Zinc Inhibits HIF-Prolyl Hydroxylase Inhibitor-Aggravated VSMC Calcification Induced by High Phosphate

Abstract

Vascular calcification is a life-threatening clinical condition in chronic kidney disease (CKD) and is associated with reduced zinc serum levels. Anemia is another frequent complication of CKD. Hypoxia-inducible factor (HIF) stabilizers, also known as HIF prolyl hydroxylase inhibitors (PHI), are promising candidates to treat CKD-associated anemia by increasing erythropoietin synthesis. Recent evidence suggests that HIFs play a pivotal role in vascular calcification. Our study explored feasible impacts of HIF PHI on phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs) and tested whether zinc might inhibit this mineralization process. Treatment of VSMCs with PHI aggravated Pi-induced calcium deposition and Pi uptake. PHI promoted Pi-induced loss of smooth muscle cell markers (ACTA-2, MYH11, SM22α) and enhanced osteochondrogenic gene expression (Msx-2, BMP-2, Sp7) triggering osteochondrogenic phenotypic switch of VSMCs. These effects of PHI paralleled with increased pyruvate dehydrogenase kinase 4 (PDK4) expression, decreased Runx2 Ser451 phosphorylation, and reduced cell viability. Zinc inhibited Pi-induced mineralization of VSMCs in a dose-dependent manner and also attenuated the procalcification effect of PHI in Pi-induced mineralization. Zinc inhibited osteochondrogenic phenotypic switch of VSMCs reflected by lowering Pi uptake, decreasing the expressions of Msx-2, BMP-2, and Sp7 as well as the loss of smooth muscle cellspecific markers. Zinc preserved phosphorylation state of Runx2 Ser451, decreased PDK4 level, and restored cell viability. PHI alone reduced the expression of smooth muscle markers without inducing mineralization, which was also inhibited by zinc. In addition, we observed a significantly lower serum zinc level in CKD as well as in patients undergoing carotid endarterectomy compared to healthy individuals. Conclusion-PHI promoted the loss of smooth muscle markers and augmented

Association between Serum Zinc and Calcification Propensity (T50) in Patients with Type 2 Diabetes Mellitus and In Vitro Effect of Exogenous Zinc on T50

“…That study also demonstrated that zinc preserves the phosphorylation state of Runx2 and Ser451, decreases the level of pyruvate dehydrogenase kinase 4 (PDK4) level, and restores cell viability (Supplemental Figure S1). Together, the present findings of the effect of zinc on serum T 50 , and the previous reported in vitro effects of zinc on phosphate-induced calcification in VSMCs [38,51], are similar to the effects of magnesium [50]. Although approximately 40% of magnesium binds to albumin or forms a complex with anions, including bicarbonate, phosphate, and citrate, in blood [52], a previous in vitro experiment demonstrated that addition of exogenous magnesium from 0.5 to 1.5 mmol/L increased serum T 50 levels [19].…”

“…Similarly, zinc increases zinc finger protein TNF-α-induced protein 3 (TNFAIP3) expression, which subsequently inhibits NF-kB activation and osteo-/chondrogenic reprograming, resulting in suppression of phosphate-induced VSMCs calcification [38]. Another report noted that zinc also inhibits osteochondrogenic phenotypic switch of VSMCs, reflected by reduced phosphate uptake, thus decreasing osteochondrogenic gene expressions of Msx-2, BMP-2, and Sp7, as well as loss of smooth muscle cell-specific markers [51]. That study also demonstrated that zinc preserves the phosphorylation state of Runx2 and Ser451, decreases the level of pyruvate dehydrogenase kinase 4 (PDK4) level, and restores cell viability (Supplemental Figure S1).…”

“…The following are available online at , Figure S1: Schematic illustration of zinc and calcification; Table S1: Clinical characteristics of the pooled serum from healthy volunteers and patients undergoing hemodialysis. References [ 38 , 51 ] are cited in the supplementary materials.…”

Association of Zinc Deficiency with Development of CVD Events in Patients with CKD

“…In a recent in vitro study, zinc attenuated phosphate-induced osteochondrogenic phenotypic switch of vasculature smooth muscle cells (VSMCs) leading to development of vascular calcification [ 16 ]. Additionally, an in vivo study demonstrated that zinc can protect against phosphate-induced arterial calcification by inducing production of a zinc-finger protein, tumor necrosis factor (TNF)-a-induced protein 3 (TNFAIP3), and suppressing activation of nuclear factor kappa-light-chain-enhancer of activated B (NF-k B) [ 17 ].…”

Inflammation: a putative link between phosphate metabolism and cardiovascular disease

“…Zinc supplementation is able to blunt activation of NF-kB and osteo-/chondrogenic transdifferentiation in VSMCs as well as vascular calcification during high Pi conditions, via G protein-coupled receptor 39 (GPR39)-mediated induction of TNF α-induced protein 3 (TNFAIP3) [ 274 ]. Zinc treatment further abrogates the pro-calcific effects of HIF prolyl hydroxylase inhibitors on VSMCs [ 275 ]. A similar anti-calcific effect of zinc was observed in valvular interstitial cells [ 276 ].…”