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(Circulation. 2000;102:225.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Shear Stress Downregulation of Platelet-Derived Growth Factor Receptor-ß and Matrix Metalloprotease-2 Is Associated With Inhibition of Smooth Muscle Cell Invasion and Migration

Roberta Palumbo, PhD; Carlo Gaetano, MD; Guido Melillo, MD; Elena Toschi, PhD; Andrea Remuzzi, EngD; Maurizio C. Capogrossi, MD

From the Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome (R.P., C.G., G.M., E.T., M.C.C.), and the Istituto di Ricerche Farmacologiche Mario Negri, Bergamo (A.R.), Italy.

Correspondence to Dr Maurizio C. Capogrossi, Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, Via dei Monti di Creta, 104, 00167 Rome, Italy. E-mail capogrossi{at}idi.it

	Abstract

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Background—After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study.

Methods and Results—Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm² for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB–directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (P<0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-ß (PDGF-Rß) mRNA and protein (P<0.05), and the SS-dependent decrease in PDGF-BB–directed cell migration was rescued by overexpressing PDGF-Rß.

Conclusions—SS inhibits SMC migration and invasion via diminished PDGF-Rß expression. This effect of SS is associated with decreased MMP-2 secretion and MT-MMP downregulation.

Key Words: stress • muscle, smooth • metalloproteinases • platelet-derived factors • restenosis

	Introduction

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Under physiological conditions, smooth muscle cells (SMCs) are localized predominantly in the medial layer of blood vessels and are not in contact with the bloodstream. After vascular injury, however, SMCs may be exposed to hemodynamic shear stress, and in balloon-injured carotid arteries, low flow enhanced but high flow inhibited neointima accumulation.^{1 2} Furthermore, intimal thickening in injured carotids exposed to low flow was associated with increased matrix metalloprotease 2 (MMP-2),² a member of a family of enzymes that digest extracellular matrix (ECM) components³ and play a key role in neointima accumulation after vascular injury.^{4 5 6} MMP-2 is secreted in a latent form, and membrane-type MMP (MT-MMP or MMP-14), which is an integral membrane protein rather than a secreted protein, is responsible for the activation of pro-MMP-2 on the cell surface.⁷ Furthermore, MMP-2 binding to the integrin _vß₃ on the cell surface leads to MMP-2 activation.⁸ All active MMPs are inhibited by a family of natural inhibitors called tissue inhibitors of matrix metalloprotease (TIMPs), which form a 1-to-1 complex with MMPs.³

In addition to MMPs, vascular injury modulates SMC expression of platelet-derived growth factor (PDGF) and of its receptors⁹ ; these effects are time-dependent, differ between SMCs from the neointima and the media, and are associated with functional differences in SMC responses to PDGF.^{10 11 12 13} PDGF ligands consist of homodimers or heterodimers of A and B chains, and there are 3 PDGF isoforms: AA, AB, and BB. These PDGF isoforms bind PDGF- or -ß receptor (PDGF-R) subunits with different affinities and induce their dimerization. PDGF-R can bind either the A or B chain, whereas PDGF-Rß binds only B chain with high affinity.⁹ The key role of PDGF in neointima accumulation after vascular injury has been shown by in vivo experiments. Exogenous PDGF-BB enhanced intimal thickening,¹⁴ whereas antibodies to PDGF inhibited SMC accumulation after angioplasty,^{15 16} and similar results were obtained with an antisense oligonucleotide against the PDGF-Rß subunit¹⁷ and a specific receptor tyrosine kinase inhibitor.¹⁸ Furthermore, it has been shown that endothelial cell PDGF expression was modulated by shear stress both in vitro¹⁹ and in vivo.²⁰ In light of the importance of shear stress in vascular wall remodeling after injury, the objective of the present study was to determine in vitro whether, and eventually by what mechanism, shear stress modulates SMC migration and invasion. The results show that laminar shear stress inhibited both SMC migration and invasion through a reconstituted ECM layer. These responses were associated with diminished MMP-2 secretion, as well as reduced MT-MMP and PDGF-Rß mRNA and protein. The shear-dependent decrease in cell migration was abolished in SMCs forced to overexpress PDGF-Rß.

	Methods

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SMC Isolation
Bovine aorta SMCs were isolated as described.²¹ Cells between passages 3 and 12 were used in all experiments, and positive staining for anti-SMC -actin (Dako A/S) demonstrated >95% SMC population purity.

Shear Stress Apparatus
Near-confluent SMC monolayers in DMEM with 10% FCS were exposed to uniform fluid shear stress of 12 dyne/cm² for either 3 (SS3) or 15 (SS15) hours in a cone-and-plate apparatus²² maintained at 37°C in humidified air with 5% CO₂. Control SMCs (C3 and C15) were kept under static conditions. Cells were detached with trypsin and used in migration/invasion assays; alternatively, cells were lysed and used for either Northern or Western blots.

Invasion and Migration Assay
Previous studies have validated the use of a modified Boyden chamber assay to differentiate between cell migration and cell invasion, demonstrating the key role of MMPs in these processes.^{23 24} Polycarbonate filters (8-µm pores; Nucleopore Costar Scientific Corp) were coated with either 5 mg/L gelatin (Sigma Chemical Co) or reconstituted basement membrane proteins (Matrigel; Collaborative Research) for migration and invasion assays, respectively.

PDGF-BB, 1 to 10 ng/mL (Collaborative Research), was the chemoattractant. After 4 hours of incubation, cells on the filter were fixed with ethanol and stained with toluidine. Cells from 5 randomly chosen high-power (x400) fields on the lower side of the filter were counted, and all experiments were run in triplicate.

Gelatin Zymography
SDS polyacrylamide 10% gels containing gelatin (1 mg/mL) were used to identify proteins with gelatinolytic activity.²⁴ Latent collagenases were not activated with aminophenyl mercuric acetate, and by gelatin zymography, only zymogen forms of MMPs were identified. In the presence of SDS, otherwise inactive forms can lyse the substrate in the gel because of detergent-induced conformational change of the enzyme.

ELISA Test
TIMP-2 in cell culture supernatant was measured by ELISA according to the manufacturer’s instructions (Amersham).

Northern Blot Analysis
Total RNA was isolated from SMCs and analyzed by gel electrophoresis.⁵ The cDNA fragment of human MMP-2, MMP-9 (kindly provided by W.G. Stetler-Stevenson, Laboratory of Pathology, National Institutes of Health, Bethesda, Md), MT-MMP (kindly provided by M.T. Crow), and PDGF-Rß (kindly provided by C.H. Heldin, Biomedical Center, Ludwig Institute for Cancer Research, Uppasala, Sweden) were labeled with [-³²P]dCTP by the random-primer method.

Either the housekeeping GAPDH gene or the 18S rRNA was used for normalization.

Flow Cytometry
SMCs exposed to laminar flow and control cells were analyzed by flow cytometry (fluorescence-activated cell sorter [FACS], Becton Dickinson).

Briefly, 2x10⁵ cells/mL were incubated with primary antibodies against _vß₃ and _vß₅ integrins, and ₁-, ₂-, and ß₁-integrin subunits (Chemicon) and FITC-conjugated secondary antibodies (Dako A/S) were fixed in 1% paraformaldehyde and analyzed.

Western Blot Analysis
Activated MMP-2 and its zymogen form as well as PDGF-Rß and MT-MMP were detected in SMC lysates by Western blot analysis. Conditioned media (CM) were examined for the presence of MMP-2. Proteins (100 µg) were separated by 6% SDS-PAGE gel under reducing conditions and blotted. The membranes were blocked in PBS/5% nonfat dry milk, washed, and incubated with the following primary antibodies: rabbit polyclonal anti-human PDGF-Rß IgG antibody (Santa Cruz Inc), mouse monoclonal anti-human MT-MMP IgG antibody (Oncogene Research Products), and mouse monoclonal anti-human MMP-2 IgG antibody (Oncogene Research Products). Horseradish peroxidase–conjugated anti-rabbit (PDGF-Rß) and anti-mouse (MT-MMP and MMP-2) secondary antibodies were used, and chemiluminescence was detected according to the manufacturer’s specifications (Amersham).

Plasmids and Transfection Methodology
These experiments were performed to examine the effect of PDGF-Rß overexpression on SMC function. CMV.PDGF-Rß (kindly provided by C.H. Heldin) or equal amounts of pCDNA3 (Invitrogen) empty vector were cotransfected with either CMV.LacZ or pEGFP-N1 (Clontech) reporter vectors (3:1 ratio). SMCs (1.7x10⁶ cells/100-mm-diameter dish) were transfected with lipofectamine plus reagent (Gibco BRL). After 48 hours, SMCs were exposed to shear stress for 15 hours and studied in migration and invasion assays. Cells were also either assayed for ß-gal activity^{25 26} or examined for GFP fluorescence by UV light microscopy.²⁷ By this approach, it was possible to normalize results for ß-gal units or to examine the effect of shear stress only on GFP-positive cells. Cotransfection with 2 independent vectors results in the internalization of both plasmids by the same cell²⁸ ; therefore, these experiments made it possible to determine the effect of shear stress on PDGF-Rß–transfected cells, overcoming the potential limitations of low transfection efficiency, ie, transfection of 5% to 10% of the total population.

Statistical Analysis
Continuous variables were analyzed by Student’s t test and 1-way ANOVA. Post hoc tests according to the Student-Newman-Keuls method were used when the ANOVA P value indicated a statistically significant difference among groups. Data are expressed as mean±SD. A value of P0.05 was deemed statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Shear Stress on SMC Migration and Invasion
Laminar shear stress of 12 dyne/cm² for 3 hours did not affect SMC migration and invasion (Figure 1). In contrast, 15 hours of exposure to shear stress inhibited both migration (23±1 versus 33±3 cells/field for SS15 and C15 cells, respectively; 31±3% inhibition; P<0.0001) and invasion (10±2 versus 22±3 cells/field for SS15 and C15 cells, respectively; 54±9.9% inhibition; P<0.0001) (Figure 1); the inhibitory effect was more pronounced on invasion than on migration (P<0.05). In the absence of PDGF-BB as a chemoattractant, there was no effect of shear stress on SMC migration (cells/field: SS3 14±3, C3 16±5, SS15 7±1, C15 8±1) and invasion (cells/field: SS3 27±9, C3 27±10, SS15 6.7±1, C15 7.5±2; n=4). This result suggests that the inhibitory effect of SS15 was limited to PDGF-BB–directed migration and invasion and was not due to nonspecific inhibition of cell motility. It is noteworthy that both SMC migration and invasion were lower in cells kept in either control or shear stress conditions for 15 hours versus those maintained for 3 hours. Because all experiments were started when cells had reached near-confluence and cells continued proliferating throughout the course of the experiment, it is possible that more marked contact inhibition of SMC motility may account for this difference between the 15- and 3-hour experiments.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effect of shear stress on SMC migration and invasion. Cells were exposed to shear stress for either 3 (SS3) or 15 (SS15) hours. Control cells (C3 and C15) were grown under static conditions. Top, SMC migration. No effect was evident in C3 vs SS3 cells, whereas after 15 hours, SMC migration was inhibited by 31±3%. Bottom, SMC invasion. No effect was evident in C3 vs SS3 cells, whereas after 15 hours, SMC invasion was inhibited by 54±9.9%. Inhibition of SMC invasion was more marked than inhibition of migration (P<0.05). Data are expressed as % of number of cells grown under static conditions that passed through filter in response to 10 ng/mL PDGF-BB. Absolute cell numbers for C3 and C15 were 71±20 and 33±3 for migration assays and 43±17 and 22±3 for invasion assays, respectively. Results represent mean±SD of 4 independent experiments. *P<0.0001 vs C15.

Effect of Shear Stress on MMPs and TIMP-2
Because MMPs are required for SMCs to migrate across an ECM layer, the effect of laminar flow on MMPs was evaluated. CM were examined for their gelatinolytic activity by zymography, and bands of gelatinase activity with molecular weights of 72 and 92 kDa, which correspond to MMP-2 and MMP-9, respectively, in their secreted forms, were identified (Figure 2). The decrease in MMP-2 in SS3 versus C3 media was not statistically significant, whereas a significant 40±7% decrease in the 72-kDa band density was present in CM from SS15 versus C15 (P<0.05) (Figure 2). SS3 and SS15 did not modulate MMP-9 gelatinase (Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of shear stress on MMP-2 and MMP-9 in SMC CM. Both 72-kDa MMP-2 and 92-kDa MMP-9 were detected by gelatin zymography in CM from SMCs and from HT-1080 cells that were used as positive controls (top left). SS3 had no effect on MMP-2 and MMP-9. In contrast, SS15 downregulated MMP-2, whereas it did not modulate MMP-9. Bar graphs (right) show average 72-kDa MMP-2 band density of gelatin zymograms. MMP-2s in CM from SS3 and SS15 cells were normalized to static controls. SS15 decreased MMP-2 by 40±7.2%, whereas effect of SS3 was not significant. Results represent mean±SD of 3 experiments. *P<0.05 vs C15. MMP-2 detection by Western analysis (bottom left) shows that SS15 decreased both 72- and 66-kDa MMP-2s, considered zymogen and active protein, respectively. Similar result was obtained in 2 separate experiments.

In these experiments, latent collagenase was not activated with aminophenyl mercuric acetate; therefore, only the 72-kDa gelatinolytic band was identified by gelatin zymography.

This band is considered to be the zymogen, and not the active MMP-2 protein (66 kDa). The same CM as used for gelatin zymography were examined by Western analysis, and SS15 was found to decrease both 72- and 66-kDa MMP-2 (Figure 2).

Because MMP-2 activity is inhibited by TIMP-2 and to a lesser extent by TIMP-1,^{3 29} the effect of shear stress on TIMP-2 in CM was evaluated by ELISA, but no differences among groups were identified (C3 9±1 ng/mL versus SS3 8±0.3 ng/mL; C15 10±3.3 ng/mL versus SS15 8±0.1 ng/mL; n= 3). In additional experiments, we determined whether SS15 induced changes in MMP-2 mRNA or MT-MMP mRNA and protein that could account for the decrease in MMP-2 reported above. Neither MMP-2 mRNA (Figure 3) nor MMP-9 mRNA (data not shown) levels were modulated by shear stress. In contrast, after 15 hours of shear stress, MT-MMP mRNA exhibited a 33±1% reduction versus C15 (P<0.001; n=3), and this effect was associated with a diminution in MT-MMP protein by Western analysis (Figure 3). These results suggest that the decrease in 66-kDa MMP-2 may be related to the effect of shear stress to diminish MT-MMP expression rather than to a direct effect on MMP-2 expression.

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of shear stress on MMP-2 and MT-MMP expression. SS15 did not modulate MMP-2 mRNA expression, whereas it decreased MT-MMP mRNA levels. Average decrease in 3 independent experiments was 33±1% (P<0.001). Western blot analysis of cell lysates showed effect of SS15 to decrease MT-MMP proteins levels vs C15. Similar result was obtained in 2 separate experiments.

Effect of Shear Stress on SMC Integrin Expression
Integrins expressed on the cell surface modulate migration, and it has also recently been shown that MMP-2 colocalizes with the integrin _vß₃ and that by this mechanism, the cell may achieve directional migration.⁸ Therefore, we examined the distribution of _vß₃ as well as of _vß₅ integrin and of ₁-, ₂-, and ß₁-integrin subunits on SMCs after shear stress. Flow cytometry showed that SMCs express these integrins on their surface but that no significant changes were induced by shear stress for 3 and 15 hours. This experiment was repeated 3 times with similar results (data not shown).

Effect of Shear Stress on PDGF-Rß
The results presented so far suggest that a decrease in MMP-2 may be responsible for the effect of shear stress to inhibit SMC invasion across a layer of reconstituted basement membrane proteins but do not explain the diminished SMC migration in response to PDGF-BB. Therefore, both PDGF-Rß mRNA and protein levels were evaluated; after 15 hours of exposure to shear stress, PDGF-Rß mRNA levels decreased by 35±8% versus control (Figure 4A), and PDGF-Rß protein expression decreased by 70±3% (P<0.05) (Figure 4B).

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of shear stress on PDGF-Rß mRNA and protein levels. A, Northern blot analysis shows that SS15 decreased PDGF-Rß mRNA. Average decrease in 3 independent experiments was 35±8% (P<0.05). B, Western blot analysis of cell lysate. Representative example (inset) and average results show that SS15 decreased PDGF-Rß protein expression by 70±3% (top left) vs C15 (P<0.05). Results were quantified by densitometry scan and represent mean±SD of 3 independent experiments.

The functional role of the shear stress–mediated decrease in PDGF-Rß was further characterized in SMCs transfected with an expression vector carrying the cDNA for PDGF-Rß. These cells overexpressed PDGF-Rß, as shown by Western analysis (Figure 5A, inset), and after SS15, they exhibited no decrease in migration in response to 5 and 10 ng/mL PDGF-BB (Figure 5A). A similar result was obtained in invasion assays in response to 10 ng/mL PDGF-BB (not shown). In contrast, cells transfected with a control plasmid responded to SS15 with a decrease in migration and invasion comparable to that described in untransfected cells (Figure 1). To avoid the potential limitations posed by a background of untransfected SMCs, additional experiments were performed in which PDGF-Rß was cotransfected with a green fluorescent protein (GFP) expression vector. After migration, only GFP-positive cells, which are expected to have been transformed also with PDGF-Rß or the control expression vector,²⁸ were counted. Figure 5B shows that PDGF-BB–directed migration of SMCs transfected with PDGF-Rß was enhanced versus control both under static conditions and after SS15. Furthermore, shear stress–mediated inhibition of SMC migration was abolished. Taken together, these results confirm that shear stress downregulation of PDGF-Rß plays a key role in the inhibition of PDGF-BB–directed SMC migration.

View larger version (25K): [in this window] [in a new window]   Figure 5. Effect of shear stress on SMCs overexpressing PDGF-Rß. SMCs transfected with cDNA for PDGF-Rß overexpressed receptor, as shown by Western analysis (inset). A, Cells overexpressing PDGF-Rß did not exhibit a decrease in migration and invasion in response to 5 and 10 ng/mL PDGF-BB. In contrast, cells transfected with a control plasmid responded to SS15 with a decrease in migration comparable to that observed in untransfected cells and reported in Figure 1. After migration, number of migrated cells in each condition was normalized by ß-gal units reflecting transfection efficiency. For cells transfected with a control plasmid, normalized cell numbers for C15 were 14±3, 26±4, and 32±14 at 1, 5, and 10 ng/mL PDGF-BB, respectively. For cells transfected with PDGF-Rß, normalized cell numbers for C15 were 14±5, 20±4, and 23±6. All results represent mean±SD of 4 independent experiments in each condition. *P<0.05 vs C15. B, This experiment was performed with cells cotransfected with GFP and either PDGF-Rß or control expression vectors; only GFP-positive cells were counted in migration assay. Cells overexpressing PDGF-Rß exhibited enhanced migration vs controls both under static conditions (P<0.001) and after shear stress (P<0.001). Furthermore, ability of SS15 to inhibit PDGF-BB–directed (5 ng/mL) SMC migration vs C15 was abolished in cells overexpressing PDGF-Rß. Results represent mean±SD of 4 independent experiments.

	Discussion

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The present study shows that SMC exposure to laminar flow, at arterial levels of shear stress, reduced PDGF-BB–directed cell migration and invasion. A major mechanism for this effect was the decrease in PDGF-Rß mRNA expression and protein levels, because shear-dependent inhibition of SMC migration was abolished in cells forced to overexpress PDGF-Rß. It is of interest that diminished PDGF binding¹⁰ and mitogenic response to PDGF^{10 13} have also been observed in SMCs derived from the intima of injured carotid arteries, suggesting the possibility that SMC exposure to hemodynamic shear stress in vivo may be a mechanism for these effects.

A second mechanism that may contribute to the effect of shear stress to diminish SMC invasiveness across Matrigel is the decrease in MMP-2. MMPs are required for SMCs to invade a barrier constituted by ECM components,^{3 24} and lower MMP-2 levels without a change in TIMP-2, the main MMP-2 inhibitor,²⁹ may account, at least in part, for the results shown in Figure 1B. The decrease in MMP-2 occurred without an apparent concomitant decrease in MMP-2 mRNA levels but was associated with a significantly decreased expression of MT-MMP, a membrane activator of MMP-2.⁷ In addition, some integrins modulate SMC migration in vitro³⁰ and in vivo.³¹ We examined the effect of shear stress on _vß₃ and _vß₅ integrins as well as on ₁-, ₂-, and ß₁-integrin subunits; all integrins tested were expressed in SMCs, but none were modulated by shear stress.

Although the possibility that cell selection may occur during experimental shear stress cannot be excluded, our results suggest that shear stress–mediated inhibition of SMC migration may act in synergy with the previously shown effect of shear stress to decrease SMC proliferation^{21 32} to prevent intima thickening after vascular injury.

The physiological significance of our results is related to the ability of shear stress to modulate neointima accumulation after vascular injury and in vascular grafts. It has been shown that high blood flow inhibits neointima accumulation in the balloon-injured rat¹ and rabbit² carotid artery, and MMP-2 activity was lower in the blood vessels exposed to high flow versus the low-flow group.² Other studies have shown that shear stress inhibits neointima accumulation in vascular grafts.^{33 34} An important role for blood flow in neointima accumulation is also supported by clinical observations that restenosis after balloon angioplasty is less likely to occur in lesions that have been widely dilated and in which the intima has been dissected,³⁵ ie, under conditions in which SMCs are exposed to the blood stream and shear stress is more likely to be laminar than in a stenotic blood vessel. The issue could be raised that SMC migration from the media to the intima occurs in response to endovascular injury and that if only SMCs exposed to shear stress were inhibited in their migratory ability, this would be irrelevant, because these cells are on the luminal surface of vascular wall. However, because MMP-2 is secreted, it is likely that it may have functional effects not only on cells that secrete it but on all cells that are exposed to it. Thus, inhibition of MMP-2 secretion by cells exposed to shear stress may diminish migration of SMCs not directly in contact with the blood stream and may affect ECM volume and composition in neointima.

In conclusion, our results suggest that SMC exposure to shear stress may minimize the ability of SMCs to migrate from the media toward the lumen of the vessel and lead to the accumulation of neointima because of diminished SMC responsiveness to both PDGF-BB and MMP-2 activity.

	Acknowledgments

 
This work was supported in part by Biomed 2 grants BMH4-CT95-1160 and BMH4-CT97-2270. The authors thank Gabriella Ricci and Cinzia Carloni for excellent secretarial assistance.

Received September 29, 1999; revision received December 31, 1999; accepted February 7, 2000.

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