Knockout of the NONO Gene Inhibits Neointima Formation in a Mouse Model of Vascular Injury

Abstract

Objective: The NONO (non-POU domain-containing octamer-binding protein) is a multifunctional nuclear protein involved in RNA synthesis, transcriptional regulation, and DNA repair. However, the effect of NONO on neointima induced by vascular injury or restenosis remains unclear. We hypothesized that NONO is required for maintaining vascular integrity and NONO knockout may inhibit neointima formation. Approach and Results: NONO gene KO (knockout; NONO KO or NONO gt/0 ) mice were produced from C57BL/6J mice using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) technique. The left common carotid artery of mice was ligated to induce carotid neointima formation. The primary mouse aortic vascular smooth muscle cells (VSMCs) were derived from the media of wide-type and NONO gt/0 mouse aortas for in vitro studies. Human coronary arteries containing atherosclerotic plaque and normal coronary arteries were obtained from body donors. Histological staining demonstrated that NONO expression was increased in human coronary atherosclerotic lesions and mouse ligated carotid arteries. Moreover, the increased NONO was primarily from VSMCs of neointima. Mice with NONO deficiency showed no significant difference in carotid artery structure from control mice. However, after carotid artery ligation, NONO gt/0 mice exhibited reduced neointima thickness in ligated arteries. NONO deficiency led to decreased proliferation and migration of VSMCs and increased expression of contractile marker genes in neointima and VSMCs. The mechanistic study indicated that NONO interacted with Erk (extracellular regulated kinase) 1/2 in VSMCs and affected its activation in VSMCs, implying Erk signaling cascade might mediate the roles of NONO in VSMCs. Conclusions: NONO was not required for maintaining vascular integrity, but NONO knockout reversed the pathological processes mediated by increased proliferation, migration, and phenotypic switching of VSMCs. The mechanism of these effects involved an interaction of NONO and Erk signaling cascade. Thus, inhibition of NONO may provide a novel therapeutic strategy in cardiovascular disease associated with intimal thickening.