Abstract
Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.
Highlights
Maintenance of the endothelial homeostasis is largely dependent on the activity of endothelial nitric oxide synthase and the bioa-vailability of nitric oxide (NO). eNOS is localized in caveolae, plasma membrane microdomains rich in caveolin-1 protein, which when bound to eNOS attenuates its enzymatic activity [1]
To assess eNOS activating capacity of Endothelial lipase (EL)-High-density lipoprotein (HDL), we examined the rate of eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells incubated with empty virus (EV)-HDL or EL-modified HDL (EL-HDL) for 5 and 16 h, respectively
The impact of EV-HDL and EL-HDL on eNOS phosphorylation was not studied at time points earlier than 1 h of exposure because mechanical forces induced by changing of cell culture medium and/or washing of cells strongly and rapidly induced eNOS phosphorylation
Summary
Maintenance of the endothelial homeostasis is largely dependent on the activity of endothelial nitric oxide synthase (eNOS) and the bioa-vailability of nitric oxide (NO). eNOS is localized in caveolae, plasma membrane microdomains rich in caveolin-1 protein, which when bound to eNOS attenuates its enzymatic activity [1]. Not less important is the role of HDL in the maintenance of endothelial function, which is accomplished by the induction of eNOS activity and increased vascular NO bioavailability [2,3,4]. This process involves interaction of HDL with endothelial receptors, the scavenger receptor class B type 1 (SR-BI) and sphingosine-1 phosphate (S1P) receptors, leading to eNOS phosphorylation at Ser1177 and its activation [3,5,6,7]
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