SUMMARY Atherosclerosis is a chronic inflammatory disease that underlies heart attacks and stroke. The disease preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), in part by altering expression of flow-sensitive genes. While looking for flow-sensitive long non-coding RNAs, we identified the flow-sensitive heart of glass (HEG1) gene as a potential target. Previous studies using HEG1 knockouts in zebra fish and mice have demonstrated its critical role in cardiovascular development and vascular integrity, but its role and mechanisms of action in vascular biology and atherosclerosis are far from clear. Recently, we generated exciting preliminary data, including a single-cell RNAseq study using the mouse partial carotid ligation (PCL) model, demonstrating that HEG1 expression is increased by stable flow (s-flow) and decreased under disturbed flow (d- flow) conditions. HEG1 knockdown in human aortic endothelial cells (HAECs) induces inflammation, barrier dysfunction, and endothelial-mesenchymal transition (EndMT), key pro-atherogenic processes. HEG1 has a long N-terminal extracellular structure containing two highly glycosylated (Gly) domains (potential flow sensing domain), three EGF-like (EGFL) domains containing highly conserved Cys clusters (potential redox-sensitive, flow-sensing domain),a transmembrane (TM) domain, and the cytosolic C-terminal (C-term signal transduction) domain. Our preliminary results show that HEG1 1) can be pulled with a HEG1 antibody or sheared to induce Ca++ flux, and 2) is redox-sensitive in a Poldip2-dependent manner. Based on these exciting data, we hypothesize that HEG1 protein is a redox-sensitive mechanosensor, mediating the atheroprotective effects of stable flow, while HEG1 loss and malfunction by d-flow induces endothelial dysfunction leading to atherosclerosis. We will test this hypothesis in three aims: Aim 1 will determine the role of HEG1 in flow- dependent EC function (inflammation, EndMT, and permeability) using siRNA-mediated knockdown or overexpression of HEG1 (using AAV-HEG1 expressing WT or 3 truncation mutants ΔGly, ΔEGFL or ΔTM+C- term) in HAECs and immortalized mouse aortic ECs (iMAECs). EC-targeted HEG1-null mice (HEG1-EC-/-) will be used without or with the AAV-HEG1 constructs for in vivo validation of EC function. Aim 2 will test if HEG1 is a redox-sensitive mechanosensor by focusing on immediate changes (seconds) in intracellular calcium, acute activation (seconds-minutes) of signaling proteins, and slow (>hours) cell changes in response to shear stress or tensional force using magnetic beads coated with HEG1 mAb. For these studies, HAECs and iMAECs treated with siHEG1 or the same AAV9-HEG1 constructs described in Aim 1 will be used. Aim 3 will determine the role of HEG1 in atherosclerosis using HEG1-EC-/- mice injected with AAV-PCSK9. We will further test if transduction with the AAV-HEG1 constructs can prevent atherosclerosis in HEG1-EC-/- mice. These studies will define if HEG1 mediates the atheroprotective effects of stable flow by serving as a redox-sensitive mechanosensor and may reveal novel therapeutic strategies for atherosclerosis.

PROJECT NARRATIVE Atherosclerosis is an inflammatory disease, occurring preferentially in arterial branch points exposed to pro- atherogenic unstable blood flow while the straight parts of arteries exposed to uni-directional athero-protective stable blood flow are spared. Here, we propose a novel hypothesis that atheroprotective stable flow stimulates expression of HEG1 in endothelial cells, which in turn inhibits endothelial inflammation and protects endothelial barrier function, preventing atherosclerosis development. Our study purports that HEG1 is a novel flow sensor (mechanosensor), which plays a key role in atherosclerosis development and is a novel therapeutic target in prevention and treatment of atherosclerosis.