Abstract
Low density lipoprotein (LDL) exists in various forms that possess unique characteristics, including particle content and metabolism. One circulating subfraction, electronegative LDL (LDL(-)), which is increased in familial hypercholesterolemia and diabetes, is implicated in accelerated atherosclerosis. Cellular responses to LDL(-) remain poorly described. Here we demonstrate that LDL(-) increases tumor necrosis factor alpha (TNFalpha)-induced inflammatory responses through NF kappa B and AP-1 activation with corresponding increases in vascular cell adhesion molecule-1 (VCAM1) expression. LDL receptor overexpression increased these effects. In contrast, exposing LDL(-) to the key lipolytic enzyme lipoprotein lipase (LPL) reversed these responses, inhibiting VCAM1 below levels seen with TNFalpha alone. LPL is known to act on lipoproteins to generate endogenous peroxisomal proliferator-activated receptor alpha (PPAR alpha) ligand, thus limiting inflammation. These responses varied according to the lipoprotein substrate triglyceride content (very low density lipoprotein >> LDL > high density lipoprotein). The PPAR alpha activation seen with LDL, however, was disproportionately high. We show here that MUT LDL activates PPAR alpha to an extent proportional to its LDL(-) content. As compared with LDL(-) alone, LPL-treated LDL(-) increased PPAR alpha activation 20-fold in either cell-based transfection or radioligand displacement assays. LPL-treated LDL(-) suppressed NF kappa B and AP-1 activation, increasing expression of the PPAR alpha target gene I kappa B alpha, although only in the genetic presence of PPAR alpha and with intact LPL hydrolysis. Mass spectrometry reveals that LPL-treatment of either LDL or LDL(-) releases hydroxy-octadecadienoic acids (HODEs), potent PPAR alpha activators. These findings suggest LPL-mediated PPAR alpha activation as an alternative catabolic pathway that may limit inflammatory responses to LDL(-).
Highlights
Extensive data links low density lipoprotein (LDL)1 to atherosclerosis [1, 2]
lipoprotein lipase (LPL) Decreases LDL(Ϫ)-mediated VCAM-1 Induction in a PPAR␣-dependent Manner—We compared the effect of native LDL and LDL(Ϫ) on TNF␣-induced vascular cell adhesion molecule-1 (VCAM1) expression in human endothelial cells (ECs)
LDL(Ϫ) treatment induced the VCAM1 promoter 120 –180% in bovine aortic EC transfections, this same stimulation in the presence of LPL repressed this response by 60 – 80% as compared, in both cases, with TNF␣ alone (Fig. 1B). These effects of LPL/LDL(Ϫ) on TNF␣-induced VCAM1 expression equaled those seen with synthetic PPAR␣ ligands (Wy14163 or fenofibric acid, Fig. 1B)
Summary
Reagents—All reagents were purchased from Sigma-Aldrich unless otherwise indicated. All media were obtained from BioWhittaker (Walkersville, Maryland) and contained fungizone/penicillin/streptomycin. Western Blotting—Western blotting was performed on proteins extracted from cells harvested in phosphate-buffered saline lysis buffer containing freshly added 1 mM phenylmethylsulfonyl fluoride at 4 °C. Treated cells were kept on ice for 10 min, washed with cold phosphate-buffered saline, incubated with human VCAM1 monoclonal antibodies Flow Cytometry—Flow cytometry was performed using confluent mouse ECs obtained from heart of PPAR␣ϩ/ϩ and PPAR␣Ϫ/Ϫ mice. Cells were washed in phosphate-buffered saline, harvested by trypsinization, and incubated (1 h at 4 °C) with fluorescein isothiocyanate-conjugated anti-mouse VCAM1 antibody (BD PharMingen). Cells were transfected 3 h after replating in Dulbecco’s modified Eagle’s medium containing 1% of delipidated fetal calf serum (for PPAR ligand binding domain (LBD) studies) or 1% Nutridoma SP Hoffmann-La Roche) were measured according to manufacturer’s protocols
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