Sphingosine 1-phosphate activation of ERM contributes to vascular calcification

Abstract

Vascular calcification is the deposition of mineral in the artery wall by vascular smooth muscle cells (VSMCs) in response to pathological stimuli. The process is similar to bone formation and is an independent risk factor for cardiovascular disease. Given that ceramide and sphingosine 1-phosphate (S1P) are involved in cardiovascular pathophysiology and biomineralization, their role in VSMC matrix mineralization was investigated. During phosphate-induced VSMC mineralization, endogenous S1P levels increased accompanied by increased sphingosine kinase (SK) activity and increased mRNA expression of SK1 and SK2. Consistent with this, mineralization was increased by exogenous S1P, but decreased by C2-ceramide. Mechanistically, exogenous S1P stimulated ezrin-radixin-moesin (ERM) phosphorylation in VSMCs and ERM phosphorylation was increased concomitantly with endogenous S1P during mineralization. Moreover, inhibition of acid sphingomyelinase and ceramidase with desipramine prevented increased S1P levels, ERM activation, and mineralization. Finally, pharmacological inhibition of ERM phosphorylation with NSC663894 decreased mineralization induced by phosphate and exogenous S1P. Although further studies will be needed to verify these findings in vivo, this study defines a novel role for the SK-S1P-ERM pathways in phosphate-induced VSMC matrix mineralization and shows that blocking these pathways with pharmacological inhibitors reduces mineralization. These results may inform new therapeutic approaches to inhibit or delay vascular calcification.

FTI-277 inhibits smooth muscle cell calcification by up-regulating PI3K/Akt signaling and inhibiting apoptosis

“…We did this using active Ras pull-down assays as decreased Ras activation is a well-accepted read-out for this inhibitor ( S1 Fig ). Next, we investigated the effects of FTI-277 on the osteogenic differentiation and mineralization of VSMC by using a well-established in vitro model of vascular calcification in which confluent cells are incubated in the presence of βGP [ 17 , 18 , 20 , 21 ]. These studies demonstrated that FTI-277 prevents the βGP-induced increase in the mRNA expression of the osteogenic transcription factors Runx2 ( Fig 1A ) and Msx2 ( Fig 1B ) , reduces the βGP-induced decrease in αSMA mRNA expression ( Fig 1C ) and significantly increases the mRNA levels of matrix Gla protein (an inhibitor of mineralization [ 26 ]) ( Fig 1D ).…”

“…VSMC were plated in 6-well plates at 2 x 10 4 cells / cm 2 and maintained in 10% FCS-DMEM until confluent (day 0). At this point, the cells were cultured in 10% FCS-DMEM containing β-glycerophosphate (βGP, 3–5 mM [ 17 , 18 , 20 , 21 ]) and either FTI-277 (1–20 μM), manumycin A (10 or 20 μM), SH6 (Akt inhibitor, 10 μM) or combinations thereof for up to 12 days; control cells were incubated with βGP but with vehicle. Additional controls included cells incubated in 10% FCS-DMEM plus vehicle but without βGP.…”

“…Mineralization was assessed using alizarin red staining; images were captured using a digital camera and analyzed using AnalySIS software (Soft Imaging System, Germany). Mineralization was quantified by eluting the alizarin red dye as previously described [ 17 , 18 , 20 , 21 ].…”

Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice

“…Morris et al determined the role of ezrin-radixin-moesin (ERM) proteins as important downstream effectors of sphingosine-1-phosphate (S1P)-induced VSMC matrix mineralization in vitro. The study used pharmacological inhibitors such as desipramine for the inhibition of acid sphingomyelinase (ASM) and ceramidase to elucidate the role of sphingolipids in vitro, but lacking genetic manipulations and in vivo animal model approach in this context 49 . Of course, involvement of various other pathways cannot be excluded, including inflammatory pathways and oxidative stress pathways involving in ceramide-induced vascular complications.…”

Conserved and Divergent Modulation of Calcification in Atherosclerosis and Aortic Valve Disease by Tissue Extracellular Vesicles

“…(39) Pathway analysis on the predicted targets of these miRs strongly implicated RUNX2, toll-like receptors, and mechanotransduction as key downstream pathological effectors of vascular EVs, while valvular EV miRs were linked to IL-17 and sphingolipid signaling pathways that are involved in valvular inflammatory cell infiltration and osteogenesis/calcification. (40)…”