Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, C18 and PC18
Induction of calcium-sensing receptor expression in human vascular smooth muscle cells by mechanical strain prevents calcification
G. Molostvov1, D. Zehnder1, R. Bland1
1. Warwick Medical School, The University of Warwick, Coventry, United Kingdom.
Vascular smooth muscle cells (SMC) play a crucial role in the development of medial arterial calcification, contributing to premature cardiovascular mortality in chronic kidney disease patients. We have previously shown that human aortic SMC (HAoSMC) express a functional calcium-sensing receptor (CaSR) and we have demonstrated a correlation between CaSR expression and calcification(1). This study investigated the effect of mechanical strain on CaSR expression, SMC phenotype and the development of vascular calcification. HAoSMC were cultured under static or cyclic strain conditions using Flexcell apparatus (7% stretch, 30 cycles/min) for up to 14 days. HAoSMC phenotype was quantified by expression of smooth muscle α-actin (α-SM-actin; western blotting), alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion. Calcification was assessed by alizarin red staining. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison tests; data are presented as mean ± S.D. (n=3). Culture of HAoSMC in static conditions resulted in a gradual decline in α-SM-actin expression, which decreased by 35% (p<0.05) after 2 weeks and >60% (p<0.01) after 4 weeks of culture. This was accompanied by a reduction in CaSR protein expression (p<0.05). Culture under cyclic strain prevented these changes and resulted in up-regulation of α-SM-actin expression by days 7 and 10 (23%, p<0.05) and a 45% increase in CaSR expression (p<0.05) compared to static cultures. Alizarin red staining of day 7 and 14 cultures revealed significantly smaller areas of calcification in strained cells compared to control cultures (p<0.05). To assess the role of the CaSR, cells were treated with CaSR agonists. Treatment of static HAoSMC with 2mM and 5mM Ca2+, 50μM Gd3+ alone or in combination for 7 days induced a significant down-regulation (p<0.01) of CaSR expression and a dramatic up-regulation of HAoSMC calcification (p<0.01). ALP and OC were increased in cells treated with both CaSR agonists (p<0.05). In HAoSMC cultured under cyclic strain, the Ca2+ and Gd3+-induced down-regulation of CaSR expression and increased calcification were significantly attenuated (p<0.05 to p<0.001). In addition, cyclic strain induced a significant down-regulation of ALP and OC production in both control and CaSR agonist-treated cells (p<0.01). CaSR knockdown with CaSR siRNA resulted in a further increase in calcification in Ca2+ and Gd3+-treated cells compared to untransfected cells (p<0.05). Importantly, this was accompanied by up-regulation of ALP and OC production in control and agonist-treated CaSR knockdown cells (p<0.01). Our findings indicate that maintaining expression of a functional CaSR may serve to prevent a shift towards calcifying SMC phenotype and protect against calcification.
Where applicable, experiments conform with Society ethical requirements