Abstract
Enzyme modified non-oxidative LDL (ELDL) is effectively taken up by vascular smooth muscle cells (SMC) and mediates transition into foam cells and produces phenotypic changes in SMC function. Our data show that incubation of human coronary artery SMC (HCASMC) with low concentration of ELDL (10 μg/ml) results in significantly enhanced foam cell formation compared to oxidized LDL (200 μg/ml; p < 0.01) or native LDL (200 μg/ml; p < 0.01). Bioinformatic network analysis identified activation of p38 MAPK, NFkB, ERK as top canonical pathways relevant for biological processes linked to cell migration and osteoblastic differentiation in ELDL-treated cells. Functional studies confirmed increased migration of HCASMC upon stimulation with ELDL (10 μg/ml) or Angiopoietin like protein 4, (ANGPTL4, 5 μg/ml), and gain in osteoblastic gene profile with significant increase in mRNA levels for DMP-1, ALPL, RUNX2, OPN/SPP1, osterix/SP7, BMP and reduction in mRNA for MGP and ENPP1. Enhanced calcification of HCASMC by ELDL was demonstrated by Alizarin Red staining. In summary, ELDL is highly potent in inducing foam cells in HCASMC and mediates a phenotypic switch with enhanced migration and osteoblastic gene profile. These results point to the potential of ELDL to induce migratory and osteoblastic effects in human smooth muscle cells with potential implications for migration and calcification of SMCs in human atherosclerosis.
Highlights
Enzyme-modified non oxidized LDL (ELDL) and oxidized LDL (OxLDL) are two prominent post-translational modification of low density lipoproteins (LDL) and are well characterized in their ability to mediate atherosclerosis[1,2,3,4]
Enzyme modified non-oxidative LDL (ELDL) was previously shown to be more potent than OxLDL in inducing foam cells in cultured macrophages[9]; and more recently, our laboratory demonstrated its ability to foam murine aortic smooth muscle cells (SMC) at lower concentrations compared to OxLDL or native LDL7
ELDL uptake and foaming of human coronary artery smooth muscle cells (HCASMC) induced gene expression of some adipocyte markers (FABp4, perilipin), many others genes for adipocytes or smooth muscle cell were expressed in ELDL and BSA control cells (Supplementary Fig. 3)
Summary
Enzyme-modified non oxidized LDL (ELDL) and oxidized LDL (OxLDL) are two prominent post-translational modification of low density lipoproteins (LDL) and are well characterized in their ability to mediate atherosclerosis[1,2,3,4]. ELDL and OxLDL are rapidly taken up by macrophages, the mechanism how lipids transforms SMCs into foam cells is less studied. BSA, ELDL, OxLDL or Native LDL for 24 h at the indicated amounts (concentration by protein content) and lipid accumulation in SMC is visualized by Oil red O staining. Potential candidates for proteolytic enzymes that may modify LDL in vivo, similar to that by trypsin in vitro, include plasmin, chymases, matrix metalloproteinases (MMPs) and cathepsins; all are highly expressed in atherosclerotic plaques[1,17,18], with studies showing that both, plasmin and MMPs, are sufficient to produce ELDL in vitro[1]. Multiple factors contribute to SMC migration and calcification including growth factors, cytokines, and remodeling of extracellular matrix by proteases released from inflammatory cells present within the atherosclerotic milieu[19,22,23,24]. OxLDL has been shown to activate SMC migration and calcification[25,26]; the role of ELDL in SMC calcification is not studied
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