Role of class IA PI 3-kinase isoforms in neointima formation following balloon angioplasty: only p110alpha is important

Abstract

Growth factor induced proliferation and migration of vascular smooth muscle cells (SMCs) are essential for neointima formation following balloon angioplasty. Growth factors such as platelet-derived growth factor (PDGF) activate receptor tyrosine kinases (RTKs). RTK-induced responses are largely mediated by activation of class IA phosphatidylinositol 3'-kinase (PI3K) isoforms. Class IA PI3Ks comprise one of three different catalytic isoforms (p110alpha, p110beta, p110delta). Here, we sought to clarify, which p110 subunits contribute to neointima formation following balloon injury. Western blot analysis of lysates from cultured human, mouse and rat SMCs revealed that p110alpha, p110beta, and p110delta are expressed in these cells. However, co-immunoprecipitation experiments using an antibody against the PDGF beta receptor indicate that only p110alpha and p110delta bind to activated RTKs. p110beta signals downstream of G-protein coupled receptors (GPCR) as p110beta inhibition by TGX-221 (1μM) completely abrogated LPA (GPCR agonist) induced AKT activation downstream of PI3K. TGX-221 had no influence on RTK induced AKT activation. Inhibition (PIK75, 500 nM) or siRNA mediated knockdown of p110alpha completely abrogated PDGF mediated proliferation (BrdU incorporation assay) and migration (modified Boyden-chamber) of SMCs. p110beta (TGX-221, 3μM) and p110delta (IC87114, 3μM) inhibition had no influence. However, knockdown of p110delta using a specific siRNA significantly reduced PDGF induced SMC proliferation without effecting SMC migration. As expected, siRNA against p110beta was noneffective. By means of SMC-specific p110alpha deficient mice and p110delta knockout mice, we analysed in vivo the relevance of p110alpha and p110delta for restenosis formation following balloon angioplasty. The extent of neointima formation was quantified 4 weeks following balloon angioplasty of carotid arteries from wild-type (WT) and p110alpha ko as well as from WT and p110delta ko mice (n=7, respectively). Whereas p110alpha deficiency largely suppressed neointima formation (p<0.05), p110delta deficiency was noneffective. In conclusion, our results indicate that the p110alpha subunit of PI3K, is crucial for growth factor-mediated proliferation and migration of SMCs in restonosis following balloon angioplasty. Therefore, p110alpha represents a promising therapeutic target.