Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

Abstract

Vascular calcification is associated with significant cardiovascular morbidity and mortality, and has been demonstrated as an actively regulated process resembling bone formation. Oxidized low density lipoprotein (Ox-LDL) has been identified as a regulatory factor involved in calcification of vascular smooth muscle cells (VSMCs). Additionally, over-expression of recombinant human neutral sphingomyelinase (N-SMase) has been shown to stimulate VSMC apoptosis, which plays an important role in the progression of vascular calcification. The aim of this study is to investigate whether ceramide regulates Ox-LDL-induced calcification of VSMCs via activation of p38 mitogen-activated protein kinase (MAPK) pathway. Ox-LDL increased the activity of N-SMase and the level of ceramide in cultured VSMCs. Calcification and the osteogenic transcription factor, Msx2 mRNA expression were reduced by N-SMase inhibitor, GW4869 in the presence of Ox-LDL. Usage of GW4869 inhibited Ox-LDL-induced apoptosis in VSMCs, an effect which was reversed by C2-ceramide. Additionally, C2-ceramide treatment accelerated VSMC calcification, with a concomitant increase in ALP activity. Furthermore, C2-ceramide treatment enhanced Ox-LDL-induced VSMC calcification. Addition of caspase inhibitor, ZVAD-fmk attenuated Ox-LDL-induced calcification. Both Ox-LDL and C2-ceramide treatment increased the phosphorylation of p38 MAPK. Inhibition of p38 MAPK by SB203580 attenuated Ox-LDL-induced calcification of VSMCs. These data suggest that Ox-LDL activates N-SMase-ceramide signaling pathway, and stimulates phosphorylation of p38 MAPK, leading to apoptosis in VSMCs, which initiates VSMC calcification.

Suppression of EC‐SOD by oxLDL During Vascular Smooth Muscle Cell Proliferation

“…C). Previous studies reported that a treatment with 100 μg/mL oxLDL induced cell apoptosis and calcification, while that with 50 μg/mL oxLDL induced cell migration and proliferation in VSMCs [Zhao et al, ; Ding et al, ; Liao et al, ]. In the present study, 100 μg/mL oxLDL, but not 50 μg/mL significantly induced cell damage from the results of MTT assay, LDH release and trypan blue staining (Fig.…”

“…and ). A treatment with 100 μg/mL oxLDL has been reported to induce apoptosis in and the calcification of VSMCs, whereas that with 50 μg/mL oxLDL induced cell proliferation in human VSMCs [Zhao et al, ; Ding et al, ; Liao et al, ]. In the present study, 100 μg/mL, but not 50 μg/mL oxLDL significantly induced cell damage (Fig.…”

Roles of platelet‐derived growth factor in vascular calcification

“…In another study, oxidized low density lipoprotein (Ox‐LDL) stimulated phosphorylation of p38 in cultured human VSMCs, resulting in apoptosis and VC. By contrast, inhibitor for p38 significantly attenuated these effects (Liao et al, ). Speer et al () found that VSMCs incubated with indoxyl sulfate (IS) showed a significant increase of calcium deposition, BMP2, and Pit‐1 expression, paralleled by activating JNK signaling.…”

Lipoprotein(a) and oxidized phospholipids in calcific aortic valve stenosis

“…BMP2 [69, 75–77] as well as RUNX2 [78] and MSX2 [79] expression in vascular cells, including AV derived vascular interstitial cells (VIC), are up-regulated following exposure to OxLDL. OxLDL exposure also suppresses osteoprotegerin [80], an inhibitor of vascular calcification via the RANK-L pathway [81, 82].…”

“…OxLDL exposure also suppresses osteoprotegerin [80], an inhibitor of vascular calcification via the RANK-L pathway [81, 82]. Finally, exposure to OxLDL in vitro stimulates extracellular matrix calcium deposition by vascular cells [78, 79, 83, 84] as well as up-regulation of alkaline phosphatase (ALP) [67, 79, 83, 85, 86] promoting mineralization.…”