Endothelial Shear-Stress-Responsive Gene Adenylate Cyclase 4 Suppresses Atherosclerosis by Inhibiting cAMP/PKA-NF-κB Mediated Vascular Inflammation

BACKGROUND: Endothelial dysfunction is critical for the pathogenesis of atherosclerosis, particularly in arterial regions exposed to disturbed flow (DF). ADCY4 (adenylate cyclase 4) catalyzes the production of cAMP (cyclic adenosine monophosphate), a ubiquitous second messenger that regulates cellular and physiological processes. This study investigated ADCY4 as a shear-stress-responsive gene in endothelial cells (ECs) for the regulation of vascular endothelial inflammation during atherogenesis. METHODS: Integrated analysis of RNA sequencing data sets from human vascular ECs exposed to unidirectional flow or DF, and single-cell RNA-seq data from mouse partial ligation carotid arteries, was performed. ADCY4 expression was quantified in cultured ECs under various flow conditions. Nanoparticles carrying Cdh5 promoter-driven CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and Adcy4 -specific guide RNA plasmids were used to achieve EC-specific Adcy4 knockout in ApoE −/− mice for atherogenesis studies. RESULTS: ADCY4 was upregulated in ECs under unidirectional flow but downregulated under DF. We identified that KLF2 (Krüppel-like factor 2) directly bound to the ADCY4 promoter and transcriptionally enhanced its expression in human aortic ECs. ADCY4 knockdown in ECs under unidirectional flow increased proinflammatory gene expression and monocyte adhesion, whereas ADCY4 overexpression under DF produced opposite changes. Further studies showed ADCY4 attenuated endothelial inflammation by inhibiting NF-κB (nuclear factor κB) signaling. We performed partial carotid ligation in the high-fat diet-fed ApoE −/− mouse atherosclerosis model, demonstrating that EC-specific Adcy4 knockout increased vascular endothelial inflammation and monocyte infiltration, thus promoting atherosclerotic plaque formation. Aggravated atherogenesis was also observed in EC- Adcy4 knockout high-fat diet-fed ApoE − /− mice, with increased atherosclerotic lesions in en face aortas and aortic arches. Finally, semaglutide increased ADCY4 expression in human aortic ECs under DF and reduced vascular endothelial inflammation, monocyte accumulation, and atherosclerotic plaque formation. These beneficial effects were substantially impaired when EC ADCY4 was absent. CONCLUSIONS: ADCY4 regulation of the cAMP/PKA (protein kinase A)-NF-κB pathway in the endothelium advances our understanding of vascular inflammation in atherogenesis and provides opportunities for therapeutic intervention of atherosclerotic cardiovascular disease.