Crosstalk Of Integrin β₃ And PDGF Receptor Regulates Adhesion And Migration Of VSMCs

The migration and proliferation of vascular smooth muscle cells (VSMCs) are the pathological basis of cardiovascular diseases, such as hypertension, atherosclerosis, and restenosis after percutaneous transluminal coronary angioplasty (PTCA).It has been demonstrated that VSMC phenotype is regulated by cytokines, growth factors, and extracellular matrix (ECM). Platelet-derived growth factor (PDGF) is a potent cytokine for VSMC phenotypic change from the contractile (differentiated) state to the synthetic (dedifferentiated) state. PDGF activates signal transductive pathway for migration and proliferation via its receptor. Integrins, as ECM receptors, not only mediate the interaction of ECM-cell and cell-cell, but also initiate cell proliferation and migration in response to ECM and growth factors. Our previous work has shown that osteopontin (OPN), as a member of ECM, can significantly induce expression of integrin β₃ and promote migration of VSMCs. After blocking the interaction between ECM and integrin, migration and proliferation of VSMCs stimulated by growth factor were inhibited. Our data suggest that there are the synergistic effects of ECM and growth factors on migration and proliferation of VMSCs. But now, little is known about the mechanism of synergistic effect of integrin β₃ and growth factor in modulating VSMC adhesion and proliferation. In the present study, we examined the effects of integrin β₃ on adhesion and migration of VSMCs mediated by PDGF, after the interaction of ECM with integrin β₃ was blocked by anti-integrin β₃ antibody. Furthermore, to study the mechanism of signal transdution induced by PDGF, we tested adhesion, migration and proliferation of VSMCs stimulated by PDGF, after PDGFR pathway was blocked using LY 294002 or PD 98059,which are specific inhibitors of PI3K and MEK/ERK. In addition, we investigated the relationship between neointima formation and the expression of OPN, integrin 3 3, PDGFR and MMP-2 following de-endothelium. We try to confirm whether PDGFR pathway plays a key role in VSMC migration. 1 Effects of integrin J3 3 on adhesion and migration of VSMCs induced by PDGF.To elucidate the signaling mechanism of migration and adhesion stimulated by PDGF and OPN, we detected the expression of integrin 33 induced by PDGF, and the effect of blocking integrin 3 3 on VSMC adhesion and migration. The results were as follows.1.1 VSMCs were exposed to anti- integrin 3 3 antibody (5, 10, 15 mg/L) for 45 minutes and then treated with 15 u g/L PDGF for 12 hours. [3H]-thymidine incorporation assay showed that under either basal (0 ml/L) or antibody-blocked conditions, the ability of VSMC proliferation did not changed (p>0.05). The data indicated that integrin 3 3 was not involved in VSMC proliferation stimulated by PDGF.1.2 After VSMCs were pretreated with anti- integrin 3 3 antibody (5, 10, 15 mg/L) for 45 minutes, cells were cultured in 96-well plate coated by OPN and then incubated with 15 u g/L PDGF for 12 hours. Adhesion assay indicated that adhesion rate decreased with increasing antibody concentration. At dose of 10 mg/L anti- integrin 3 3, there was a significant difference (p<0.05), compared with control (0 mg/L). The migration activity of VSMCs stimulated by PDGF was observed using a model of wounding injury of confluent cells. Migration activity was significantly inhibited by anti- integrin 3 3 antibody at 10 mg/L (p<0.05), compared with the control. This result suggested that the interaction between integrin 3 3 and OPN plays a critical role in adhesion and migration of VSMCs stimulated by PDGF.1.3 Total RNA was extracted from VSMCs treated with PDGF (15 P g/L ) for 6, 12, 24, 36 and 48 hours. Compared with the control ( 0 h), the level of integrin 3 3 mRNA was robustly increased by 1.87-, 1.75-fold at 6, 12 hours after stimulating by PDGF ( p<0.05), and reached to a peak at 6 hours. Thelevel of mRNA then returned to the control level after 48 hours. Similarly, flow cytometry and immunofluorescence analysis confirmed that PDGF could induce expression of integrin ï¿¡ 3. The fluorescence percent was 26.79%, 58.71%, 67.84% and 66.10% after PDGF stimulated VSMCs for 0, 6, 12, 24 hours, respectively. The integrin $ 3 expression increased by 2.53-fold in VSMCs stimulated by PDGF for 12 hours , compared with the control.In summary, PDGF can promote proliferation, adhesion and migration of VSMCs. The interaction of ECM and integrin ï¿¡ 3 plays an important role in adhesion and migration of VSMCs induced by PDGF. PDGF enhances the interaction between ECM and the cells via inducing integrin ï¿¡ 3 expression. 2 The mechanism of signal transduction triggered by PDGF in proliferation and adhesion of VSMCTo further study the signaling mechanism of adhesion and migration stimulated by PDGF, two specific inhibitors (LY 294002, PD 98059) were used to block PI3K or MEK/ERK signal pathway that triggered by PDGFR.2.1 After VSMC was treated with either 10 uMLY 294002 or 25 u M PD 98059 and PDGF for 45 minutes, the adhesion assay was carried out. Upon treatment with LY 294002 and PD 98059, the rate of VSMC adhesion was reduced by 17.6% and 33.9% (p<0.05), respectively, in VSMC induced by PDGF. This data suggested that induction of adhesion was mainly mediated through activation of MEK/ERK signal pathway. In contrast, LY 294002 and PD 98059 resulted in a decrease in adhesion rate by 3.4% and 29.1%, respectively, in VSMC stimulated by PDGF. The adhesion rate of VSMC stimulated by PDGF was significantly higher than that of the cells unstimulated. Our results showed that PDGF promoted cell adhesion through activation of PI3K and /or MEK/ERK.2.2 In this study, we found that PDGF significantly induced OPN synthesis and secretion. After the VSMCs were incubated with PDGF for 6, 12, 24 hours, RT-PCR analysis showed that OPN mRNA was significantly increased by 1.78-, 2.6-, 1.47-fold, compared with control (0 hour), respectively. Western blot also confirmed that there was an increase in OPN level in atime-dependent manner both in VSMCs and culture medium.2.3 The VSMCs were pretreated with the two kinase inhibitors for 45 minutes and then incubated with PDGF for an additional 12 hours. The level of OPN mRNA was reduced by 24% and 52% in VSMC treated by LY 294002 and PD 98059, compared with the control, respectively, coinciding with a reduction in OPN level in culture medium. The study suggested that inhibition of either PI3K or MEK/ERK signal pathway could decrease OPN expression induced by PDGF.2.4 Flow cytometry analysis revealed that both LY 294002 and PD 98059 could inhibite the PDGF-induced surface expression of integrin P 3 in VSMCs induced by PDGF.2.5 Matrix metalloproteinases (MMPs) were synthesized during VSMC migrating. Gelatin zymography assay demonstrated the elevation of MMP-2 activity in medium of VSMCs stimulated by PDGF. Compared with control, MMP-2 activity was increased by 1.77-, 2.34-, 3.12-fold at 6, 12, 24 hours after PDGF stimulation, respectively. However, LY 294002 and PD 98059 could not inhibit MMP-2 activity. This result implied that PDGF improved MMP-2 activity of VSMCs, and the blocking either PI3K or MEK/ERK pathways had a minimally inhibitory effect on MMP-2 synthesis and secretion induced by PDGF.All above results indicated that PDGF significantly improved VSMC adhesion and synthesis of OPN. The MEK/ERK pathway was more important than PI3K pathway in OPN and integrin J3 3 expression mediated by PDGFR. 3 The dynamic changes of expression of integrin ï¿¡ 3 and PDGFR in neointimal formation.In first two parts of studies, we demonstrated that PDGF promoted VSMC proliferation, adhesion, synthesis of OPN and MMP-2 in vitro. In order to investigate the relationship between neointima formation and the expression of OPN, integrin $ 3, PDGFR and MMP-2 in denuded aorta endothelium rats, we further examined the dynamic changes of OPN, MMP-2, integrin ï¿¡ 3 and PDGFR during neointimal formation after balloon injury.3.1 We established intimal hyperplasia model by denuding the rat aorta endothelium with balloon catheter. The aortic sections were stained with HE at 3, 7, 14, 21 days after de-endothelium. Compared with smooth intima and orderly arranged medial VSMCs of aorta in the normal rats, rough intima was first seen at day 3 after de-endothelialization. VSMCs appeared in intima, and proliferation and disorder of VSMC in media were observed at day 7 after operation. The progressive and widespread intimal thickening and lumen stenosis were observed at day 14 and 21 after operation. The morphological changes suggested that migration and proliferation of VSMCs were main causes for neointimal formation after denuding the endothelium.3.2 Expression of integrin ï¿¡3 in balloon-injuried arteries was detected with immunohistochemistry. The expression of integrin ï¿¡ 3 was not detected in the media VSMCs of normal aorta. At day 3-21 after balloon injury, the positive stain for integrin ï¿¡ 3 was observed in neointima. The expression pattern of integrin ï¿¡ 3 was positively related to VSMC migration during neointima formation.Immunohistochemistry with anti-PDGFR antibody showed that expression of PDGFR was not detected in normal aorta. At day 3 after balloon injury, the weak stain for PDGFR appeared in media. At 7-21 days after balloon injury, the positive VSMCs for PDGFR were increased in neointima and media. The expression pattern of PDGFR was positively related to VSMC migration during neointima formation.3.3 Extracts from aortic tissues were analyzed by Western blotting with anti-OPN antibody. OPN was undetected in normal arteries. However, expression of OPN was induced at day 3 after de-endothelialization, peaked at day 14 and declined thereafter. Expression of OPN induced by de-endothelium was parallel to VSMC migration from media to intima at different time points.3.4 VSMC migration in vivo depends on ECM remodeling. The matrix metalloproteinase 2 (MMP-2) is a key enzyme during ECM remodeling. When extracts from both normal and balloon-injuried arteries were subjected to...