Calcium/Calmodulin-Dependent Protein Kinase II Gamma (CaMKII-gamma) Function in Vascular Remodeling

Ca2+ dependent signaling pathways have been known to regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimai accumulation of VSM, a hallmark of vascular diseases such as atherosclerosis, post-angioplasty/in-stent restenosis, and graft arteriosclerosis. The multifunctional serine/threonine protein kinase Ca2+/Calmodulin-dependent protein Kinase II (CaMKII) is Ca2+ mediator. VSM cells express primarily CaMKIIgamma and -delta isoforms. Previous studies using molecular and genetic approaches demonstrated that CaMKIIdelta promotes VSM synthetic phenotype cell function and neointima formation following vascular injury. CaMKIIgamma expression is downregulated with yet unknown functional consequences. In this study, we investigated the function of CaMKIIgamma in the regulation of VSM proliferation and vascular remodeling. We first generated CaMKIIdelta and CaMKIIgamma knockout mice under the transgelin (SM22alpha) promoter in order to study CaMKII in smooth muscle and in an isoform specific manner. Chapter 1 shows that under baseline conditions, smooth muscle targeted deletion of CaMKIIdelta and CaMKIIgamma had no effect on arterial morphology, hemodynamics or smooth muscle differentiation marker gene expression. Interestingly, double deletion of both isoforms (Camk2g/dSMKO) resulted in loss of one lamellar unit in the aortic vessel wall and increased systolic blood pressure. These data indicate that CaMKIIdelta and CaMKII isoforms are dispensable to VSM growth and development whereas CaMKII activity is required for normal vascular development.;To test the function of CaMKIIgamma in vascular remodeling following injury, we used the mouse carotid artery ligation model. Chapter 2 demonstrates that CaMKIIgamma mRNA and protein expression are down-regulated after mouse carotid artery ligation and correlates with loss of smooth muscle contractile phenotype markers. Carotid artery neointima formation was markedly enhanced in smooth muscle targeted CaMKIIgamma knockout mice compared to littermate controls 3 weeks post-ligation. CaMKIIgamma deletion resulted in increased cellular proliferation associated with decreased expression of the cell cycle inhibitor CDKN1A (p21) with no change in apoptosis. In cultured VSM, CaMKIIgamma over-expression decreased cell proliferation, induced p53 and its target gene p21 expression, and down-regulated cell cycle genes known to promote cell proliferation through the G2/M. Over-expression of comparable levels of CaMKIIdelta had no significant effect. Intraluminal transduction of adenovirus encoding CaMKIIgamma following balloon angioplasty in rat carotid artery rescued injury-induced decreases in CaMKIIgamma expression, inhibited neointima formation, inhibited vascular cell proliferation and increased expression of p21 while comparable doses of CaMKIIdelta had no effect.;Coincident loss of CaMKIIgamma and smooth muscle differentiation markers suggests some level of co-regulated expression. Myocardin, a transcriptional co-factor of SRF supports smooth muscle differentiation and contractile phenotype. We hypothesized that CaMKIIgamma expression is dependent on the myocardin-SRF-CArG switch. Chapter 3 demonstrates that endogenous CaMKIIgamma expression increased upon myocardin over-expression whereas knockdown or knockout of SRF in cultured VSM cells failed to affect basal CaMKIIgamma expression. Moreover, knockdown of SRF followed by over-expression of myocardin did not inhibit myocardininduced expression of CaMKIIgamma. Collectively, the data in this thesis show that CaMKIIgamma and Sisoforms have non-equivalent functions in VSM and play opposing roles in regulating the vasculoproliferative response to injury. CaMKIIgamma expression is CArG-SRF independent, but myocardin dependent through yet an unknown mechanism.