O‐GlcNAc Transferase Plays a Direct Role in Vascular Smooth Muscle Cell Phenotypic Switching

Abstract

Atherosclerotic complications account for increased morbidity and mortality in diabetic patients. Vascular smooth muscle cell (VSMC) transformation from quiescent contractile to synthetic proliferative phenotype is central to the evolution of atherosclerosis. Diabetic patients show increased propensity for VSMC migration and proliferation, a hallmark of SMC phenotypic de‐differentiation. Using specific pharmacological activators and inhibitors, we previously reported that augmented signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins, controls glucose‐induced VSMC proliferation. However, the mechanistic link between OGT and VSMC activation remains unknown. The goal of the current study was to investigate whether OGT plays a direct role in VSMC phenotypic transition. Using siRNA gene silencing in primary human aortic SMC (HASMC) cultures, we demonstrated that OGT deletion increased SM‐MHC and α‐SMA (SM contractile markers) expression concomitant to attenuated PCNA (proliferation marker) expression in response to high glucose in vitro, shown via immunoblotting and immunocytochemistry. Moreover, under glucose‐stimulated conditions, OGT knockdown decreased the expression of Cyclin E (cell cycle regulator) in OGT siRNA‐transfected HASMC vs. cells transfected with control siRNA. To interrogate the role of OGT in VSMC activation in vivo, we next developed the tamoxifen‐inducible VSMC‐specific OGT knockout mice by crossing OGTfl/fl female mice with tamoxifen‐inducible Myh11‐CreERT2 male mice; the resulting Cretg/OGTfl/Y male mice (produced in F1 generation) were used for Cre recombinase activation. Specifically, 6 wks old male mice were treated with tamoxifen (40 mg/Kg) or vehicle (peanut oil) i.p. once daily for 5 consecutive days. Aorta and heart were harvested from the mice 21 days after the last tamoxifen injection. Immunoblotting experiments confirmed loss of OGT expression in aortic vessels of tamoxifen‐treated Cretg/OGTfl/Y mice (smOGT−/Y) compared to vehicle‐treated Cretg/OGTfl/Y littermates and tamoxifen‐treated OGTfl/Y mice (smOGT+/Y, with intact OGT). In contrast, OGT expression remained unaffected in left ventricular tissue lysates derived from smOGT−/Y vs. smOGT+/Y mice, validating our SMC‐specific OGT knockout mouse model. Importantly, immunoblotting revealed increased SM‐MHC and α‐SMA expression in aortic vessels of smOGT−/Y mice compared to smOGT+/Y, with intact OGT; this increase in SM contractile marker expression was further accompanied with attenuated PCNA and IL1β (pro‐inflammatory marker) expression in smOGT−/Y mice. Together, these data demonstrate a direct role of OGT in VSMC phenotypic de‐differentiation. Our findings suggest a putative fundamental role of OGT in the etiology of diabetic vascular disease.Support or Funding InformationAHA‐Grant‐in‐Aid 16GRNT31200034; NIH‐NHLBI 1R56HL141409‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.