(Verbatim from the application): Atherosclerosis and arterial restenosis following balloon angioplasty is enhanced in insulin resistant conditions, where migration of vascular smooth muscle cells (VSMCs) from the media to the neointima is increased. Insulin can inhibit VSMC migration, and insulin's failure to do so in insulin resistant states might contribute to enhanced atherosclerosis/restenosis in these conditions. The mechanism of insulin's inhibition of normal VSMC migration is not known. We will test the overall hypothesis that insulin acts synergistically with NO to stimulate cyclic GMP production which inhibits VSMC migration in-vitro, and test whether insulin inhibits VSMC migration in-vivo after balloon catheter injury of carotidarteries in normal and insulin resistant obese dogs. We propose the following hypothetical scheme: 1) Insulin inhibits platelet-derived growth factor (PDGF-AB)-stimulated migration of VSMCs after they have acquired inducible nitric oxide synthase (iNOS). 2) To achieve this, insulin stimulates aerobic glycolysis, thereby raising the redox potential which stimulates cyclic GMP production induced by the NO already present in the cell. 3) Insulin-stimulated cyclic GMP production attenuates the PDGF-AB-induced Cai2+ transient which inhibits activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) which is necessary for migration. 4) Insulin-stimulated cyclic OMP production stimulates induction of mitogen-activated protein kinase phosphatase-1 (MKP-I), which inhibits PDGF-AB-stimulated mitogen-activated protein kinase (MAPK) which is also necessary for mig2ration. The effects of insulin on cultured VSMC aerobic glycolysis, redox potential, cyclic GMP levels, Cai +, CaM kinase LI and MAPK phosphorylation and activities and MKP-1 expression will be assessed. VSMC migration will be measured via Boyden Chamber and wound migration assays. The role of MKP-l and potential cross talk between CaM kinase II and MAPK will be studied using antisense oligonucleotides and/or transfections with dominant negative or constitutively active mutants. Migration of VSMCs following balloon injury of dog carotid artery will be assessed by measuring the progression of BrdU, smooth muscle cc-actin and iNOS labelled VSMCs from the media to the neointima. These studies will determine the mechanism for insulin's inhibition of VSMC migration and help explain the link between enhanced atherosclerosis/restenosis and insulin resistance.
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