In chronic kidney disease, hyperphosphatemia is a key pathological factor promoting medial vascular calcification, a common complication associated with cardiovascular events and mortality. This active pathophysiological process involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) via complex intracellular mechanisms that are still incompletely understood. Little is known about the effects of phosphate on the bioenergetic profile of VSMCs during the onset of this process. Therefore, the present study explored the effects of the phosphate donor β-glycerophosphate on cellular bioenergetics of VSMCs. Mitochondrial and glycolytic functions were determined utilizing extracellular flux analysis in primary human aortic VSMCs following exposure to β-glycerophosphate. In VSMCs, β-glycerophosphate increased basal respiration, mitochondrial ATP production as well as proton leak and decreased spare respiratory capacity and coupling efficiency, but did not modify non-mitochondrial or maximal respiration. β-Glycerophosphate-treated VSMCs had higher ability to increase mitochondrial glutamine and long-chain fatty acid usage as oxidation substrates to meet their energy demand. β-Glycerophosphate did not modify glycolytic function or basal and glycolytic proton efflux rate. In contrast, β-glycerophosphate increased non-glycolytic acidification. β-Glycerophosphate-treated VSMCs had a more oxidative and less glycolytic phenotype, but a reduced ability to respond to stressed conditions via mitochondrial respiration. Moreover, compounds targeting components of mitochondrial respiration modulated β-glycerophosphate-induced oxidative stress, osteo-/chondrogenic signalling and mineralization of VSMCs. In conclusion, β-glycerophosphate modifies key parameters of mitochondrial function and cellular bioenergetics in VSMCs that may contribute to the onset of phenotypical transdifferentiation and calcification. These observations advance the understanding of the role of energy metabolism in VSMC physiology and pathophysiology of vascular calcification during hyperphosphatemia. Key messages: β-Glycerophosphate modifies key parameters of mitochondrial respiration in VSMCs. β-Glycerophosphate induces changes in mitochondrial fuel choice in VSMCs. β-Glycerophosphate promotes a more oxidative and less glycolytic phenotype of VSMCs. β-Glycerophosphate triggers mitochondrial-dependent oxidative stress in VSMCs. Bioenergetics impact β-glycerophosphate-induced VSMC calcification.

In diabetes mellitus, hyperglycemia promotes the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) to enhance medial vascular calcification, a common complication strongly associated with cardiovascular disease and mortality. The mechanisms involved are, however, still poorly understood. Therefore, the present study explored the potential role of serum- and glucocorticoid-inducible kinase 1 (SGK1) during vascular calcification promoted by hyperglycemic conditions. Exposure to high-glucose conditions up-regulated the SGK1 expression in primary human aortic VSMCs. High glucose increased osteogenic marker expression and activity and, thus, promoted the osteogenic transdifferentiation of VSMCs, effects significantly suppressed by additional treatment with the SGK1 inhibitor EMD638683. Moreover, high glucose augmented the mineralization of VSMCs in the presence of calcification medium, effects again significantly reduced by SGK1 inhibition. Similarly, SGK1 knockdown blunted the high glucose-induced osteogenic transdifferentiation of VSMCs. The osteoinductive signaling promoted by high glucose required SGK1-dependent NF-κB activation. In addition, advanced glycation end products (AGEs) increased the SGK1 expression in VSMCs, and SGK1 inhibition was able to interfere with AGEs-induced osteogenic signaling. In conclusion, SGK1 is up-regulated and mediates, at least partly, the osteogenic transdifferentiation and calcification of VSMCs during hyperglycemic conditions. Thus, SGK1 inhibition may reduce the development of vascular calcification promoted by hyperglycemia in diabetes.

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.

Medial vascular calcification is common in chronic kidney disease (CKD) and is closely linked to hyperphosphatemia. Vascular smooth muscle cells (VSMCs) can take up pro-calcific properties and actively augment vascular calcification. Various pro-inflammatory mediators are able to promote VSMC calcification. In this study, we investigated the effects and mechanisms of periostin, a matricellular signaling protein, in calcifying human VSMCs and human serum samples. As a result, periostin induced the mRNA expression of pro-calcific markers in VSMCs. Furthermore, periostin augmented the effects of β-glycerophosphate on the expression of pro-calcific markers and aggravated the calcification of VSMCs. A periostin treatment was associated with an increased β-catenin abundance as well as the expression of target genes. The pro-calcific effects of periostin were ameliorated by WNT/β-catenin pathway inhibitors. Moreover, a co-treatment with an integrin αvβ3-blocking antibody blunted the pro-calcific effects of periostin. The silencing of periostin reduced the effects of β-glycerophosphate on the expression of pro-calcific markers and the calcification of VSMCs. Elevated serum periostin levels were observed in hemodialysis patients compared with healthy controls. These observations identified periostin as an augmentative factor in VSMC calcification. The pro-calcific effects of periostin involve integrin αvβ3 and the activation of the WNT/β-catenin pathway. Thus, the inhibition of periostin may be beneficial to reduce the burden of vascular calcification in CKD patients.

In chronic kidney disease (CKD), hyperphosphatemia promotes medial vascular calcification, a process augmented by osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). VSMC function is regulated by sympathetic innervation, and these cells express α- and β-adrenergic receptors. The present study explored the effects of β2-adrenergic stimulation by isoproterenol on VSMC calcification. Experiments were performed in primary human aortic VSMCs treated with isoproterenol during control or high phosphate conditions. As a result, isoproterenol dose dependently up-regulated the expression of osteogenic markers core-binding factor α-1 (CBFA1) and tissue-nonspecific alkaline phosphatase (ALPL) in VSMCs. Furthermore, prolonged isoproterenol exposure augmented phosphate-induced calcification of VSMCs. Isoproterenol increased the activation of PKA and CREB, while knockdown of the PKA catalytic subunit α (PRKACA) or of CREB1 genes was able to suppress the pro-calcific effects of isoproterenol in VSMCs. β2-adrenergic receptor silencing or inhibition with the selective antagonist ICI 118,551 blocked isoproterenol-induced osteogenic signalling in VSMCs. The present observations imply a pro-calcific effect of β2-adrenergic overstimulation in VSMCs, which is mediated, at least partly, by PKA/CREB signalling. These observations may support a link between sympathetic overactivity in CKD and vascular calcification.