Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an α-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses α-smooth muscle actin, β-actin, nonmuscle γ-actin, and smooth muscle γ-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during α-agonist contractions involves the remodeling of primarily γ-actin and, to a lesser extent, β-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

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.

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.

Abstract-Subcellular targeting of kinases controls their activation and access to substrates. Although Ca 2ϩ /calmodulindependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the ␥ isoform of CaMKII that contains a potential targeting sequence in its association domain . Antisense knockdown of CaMKII␥ G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKII␥ G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKII␥ G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKII␥ G-2 activation, and before CaMKII␥ G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKII␥ G-2 inhibited targeting. Furthermore, prevention of CaMKII␥ G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization. Key Words: CaMKII Ⅲ smooth muscle Ⅲ contractility Ⅲ targeting Ⅲ extracellular signal-regulated kinase C a 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase that is expressed as 4 different isoforms (␣, ␤, ␥, and ␦). Each member of this kinase family possesses 3 major domains ( Figure 1A): an N-terminal catalytic/regulatory domain containing a Ser/Thr kinase region and overlapping autoinhibitory and Ca 2ϩ /calmodulin binding regions, a central-linker domain containing variable regions, and a C-terminal association domain that promotes oligomerization. 1 CaMKII is not active until Ca 2ϩ /calmodulin binds to its regulatory domain. This binding allows CaMKII to undergo autophosphorylation at Thr287 (numbering according to the ␥ isoform), which, in turn, increases its affinity for Ca 2ϩ /calmodulin and allows kinase activity even in the absence of Ca 2ϩ /calmodulin. The ␣ and ␤ isoforms of CaMKII are predominantly expressed in neural tissue, 2 where they have been demonstrated to be involved in synaptic plasticity, memory, and learning. 3,4 In contrast, differentiated smooth muscle cells (dSMCs) express mainly the ␥ isoform of CaMKII, 5 which has been linked to contractile activity by the use of antisense and phar...

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with alpha-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with alpha-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.