Abstract 1701: Deletion of β PDGF Receptor Dependent PI-3 Kinase and PLCγ Signaling Attenuates Vascular Smooth Muscle Cell Proliferation and Inhibits Neointima Formation in Mice

Platelet-derived growth factor β-receptor (βPDGFR)-mediated proliferation of vascular smooth muscle cells (VSMC) plays a pivotal role in the development of restenosis. The βPDGFR binds and activates cytoplasmic signaling molecules such as Src, PI 3-kinase (PI3K), RasGAP, SHP-2 and phospholipase C-gamma (PLCγ). However, the βPDGFR-induced signaling pathways leading to cell cycle progression are largely unknown. In order to characterize the signaling molecules, which are important for βPDGFR-induced VSMC proliferation, we generated several stable VSMC lines with mutated βPDGFRs which were unable to bind/activate each individual signaling molecule. BrdU incorporation assays revealed PI3K and PLCγ as the main mediators of βPDGFR-mediated VSMC proliferation. Deletion of the binding sites for PI3K or PLCγ diminished DNA-synthesis about 47 ± 6% and 54 ± 5%, respectively, whereas the deletion of Src, RasGAP, or SHP-2 binding to the βPDGFR had no influence on the mitogenic response. Mutant cell lines which were only able to bind/activate PI3K or PLCγ induced 43 ± 4% and 52 ± 5% of the WT response, respectively. We further investigated the influence of PI3K and PLCγ on the distinct expression of the key players of cell cycle progression. Western blot analysis revealed that βPDGFR-activated PI3K mainly mediated the up-regulation of cyclin D1, whereas activation of PLCγ mediated the down-regulation of the cyclin-dependent kinase-inhibitor p27 kip1 . Consistently, βPDGFR-dependent phosphorylation of the retinoblastoma protein which is important for the G1/S transition was attenuated when either the binding of PI3K or PLCγ to the βPDGFR was abolished. Moreover, we confirmed our in vitro results in an in vivo model of balloon-induced carotid artery neointima formation. Mice lacking binding of PI3K and PLCγ to the βPDGFR (F3 mice) developed ~50% less neointima formation after balloon angioplasty compared to control WT mice after 3 weeks (p < 0.05). These results indicate that the mitogenic signal of the β PDGFR is mediated by PI3K and PLCγ affecting distinct targets of cell cycle progression. Targeting specific downstream molecules of the βPDGFR in VSMCs might provide a novel approach to reduce neointima formation in patients undergoing balloon angioplasty.