Acid sphingomyelinase promotes SGK1-dependent vascular calcification.

In chronic kidney disease (CKD), hyperphosphatemia is a key factor promoting medial vascular calcification, a common complication associated with cardiovascular events and high mortality. Vascular calcification involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), but the complex signaling events inducing pro-calcific pathways are incompletely understood. The present study investigated the role of acid sphingomyelinase (ASM)/ceramide as regulator of VSMC calcification. In vitro, both, bacterial sphingomyelinase and phosphate increased ceramide levels in VSMCs. Bacterial sphingomyelinase as well as ceramide supplementation stimulated osteo-/chondrogenic transdifferentiation during control and high phosphate conditions and augmented phosphate-induced calcification of VSMCs. Silencing of serum- and glucocorticoid-inducible kinase 1 (SGK1) blunted the pro-calcific effects of bacterial sphingomyelinase or ceramide. Asm deficiency blunted vascular calcification in a cholecalciferol-overload mouse model and ex vivo isolated-perfused arteries. In addition, Asm deficiency suppressed phosphate-induced osteo-/chondrogenic signaling and calcification of cultured VSMCs. Treatment with the functional ASM inhibitors amitriptyline or fendiline strongly blunted pro-calcific signaling pathways in vitro and in vivo. In conclusion, ASM/ceramide is a critical upstream regulator of vascular calcification, at least partly, through SGK1-dependent signaling. Thus, ASM inhibition by repurposing functional ASM inhibitors to reduce the progression of vascular calcification during CKD warrants further study.

Fibulin-3 Attenuates Phosphate-Induced Vascular Smooth Muscle Cell Calcification by Inhibition of Oxidative Stress.

BACKGROUND/AIMS: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. METHODS: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. RESULTS: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. CONCLUSIONS: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.