PAR-1 and tPA are shown. PAR-1 mRNA expression was transiently upregulated by low shear (5 dyn/cm²) compared with stationary conditions, being increased 2.1-fold at 2 hours before returning to baseline (Figure 3A). Under 25 dyn/cm², PAR-1 expression was significantly decreased at all examined time points (P<0.05). The opposite pattern was seen for tPA mRNA expression (Figure 3B). A trend toward increased tPA mRNA expression under 25 dyn/cm² was seen at 24 hours compared with 5 and 15 dyn/cm² (P<0.05). Conversely, low-shear conditions decreased tPA mRNA levels, with a significant decrease to 8.4% of stationary conditions seen at 12 hours (P<0.05). Taken together, these results indicate that shear stress differentially regulates PAR-1 and tPA expression in VSMCs. Under low-shear conditions, similar to those seen in atherosclerosis-prone arterial segments, tPA mRNA expression is decreased and PAR-1 expression is increased, whereas under high–shear stress conditions, the converse is true.

View larger version (30K): [in this window] [in a new window]   Figure 3. Time course of shear stress–induced gene expression in HASMCs. HASMCs were exposed to different levels of shear stress for different times and the mRNA levels of (A) PAR-1 and (B) tPA were assessed by RNase protection assays. Autoradiographs were analyzed by densitometry, and bands were normalized to GAPDH. Bars represent fold differences compared with stationary control cells. Results are shown as mean±SEM. For PAR-1 experiments, n=3 to 5, and for tPA experiments, n=3. A, Significantly different from: *stationary controls; 5 and 15 dyn/cm2 at 2 hours; 5 dyn/cm2; §5 dyn/cm2 at 4, 6, 12, and 24 hours; and ¶15 dyn/cm2 at 2, 4, 6, and 24 hours (P<0.05). B, Significantly different from *stationary controls and 5 and 15 dyn/cm2 at 24 hours (P<0.05).

mRNA Stability
To determine whether shear stress influenced human PAR-1 mRNA stability, the half-life of human PAR-1 mRNA in the presence or absence of shear stress was evaluated. PAR-1 mRNA levels from control and shear-stressed HASMCs were quantified by Northern blot analysis at 0, 2, 4, and 6 hours of actinomycin-D treatment, and PAR-1 mRNA levels were corrected to the level of GAPDH mRNA by densitometric analysis (Figure 4). Actinomycin-D treatment did not influence the stability of the GAPDH mRNA (results not shown). As illustrated in Figure 4, there was a gradual decline in normalized PAR-1 mRNA levels with actinomycin-D treatment. The half-life of PAR-1 mRNA in actinomycin-D–treated stationary control HASMCs was 4.8±0.7 hours, and this was unaffected by exposure to shear stress (4.4±0.8 hours), indicating no significant effect of shear stress on human PAR-1 mRNA stability.

View larger version (16K): [in this window] [in a new window]   Figure 4. Exposure to shear stress does not affect human PAR-1 mRNA half-life. Shear-stressed HASMCs (25 dyn/cm2 for 12 hours) were incubated with 4 µg/mL actinomycin-D for 0, 2, 4, and 6 hours. The PAR-1 mRNA levels were quantified by Northern blot analysis, and the band intensity of autoradiographs was quantified by scanning densitometry with results normalized to GAPDH. The results are presented as percentage of the PAR-1 mRNA present at the time of actinomycin-D addition (n=4); slope for stationary control HASMCs was 4.83±0.67% decrease in PAR-1 mRNA per hour, whereas for sheared stressed HASMCs, it was 4.41±0.8% decrease in PAR-1 mRNA per hour.

Effects of Shear Stress on Cell Surface–Associated PAR-1 Expression
To confirm that the decrease in PAR-1 mRNA induced by shear stress was accompanied by a decrease in functional cell surface–associated PAR-1 protein, we measured immunoreactive PAR-1 through flow cytometry. Because of the species specificity of the available antibody, we used RASMCs for these experiments. RASMCs exposed to 25 dyn/cm² for 24 hours showed a 3-fold downregulation of PAR-1 mRNA compared with control cultures (data not shown), indicating that effects of shear stress on PAR-1 expression are not species-specific. In the absence of -thrombin pretreatment, exposure of RASMCs to shear stress (25 dyn/cm²) for 24 hours had no effect on cell surface PAR-1 expression (Figure 5), whereas exposure to shear stress for 48 hours modestly reduced PAR-1 expression by 25% in comparison to stationary conditions (results not shown).

View larger version (44K): [in this window] [in a new window]   Figure 5. Downregulation of membrane-associated PAR-1 protein by shear stress. RASMCs were exposed to shear stress (25 dyn/cm2) for 24 hours without pretreatment and for 24 hours after pretreatment (15 minutes) with -thrombin (4 U/mL), resulting in intracellular rat PAR-1 turnover. Cells were analyzed with flow cytometry using a polyclonal rat PAR-1 antibody. Equal numbers of cells (6000) were analyzed with flow cytometry using a polyclonal rat PAR-1 antibody. Bars represent percentage differences in membrane-associated PAR-1 expression between stationary control and shear-stressed cultures. Results are shown as mean±SEM (n=3). *Significantly different from cultures exposed to 25 dyn/cm2 for 24 hours in the absence of -thrombin.

Because PAR-1 exhibits a complex mechanism of internalization, pooling, and restoration,^{26 27} we repeated the same experiments with brief -thrombin pretreatment to deplete intracellular receptor pools that could replace surface receptors and therefore mask shear stress–induced down-regulation. A brief pretreatment of 15 minutes was chosen, because -thrombin downregulates its own receptor after longer exposure times.³⁸ The -thrombin pretreatment–enhanced cell surface–associated PAR-1 downregulation measured through flow cytometry in shear-stressed RASMCs cultures after 24 hours (44.7% decrease, represented by 42.7±6.6 fluorescence units for cells under stationary conditions versus 23.6±3.7 fluorescence units for shear stress–exposed cells). There was a significant difference compared with results obtained without pretreatment with -thrombin (P<0.05; Figure 5). These values were obtained after subtraction of the negative control values without primary antibody (4.98±0.01 fluorescence units). These experiments indicate that: (1) downregulation of PAR-1 mRNA by high levels of shear stress (25 dyn/cm²) is accompanied by a delayed downregulation of cell surface–associated PAR-1; and (2) this effect is probably a reflection of de novo PAR-1 protein production, because intracellular pools of PAR-1 can partly blunt the effect of shear stress.

Effects of Shear Stress on tPA Secretion
We measured tPA protein expression by ELISA in conditioned media of HASMCs exposed to stationary conditions or high levels of shear stress (25 dyn/cm²) to determine whether the effects of shear stress on tPA mRNA resulted in similar increases in tPA secretion (Figure 6). After 12 and 24 hours of exposure to high–shear stress conditions (25 dyn/cm²), tPA secretion was increased significantly in the conditioned media of HASMCs compared with cells grown in stationary conditions (3.6-fold and 4.0-fold, respectively; P<0.05; Figure 5). Although exposure to 5 dyn/cm² shear stress caused downregulation of tPA mRNA, no effects were observed on the secretory tPA protein at any examined time point.

View larger version (19K): [in this window] [in a new window]   Figure 6. Increased tPA secretion in conditioned media in response to shear stress. tPA levels in the conditioned media of HASMCs were measured by ELISA. Results are shown as mean±SEM (n=3). Significantly different from *stationary controls and 25 dyn/cm2 at 2, 4, and 6 hours (P<0.05).

Functional Studies
To investigate the functional consequences of shear stress–induced downregulation of human PAR-1, we studied the effects of -thrombin on [Ca²⁺] mobilization (Figure 7A) and cell proliferation (Figure 7B) in control and shear-stressed HASMCs. The thrombin concentration used (5 U/mL) has been shown to induce maximal increases in cytosolic [Ca²⁺] and mitogenic responses.^{19 25}

View larger version (29K): [in this window] [in a new window]   Figure 7. Effects of -thrombin on the rise in [Ca2+] and cell proliferation in shear-stressed HASMCs. HASMCs were exposed to 25 dyn/cm2 for 24 hours, and Ca2+ release and cell proliferation by -thrombin were compared with untreated cells. A, Shear stress attenuated Ca2+ responses in response to -thrombin in HASMCs. Data are presented as ratio of Fura-2 (340 nm/380 nm)t=i at each time point to Fura-2 at time zero (340 nm/380 nm)t=0, (n>20). B, Shear stress inhibited cell proliferation in HASMCs in response to -thrombin. Results are shown as mean±SEM (n=4). Significantly different from *respective unstimulated controls and thrombin-stimulated shear-stressed cultures (P<0.05).

The effects of thrombin on [Ca²⁺] were studied using cells loaded with the Ca²⁺-sensitive dye, Fura-2 (Figure 7A). Thrombin induced a rapid increase in [Ca²⁺] in both control and shear-stressed HASMCs, which reached a peak within 50 to 100 s after addition, followed by a rapid decline to resting levels. Exposure to 25 dyn/cm² for 24 hours resulted in a significant decrease in Ca²⁺ mobilization in response to thrombin compared with the response of stationary control cells, at 160 and 180 s (P<0.02).

Stimulation with 5 U/mL -thrombin induced a significant increase in cell proliferation in HASMCs (P<0.03; Figure 7B). The percentage increase in number of cells after addition of thrombin was lower in cells exposed to 25 dyn/cm² for 24 hours compared with stationary control cells after thrombin treatment, but this trend was not statistically significant. In the absence of thrombin stimulation, cell numbers were not significantly different between HASMCs exposed to 25 dyn/cm² for 24 hours and unstimulated controls, indicating that the previously reported shear stress–induced growth inhibition was a reversible event.¹¹ Thrombin-induced increases in number of cells were accompanied by similar increases in thymidine incorporation (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular homeostasis is maintained by wound healing, vascular tone, immunological responses, hemostasis and fibrinolysis, changes in cell shape, and cell-cell interactions. After injury or immunological activation, this equilibrium can be altered by changes in gene expression, ultimately resulting in cell proliferation and contributing to pathogenic processes such as atherogenesis. On the basis of the observation that lesion-prone areas of the vasculature are correlated with sites of low-wall shear stress and often-disturbed blood flow, it has been suggested that hemodynamic forces, such as fluid shear stress, may also play a role in vascular homeostasis and in the response of vascular cells to injury.^{39 40}

In contrast to EC, VSMCs normally are not exposed directly to blood flow in vivo, except after endothelial denudation by mechanical means, such as balloon angioplasty. However, it is likely, on the basis of recent modeling studies, that under physiological conditions, the transmural interstitial fluid flow imposes shear forces on VSMCs of a magnitude that is already known to affect the function of EC.¹⁶ Because VSMCs account for the majority of proliferating cells in atherosclerotic lesion formation⁴¹ and after balloon angioplasty,⁴² the effects of shear stress on regulation of molecules are important for growth, migration, and thrombosis are likely of physiological importance. Recent in vitro studies demonstrated that shear stress–induced upregulation of TGF-ß₁ mRNA and that protein was responsible partially for the growth inhibition in VSMCs.¹² In addition, it has been shown recently that shear stress stimulated the production of nitric oxide, heme oxygenase-1, and prostaglandins in VSMCs, molecules that play major regulatory roles in vascular wall homeostasis.^{13 14 15}

The findings that tPA mRNA decreased and human PAR-1 mRNA increased under low shear stresses are consistent with the known predilection of low shear stress and/or disturbed flow areas within the vasculature result in thrombus formation and vascular cell proliferation.^{43 44 45 46} The observed up-regulation of tPA and downregulation of PAR-1 mRNA and protein by high shear stress are also consistent with the relative paucity of lesions in areas of high shear stress.⁴⁷ The fact that tPA expression in response to shear stress is qualitatively similar between EC and VSMCs, as reported here, suggests that at least some of the same mechanisms that regulate gene expression in EC in response to shear stress also exist in VSMCs. In agreement with our findings on tPA, Ueba et al¹² recently have shown that exposure to 28 dyn/cm² induced 2- to 5-fold increases in tPA mRNA levels in human umbilical artery smooth muscle cells.⁹ Increases in tPA have been related to the release of an active form of TGF-ß₁ under shear stress, because TGF-ß₁ is known to be activated by plasmin in VSMCs.¹²

The lack of any effect of shear stress on the stability of human PAR-1 mRNA indicates that the suppression of PAR-1 probably is due to a transcriptional mechanism. Preliminary data from our laboratories, using a panel of human PAR-1 promoter-luciferase constructs transfected into RASMCs, provided evidence that a shear stress–sensitive element is present in the PAR-1 promoter between –300 and –160 bp upstream of the transcription initiation site.⁴⁸ Although the human PAR-1 promoter contains consensus binding sequences for 2 shear stress–sensitive elements (-3.76 and -2.93 kb), neither is present between -300 and -160 bp, indicating that unique transcriptional mechanisms are likely to be involved in shear stress–mediated PAR-1 downregulation.

HASMCs function in response to thrombin stimulation was measured to explore the physiological consequences of shear stress–induced downregulation of PAR-1, because most of the effects of thrombin on VSMCs are mediated through a proteolytically activated receptor.²¹ Exposure to 25 dyn/cm² shear stress for 24 hours significantly reduced the rise in [Ca²⁺] in response to thrombin and resulted in a slight decrease in cell proliferation and DNA synthesis. These findings indicate that the decrease in PAR-1 gene and protein levels was coupled to an attenuation of thrombin-induced VSMCs activation responses.

In summary, the data reported here indicate that HASMCs and RASMCs are responsive to shear stress in vitro. The data are consistent with the hypothesis that VSMCs, such as vascular EC, require exposure to certain levels of mechanical stimulation by flow or strain for vessel wall homeostasis, as expressed by a passivity relative to proliferation and compatibility relative to homeostatic functions. Additionally, linking molecular mechanisms of atherogenesis and restenosis to blood fluid mechanics provides future directions for in vivo shear stress assessment⁴⁹ and the development of novel therapeutic approaches based on these findings.

	Acknowledgments

 
This work was supported in part by NIH grants HL18672 and NS23326, NASA grant NAGW-5007, Welch Foundation grant C-0938, and TATP grant 003604 (L.V.M.); Texas Biotechnology Corporation, the National Heart, Lung, and Blood Institute grants HL48667 and HL57352 (M.S.R) and HL03658 (C.P.); and scholarship Ru 620/1-1 from the German Research Foundation DFG (J.R.). The authors are grateful to Larry Sperling, Christoph Bode, Carol Ballinger, Georgios Stamatas, and Susan Peter for help in this work.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received June 10, 1998; accepted August 17, 1998.

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