cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling

Fluid shear stress (FSS) from blood flow sensed by vascular endothelial cells (ECs) determines vessel behavior but regulatory mechanisms are only partially understood. We used cell State Transition Assessment and Regulation (cSTAR), a powerful new computational method, to elucidate EC transcriptomic states under low shear stress (LSS), physiological shear stress (PSS), high shear stress (HSS), and oscillatory shear stress (OSS) that induce vessel inward remodeling, stabilization, outward remodeling or disease susceptibility, respectively. Combined with publicly available EC transcriptomic responses to drug treatments from the LINCS database, this approach inferred a regulatory network controlling EC states and made several novel predictions. Particularly, inhibiting cell cycle dependent kinase (CDK) 2 was predicted to initiate inward vessel remodeling and promote atherogenesis. In vitro, PSS activated CDK2 and induced late G1 cell cycle arrest. In mice, EC deletion of CDK2 triggered inward artery remodeling, pulmonary and systemic hypertension and accelerated atherosclerosis. These results validate use of cSTAR and identify key determinants of normal and pathological artery remodeling. Overall design: Human umbilical vein ECs (HUVECs) were seeded on tissue culture plastic slides coated with 20 µg/mL fibronectin for two hours at 37°C and grown to confluence. Shear stress (FSS) was applied in parallel flow chambers. HUVECs were subjected to oscillatory FSS (OSS, 0.5±4 dynes/cm^2), low FSS (LSS, 3 dynes/cm^2), physiological FSS (PSS, 16 dynes/cm^2) and high FSS (HSS, 40 dynes/cm^2) for 24 hours, in each case in comparison to STAT as an internal control. Total RNA was extracted and prepared for RNA sequencing (RNA-seq). To evaluate how CDK2 affects cell cycle state, we performed CDK2 knockdown in HUVECs in static and PSS conditions.