| In this study we investigated the role of the multifunctional serine/threonine kinase, CaMKII, as a mediator of Ca2+ signals regulating VSM cell proliferation. Specifically, we identified CaMKII isoform composition modulation (downregulation of CaMKIIγ and upregulation of CaMKIIδ2) as an important mediator of cell proliferation in phenotypically modulating cells following primary dispersion of rat aortic VSM cells as well as following vascular injury in a rat carotid artery balloon angioplasty model. Attenuating the initial upregulation of CaMKIIδ 2 in primary cultured cells using siRNA resulted in a significant decrease in serum-stimulated DNA synthesis and cell proliferation. In passaged arotic VSM cells, which express CaMKIIδ2, suppression of CaMKIIδ 2 activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using siRNA, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated a significant increase in proportion of cells in G2/M. Accumulation of cells in G2/M is potentially due to a significant suppression of phosphorylation of the G2/M cell cycle regulatory protein phosphatase Cdc25C. Utilizing a rat carotid artery balloon angioplasty model of vascular injury in vivo we described acute upregulation of CaMKIIδ 2 in medial and neointimal VSM cells. Using siRNA at the time of injury, we prevented the upregulation of the δ2 isoform and inhibited intimal hyperplasia by 80% following incubation of the injured vessel with siRNA to the δ2 isoform. A significant reduction in proliferation in VSM cells was observed in vessels incubated with siRNA to the δ 2 isoform 3 and 7 days after injury. In addition, CaMKIIδ 2 potentially regulates proteins involved in triggering the cellular changes observed in phenotypically modulating VSM cells as suppression of CaMKIIδ2 rescued the loss of REST mRNA in freshly dispersed cells. In vivo loss of REST protein associated with vascular injury was abrogated in carotid arteries treated with siRNA directed against the δ2 isoform. These results indicate that CaMKIIδ 2 is a negative regulator of REST in phenotypically modulating VSM cells and in this context, CaMKIIδ2 may serve as a regulator of the gene program supporting increased VSM cell proliferation following VSM cell culture and vascular injury. |
