ANTI-PROLIFERATIVE EFFECTS OF PRAVASTATIN (PVS) AND CYCLOSPORIN (CS) ON VASCULAR SMOOTH MUSCLE CELLS (VSMC) - IS INHIBITION OF RAS ACTIVITY THE MECHANISM?

Abstract

388 Transplant coronary vasculopathy (TCV) in heart allografts is the most important causes of late graft loss and is caused by endothelial cell injury leading to proliferation of VSMCs. We have shown that PVS-treated heart transplant (HT) recipients have decreases in the incidence and progression of TCV. It has been hypothesized that the combination of PVS, an HMGCoA reductase inhibitor, and CS in these patients leads to this reduction in TCV. The purpose of this study is to examine the effects of PVS and CS on the proliferation of VSMCs and to gain insight into the mechanism of the clinical effects noted above. A10 cultured rat aortic VSMCs were grown to confluence and then serum-starved for 2 days. Test VSMCs were treated with CS (200 ng/ml) and/or PVS (100mcM) prior to stimulation with PDGF (30 ng/ml). The cells were pulsed with 3H-thymidine for 8 hours and radioactivity assessed by liquid scintillation. PDGF-stimulated DNA synthesis was demonstrated by a 12-fold increase in 3H thymidine incorporation over control. PVS(p=.04) and CS (p=.06) alone inhibited VSMC DNA synthesis by 46% and 33%, respectively. PVS and CS together led to a 64% inhibition of PDGF-induced DNA synthesis (p=.008) and a 46% inhibition (p=.01) compared with CS alone. When mevalonate, an early metabolite of the cholesterol synthesis pathway, was added to the PVS-inhibited VSMCs, DNA synthesis was restored to 92% of control, confirming that the PVS-induced inhibition was specific to its effect on HMGCoA reductase. We then examined signaling through the MAP kinase pathway, which is thought to represent the mitogenic pathway of tyrosine kinase receptor-associated growth factors. Tyrosine phosphorylation, a proximal step in this pathway, as well as MAPK activity were markedly stimulated by PDGF, but were unaffected by inhibitory doses of PVS. However, the activity of Ras (an intermediate component of the MAPK pathway and a byproduct of the cholesterol synthesis pathway) was increased by PDGF but reduced to baseline with PVS. CS had no additional effect on elements of the MAPK pathway. In conclusion, PVS alone inhibits VSMC proliferation but the combination of PVS and CS inhibits proliferation even further. This inhibitory effect may occur via a PVS-induced inhibition of Ras farnesylation. These data may explain the reduction of TCV in our HT recipients taking both PVS and CS. This research was funded in part by a grant from Bristol-Myers Squibb.