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

SGK1-dependent stimulation of vascular smooth muscle cell osteo-/chondrogenic transdifferentiation by interleukin-18

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a key regulator of osteo-/chondrogenic transdifferentiation and subsequent calcification of vascular smooth muscle cells (VSMCs). The phenotypical transdifferentiation of VSMCs is associated with increased interleukin-18 (IL-18) levels and generalized inflammation. Therefore, the present study investigated the possible involvement of SGK1 in IL-18-induced vascular calcification. Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with recombinant human IL-18 protein in control or high phosphate conditions and following SGK1 knockdown by siRNA or pharmacological inhibition of SGK1, PI3K, and PDK1. As a result, IL-18 treatment increased SGK1 mRNA and protein expression in HAoSMCs. IL-18 upregulated SGK1 mRNA expression in a dose-dependent manner. This effect was paralleled by upregulation of the mRNA expression of MSX2 and CBFA1, osteogenic transcription factors, and of tissue-nonspecific alkaline phosphatase (ALPL), an osteogenic enzyme, as markers of increased osteo-/chondrogenic transdifferentiation. Phosphate treatment increased SGK1 and osteogenic markers mRNA expression as well as ALPL activity and induced calcification of HAoSMCs, all effects significantly augmented by additional treatment with IL-18. Conversely, silencing of SGK1 or cotreatment with the SGK1 inhibitor EMD638683 blunted the effects of IL-18 on osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. The procalcific effects of IL-18 were similarly suppressed in the presence of PI3K or PDK1 inhibitors. In conclusion, SGK1 expression is upregulated by IL-18 in VSMCs and SGK1 participates in the intracellular signaling of IL-18-induced osteo-/chondrogenic transdifferentiation of VSMCs. Thus, SGK1 may serve as therapeutic target to limit the progression of medial vascular calcification during vascular inflammation.(VLID)4848456Version of recor

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

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

International audienceAlthough 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