Abstract
Abstract Background Chronic treatment with fetal bovine serum (FBS) causes gradual vasoconstriction, vascular wall thickening, and contractility reduction in organ-cultured vascular tissues. We have previously demonstrated that Rho-associated kinase (ROCK) inhibitors prevent the functional alterations of small arteries in response to the FBS treatment. Here, we tested a further hypothesis that the chronic inhibition of ROCK has a protective effect on FBS-induced structural alterations. Methods To verify the new hypothesis, the rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor and then western blot, immunohistochemistry, apoptosis assay, and electron microscopy were performed using organ-cultured arterial rings. Results Chronic treatment with Y-27632 maintained the arterial diameter by preventing FBS-induced gradual arterial constriction during organ culture. Y-27632 also reduced the apoptosis and the loss of contractile myosin and actin filaments of smooth muscle cells. In addition, Y-27632 protected the morphological integrity between the endothelial cell layer and smooth muscle cell layer by preventing endothelial cell detachment and platelet endothelial cell adhesion molecule (PECAM) expression decrement. Conclusions Chronic ROCK inhibition provides protective effects against FBS-stimulated structural in addition to functional alterations of vascular smooth muscle cells and endothelial cells. These results strongly suggest that the RhoA/ROCK signaling is crucial for maintaining the structural and functional phenotypes of vasculature, and hence, chronic ROCK inhibition may provide protective effects on excessive growth factor-related vascular diseases including hypertension and atherosclerosis.
Highlights
Chronic treatment with fetal bovine serum (FBS) causes gradual vasoconstriction, vascular wall thickening, and contractility reduction in organ-cultured vascular tissues
We report that the Rho-associated kinase (ROCK) inhibitor Y27632 prevents the detachment and loss of the endothelial cell layer, the disappearance of contractile thick and thin filaments, and apoptosis and necrosis. These results strongly suggest that the Ras homolog gene family member A (RhoA)/ROCK signaling is crucial for maintaining the structural and functional phenotypes of the vasculature and has the potential to be a therapeutic target in cardiovascular diseases
Effect of Y-27632 on the FBS-induced contraction of the organ-cultured artery To examine the morphological changes in the arterial rings during the FBS-supplemented organ culture, the isolated arterial rings were cultured for 7 days with 10% FBS-supplemented Dulbecco's modified Eagle's medium (DMEM) with or without 10 μM Y27632 (Figure 2A)
Summary
Chronic treatment with fetal bovine serum (FBS) causes gradual vasoconstriction, vascular wall thickening, and contractility reduction in organ-cultured vascular tissues. To address the underlying mechanisms by which the upregulation of RhoA/ROCK signaling causes abnormal contractility of the vascular smooth muscle and following vascular disease, various studies were carried out by the use of an animal model and isolated or cultured cell model. In vitro cell culture models have been used extensively to elucidate the molecular mechanism governed by RhoA/ROCK signaling with finely tuned culture conditions (Chen et al 2010; Singh et al 2011; Pagiatakis et al 2012). Organ culture of the vasculature has been used as an alternative method that can complement the limitations of animal- or cultured cell-based studies for investigating the chronic treatment effects of various inhibitors in arterial tissues (Ozaki and Karaki 2002; Thorne and Paul 2003; Huh et al 2011)
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