Smooth muscle‐specific deletion of O‐GlcNAc transferase inhibits SMC de‐differentiation in STZ‐induced hyperglycemic mice

Smooth muscle‐specific deletion of O‐GlcNAc transferase inhibits SMC de‐differentiation in STZ‐induced hyperglycemic mice

Khanal S;Mathias A;Lallo Jason;Ferrell J;Ohanyan V;Raman P

Faseb Journal

2020

2020-04

Vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC phenotypic switching central to the evolution of atherosclerosis, is profoundly enhanced in diabetic patients. Hyperglycemia, characteristic of diabetes, increases glucose flux through the hexosamine metabolic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), key regulator of protein O‐GlcNAcylation. Multiple studies, including ours, support an association between O‐GlcNAcylation and diabetes‐related complications. However, the role of OGT in VSMC activation in diabetes remains elusive. The goal of the present study was to interrogate whether OGT plays a direct role in SMC de‐differentiation in hyperglycemic mice in vivo. Briefly, we crossed OGTfl/fl female mice with tamoxifen‐inducible Myh11‐Cre ERT2 male mice (Cretg); the resulting Cretg/OGTfl/Y males (produced in F1 generation) were used for Cre recombinase activation. Specifically, Cretg/OGTfl/Y and age‐matched Cretg/OGT+/Y littermates at 6 wks age were injected i.p. with tamoxifen (60mg/kg/day) or peanut oil (vehicle control) once daily for 5 consecutive days. Two weeks post‐tamoxifen, mice genotypes received low‐dose STZ (50mg/Kg/day, i.p) or vehicle (citrated buffer) once daily for 5 consecutive days for induction of hyperglycemia. At 16 wks age, mice were subjected to metabolic profiling and cardiac function studies followed by plasma, heart and aortic tissue harvests. Immunoblotting of aortic lysates confirmed loss of OGT expression in tamoxifen‐treated Cretg/OGTfl/Y (smOGT−/Y) mice compared to vehicle‐treated Cretg/OGTfl/Y littermates and tamoxifen‐treated OGTfl/Y mice (smOGT+/Y, with intact OGT). In contrast, OGT expression was unaltered in left ventricular tissue lysates derived from smOGT−/Y vs. smOGT+/Y mice, validating our SMC‐specific OGT knockout mouse model. Immunoblotting further revealed augmented α‐SMA and Calponin expression (SM contractile markers) in aortic vessels of hyperglycemic smOGT−/Y vs. hyperglycemic smOGT+/Y mice. This was accompanied with attenuated PCNA (proliferation marker) and Vimentin (SM synthetic marker) expression in aortic vasculature of smOGT−/Y vs smOGT+/Y mice, under conditions of hyperglycemia. Importantly, increased α‐SMA and Calponin expression occured concomitant to reduced YY1 (transcriptional repressor of SM contractile genes) expression in aortic vessels of smOGT−/Y vs. smOGT+/Y mice, in response to STZ‐induced hyperglycemia. Interestingly, SMC‐specific OGT deletion had no effect on cardiac function of the mice genotypes under basal and hyperglycemic conditions, shown via Echocardiography. Moreover, hyperglycemic smOGT−/Y mice revealed a time‐dependent increase in energy expenditure reflected in differences in O2 consumption and CO2 production compared to hyperglycemic smOGT+/Y mice, depicted via CLAMS studies. Taken together, our results demonstrate a direct regulatory role of OGT on VSMC phenotypic transformation in response to hyperglycemia. Overall, the current study suggests OGT as a potential target in diabetic atherosclerotic disease.

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NEOMED College of Medicine

NEOMED Student Publications

Department of Integrative Medical Sciences

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