Table 1.
Clinical characteristics of the patients donating medium sized artery.
Fig 1.
Effect of cyclic biaxial strain and CaSR agonists on HAoSMC CaSR expression and phenotype.
A) Calcium deposition was measured with alizarin red staining (n = 4) for each condition. Data are represented on a logarithmic y-axis. Exposure to CaSR agonists (Ca5, Gd, Gd2+Ca5) significantly increased calcification, and this was reduced by cyclic strain. B) Representative photographs of alizarin red staining (magnification x100). C) OC expression was determined by ELISA, and corrected for protein concentration (n = 4 for each condition). In static HAoSMC, OC expression was significantly increased by Ca5, Gd, and Gd+Ca2. In comparison, cyclic strain significantly reduced OC expression compared to static cells under all conditions (Control p = 0.008; Ca2 p = 0.004; Ca5 p = 0.004; Gd p = 0.0004; Gd+Ca2 p = 0.002). D) ALP activity (n = 5 for each condition) increased with Ca5 in static cells, but decreased in the presence of Gadolinium. Under all conditions, cyclic strain reduced ALP activity compared to static HAoSMC (Control p = 0.008; Ca2 p = 0.0004; Ca5 p = 0.004; Gd p = 0.0002; Gd+Ca2 p = 0.001). E) CaSR protein expression (n = 4 for each condition) was reduced by Ca5, Gd and GD+Ca2, whereas cyclic strain increased CaSR expression and attenuated CaSR agonist-induced reductions in CaSR expression. OC–osteocalcin; ALP–alkaline phosphatase; CaSR–calcium-sensing receptor.
Fig 2.
Effect of CaSR silencing on HAoSMC calcification and phenotype.
A) Calcium deposition (measured by alizarin red, n = 4 per condition, logarithmic y-axis) was significantly higher after CaSR silencing in Ca5, Gd and Gd2+Ca2 HAoSMC. B) Representative photographs of alizarin red staining (magnification x100). C) OC expression (n = 5 per condition) was significantly increased by CaSR knockdown across all conditions (Control p = 0.004; Ca2 p = 0.004; Ca5 p = 0.0003; Gd p = 0.0003; Gd+Ca2 p = 0.0001). D) ALP activity (n = 4 per condition) was significantly higher with CaSR silencing compared to control in the presence of Gadolinium (Gd, p = 0.0005; Gd+Ca2, p = 0.01). OC–osteocalcin; ALP–alkaline phosphatase; CaSR–calcium-sensing receptor.
Fig 3.
Effect of CaSR over-expression and CaSR agonists on HAoSMC calcification and phenotype.
HAoSMC were transfected with pcDNA 3.1 CaSR+. A) An increase in CaSR mRNA and B) CaSR protein expression in HAoSMC transfected with pcDNA 3.1 CaSR+ after 7 and 14 day culture. C) Calcium deposition (measured by alizarin red, n = 4 per condition, logarithmic y-axis) was significantly lower after CaSR over-expression compared to controls in Ca5, Gd and Gd2+Ca2 HAoSMC. F) Representative photographs of alizarin red staining (magnification x100). D) OC expression (n = 4 per condition) was markedly lower with CaSR over-expression across all conditions. E) ALP activity (n = 4 per condition) was lower overall with CaSR over-expression, and significantly lower than the respective control in the presence of Ca5. OC–osteocalcin; ALP–alkaline phosphatase; CaSR–calcium-sensing receptor.
Fig 4.
Effect of calcimimetic R568 and extracellular calcium on HAoSMC calcification and phenotype.
Shading of box plots represents increasing concentrations (10, 100 and 1000 nmol/L) of R568. A) Calcium deposition (measured by alizarin red, n = 4 per condition, logarithmic y-axis) was reduced by R568 in a dose-dependent manner in the presence of Ca5, whereas the inactive enantiomer S568 had no effect on calcification. The effect of R568 was less pronounced under cyclic strain, where basal calcification is lower. E) Representative photographs of cells stained with alizarin red (magnification x100). B) OC expression (n = 4 per condition) was reduced by increasing concentrations of R568 in the presence of both Ca2 and Ca5. These effects were also evident under cyclic strain, although less pronounced. C) ALP activity (n = 4 per condition) was not significantly influenced by R568. D) CaSR expression (n = 4 per condition) was not convincingly altered by exposure to R568, alhough low doses (10nmol/L) appeared to attenuate Ca5-induced reduction in CaSR expression in static HAoSMC. OC–osteocalcin; ALP–alkaline phosphatase; CaSR–calcium-sensing receptor.
Fig 5.
Effect of R568 and CaSR agonists on calcium deposition and osteogenic markers in arterial explants.
Medium sized arterial explants from healthy individuals donating a kidney (control, n = 9) and patients with CKD (n = 11) undergoing a kidney transplant were cultured for 7 days. Shaded boxes represent exposure to R568 (100nmol/L). A) CaSR and B) Runx2 expression in Control and CKD artery (n = 4 per sample). CaSR expression was significantly lower in CKD patients (p = 0.0003), whereas Runx2 was significantly higher (p = 0.008). C) Total artery calcium content was not significantly different between control and CKD patients, and not significantly altered by R568. Expression of D) Runx2 (p = 0.0003) and E) DMP–1 (p = 0.0003) was significantly higher in CKD arteries compared to controls. R568 exposure did not significantly alter expression of these proteins. DMP–1 –dental matrix protein–1; CaSR–calcium-sensing receptor.