Caveolae And Caveolin-1 Mediate The Uptake Of Oxidized Low Density Lipoproteins And The Transcytosis Of Oxidized Low Density Lipoproteins Across Endothelium

Objective: To explore the mechanisms involved in cholesterol transcytosis across endothelial cells and the effects of caveolae in this process,and determine the relation between caveolin-1 expression and cholesterol efflux in human umbilical vein endothelial cellsMethod: Human umbilical vein Endothelial Cells(HUVEC) were incubated with ox-LDL(40μg/ml) for different times,and depleted ox-LDL from medium after lipid-loaded 24h for different times, we used fluorescence microscope and HPLC to determine the cholesterol content in HUVEC at varies times. We first incubated HUVEC with carrageenan, filipin, or nocodazole for 1h, and a 24h incubation was followed after addition of DiI-ox-LDL(40μg/ml), the DiI fluorescent was observed with fluorescent microscope after incubation. We grown HUVEC on a Transwell insert to obtain a tight monolayer and preincubated with carrageenan, filipin, or nocodazole in the apical reservoir for 1h, then incubated 24h after addition of DiI-ox-LDL(40μg/ml), at the end of incubation period, the fluorescence intensity of DiI-ox-LDL that transcytosised up to the bottom reservoir was determined by Fluorescence spectrophotometer. We preincubated HUVEC with or without nocodazole for 1h, and incubated for another 24h with ox-LDL, use Indirect Immunofluorescence to determine the location of LOX-1. We Knocked down cavoelin-1 by caveolin-1 siRNA in HUVEC, then determined the activation of NF-κB and expression of LOX-1.Result: We incubated HUVEC with ox-LDL(40μg/ml) for different times and depleted ox-LDL from medium after lipid-loaded 24h for different times, we found that HUVEC intra-cellular cholesterol contents increased in a time-dependented manner, and decreased gradually after depletion of ox-LDL from medium. We first incubated HUVEC with carrageenan, filipin, or nocodazole for 1h, and a second 24h incubation was performed after addition of DiI-ox-LDL(40μg/ml), we found that carrageenan, filipin, and nocodazole could inhibit ox-LDL uptake by HUVEC significantly. We also found that, after pretreated with those drugs, the Fluorescence intensity of DiI-ox-LDL that transcytosised up to the bottom reservoir was decreased dramatically to 49%, 72% and 80% respectively, comparing to control group. When used Immunofluorescence to locate LOX-1,we found that LOX-1 focus in endochylema, but when preincubated with nocodazole, then incubated with ox-LDL, LOCX-1 still focus at plasma membrane. After silenced the cavoelin-1 in HUVEC, we found that NF-κB was inactivated and the expression of LOX-1 was decreased.Conclusion1. HUVEC could uptake lipid, also could release lipid by some mechanism inherent exist, and this process is spontaneous. LOX-1 associated with caveolae in HUVEC, it located with or within cavoelae. Caveolae could be a carrier for ox-LDL involved in uptake of ox-LDL by HUVEC. HUVEC was a post-house in ox-LDL translocation from blood plasma to subendothelium, and cavoelae played a important role in this process.2. Ox-LDL up-regulated caveolin-1 expression, which triggered NF-κB activation and nuclear translocation, subsequent promoted LOX-1 gene transcription, at the end, LOX-1 expression up-regulated. In this pathway, caveolin-1 as a regulator play a important role in ox-LDL uptake by modulation of LOX-1's expression in HUVEC.