In this study, we demonstrated several significant or novel findings. Firstly, cyclic stretch upregulates myocardin expression in rat VSMCs; secondly, cyclic stretch induces AngII expression in VSMCs; thirdly, AngII acts as an autocrine factor to mediate the increased myocardin expression induced by cyclic stretch; fourthly, ERK MAP kinase and SRF transcriptional factor are involved in the signaling pathway of myocardin induction; and fifthly, in vivo acute hemodynamic overload increases aortic myocardin expression. Myocardin was upregulated in both a time- and load- dependent manner by cyclic stretch. Cyclic stretch of VSMCs increased both myocardin protein and mRNA expression.
In our study, exogenous addition of AngII to non-stretched VSMCs was also sufficient to induce similar myocardin protein expression as that observed in stretched VSMCs. These results provide the first evidence that AngII mediates cyclic stretch-induced expression of myocardin in VSMCs. Our study revealed that AngII acts as an autocrine mediator in response to cyclic stretch in VSMCs. Previously, another study identified that AngII enhanced myocardin expression through AngII type 1 (AT1) receptor results in VSMC hypertrophy . We also have previously demonstrated that hypoxia in cardiomyocytes increased AngII secretion and myocardin expression and finally resulted in cardiac myocyte hypertrophy through the ERK pathway . In this study, we found that cyclic stretch also enhanced myocardin expression by AngII secretion and the ERK pathway, which had not been identified by previous studies.
However, one study found no increased concentration of AngII in the medium collected from porcine VSMCs at 24 and 48 h after 25% stretch . Sotoudeh et al. used pulmonary VSMCs, whereas our study used rat aortic VSMCs. Different species, stretch intension, and stretch time may explain the discrepancy. Our results suggest that AngII is responsible for myocardin-DNA binding in VSMCs. In this study, we demonstrated that cyclic stretch stimulation of myocardin-DNA binding activity required at least phosphorylation of ERK since ERK pathway inhibitor (PD98059) and ERK siRNA abolished the myocardin/SRF binding activity. PD98059, a potent and specific inhibitor of ERK MAP kinase, also inhibited the myocardin expression induced by stretch, whereas inhibitors of p42/p44, p38, and c-JUN MAP kinase did not have this inhibitory effect. Thus, ERK MAP kinase is an important intracellular signaling pathway that regulates myocardin expression. We also demonstrated that ERK siRNA significantly inhibited myocardin expression induced by stretch. ARB likewise had an inhibitory effect on the stretch-induced myocardin expression. Since ARB is an AngII inhibitor, and mechanical stretch is known to affect the production of AngII, , our findings potentially indicate that AngII has a role in the induction of myocardin by mechanical stretch. In this study, we demonstrated via promoter activity assay that increased transcriptional activity of myocardin promoter by cyclic stretch was SRF dependent. These data imply that the ERK MAP kinase pathway, but not the other MAP kinase pathway, is the major pathway involved in the induction of myocardin by stretch and that it mediates the increased binding activity of myocardin and transcription to VSMCs.
Mechanical stretch can modulate several different cellular functions in VSMCs. These functions include cell alignment and differentiation, migration, survival or apoptosis, vascular remodeling, and autocrine or paracrine functions . However, use of different kinds of VSMCs (venous or arterial) and various species of animals used in different studies (mouse, rat, rabbit, swine and others), have resulted in sometimes controversial findings . Most of these studies used in vitro models. However, the cellular functions induced by in vitro mechanical stretch may not accurately represent cellular function in vivo. So, more studies are necessary to identify the real effects of mechanical stretch on VSMC functions and the mechanisms by which they occurr. Our study further confirmed the increased aortic myocardin expression in acute hemodynamic overload as that occurring with aorta-caval shunts. It has been previously reported that myocardin protein expression increased in the carotid artery balloon injury model in rats , suggesting myocardin may be enhanced during acute hemodynamic overload in vivo. The increased myocardin protein expression following acute hemodynamic overload may contribute to the regulation of vascular repair and remodeling, which involves VSMC proliferation .
With regard to the clinical application of cyclic stretch on VSMCs, mechanical stretch activates multiple intracellular signaling networks and regulates gene expressions and functional responses in VSMCs. The cellular and molecular effects of mechanical stretch on vascular cells may provide new insights in the pathogenesis of vascular diseases and therapeutic potentials. Mechanical stretch can modulate several different cellular functions in VSMCs, including cell alignment and differentiation, migration, survival or apoptosis, vascular remodeling, and autocrine and paracrine functions. Arterial VSMCs are aligned primarily in the circumferential direction in the media of the artery. Mechanical stretch from pulsatile blood flow is one of the key factors in regulating vascular remodeling. VSMC migration is important in the development of vascular diseases, including atherosclerosis and post-angioplasty restenosis. VSMC migration is found more frequently in curved and bifurcating blood vessels, which are exposed to non-laminar blood flow, than in straight arterial segments exposed to laminar blood flow. In this, we have also demonstrated that mechanical stretch increased the migration of VSMCs. The gene expression induced by mechanical stretch may be relevant to pathological complications in the cardiovascular system, including atherosclerosis, plaque instability and hypertension. The induction of genes by mechanical stretch may play a role in vascular remodeling. Understanding the molecular mechanisms regulating VSMC remodeling, migration, and proliferation under mechanical stretch supports the clinical application of ACEI (angiotensin-converting enzyme inhibitors), ARB, and statin in cardiac protection and in the prevention of vascular diseases. Therefore knowledge of the impact of mechanical stretch on VSMCs is vital in the understanding of the pathogenesis of cardiovascular diseases and is crucial in providing new insights into the prevention and therapy of cardiovascular diseases.