P221
LONG-TERM MECHANICAL STRAIN OF HUMAN VASCULAR SMOOTH MUSCLE
CELLS MAINTAINS CONTRACTILE PHENOTYPE, INHIBITS ALKALINE
PHOSPHATASE AND OSTEOCALCIN PRODUCTION AND PREVENTS
CALCIFICATION
Molostov, G¹, Bland, R² Zehnder, D¹
¹Clinical Sciences Research Institute and ²BioMedical Research Institute, University of
Warwick
Vascular smooth muscle cells (SMC) play a key role in the development of progressive arterial
calcification, which is responsible for premature cardiovascular mortality in patients with
chronic kidney disease. We have previously shown that SMC express a functional calciumsensing receptor (CaSR) and this study investigates the interaction of the CaSR and mechanical
strain on the SMC phenotype and calcification.
Human aortic SMC (HAoSMC) were cultured under static or cyclic biaxial strain (7% stretch,
30 cycles/min) conditions using Flexcell apparatus and collagen I coated dishes for up to 14
days in the presence of 5mM -glycerophosphate. CaSR and -actin (SMC marker) protein
expression were analysed by Western blot. Cells were stained with alizarin red to assess
calcification. Alkaline phosphatase (ALP) activity was measured using SensoLyte pNitrophenyl assay kit (AnaSpec). Osteocalcin (OC) production was assayed by N-MID
osteocalcin ELISA (IDS). Statistical analysis was performed using one-way ANOVA followed
by Tukey’s multiple comparison tests.
Culture of HAoSMC under cyclic strain for 14 days resulted in a significant up-regulation of actin expression by days 7 and 10 (maximum 23%, p<0.05) compared to control static cultures.
This was accompanied by a 45% increase by day 14 of CaSR expression (p<0.05). 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: 2 and 5mM Ca2+ or
50M Gd3+ alone or in combination. Treatment of static HAoSMC with Ca2+, Gd3+ or both for 7
days induced a marked down-regulation (p<0.01) of CaSR expression and a dramatic upregulation (p<0.01) of HAoSMC calcification. Interestingly, ALP production was increased in
cells treated with Ca2+ and decreased in Gd3+-treated cultures (p<0.05), whereas OC levels were
markedly induced by both 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).
To further examine the role of the CaSR, its expression was knocked-down using CaSR siRNA
(Santa Cruz). This resulted in a further increase in calcification in Ca2+ and Gd3+-treated cells
when compared to untransfected cells (p<0.05). ALP levels tended to be higher in CaSR siRNA
transfected cells, however, this was only significant in cells treated with 50M Gd3+ alone or in
combination with 2mM Ca2+ (p<0.05). Importantly, there was a pronounced up-regulation of
OC production in CaSR knockdown cells, both in control and in agonist-treated HAoSMC
(p<0.01).
These findings indicate that long-term static culture of HAoSMC results in a phenotypic change
towards a calcification-promoting phenotype, which is accompanied by increased ALP and OC,
increased calcification and reduced CaSR expression. Cyclic strain protected against these
changes and maintained a contractile SMC phenotype. In contrast, CaSR knockdown enhanced
Ca2+ and Gd3+-induced ALP and OC production and the concomitant calcification. This suggests
that a functional CaSR may serve to prevent a shift towards a calcifying SMC phenotype and
therefore protect against calcification.