The mechanisms by which high density lipoprotein (HDL) cholesterol is atheroprotective are not well understood. We have shown that HDL is a potent agonist for endothelial NO synthase (eNOS) via binding to scavenger receptor-BI (SR-BI) in both cultured and intact endothelium. Further recent work indicates that HDL also stimulates endothelial cell migration, and that common upstream signaling events may be involved. The OBJECTIVE of this proposal is to determine the molecular mechanisms by which HDL and SR-BI mediate endothelial cell signaling and migration, and the role of these processes in reendothelialization in vivo.
Four Aims will be addressed in cultured cell models and mice. We have preliminary evidence that in contrast to SR-BI, the splice variant SR-BII and CD36 are incapable of stimulating eNOS.
Aim 1 is to determine the domains of SR-BI required for signaling by HDL by testing mutant or chimeric receptors cotransfected with eNOS in COS-M6 cells. Based on preliminary findings, including evidence of photocholesterol binding to the SR-BI C-terminal transmembrane (TM) domain, we will test the hypothesis that the C-terminal TM domain and cytoplasmic tail are critical to signaling by HDL. We also have initial evidence that signaling to eNOS involves the PDZ domain-containing protein PDZK1, which is known to interact with the SR-BI C-terminus. We have recently found that PDZK1 is expressed in endothelium.
Aim 2 is to determine the role of PDZK1 in HDL-SR-BI signaling in gain-of-function and loss-of-function studies in cultured cells, and in studies of endothelium-dependent relaxation with HDL in arteries from PDZK1 /- mice.
Aim 3 is to determine the basis for HDL activation of endothelial migration in a cultured cell wound model. Initial studies indicate mediation by PI3 kinase, which is also required for signaling to eNOS, but the migration is NO- independent. We will test the hypothesis that HDL activation of migration is through SRBI coupling to downstream kinases and small GTPases.
Aim 4 is to determine the role of HDL in reendothelialization in vivo, and this will be done in studies of the mouse carotid artery following perivascular electric Injury. Reendothelializatlon will be compared in wild-type versus apoA-/- , SR-BI-/- and PDZK1 -/- mice receiving liver-directed gene transfer of apoA-I or control virus. The hypotheses raised are that HDL enhances reendothelialization, and that this process is mediated by SR-BI and PDZK1. These studies will provide valuable new information about novel direct actions of HDL and SR-BI in endothelium, thereby expanding our knowledge of the role of HDL in vascular health and disease.
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