Abstract 5923: Endothelial Lipase Participates in the High Density Lipoprotein Transport Through Aortic Endothelial Cells
In the reverse cholesterol transport pathway cholesterol acceptors like high density lipoproteins (HDL) and apolipoprotein A-I (apoA-I), must cross the endothelium to get access to the donor cells in the arterial initima. However, it is unknown how they get translocated from the blood stream into the vascular wall. Aortic endothelial cells (ECs) cultivated on inserts bind, internalize and translocate apoA-I and HDL from the apical to the basolateral compartment in a specific, and temperature dependent manner. Both cell surface biotinylation experiments and immunofluorescence microscopy of apoA-I and HDL recovered both intracellularly. Using pharmacological inhibitors and RNA interference we have demonstrated that ECs transcytose HDL and lipid-free apoA-I by distinct mechanisms involving either SR-BI and ABCG1 (HDL) or ABCA1 (apoA-I). Moreover substituting wildtype apoA-I with apoA-I mutants unable to trigger cholesterol efflux resulted in diminished transport through ECs corroborating a specific transport through ECs. Furthermore, during this transport the size of the HDL-particle was reduced but the protein moiety remained intact, in contrast apoA-I changed its mobility in the electrical field but the protein remained intact. These prompted us to analyze the putative involvement of endothelial lipase (EL) in the HDL transport. Binding, cell association and transport of HDL was reduced in cells treated with heparinase. The presence of tetrahydro-lipstatin did not change HDL binding but cell association and transport was reduced. Using RNA interference to modulate EL expression revealed reduced HDL binding, cell association and transport. Furthermore, addition of tetrahydrolipstatin or reduced expression levels of EL resulted in diminished transport rate and the size of the translocated particle remained in contrast to the control. Moreover, silencing SR-BI resulted in diminished HDL transport but the size of the particle was reduced. We conclude that EL participates in HDL transport through ECs.