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.

Role of Cytosolic Serine Hydroxymethyl Transferase 1 (SHMT1) in Phosphate-Induced Vascular Smooth Muscle Cell Calcification.

BACKGROUND/AIMS: Hyperphosphatemia promotes medial vascular calcification, at least partly, by induction of osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). The complex signaling pathways regulating this process are still incompletely understood. The present study investigated the role of cytosolic serine hydroxymethyl transferase 1 (SHMT1) in phosphate-induced vascular calcification. METHODS: Endogenous expression of SHMT1 was suppressed by silencing in primary human aortic smooth muscle cells (HAoSMCs) followed by treatment without and with phosphate or antioxidants. RESULTS: In HAoSMCs, SHMT1 mRNA expression was up-regulated by phosphate. Silencing of SHMT1 alone was sufficient to induce osteo-/chondrogenic transdifferentiation of HAoSMCs, as shown by increased tissue-nonspecific alkaline phosphatase (ALPL) activity and osteogenic markers MSX2, CBFA1 and ALPL mRNA expression. Furthermore, phosphate-induced ALPL mRNA expression and activity as well as calcification were augmented in SHMT1 silenced HAoSMCs as compared to negative control siRNA transfected HAoSMCs. Silencing of SHMT1 decreased total antioxidant capacity and up-regulated NADH/NADPH oxidase system components NOX4 and CYBA mRNA expression in HAoSMCs, effects paralleled by increased mRNA expression of matrix metalloproteinase MMP2 as well as BAX/BCL2 ratio. More importantly, additional treatment with antioxidants TEMPOL or TIRON blunted the increased osteogenic markers mRNA expression in SHMT1 silenced HAoSMCs. CONCLUSION: Silencing of SHMT1 promotes osteo-/chondrogenic signaling in VSMCs, at least in part, by inducing cellular oxidative stress. It thus aggravates phosphate-induced calcification of VSMCs. The present findings support a regulatory role of SHMT1 in vascular calcification during conditions of hyperphosphatemia such as chronic kidney disease.

Periostin Augments Vascular Smooth Muscle Cell Calcification via ß-Catenin Signaling.

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 ß-glycerophosphate on the expression of pro-calcific markers and aggravated the calcification of VSMCs. A periostin treatment was associated with an increased ß-catenin abundance as well as the expression of target genes. The pro-calcific effects of periostin were ameliorated by WNT/ß-catenin pathway inhibitors. Moreover, a co-treatment with an integrin αvß3-blocking antibody blunted the pro-calcific effects of periostin. The silencing of periostin reduced the effects of ß-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 αvß3 and the activation of the WNT/ß-catenin pathway. Thus, the inhibition of periostin may be beneficial to reduce the burden of vascular calcification in CKD patients.

Inhibition of vascular smooth muscle cell calcification by vasorin through interference with TGFß1 signaling.

Elevated transforming growth factor ß1 (TGFß1) levels are frequently observed in chronic kidney disease (CKD) patients. TGFß1 contributes to development of medial vascular calcification during hyperphosphatemia, a pathological process promoted by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Vasorin is a transmembrane glycoprotein highly expressed in VSMCs, which is able to bind TGFß to inhibit TGFß signaling. Thus, the present study explored the effects of vasorin on osteo-/chondrogenic transdifferentiation and calcification of VSMCs. Primary human aortic smooth muscle cells (HAoSMCs) were treated with recombinant human TGFß1 or ß-glycerophosphate without or with recombinant human vasorin or vasorin gene silencing by siRNA. As a result, TGFß1 down-regulated vasorin mRNA expression in HAoSMCs. Vasorin supplementation inhibited TGFß1-induced pathway activation, SMAD2 phosphorylation and downstream target genes expression in HAoSMCs. Furthermore, treatment with exogenous vasorin blunted, while vasorin knockdown augmented TGFß1-induced osteo-/chondrogenic transdifferentiation of HAoSMCs. In addition, phosphate down-regulated vasorin mRNA expression in HAoSMCs. Phosphate-induced TGFß1 expression was not affected by addition of exogenous vasorin. Nonetheless, the phosphate-induced TGFß1 signaling, osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs were all blunted by vasorin. Conversely, silencing of vasorin aggravated osteoinduction in HAoSMCs during high phosphate conditions. Aortic vasorin expression was reduced in the hyperphosphatemic klotho-hypomorphic mouse model of CKD-related vascular calcification. In conclusion, vasorin, which suppresses TGFß1 signaling and protects against osteo-/chondrogenic transdifferentiation and calcification of VSMCs, is reduced by pro-calcifying conditions. Thus, vasorin is a novel key regulator of VSMC calcification and may represent a potential therapeutic target for vascular calcification during CKD.

Impact of C-reactive protein on osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells.

Medial vascular calcification occurs during the aging process and is strongly accelerated by chronic kidney disease (CKD). Elevated C-reactive protein (CRP) levels are associated with vascular calcification, cardiovascular events and mortality in CKD patients. CRP is an important promoter of vascular inflammation. Inflammatory processes are critically involved in initiation and progression of vascular calcification. Thus, the present study explored a possible impact of CRP on vascular calcification. We found that CRP promoted osteo-/chondrogenic transdifferentiation and aggravated phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary human aortic smooth muscle cells (HAoSMCs). These effects were paralleled by increased cellular oxidative stress and corresponding pro-calcific downstream-signaling. Antioxidants or p38 MAPK inhibition suppressed CRP-induced osteo-/chondrogenic signaling and mineralization. Furthermore, silencing of Fc fragment of IgG receptor IIa (FCGR2A) blunted the pro-calcific effects of CRP. Vascular CRP expression was increased in the klotho-hypomorphic mouse model of aging as well as in HAoSMCs during calcifying conditions. In conclusion, CRP augments osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells through mechanisms involving FCGR2A-dependent induction of oxidative stress. Thus, systemic inflammation may actively contribute to the progression of vascular calcification.

Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment.

Although cardiovascular disease (CVD) is the leading cause of morbimortality worldwide, promising new drug candidates are lacking. We compared the arterial high-resolution proteome of patients with advanced versus early-stage CVD to predict, from a library of small bioactive molecules, drug candidates able to reverse this disease signature. Of the approximately 4000 identified proteins, 100 proteins were upregulated and 52 were downregulated in advanced-stage CVD. Arachidonyl trifluoromethyl ketone (AACOCF3), a cytosolic phospholipase A2 (cPLA2) inhibitor was predicted as the top drug able to reverse the advanced-stage CVD signature. Vascular cPLA2 expression was increased in patients with advanced-stage CVD. Treatment with AACOCF3 significantly reduced vascular calcification in a cholecalciferol-overload mouse model and inhibited osteoinductive signaling in vivo and in vitro in human aortic smooth muscle cells. In conclusion, using a systems biology approach, we have identified a potentially new compound that prevented typical vascular calcification in CVD in vivo. Apart from the clear effect of this approach in CVD, such strategy should also be able to generate novel drug candidates in other complex diseases.

SGK1 induces vascular smooth muscle cell calcification through NF-κB signaling.

Medial vascular calcification, associated with enhanced mortality in chronic kidney disease (CKD), is fostered by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Here, we describe that serum- and glucocorticoid-inducible kinase 1 (SGK1) was upregulated in VSMCs under calcifying conditions. In primary human aortic VSMCs, overexpression of constitutively active SGK1S422D, but not inactive SGK1K127N, upregulated osteo-/chondrogenic marker expression and activity, effects pointing to increased osteo-/chondrogenic transdifferentiation. SGK1S422D induced nuclear translocation and increased transcriptional activity of NF-κB. Silencing or pharmacological inhibition of IKK abrogated the osteoinductive effects of SGK1S422D. Genetic deficiency, silencing, and pharmacological inhibition of SGK1 dissipated phosphate-induced calcification and osteo-/chondrogenic transdifferentiation of VSMCs. Aortic calcification, stiffness, and osteo-/chondrogenic transdifferentiation in mice following cholecalciferol overload were strongly reduced by genetic knockout or pharmacological inhibition of Sgk1 by EMD638683. Similarly, Sgk1 deficiency blunted vascular calcification in apolipoprotein E-deficient mice after subtotal nephrectomy. Treatment of human aortic smooth muscle cells with serum from uremic patients induced osteo-/chondrogenic transdifferentiation, effects ameliorated by EMD638683. These observations identified SGK1 as a key regulator of vascular calcification. SGK1 promoted vascular calcification, at least partly, via NF-κB activation. Inhibition of SGK1 may, thus, reduce the burden of vascular calcification in CKD.