Abstract
Background: Enzyme modified LDL (ELDL) is present in human atherosclerotic lesions and is a major foam cell-forming modified LDL for murine vascular smooth muscle cells (SMC) as reported by us previously. Here we study ELDL and its effects on human coronary artery SMC (HCASMC) in vitro. Methods and Results: Incubation of HCASMC with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) resulted in significant foam cell formation (analyzed by Oil Red O, lipid measurement) compared to HCASMC incubated with oxidized LDL (200 μg/ml; -75%, p<0.01) or native LDL (200 μg/ml; -50%, p<0.01). Whole genome gene expression (Illumina Bead Chip HT12v4, analyzed by DAVID v6.8 and IPA) of HCASMC treated with ELDL, oxLDL, LDL, and control (cell culture medium only) showed several top canonical pathways specifically induced by ELDL, together with activated upstream regulators including p38MAPK, NFkB. ERK. Upregulation of ANGPTL4 and BMP-2 -mRNA (22 and 2 fold respectively over native LDL) was verified by qRT-PCR and immunoblotting. ELDL-induced foam cells showed dose dependent (1-20 μg/ml ELDL) increase in migration in collagen coated trans well dishes, which was attenuated by Lacidepine, a known inhibitor of ELDL uptake in murine SMCs. Furthermore, rANGPTL4 also upregulated HCASMC migration dose dependently (1-5 μg/ml for 24 h) and was comparable with the migration induced by ELDL. However, Lacidipine had no effect on rANGPLT4 mediated migration, suggesting that ANGPLT-4 independently of ELDL uptake promotes migration of HCASMC. In calcification assays using MEM with 0.2% FCS and 1.5 mM phosphate, ELDL at 2.5 μg/ml induced more calcification native LDL (>25%, p<0.01, analyzed by alizarin red staining and organic extraction, and this was proceeded by increase in BMP-2 mRNA. Conclusions: ELDL is highly potent in inducing foam cells in cultured HCASMC. Whole genome expression and bioinformatics analysis indicate up-regulation of pathways linked to osteochondrogenic transformation. BMP-2 and ANGPTL4 are significantly upregulated in ELDL-induced HCASMC foam cells. These results point to the potential of ELDL to induce migratory and osteoblastic effects in HCASMC with potential implications in SMC migration and calcification in human atherosclerosis.
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