Uptake of HDL unesterified and esterified cholesterol by human endothelial cells. Modulation by HDL phospholipolysis and cell cholesterol content

Human HDL (1.070-1.210), doubly labelled with ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol were incubated for 1–5 h with monolayer cultures of human endothelial cells. HDL were preincubated for 60–120 min the presence of albumin and with/without purified phospholipase A₂ (control HDL, phospholipase A₂ HDL) before dilution in the cell culture medium. Average phosphatidyl-choline (PC) degradation was 62.10% ± 2.57% (range 45–80%). A purified lipase /phospholipase A₁ from guinea pig pancreas was used in some experiments (range of PC hydrolysis: 16–70%). (1) ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol appeared in cells during 0–5 h incubations. Trypsin treatment allowed a simple adsorption of HDL onto the cell surface to be avoided, and most of the ³H-labelled esterified cholesterol transferred to cells was hydrolysed. Cell uptake of radioactive cholesterol increased as a function of HDL concentration but no saturation was achieved at the highest lipoprotein concentration used (200 μg cholesterol/ml). Flux of ³H/¹⁴C-labelled unesterified cholesterol was related to the cell cholesterol content, suggesting that it might partly represent an exchange process. The cell cholesterol content was slightly increased after 5 h incubation with HDL (+16%). (2) Pretreatment of HDL with purified phospholipase A₂ doubled on average the amount of cell recovered ³H-labelled esterified cholesterol, while the flux of ³H/¹⁴C-labelled unesterified cholesterol was enhanced by 15–25%. Both transfer and cell hydrolysis of ³H-labelled esterified cholesterol were increased. A stimulation was also observed using purified lipase/phospholipase A₁, provided that a threshold phospholipid degradation was achieved (between 27 and 45%). (3) Endothelial cells were conditioned in different media so as to modulate their charge in cholesterol. The uptake of ³H-labelled esterified cholesterol was found to be significantly higher in cholesterol-enriched cells compared to the sterol-depleted state. Finally, movements of ³H-labelled esterified cholesterol from HDL to endothelial cells were essentially unaffected by cell density or by the presence of partially purified cholesterol ester transfer protein. The possible roles of the transfer of HDL esterified cholesterol to endothelial cells and its modulation by phospholipases are discussed.