Endothelial Cell SR-BI Regulation and Novel Protein Partners in Atherosclerosis - Project Summary/Abstract Atherosclerosis underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke. We previously discovered that scavenger receptor class B type I (SR-BI, encoded by Scarb1) drives LDL transcytosis in endothelial cells, delivering LDL to the subendothelial space where its accumulation by macrophages promotes atherosclerosis. We recently determined by single cell RNAseq in human coronary arteries that SR-BI is upregulated in endothelial cells associated with atheromas versus in cells from segments lacking atheroma. Gene signatures for responses to varying blood flow revealed that disturbed flow may also upregulate SR-BI. We have revealed that endothelial SR-BI is upregulated in mice with either hypercholesterolemia or disturbed blood flow induced by carotid artery partial ligation. Using mass spectrometry and LDL transcytosis assays, we have determined that the E3 ligase c-Cbl and the Zn transporter ZIP10 interact with SR-BI and are required for LDL transcytosis via mechanisms likely shared with the receptor. Springboarding from these discoveries, the Overall Goal of the proposed research is to determine how endothelial SR-BI expression is regulated in vivo, and how SR-BI partners with other proteins to mediate LDL transcytosis. Aim 1 will determine how hypercholesterolemia and disturbed blood flow upregulate endothelial SR-BI. Transcription factor networks derived from the coronary endothelial cell RNAseq reveal parallel upregulation of HIF-1α and SR-BI and possible regulatory interaction in the setting of atheroma formation or disturbed flow. Using ChIP-seq, CHIP-qPCR and CRISPR, we have identified HIF-1α binding sites in intron 1 of human Scarb1 and 28kb 5’ of mouse Scarb1 that mediate HIF-1α upregulation of SR-BI. We will test the hypothesis in mouse models that there is a unifying mechanism in which HIF-1α mediates endothelial cell SR-BI upregulation by hypercholesterolemia and by disturbed blood flow to promote endothelial cell LDL uptake and atherosclerosis. Studies will include a test of the impact of CRISPR-based excision of the HIF-1α binding site 5’ to mouse Scarb1 in endothelium. Aim 2 will determine how the new partner proteins c-Cbl and ZIP10 modulate LDL transcytosis by SR-BI. In culture we will use mutagenesis to identify the specific functions of c-Cbl and ZIP10 and modes of SR-BI interaction required to govern LDL transcytosis. In vivo studies will determine how endothelial cell knockdown of c-Cbl or ZIP10 impacts artery LDL uptake and atherosclerosis. Guided by the findings in culture, using nanoparticles to reconstitute endothelial cell genes in mice, in vivo structure-function studies will be done of SR-BI interaction with c-Cbl or ZIP10 and their specific functions. Aim 2 will test the hypothesis that via discrete capacities and interactions with SR-BI, c-Cbl and ZIP10 partner with SR-BI to invoke artery wall LDL delivery and atherosclerosis. By accomplishing these Aims and unraveling how endothelial SR-BI expression is regulated and how SR-BI works with partner proteins to drive endothelial LDL transport, it is expected that novel therapies can then be developed to disrupt SR-BI-mediated artery entry of LDL for cardiovascular benefit.
