The present study was undertaken to determine whether Ca2+‐calmodulin‐dependent protein kinase II (CaMKII) participates in the regulation of vascular smooth muscle contraction, and if so, to investigate the nature of the downstream effectors. The contractility of isolated ferret aorta was measured while inhibiting CaMKII either with antisense oligodeoxynucleotides against CaMKII or with the CaMKII inhibitor KN93. Treatment with antisense oligodeoxynucleotides against CaMKII resulted in, on average, a decrease in protein levels of CaMKII to 56 % of control levels and significantly decreased the magnitude of the contraction in response to 51 mm potassium physiological saline solution (KCl). Contraction in response to the phorbol ester DPBA was not significantly affected. The CaMKII blocker KN93 also resulted in a significant decrease in the force induced by 51 mm KCl but caused no significant change in the contraction in response to DPBA or the α‐adrenoceptor agonist phenylephrine. During contraction with 51 mm KCl, both CaMKII and mitogen‐activated protein kinase (MAPK) activity increased, as determined by phospho‐specific antibodies. The MAPK phosphorylation level was inhibited by KN93, PD098059 (a MAPK kinase (MEK) inhibitor) and calcium depletion. Myosin light chain (LC20) phosphorylation also increased during contraction with KCl and the increase was significantly blocked by PD098059 as well as by both KN93 and antisense oligodeoxynucleotides to CaMKII. The data indicate that CaMKII plays a significant role in the regulation of smooth muscle contraction and suggest that CaMKII activates a pathway by which MAPK activation leads to phosphorylation of LC20 via activation of myosin light chain kinase.

Cerebral vascular dysfunction and associated diseases often occur in type-1 diabetes, but the underlying mechanisms are largely unknown. In this study, we sought to determine whether big-conductance, Ca(2+)-activated K(+) (BK) channels were impaired in vascular (cerebral artery) smooth muscle cells (CASMCs) from streptozotocin-induced type-1 diabetic mice using patch clamp, molecular biologic, and genetic approaches. Our data indicate that the frequency and amplitude of spontaneous transient outward currents (STOCs) are significantly decreased, whereas the activity of spontaneous Ca(2+) sparks is increased, in diabetic CASMCs. The sensitivity of BK channels to voltage, Ca(2+), and the specific inhibitor iberiotoxin are all reduced in diabetic myocytes. Diabetic mice show increased myogenic tone and decreased contraction in response to iberiotoxin in cerebral arteries and elevated blood pressure. The expression of the BK channel beta1, but not alpha-subunit protein, is markedly decreased in diabetic cerebral arteries. Diabetic impairment of BK channel activity is lost in CASMCs from BK channel beta1-subunit gene deletion mice. In conclusion, the BK channel beta1-subunit is impaired in type-1 diabetic vascular SMCs, resulting in increased vasoconstriction and elevated blood pressure, thereby contributing to vascular diseases in type-1 diabetes.

Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-d and -g isoforms. CaMKIId promotes VSM proliferation and vascular remodeling. We tested CaMKIIg function in vascular remodeling after injury. CaMKIIg protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIg C rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIId 2 adenovirus had no effect. Similar dynamics in CaMKIIg mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIg in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIg overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIg and -d isoforms have nonequivalent, opposing functions.

The multifunctional Ca2+/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3′UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis.

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