Background: Increased transcytosis of low-density lipoprotein (LDL) across the endothelium and oxidative modification of LDL by activated endothelial cells (EC) constitute pivotal early events to initiate atherogenesis. C1q/TNF-related protein (CTRP) 5 is a novel secreted glycoprotein and its biological functions are largely undefined. In this study, we sought to investigate the role of CTRP5 in modulating transcytosis and oxidation of LDL in EC and the underlying mechanisms.
Methods and Results: Analysis of human coronary endarterectomy specimens and atherosclerotic femoral arteries revealed greater expression of CTRP5 in EC within atherosclerotic lesions as compared to non-atherosclerotic samples. By immunofluorescence analysis, we found CTRP5 led to significantly increased transcytosis and sub-endothelial deposition of DiI-LDL with no permeation to dextran of smaller size after perfusion of mouse aorta. In cultured EC, z-stack analysis by confocal microscopy exhibited accelerated progression of DiI-LDL from the epical membrane downwards over time by CTRP5. Furthermore, CTRP5 treatment led to markedly enhanced passage of biotin-LDL across the endothelial monolayer cultured on transwells as measured by a highly sensitive ELISA. Importantly, pronounced oxidation of the biotin-LDL by CTRP5 after transcytosis was found by detecting the malondialdehyde (MDA)-epitopes with the specific antibody. Mechanistically, we found CTRP5 treatment markedly increased the expression of 12/15-lipoxygenase (LOX), a critical enzyme that catalyzes LDL oxidation in EC. Immunofluorescence studies revealed the nuclear export of 12/15-LOX and partial co-localization with DiI-LDL upon CTRP5 stimulation. Inhibition of 12/15-LOX either by specific-siRNA or the inhibitor Baicalein evidently attenuated CTRP5-elictied oxidation of LDL. Finally, intraperitoneal injection of CTRP5 resulted in enhanced progression of atherosclerosis in ApoE knockout mice fed with high-fat diet, which was markedly reversed after knockdown of 12/15-LOX by lentivirus.
Conclusions: CTPR5 is a novel pro-atherosclerotic factor by facilitating LDL transcytosis across the endothelial monolayers and oxidation of LDL through promoting 12/15-LOX expression.