Abstract
Rationale and Hypothesis: Cardiovascular disease is the top cause of death worldwide. Atherosclerosis is a chronic cardiovascular disease, caused by the accumulation of lipids in large and medium arteries. Plaques develop preferentially at arterial branches and bends. This asymmetric distribution of plaque is controlled by shear stress, a force exerted throughout the vascular beds, at different magnitudes, by flowing blood. High-magnitude uniform shear stress promotes vascular integrity and is thus atheroprotective. Protective high shear stress (HSS) promotes a number of well-known cytoprotective genes, including endothelial nitric oxide synthase (eNOS), prostacyclin synthase (PTGIS) and kruppel-like factors 2 (KLF2) while suppressing inflammation. On the contrary low shear stress (LSS) promotes the development of inflammation and plaque growth. Our laboratory has recently revealed that ankyrin repeats and single KH domain 1 (ANKHD1), which is highly expressed in endothelial cells, is an RNA-binding protein. Yet the role of ANKHD1 in controlling vascular function remains largely unknown. I hypothesize that ANKHD1 may have a major vascular role, via controlling a number of cytoprotective molecules.
Methodology: To identify ANKHD1 functions, loss-of-function of ANKHD1 was performed using ANKHD1-specific siRNA via lipotransfection in HSS-stimulated human coronary artery endothelial cells (HCAECs) followed by RNA-sequencing. The effect of different shear stress conditions on ANKHD1 expression was examined both in vivo, in the mouse aortic arch, and in vitro in primary human endothelial cells. Primary human umbilical vein endothelial cells (HUVECs) and HCAECs were exposed to either protective (~13 dyne/cm2) or athero-promoting (~4 dyne/cm2) shear stress using an orbital shaker for 72 hours. ANKHD1 levels were quantified by RT-qPCR, immunoblotting and immunofluorescence staining. The molecular mechanism of ANKHD1 in regulating the cytoprotective molecules was studied using RNA immunoprecipitation (RIP) assays. Effects on message stability were examined by RNA stability assay and by in vitro synthesis of biotinylated RNA followed by RNA pulldown. To examine the potential role of ANKHD1 in the control of vascular integrity in vivo, ANKHD1-deficient and wild-type aortas were subjected to macroscopic and microscopic (en-face staining) inspection to study size and vessel morphology and any alteration in the expression of cytoprotective genes. To examine the potential systemic factors influencing atherosclerosis development in Ankhd1 mouse models, blood glucose and cholesterol levels, blood pressure, heart rate and body weight were measured. The influence of ANKHD1 on atherosclerosis was assessed in PSCK9 mice with Ankhd1+/+, Ankhd1+/-, or Ankhd1-/-.
Findings: RNA-sequencing of human endothelial cells coupled with gene ontology analysis showed that lowering the expression of ANKHD1 changes the expression of a number of genes that are involved in shear stress responses and atherosclerosis. With a series of in vivo and in vitro experiments, I demonstrated that ANKHD1 is under the control of atheroprotective shear stress, assessed by increased levels of ANKHD1 mRNA in HCAECs and HUVECs (HCAECs p= 0.0475, N= 5; HUVECs: p= 0.0043, N= 4) and ANKHD1 protein in HCAECs (p= 0.0137, N= 5) stimulated with shear stress by the orbital shaker. In mouse aortas, ANKHD1 protein was increased in areas of the mouse aorta exposed to HSS (p= 0.0266, N= 6). To further delineate the function of ANKHD1, I focused on three atheroprotective molecules that are regulated by shear stress, namely eNOS, PTGIS and KLF2. ANKHD1 silencing in HSS-induced HCAECs (N= 6) led to a significant reduction in eNOS mRNA (ANKHD1siRNA-1: adjusted p= 0.0247; ANKHD1siRNA-2: adjusted p= 0.0209), PTGIS mRNA (ANKHD1siRNA-1: adjusted p= 0.0227; ANKHD1siRNA-2: adjusted p= 0.0271) and KLF2 mRNA (ANKHD1siRNA-1: adjusted p= 0.0133; ANKHD1siRNA-2: adjusted p= 0.0130). Mechanistically, ANKHD1 binds to eNOS, PTGIS and KLF2 mRNAs. It enhances eNOS mRNA stability and binds directly to the 3’UTR of eNOS, near a region of natural instability, known as the AU-rich Element (AUR). Comparison of Ankhd1+/+ and Ankhd1-/- mice revealed that ANKHD1 positively regulated eNOS (N= 8-11, p= 0.0425) and PTGIS (N= 6-13, p= 0.0045) as well as vessel diameter (0.8343 mm in Ankhd1-/- vs 0.9318 mm in Ankhd1+/+ (20-weeks of age). Ankhd1 genetic deletion does not affect glucose levels, cholesterol levels, body weight or heart rate. However, Ankhd1-/- mice exhibited increased inflammation as observed by expression of E-selectin (CD62E), increased thrombosis and accelerated atherosclerosis.
Conclusions: This study has discovered ANKHD1 as a novel cytoprotective molecule that maintains vascular health and attenuates atherogenesis by controlling critical processes in inflammation, vasorelaxation and thrombosis.
