Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein

Abstract

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.

Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces

“…Actin polymerization can occur in 2 major ways in cytoskeletal structures: (1) by the activation of the Arp 2/3‐N‐WASP complex, which adds branched actin, or (2) by linear extension of the barbed end of actin filaments by the activity of the ena‐VASP family of cytoskeletal proteins 40. The latter has been shown by our group to occur in vascular smooth muscle in a regulated manner 26. Hence, we determined whether regulation of linear actin polymerization could be targeted to decrease ex vivo aortic stiffness.…”

“…This protein was bath loaded into aortic rings ex vivo and, as shown in Figure 2D, was effective in decreasing both stiffness (top panel) and stress (bottom panel) compared with control loading in young and aged mice. The control used was either a mutant (Phe78Ser) EVH1 domain, which has no effect on contractility,26 or sham loading with no peptide. No significant difference ( P <0.05) was seen between the use of these 2 controls on either stiffness or stress so their values are merged in Figure 2D.…”

“…VASP EVH1‐TAT recombinant protein was expressed in a pTYB1 vector (New England Biolabs, Ipswich, MA) and purified on an affinity based chitin resin column as published previously 26. Briefly, the EVH1‐TAT construct was cloned in a pTYB1 vector (New England Biolabs).…”

“…The stiffness and plasticity of this nonmuscle actin cytoskeleton are regulated by proteins that control branched and linear actin polymerization such as N‐WASP and VASP, respectively 20, 24, 25, 26. The FA complexes include transmembrane integrins that connect with the extracellular matrix of the aortic wall.…”

“…The nonmuscle actin cytoskeleton and FAs to which it is attached have been shown to display plasticity in the presence of agonists26 and biomechanical stimuli27, 28 by exhibiting stimulus‐induced increases in actin polymerization and endosomal‐dependent remodeling of a subset of FA proteins 29. Plasticity of the cortical cytoskeleton of VSMCs may contribute to the function of the healthy, compliant proximal aorta, acting as a tunable “shock absorber” that adapts in order to limit transmission of excessive pulsatile energy into the delicate downstream microvessels.…”