Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways

Abstract

BACKGROUND: A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. RESULTS: Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-beta1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-beta1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-beta1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. CONCLUSION: These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.

Figure 1

PDGF-BB increased rVSMC migration on laminin-5 in vitro. The addition of PDGF-BB increased rVSMC migration on laminin-5, in a dose-dependent manner, over rVSMC haptotactic migration induced by the presence of laminin-5. rVSMC migration on laminin-5 was inhibited by both PD98059 (MEK1 inhibitor) and TGF-β1, in a dose-dependent manner.

Figure 2

PDGF-BB reduces rVSMC adhesion on laminin-5 adhesion in vitro. The presence of laminin-5 supported greater adhesion of rVSMC over naked plastic, and this increase in adhesion was reduced by the addition of PDGF-BB. Inhibition of MEK1, using PD98059 restored rVSMC adhesion to laminin-5 in the presence of PDGF-BB. Although fibronectin also supported rVSMC adhesion, the effect of adding PDGF-BB was less pronounced and was not restored using PD98059. The addition of TGF-β1, however, completely restored rVSMC adhesion to both fibronectin and laminin-5 in the presence of PDGF-BB.

Figure 3

The effect of growth factors on rVSMC proliferation in vitro. The addition of PDGF-BB stimulated proliferation of rVSMC on laminin-5 and to some extent on naked plastic, but not on fibronectin. The presence of fibronectin alone was able to stimulate proliferation of rVSMC. The proliferative response of rVSMC to presence of laminin-5 with PDGF-BB or to fibronectin was suppressed by the addition of TGF-β1. Figure 3b The ECM induces differential rVSMC proliferation responses in vitro. The plating of rVSMC on fibronectin, but not laminin, induced increases in proliferation over four days. The addition of PDGF-BB, however, increased rVSMC migration on laminin-5, but not on fibronectin. The addition of TGF-β1 was sufficient to suppress rVSMC proliferation on both laminin-5 and fibronectin.

Figure 4

The MAPK pathway in rVSMC is activated by different stimuli. The addition of FCS or PDGF-BB or the plating of rVSMC on fibronectin was sufficient to induce measurable increases in p44/p42 activation over 30 minutes. However, the addition of TGF-β1 or the plating of rVSMC on laminin-5 was not sufficient to induce MAPK activation.

Figure 5

Integrins and growth factors activate intracellular signaling cascades in rVSMC. Intracellular signaling pathways that converge through MEK and ERK activation, may be initiated in rVSMC by the addition of PDGF-BB, as well as binding to laminin-5 or fibronectin. The MEK1 inhibitor PD98059 blocks MEK1 activation and alters rVSMC responses to PDGF-BB on laminin-5, but not fibronectin. The addition of TGF-β1, which may block later events in the cell cycle, is sufficient to block PDGF-BB induced responses of rVSMC on both laminin-5 and fibronectin.