Different Effects of High and Low Shear Stress on Platelet-Derived Growth Factor Isoform Release by Endothelial Cells
Consequences for Smooth Muscle Cell Migration
In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm2 (SS5) or 15 dyne/cm2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45±5.5 and 30±1.5 cells per field, respectively (P<0.05). Similar results were obtained with SS of 2 versus 20 dyne/cm2 and also when SS of 5 and 15 dyne/cm2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9±7 and 18±5 ng/106 cells for 16 hours, respectively (P<0.05); PDGF-BB levels in CMSS5 and CMSS15 were 38±10 and 53±10 ng/106 cells for 16 hours, respectively (P<0.05). PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ) in SMCs were phosphorylated by CMSS15>CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRα or PDGFRβ increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRα inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB (P<0.001). These results suggest that the ability of high SS to inhibit arterial wall thickening in vivo may be related to enhanced activation of PDGFRα in SMCs by PDGF isoforms secreted by the endothelium.
- shear stress
- endothelial cells
- smooth muscle cells
- platelet-derived growth factors
- platelet-derived growth factor receptors
Smooth muscle cell (SMC) migration and proliferation play key roles in neointimal accumulation,1 and in native arteries and in vascular grafts with an intact endothelium, SMC function is modulated by shear stress (SS). In fact, intimal thickness is enhanced by low blood flow, whereas it is inhibited by high blood flow.2,3⇓ Endothelial cells (ECs) are directly in contact with the bloodstream, and in response to SS, they secrete a variety of growth factors, including platelet-derived growth factor (PDGF), a potent modulator of SMC migration and proliferation. Therefore, it is possible that the different effects of high and low SS on neointimal accumulation in the presence of an intact endothelium may be partially due to PDGF secreted by ECs.4–6⇓⇓
PDGF isoforms consist of homodimers and heterodimers of A, B, C, and D chains, and at least 5 PDGF isoforms have been identified so far: AA, AB, BB, CC, and DD.7,8⇓ They bind PDGF receptor (PDGFR) α or β subunits (PDGFRα and PDGFRβ, respectively) with different affinities, inducing receptor dimerization and autophosphorylation. Receptor dimer αα binds AA, AB, BB, and CC dimers; ββ9 binds BB and DD dimers; and receptor dimer αβ binds BB and AB dimers. Once activated, PDGFRs retain distinct chemotactic properties.10 In fact, cells expressing ββ-receptor dimers migrate toward PDGF-BB,11 whereas activation of αα-receptor dimers does not elicit SMC chemotaxis.12 The molecular basis for these different responses is not completely clarified but may be the consequence of the activation of different signaling pathways.11 In fact, several reports indicate that αα- and ββ-receptor dimer signaling relies on a differential usage of intermediate signal transducers, such as Ras and phosphatidylinositol 3′-kinase (PI3K).11,13,14⇓⇓
In the present study, it is shown that the chemotactic response of SMCs to conditioned media (CM) from ECs exposed to high SS is lower than that induced by CM from cells exposed to low SS and that this effect is partially due to enhanced PDGFRα activation. These results provide a new insight into how SS may modulate SMC migration in the presence of an intact endothelium.
Bovine aortic ECs and SMCs were isolated as previously described.15 Cells between passages 3 and 10 were used in all experiments. ECs and SMCs were characterized by immunofluorescence staining for factor VIII and anti-SMC α-actin (Dako A/S), respectively. Cell populations >95% pure were used in all experiments.
Confluent EC monolayers in DMEM without FCS were exposed to laminar fluid SS of 2, 5, 15, or 20 dyne/cm2 (SS2, SS5, SS15, and SS20, respectively) for 8, 16, or 24 hours in a cone-and-plate apparatus16 maintained at 37°C in humidified air with 5% CO2. Control ECs were kept under static conditions. CM from static control cells (CMC) or from cells exposed to SS of different intensity (CMSS2, CMSS5, CMSS15, and CMSS20) was used as chemoattractant in SMC migration assays.
SMC migration was evaluated in a modified Boyden chamber assay as previously described.17 Briefly, bovine aortic SMCs were detached with trypsin, counted, centrifuged, and resuspended at 2×105 cells/800 μL in DMEM containing 0.1% BSA. Cells were plated on the upper side of a gelatin-treated polycarbonate filter (8.0-μm pores, Nucleopore Costar Scientific Corp). In the lower chamber of the Boyden apparatus, either human recombinant PDGF-AA (1 or 5 ng/mL, Collaborative Research), PDGF-BB (5 or 10 ng/mL, Collaborative Research), or CM from ECs exposed to SS were used as chemoattractants.
In migration-inhibition experiments, neutralizing antibodies against PDGF-AA and PDGF-BB were placed in the lower chamber of the Boyden apparatus at a concentration of 40 μg/mL. After 4 hours of incubation, cells on the filter were fixed with ethanol and stained with toluidine blue. Cells from 5 randomly chosen high-power (magnification×400) fields on the lower side of the filter were counted.
Determination of PDGF Isoforms in CM
PDGF-AA and PDGF-BB in CM was assayed by an inhibition antibody binding assay. Fixed amounts of polyclonal rabbit anti–PDGF-AA and anti–PDGF-BB were incubated with aliquots of CM in Eppendorf tubes precoated with PBS supplemented with 2% gelatin (PBS-gelatin 2%). After 20 hours of incubation at 4°C, Staphylococcus aureus protein A (Sigma Chemical Co) was added, and immunoaggregates were removed by centrifugation. The residual antibody-binding activity in the supernatant was measured by direct ELISA, as previously described.18
Immunoprecipitation and Western Blot Analysis
SMCs were incubated for 10 minutes with CM from ECs subjected to SS. Cells were rinsed twice with ice-cold PBS and lysed with 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 100 mmol/L NaCl, 10 μg/mL leupeptin, and 10 μg/mL pepstatin. Lysates were incubated overnight at 4°C with 1 μg anti-PDGFRα or anti-PDGFRβ antisera (Santa Cruz) with an orbital shaker. Subsequently, 50 μL of protein A–Sepharose (Sigma) was added, and orbital shaking was continued for additional 16 hours at 4°C. The immunocomplexes were washed 10 times with lysis buffer and then subjected to 6% SDS-polyacrylamide electrophoresis. For Western blotting, proteins were transferred from the gel to a nitrocellulose membrane and then blocked in PBS containing 5% nonfat dry milk, washed, and incubated with anti-phosphotyrosine antibody (4G10, Upstate Biotechnology). The membranes were then stripped and incubated with anti-PDGFRα or anti-PDGFRβ. Specific signals were visualized by using the ECL Western Blot Detection Kit according to manufacturer’s instructions (Amersham Pharmacia Biotech).
Plasmids and Transfection Methodology
SMCs (1.8×106) were transfected by using the Lipofectamine Plus reagent (GIBCO-BRL), as previously reported,19 either with 15 μg of DNA encoding dominant-negative PDGFRα (DN-PDGFRα), dominant-negative PDGFRβ (DN-PDGFRβ), wild-type PDGFRα, or PDGFRβ mutants in which single or double tyrosine residues were mutated to phenylalanine at amino acid positions 1009, 1009/1021, or 740/751. Controls received equal amounts of pCDNA3 empty vector. All transfections were performed in the presence of 5 μg (3:1 ratio) cotransfected enhanced green fluorescent protein (GFP)-N1 vector (pEGFP-N1) (Clontech).
Because cotransfection with 2 independent vectors results in the internalization of both plasmids by the same cell,20 migrated cells were counted by using fluorescence microscopy to evaluate only GFP-positive cells to overcome the limitations of low transfection efficiency, ie, transfection of 5% to 10% of the total population.
Continuous variables were analyzed by the Student t test and ANOVA. Post hoc tests according to the Student-Newman-Keuls methods were used when the P value (by ANOVA) indicated a statistically significant difference among groups. Data are expressed as mean±SD. A value of P<0.05 was deemed statistically significant.
Modulation of SMC Migration
In these experiments, the chemotactic effect of CM from ECs kept under static conditions (CMC) or exposed to different levels of SS on SMC migration was examined (Figure 1A).
In the Boyden chamber assay, all CM from ECs exposed to SS enhanced EC migration compared with CMC (P<0.05). However, the ability of CMSS15 and CMSS20 to induce SMC migration was ≈40% lower than that of CMSS2 and CMSS5 (P<0.05). The chemotactic effect of CMSS5 versus CMSS2 was enhanced (P<0.05), whereas there was no significant difference between CMSS15 versus CMSS20. It is noteworthy that the chemotactic effect of CMSS2 and CMSS5 was close to that of 10 ng/mL PDGF-BB, which, in prior studies, has been shown to be the concentration of PDGF-BB that elicits maximal or near-maximal SMC migration in the Boyden chamber assay.21,22⇓ In other experiments, the time course of the effect of SS on SMC migration was examined. After 8 hours of exposure to SS, it was found that CMSS15 versus CMC enhanced SMC migration, but this effect did not achieve statistical significance, whereas a significant difference (P<0.05) was observed after 16 and 24 hours of exposure to SS (Figure 1B). In addition, after 24 hours of exposure to SS, the chemotactic effect of CMSS5 was still stronger than that of CMSS15 (Figure 1B).
The levels of PDGF-BB, PDGF-AA, and PDGF-AB were examined in CM from ECs kept under static conditions or exposed to either SS5 or SS15 for 16 hours (Table). Significant levels of PDGF-AA and PDGF-BB were found in all CM, and both PDGF isoforms were significantly increased in response to SS15 compared with SS5 or with the static condition (P<0.05). In contrast, the increase induced by SS5 versus the static condition was not statistically significant. PDGF-AB was either undetectable or extremely low in all CM.
Induction of PDGFR Activation
PDGF family members bind cell surface α- and β-tyrosine kinase receptors and induce receptor dimerization and trans-phosphorylation.9 To investigate whether the EC CM induced receptor activation in SMCs, we examined PDGFRα and PDGFRβ phosphorylation by immunoprecipitation and Western blot analyses (Figure 2). CMSS5 enhanced α- and β-receptor phosphorylation ≈6-fold and 2-fold, respectively, compared with CMC, whereas CMSS15 enhanced PDGFRα and PDGFRβ phosphorylation ≈10-fold and 3-fold, respectively, compared with CMC (please see online Figure I, which can be accessed at http://atvb.ahajournals.org). These results indicate that PDGF isoforms present in the CM collected from ECs exposed to different SS levels activated PDGF receptors and that the activation of PDGFRα and PDGFRβ was higher in response to CMSS15 than to CMSS5.
Effects of PDGF-AA and PDGF-BB on SMC Migration
It has been shown previously that PDGF-AA inhibits the chemotactic effect of PDGF-BB on SMCs.13 Therefore, the possibility was examined that the presence of PDGF-AA in the CM may inhibit PDGF-BB–directed SMC migration. To address this issue, anti–PDGF-BB and anti–PDGF-AA antibodies were used, and their effectiveness was evaluated in preliminary experiments with human recombinant PDGF-BB and PDGF-AA. In agreement with the results of prior studies, PDGF-BB exhibited a strong chemotactic effect on SMCs (Figure 3A). In contrast, SMC migration in response to PDGF-AA was markedly lower and comparable to that observed in the absence of a chemoattractant (not shown). The chemotactic effect of PDGF-BB was inhibited by PDGF-AA in a dose-dependent manner, and under these conditions, PDGF-AA antibodies reversed the inhibitory effect of 1 and 5 ng/mL PDGF-AA on SMC migration. In contrast, anti–PDGF-BB antibodies decreased the effect further when PDGF-BB and PDGF-AA were used in a 5:1 ratio (Figure 3A). When CMSS5, CMSS15, or CMC was used as a chemoattractant, anti–PDGF-BB antibodies inhibited SMC migration 57±0.6%, 50±1%, or 13±2%, respectively. This result suggests that the ability of EC CM to induce SMC migration is largely related to the presence of PDGF-BB in the CM. In contrast, anti–PDGF-AA antibodies enhanced the chemotactic effect of CMSS15, which achieved a level comparable to that of CMSS5 in the absence of any antibody, whereas the chemotactic effect of CMSS5 and CMC were not affected by the anti–PDGF-AA antibody (Figure 3B). Taken together, these results suggest that the weaker chemotactic effect of CMSS15 compared with CMSS5 is due to the presence of PDGF-AA in CMSS15.
PDGFRα and PDGFRβ in SMC Migration
Because previous studies23,24⇓ have demonstrated that PI3K and phospholipase C-γ (PLC-γ) are required for SMC migration, we examined whether PDGFRβ mutants, in which the tyrosines residues involved in the binding of PI3K and PLC-γ were changed to phenylalanine residues, could alter SMC migration. Cells transfected with the control plasmid revealed the same migratory capacity as did mock-transfected cells (Figure 1). In contrast, SMC migration was markedly inhibited when cells were transfected with PDGFRβ mutants Y1009/Y1021F and Y1021F, which are unable to activate PLC-γ, or when cells were transfected with the Y740/751F mutant, which is unable to activate PI3K (please see online Figure II, which can be accessed at http://atvb.ahajournals.org). Το further explore this aspect, we used DN-PDGFRα and DN-PDGFRβ containing the transmembrane and extracellular domains but lacking the cytoplasmic domains. SMCs transfected with DN-PDGFRβ showed a strong inhibition of the chemotactic response to PDGF-BB, CMSS5, and CMSS15 (P<0.05), whereas there was no significant inhibition in response to CMC (Figure 4A). Cells transfected with DN-PDGFRα exhibited an enhanced chemotactic response to CMSS15 (P<0.05), whereas the effect of CMSS5, CMC, and PDGF-BB was not modulated (Figure 4B). Furthermore, the overexpression of wild-type PDGFRα inhibited SMC migration under all conditions tested (Figure 4C), and the magnitude of this effect was comparable to that of DN-PDGFRβ (Figure 4A). These data are in agreement with prior studies in other cellular systems21,25,26⇓⇓ showing that positive chemotactic signals are mediated only by PDGFRβ, whereas PDGFRα activates negative regulatory pathways. Our data suggest that the production of different PDGF isoforms by ECs in response to SS could be one of the mechanisms by which the endothelium modulates the biological responses of SMCs.
In the present study, we report that SMC migration is lower in response to the CM of ECs exposed to high SS compared with low SS. The increase in SS from 5 to 15 dyne/cm2 enhanced PDGF-BB and PDGF-AA secretion. In addition, the degree of phosphorylation of PDGFRα and PDGFRβ in SMCs was enhanced when the cells were incubated with CM from ECs exposed to low SS, and it was increased even further when the cells were incubated with CM from ECs exposed to high SS. Inhibition of PDGFRα-mediated effects either by overexpressing DN-PDGFRα or by a PDGF-AA antibody enhanced SMC migration in response to CMSS15; in contrast, these interventions failed to modulate SMC migration when CMSS5 was used as a chemoattractant. Taken together, these results indicate that PDGFRα activation on SMCs is enhanced when the cells are treated with the CM of ECs exposed to high SS and that, via this mechanism, PDGFRβ-directed SMC migration is inhibited. PDGF-BB binds ββ-, αβ-, and αα-receptor dimers, whereas PDGF-AA selectively binds αα dimers; it has been shown that PDGF-AA inhibits PDGF-BB–directed SMC migration,13,22,25,26⇓⇓⇓ and this effect has been attributed to different signaling mechanisms between PDGFRβ and PDGFRα.14 In fibroblasts, PDGF-AA inhibits the increase in cytosolic Ca2+ concentration triggered by PDGF-BB, which is a key signal in SMC migration.27 Furthermore, α- and β-receptors activate extracellular signal–regulated kinases, whereas only PDGFRα activates c-Jun NH2-terminal kinase 1, which inhibits the PDGFRβ-induced phenotypic transformation of NIH 3T3 cells.28 Previously, it has been shown that PDGFRβ, on exposure to high concentration PDGF-BB, can also inhibit SMC chemotaxis.19 However, under the conditions of the present study, the PDGFRβ-mediated inhibition of SMC migration did not appear to play a role, inasmuch as the cells overexpressing DN-PDGFRβ or cells treated with anti–PDGF-BB neutralizing antibody failed to exhibit an enhanced chemotactic response; rather, they showed a decrease in migration in response to CMSS5 and CMSS15.
In humans and in animal models, intimal-medial thickness in the presence of an intact endothelial layer is modulated by SS29,30⇓ as well as by some risk factors for atherosclerosis.31 Studies with noninvasive ultrasound techniques, performed either on human autopsy specimens or in vivo,32,33⇓ have shown that in elastic and in muscular arteries, low SS is associated with enhanced arterial wall thickness. Similar conclusions have been reached in studies using animal models.29 In endothelialized balloon vascular grafts, a reduction in flow and SS initiates subendothelial SMC proliferation, which ultimately leads to severe neointimal accumulation.34–36⇓⇓ In one in vivo study, the roles of PDGFRα and PDGFRβ in neointimal accumulation in vascular grafts were addressed34–36⇓⇓ by treating the animals with blocking antibodies either to PDGFRβ οr to PDGFRα.36,37⇓ Baboons treated with the antibody to PDGFRα exhibited a decrease in SMC nuclear density, consistent with the known effect of PDGFRα on SMC proliferation; however, despite this effect on cell number, there was not a decrease in neointima. SMC proliferation and migration from the media to the intima and extracellular matrix production are key events in arterial wall remodeling. However, the mechanisms by which blood flow affects SMC function within the arterial wall, in the presence of an intact endothelium, are still poorly characterized. Recently, it has been shown that ECs exposed to flow produce and secrete plasminogen activator inhibitor-1 and, thus, inhibit SMC migration in vitro.38 The potential role of PDGF has been examined in several prior studies because this growth factor is a powerful SMC chemotactic agent and also enhances SMC proliferation and matrix metalloproteinase-2 and -9 expression in SMCs.39 Hsieh et al40 reported that SS transiently increases PDGF-A and -B mRNA. Specifically, PDGF-A mRNA increased when SS was enhanced from 6 to 51 dyne/cm2, whereas PDGF-B mRNA was upregulated at significantly lower SS rates, ranging between 0 and 6 dyne/cm2. This effect of SS on PDGF gene expression has been confirmed by other studies,40–44⇓⇓⇓⇓ and more recently, an SS response element has been identified in the promoter region of several genes, including PDGF-B and PDGF-A.43 These results on PDGF gene expression were corroborated by PDGF-BB measurements in CM of ECs; in these experiments, SS of 3 to 9 dyne/cm2 enhanced PDGF-BB secretion.41 Furthermore, in vivo studies have shown that flow reduction enhances endothelial PDGF-A and -B gene expression in the rat carotid.45 The present report confirms that SS modulates PDGF secretion by the endothelium; in addition, it shows that different levels of SS lead to the production of different PDGF isoforms, which, in turn, exhibit different chemotactic effects on SMCs. Under the experimental conditions of the present study, SMC migration in response to the CM of ECs exposed to 15 or 20 dyne/cm2 versus 2 or 5 dyne/cm2 was ≈40% lower. It is difficult to establish the biological significance of this finding and extrapolate it to in vivo studies that have shown a protective effect of higher SS on arterial wall thickness.32 Nevertheless, these results, without excluding other contributing mechanisms, establish proof of the principle that SS levels comparable to those found in the human arterial system32 determine which PDGF isoform is preferentially secreted by the endothelium; because PDGF isoforms exhibit different binding affinities for PDGFRα and PDGFRβ, signaling via these receptors may be responsible, at least in part, for the different behavior of SMCs underlying ECs exposed to high versus low SS.
This work was partially supported by the Italian Ministero della Salute. Dr Palumbo was partially supported by an EMBO Fellowship. Dr Rönnstrand holds a position as Senior Researcher funded by the Swedish Research Council. We thank Alessandra Cucina, PhD, and Valeria Borrelli, PhD, for PDGF quantification. We acknowledge Gabriella Ricci Cinzia Carloni and Susanna Rulli for excellent secretarial assistance.
↵*These authors contributed equally to the present study.
Received July 9, 2001; revision accepted November 8, 2001.
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