EXTRACELLULAR MATRIX REMODELLING OF ENDOTHELIAL CELL WAS INDUCED BY VERY LOW DENSITY LIPOPROTEINS THROUGH NFKB ACTIVATION

Predisposing risk factors for atherosclerosis, which include hypertension, diabetes, smoking, and hypercholesterolemia, are all linked to endothelial cell (EC) dysfunction (1). However, the remodelling of the vascular extracellular matrix (vECM) and the signaling cascade involved in EC dysfunction induced by normal very low density lipoprotein (N-VLDL)injury is still unclear (2). The aim of the present work was to study the remodelling of vECM and signaling of Human Umbilical Vein Endothelial Cells (HUVEC) in response to human N-VLDL.All the protocols performed were approved by the Bioethics Committee of the University of Buenos Aires, Argentina. Human N-VLDLwere isolated by ultracentrifugation from healthy volunteers. EC were obtained and cultured as described Ulrich-Merzenic (3). After reaching 70-80%confluence, HUVEC were incubated with 75 mcg/mL of N-VLDLin serum-free medium for 24 hs. After treatment, vECM remodelling was studied through: 1) proteoglycans core protein production, specifically decorin, versican and perlecan analysis by Western blot; 2) glycosaminoglycans content studied by reverse phase HPLC; 3) matrix metalloproteinase (MMP) activity analyzed by zymography. The signaling pathways (NFKBand ERK 1/2) induced by N-VLDL were studied by immunofluorescence and Western blot. HUVEC showed a significant augmentation in the production of decorin and versican (p<0.05, C vs N-VLDL, t-Test, ri-G). This increased was accompanied by an increased secretion of chondroitin sulphate and dermatan sulphate (C4S/C6S 3.52 vs 4.56 and C4S/COS 4.97 vs 12.7, control vs N-VLDL respectively, n~3). In addition, perlecan production showed no differences. The NF-KB was accumulated in the nucleus of HUVEC after lipoprotein treatment (p<0.05, C vs N-VLDL,t-Test, n-B). No differences were detected in ERK 1/2 activation between control and N-VLDlrtreated cells. The proinflammatory state reached by HUVEC in the presence of N-VLDLwas associated with a significant increase in MMP-9 activity (p< 0.05, C vs N-VLDL). Our results suggest that human N-VLDLinduced an athero-protective remodelling of the vECM in HUVEC, through NFKB activation and dermatan sulfate proteoglycans increase. This remodelling was different from our previous results obtained for Human Umbilical Artery Endothelial Cells (4). References l. Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran 0, Sethi G, Nishigaki I.The vascular endothelium and human diseases. lnt. J. BioI. Sci. 2013; 9(10): 1057-1069. 2. ldberg U, Eckel RH, McPherson R Triglycerides and heart disease, still a hypothesis? ArteriosclerThromb Vasc BioI. 2011; 31(8): 1716-1725. Go 3. Ulrich-Merzenich G, Metzner C, Bhonde R, Maisch G, Schiermeyer B, Vetter H. Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins. 4. Oberkersch R, Rasente Y, Gualco L, Yuschak S, Calabrese G. Very low density lipoproteins induce differential vascular extracellular remodelling according to the endothelial phenotype. Angiogenesis and Leukocytes Atherosclerosis. Geneve, Switzerland. January 2014; 30-3l.