Deletion of Smooth Muscle O-GlcNAc Transferase Prevents Development of Atherosclerosis in Western Diet-Fed Hyperglycemic ApoE-/- Mice In Vivo.

Accumulating evidence highlights protein O-GlcNAcylation as a putative pathogenic contributor of diabetic vascular complications. We previously reported that elevated protein O-GlcNAcylation correlates with increased atherosclerotic lesion formation and VSMC proliferation in response to hyperglycemia. However, the role of O-GlcNAc transferase (OGT), regulator of O-GlcNAc signaling, in the evolution of diabetic atherosclerosis remains elusive. The goal of this study was to determine whether smooth muscle OGT (smOGT) plays a direct role in hyperglycemia-induced atherosclerotic lesion formation and SMC de-differentiation. Using tamoxifen-inducible Myh11-CreERT2 and Ogtfl/fl mice, we generated smOGTWT and smOGTKO mice, with and without ApoE-null backgrounds. Following STZ-induced hyperglycemia, smOGTWT and smOGTKO mice were kept on a standard laboratory diet for the study duration. In a parallel study, smOGTWTApoE-/- and smOGTKOApoE-/- were initiated on Western diet at 8-wks-age. Animals harvested at 14-16-wks-age were used for plasma and tissue collection. Loss of smOGT augmented SM contractile marker expression in aortic vessels of STZ-induced hyperglycemic smOGTKO mice. Consistently, smOGT deletion attenuated atherosclerotic lesion lipid burden (Oil red O), plaque area (H&E), leukocyte (CD45) and smooth muscle cell (ACTA2) abundance in Western diet-fed hyperglycemic smOGTKOApoE-/- mice. This was accompanied by increased SM contractile markers and reduced inflammatory and proliferative marker expression. Further, smOGT deletion attenuated YY1 and SRF expression (transcriptional regulators of SM contractile genes) in hyperglycemic smOGTKOApoE-/- and smOGTKO mice. These data uncover an athero-protective outcome of smOGT loss-of-function and suggest a direct regulatory role of OGT-mediated O-GlcNAcylation in VSMC de-differentiation in hyperglycemia.

Cognitive dysfunction and increased phosphorylated tau are associated with reduced O-GlcNAc signaling in an aging mouse model of metabolic syndrome.

Metabolic syndrome (MetS), characterized by hyperglycemia, obesity, and hyperlipidemia, can increase the risk of developing late-onset dementia. Recent studies in patients and mouse models suggest a putative link between hyperphosphorylated tau, a component of Alzheimer's disease-related dementia (ADRD) pathology, and cerebral glucose hypometabolism. Impaired glucose metabolism reduces glucose flux through the hexosamine metabolic pathway triggering attenuated O-linked N-acetylglucosamine (O-GlcNAc) protein modification. The goal of the current study was to investigate the link between cognitive function, tau pathology, and O-GlcNAc signaling in an aging mouse model of MetS, agouti KKAy+/- . Male and female C57BL/6, non-agouti KKAy-/- , and agouti KKAy+/- mice were aged 12-18 months on standard chow diet. Body weight, blood glucose, total cholesterol, and triglyceride were measured to confirm the MetS phenotype. Cognition, sensorimotor function, and emotional reactivity were assessed for each genotype followed by plasma and brain tissue collection for biochemical and molecular analyses. Body weight, blood glucose, total cholesterol, and triglyceride levels were significantly elevated in agouti KKAy+/- mice versus C57BL/6 controls and non-agouti KKAy-/- . Behaviorally, agouti KKAy+/- revealed impairments in sensorimotor and cognitive function versus age-matched C57BL/6 and non-agouti KKAy-/- mice. Immunoblotting demonstrated increased phosphorylated tau accompanied with reduced O-GlcNAc protein expression in hippocampal-associated dorsal midbrain of female agouti KKAy+/- versus C57BL/6 control mice. Together, these data demonstrate that impaired cognitive function and AD-related pathology are associated with reduced O-GlcNAc signaling in aging MetS KKAy+/- mice. Overall, our study suggests that interaction of tau pathology with O-GlcNAc signaling may contribute to MetS-induced cognitive dysfunction in aging.

Sex-specific differences in atherosclerosis, thrombospondin-1, and smooth muscle cell differentiation in metabolic syndrome versus non-metabolic syndrome mice.

Introduction: Metabolic syndrome (MetS) amplifies the risks of atherosclerosis. Despite well-known sexual dimorphism in atherosclerosis, underlying mechanisms are poorly understood. Our previous findings highlight a proatherogenic protein, thrombospondin-1 (TSP-1), in hyperglycemia- or hyperleptinemia (mimicking obesity)-induced atherosclerosis. However, the role of TSP-1 in the development of atherosclerosis prompted by co-existing hyperglycemia and obesity, characteristic of MetS, is unknown. The goal of this study was to examine sex-specific differences in lesion progression in a model of combined MetS and atherosclerosis (KKAyApoE) and interrogate how these differences relate to TSP-1 expression. Methods: Male and female KKAy+/-ApoE-/- (with ectopic agouti gene expression) and age-matched non-agouti KKAy-/-ApoE-/- littermates were placed on a standard laboratory diet from 4 to 24 weeks age followed by blood and tissue harvests for biochemical, molecular, and aortic root morphometric studies. Results: Metabolic profiling confirmed MetS phenotype of KKAy+/-ApoE-/-; however, only male genotypes were glucose intolerant with elevated VLDL-cholesterol and VLDL-triglyceride levels. Aortic root morphometry demonstrated profound lipid-filled lesions, increased plaque area, and augmented inflammatory and SMC abundance in MetS vs non-MetS males. This increase in lesion burden was accompanied with elevated TSP-1 and attenuated LMOD-1 (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in male MetS aortic vessels. In contrast, while lipid burden, plaque area, and TSP-1 expression increased in MetS and non-MetS female mice, there was no significant difference between these genotypes. Increased collagen content was noted in MetS and non-MetS genotypes, specific to female mice. Measurement of plasma testosterone revealed a link between the atherogenic phenotype and abnormally high or low testosterone levels. To interrogate whether TSP-1 plays a direct role in SMC de-differentiation in MetS, we generated KKAy+/- mice with and without global TSP-1 deletion. Immunoblotting showed increased SM contractile markers in male KKAy+/-TSP-1-/- aortic vessels vs male KKAy+/-TSP-1+/ +. In contrast, TSP-1 deletion had no effect on SM contractile marker expression in female genotypes. Conclusion: Together, the current study implicates a role of plasma testosterone in sex-specific differences in atherosclerosis and TSP-1 expression in MetS vs non-MetS mice. Our data suggest a sex-dependent differential role of TSP-1 on SMC de-differentiation in MetS. Collectively, these findings underscore a fundamental link between TSP-1 and VSMC phenotypic transformation in MetS.

TSP-1 (Thrombospondin-1) Deficiency Protects ApoE-/- Mice Against Leptin-Induced Atherosclerosis.

OBJECTIVE: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE-/- and TSP-1-/-/ApoE-/- double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE-/- mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE-/- mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE-/-; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE-/- on western diet, independent of plasma lipid alterations. CONCLUSIONS: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.