Zinc Ameliorates the Osteogenic Effects of High Glucose in Vascular Smooth Muscle Cells

Abstract

In diabetic patients, medial vascular calcification is common and associated with increased cardiovascular mortality. Excessive glucose concentrations can activate the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) and trigger pro-calcific effects in vascular smooth muscle cells (VSMCs), which may actively augment vascular calcification. Zinc is able to mitigate phosphate-induced VSMC calcification. Reduced serum zinc levels have been reported in diabetes mellitus. Therefore, in this study the effects of zinc supplementation were investigated in primary human aortic VSMCs exposed to excessive glucose concentrations. Zinc treatment was found to abrogate the stimulating effects of high glucose on VSMC calcification. Furthermore, zinc was found to blunt the increased expression of osteogenic and chondrogenic markers in high glucose-treated VSMCs. High glucose exposure was shown to activate NF-kB in VSMCs, an effect that was blunted by additional zinc treatment. Zinc was further found to increase the expression of TNFα-induced protein 3 (TNFAIP3) in high glucose-treated VSMCs. The silencing of TNFAIP3 was shown to abolish the protective effects of zinc on high glucose-induced NF-kB-dependent transcriptional activation, osteogenic marker expression, and the calcification of VSMCs. Silencing of the zinc-sensing receptor G protein-coupled receptor 39 (GPR39) was shown to abolish zinc-induced TNFAIP3 expression and the effects of zinc on high glucose-induced osteogenic marker expression. These observations indicate that zinc may be a protective factor during vascular calcification in hyperglycemic conditions.

Keywords: GPR39; NF-kB; diabetes mellitus; high glucose; osteogenic transition; vascular calcification; vascular smooth muscle cells; zinc.

Figure 1

Zinc sulfate inhibits the calcification of HAoSMCs promoted by high levels of glucose. (a). Calcium content (n = 6, µg/mg protein) in HAoSMCs treated for 11 days with control (CTR) or calcification medium (Calc.) without and with high glucose levels (HG) in the absence and presence of ZnSO₄. *** (p < 0.001) significant difference versus control group; † (p < 0.05), ††† (p < 0.001) significant difference versus Calc.-treated group; §§§ (p < 0.001) significant difference between Calc.+HG- and Calc.+HG+ZnSO₄-treated groups. (b). Calcification detected by fluorescence imaging in HAoSMCs treated for 11 days with control (CTR) or calcification medium (Calc.) without and with high glucose levels (HG) in the absence and presence of ZnSO₄. Calcified areas: white pseudocolor.

Figure 2

Zinc sulfate suppresses osteo-/chondrogenic marker expression promoted by high glucose levels in HAoSMCs. (a–c). Relative mRNA expression of CBFA1 (a), SOX9 (b), and ALPL (c) (n = 5; arbitrary units, a.u.) in HAoSMCs treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. (d). Representative Western blots and normalized CBFA1 protein expression (n = 9; a.u.) in HAoSMCs treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. (e). Normalized OPN levels (n = 10; a.u.) in conditioned medium from HAoSMCs treated for 7 days with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. (f). Normalized ALP activity (n = 5; a.u.) in HAoSMCs treated for 7 days with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. ** (p < 0.01), *** (p < 0.001) significant difference versus control group; † (p < 0.05), †† (p < 0.01), ††† (p < 0.001) significant difference versus the HG-treated group.

Figure 3

Zinc chloride inhibits osteogenic marker expression and the calcification of HAoSMCs induced by high levels of glucose. (a,b). Relative mRNA expression of CBFA1 (a) and ALPL (b) (n = 4; arbitrary units, a.u.) in HAoSMCs treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnCl₂. (c). Calcium content (n = 6, µg/mg protein) in HAoSMCs treated for 11 days with control (CTR) or calcification medium (Calc.) and high glucose levels (HG) in the absence and presence of ZnCl₂. * (p < 0.05), ** (p < 0.01) significant difference versus control group; † (p < 0.05), †† (p < 0.01) significant difference versus the HG-/Calc.+HG-treated group.

Figure 4

Zinc sulfate suppresses NF-kB activation induced by high levels of glucose in HAoSMCs. (a). Normalized NF-kB-dependent transcriptional activity (n = 4; arbitrary units, a.u.) in HAoSMCs treated for 30 min with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. (b). Relative mRNA expression of TNFAIP3 (n = 5; a.u.) in HAoSMCs treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. ** (p < 0.01), *** (p < 0.001) significant difference versus control group; † (p < 0.05) significant difference versus HG-treated group.

Figure 5

The silencing of TNFAIP3 blocks the effects of zinc sulfate on high glucose-induced NF-kB activation and osteogenic marker expression in HAoSMCs. (a). Relative mRNA expression of TNFAIP3 (n = 7; arbitrary units, a.u.) in HAoSMCs transfected with negative control (Neg.si) or TNFAIP3 (TNFAIP3si) siRNA and treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. * (p < 0.05) significant difference versus Neg.si-transfected CTR-treated group. (b). Normalized NF-kB-dependent transcriptional activity (n = 8; a.u.) in HAoSMCs transfected with negative control (Neg.si) or TNFAIP3 (TNFAIP3si) siRNA and treated for 30 min with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. (c,d). Relative mRNA expression of CBFA1 (c) and ALPL (d) (n = 7; a.u.) in HAoSMCs transfected with negative control (Neg.si) or TNFAIP3 (TNFAIP3si) siRNA and treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. * (p < 0.05), ** (p < 0.01), *** (p < 0.001) significant difference versus the Neg.si-transfected CTR-treated group; † (p < 0.05), †† (p < 0.01) significant difference between the Neg.si-transfected HG- and HG+ZnSO₄-treated groups; § (p < 0.05), §§ (p < 0.01) significant difference between the Neg.si- and TNFAIP3si-transfected HG+ZnSO₄-treated groups.

Figure 6

The silencing of TNFAIP3 interferes with the protective effects of zinc sulfate on the calcification of HAoSMCs under high-glucose conditions. Calcium content (n = 8, µg/mg protein) in HAoSMCs transfected with negative control (Neg.si) or TNFAIP3 (TNFAIP3si) siRNA and treated for 11 days with control (CTR) or calcification medium (Calc.) and high glucose levels (HG) in the absence and presence of ZnSO₄. * (p < 0.05) significant difference versus the Neg.si-transfected CTR-treated group; † (p < 0.05) significant difference between the Neg.si-transfected Calc.+HG- and Calc.+HG+ZnSO₄-treated groups; § (p < 0.05) significant difference between the Neg.si- and TNFAIP3si-transfected Calc.+HG+ZnSO₄-treated groups.

Figure 7

The silencing of GPR39 blunts the protective effects of zinc sulfate on high glucose-induced osteogenic marker expression in HAoSMCs. (a). Relative mRNA expression of GPR39 (n = 9; arbitrary units, a.u.) in HAoSMCs transfected with negative control (Neg.si) or GPR39 (GPR39si) siRNA and treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. ** (p < 0.01) significant difference versus the Neg.si-transfected CTR-treated group. (b–d). Relative mRNA expression of TNFAIP3 (b), CBFA1 (c), and ALPL (d) (n = 9; a.u.) in HAoSMCs transfected with negative control (Neg.si) or GPR39 (GPR39si) siRNA and treated for 24 h with control (CTR) or high glucose levels (HG) in the absence and presence of ZnSO₄. ** (p < 0.01), *** (p < 0.001) significant difference versus the Neg.si-transfected CTR-treated group; † (p < 0.05), ††† (p < 0.001) significant difference between the Neg.si-transfected HG- and HG+ZnSO₄-treated groups; § (p < 0.05), §§ (p < 0.01) significant difference between the Neg.si- and GPR39si-transfected HG+ZnSO₄-treated groups.