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.

Increasing adipose tissue mass in obesity directly correlates with elevated circulating leptin levels. Leptin is an adipokine known to play a role in numerous biological processes including regulation of energy homeostasis, inflammation, vascular function and angiogenesis. While physiological concentrations of leptin may exhibit multiple beneficial effects, chronically elevated pathophysiological levels or hyperleptinemia, characteristic of obesity and diabetes, is a major risk factor for development of atherosclerosis. Hyperleptinemia results in a state of selective leptin resistance such that while beneficial metabolic effects of leptin are dampened, deleterious vascular effects of leptin are conserved attributing to vascular dysfunction. Leptin exerts potent proatherogenic effects on multiple vascular cell types including macrophages, endothelial cells and smooth muscle cells; these effects are mediated via an interaction of leptin with the long form of leptin receptor, abundantly expressed in atherosclerotic plaques. This review provides a summary of recent in vivo and in vitro studies that highlight a role of leptin in the pathogenesis of atherosclerotic complications associated with obesity and diabetes.

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.

Metabolic syndrome (MetS) refers to a cluster of anomalies including type 2 diabetes, obesity, insulin resistance and dyslipidemia. Patients with MetS are 1.5 times more likely to develop late‐onset Alzheimer’s Disease (AD), with impaired glucose metabolism, defective insulin signaling, increased oxidative stress and inflammation being shared between the comorbid diseases. Notably, risk of AD is profoundly enhanced in the aging MetS population. Recent studies in AD patients and AD mouse models suggest a putative link between hyperphosphorylated tau neurofibrillary tangles, a commonly accepted AD pathology, and cerebral glucose hypometabolism. Impaired glucose metabolism is characterized by reduced glucose flux through the hexosamine metabolic pathway triggering attenuated signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), a major regulator of intracellular protein O‐GlcNAcylation. However, the role of OGT in the etiology of MetS‐induced AD remains incompletely understood. The goal of the present study was to examine the link between cognitive function, AD pathology and OGT signaling in a mouse model of MetS (KKAy+/−) that develops increased body weight, hyperglycemia, elevated total cholesterol and total triglyceride levels. Briefly, male and female obese agouti KKAy+/−, lean non‐agouti KKAy−/− and normal C57BL/6 control mice weaned at 4 wks of age on regular chow diet were subjected to periodic body weight and random blood glucose monitoring followed by a battery of behavioral tests at 12+ months of age. Plasma and brain (frontal cortex and hippocampus) tissues were then harvested from each genotype for biochemical and molecular studies. In an object recognition test of attention and memory, obese KKAy+/− mice showed a more severe impairment in discrimination between a novel and familiar object compared to lean KKAy−/− and normal C57BL/6 mice. Additionally, in a test of spontaneous activity obese KKAy+/− mice made significantly fewer rears vs lean KKAy−/− mice and wild‐type C57BL/6 controls; these results suggest diminished cognitive function in the obese KKAy+/− mice. Furthermore, immunoblotting of brain tissue lysates derived from the agouti KKAy+/− mice revealed increased ptau expression coupled with reduced pERK (signaling mediator of neuronal function) and pGSK (inactive form of tau kinase GSK3β) expression compared to non‐agouti KKAy−/−. Importantly, augmented tau phosphorylation was concomitant to attenuated OGT expression in brain lysates of obese KKAy+/− mice. Together, these data demonstrate a direct association between cognitive dysfunction, AD‐related pathology and reduced OGT expression in aged MetS KKAy+/− mice. Overall, our study implicates a role of OGT in MetS‐induced cognitive decline and AD pathogenesis. Support or Funding Information NEOMED Start‐Up Funds

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 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-inducibleMyh11-CreERT2and OGTfl/flmice, we generated smOGTWTand smOGTKOmice, with and without ApoE-null backgrounds. Following STZ-induced hyperglycemia, smOGTWTand smOGTKOmice were kept on standard laboratory diet for study duration. In a parallel study, smOGTWTApoE-/-and smOGTKOApoE-/-were kept on Western diet beginning 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 smOGTKOmice. 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-induced hyperglycemic smOGTKOApoE-/-mice. This was accompanied with 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 smOGTKOmice. These data uncover an atheroprotective outcome of smOGT loss-of-function and suggest a direct regulatory role of OGT-mediated O-GlcNAcylation in VSMC de-differentiation in hyperglycemia.

Metabolic syndrome (MetS) amplifies risks of atherosclerotic complications. MetS induces abnormal vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC de-differentiation, crucial in the pathogenesis of atherosclerosis. Despite multiple studies revealing sexual dimorphism in atherosclerosis, underlying sex-specific mechanisms are poorly understood. We previously reported a protective role of a potent proatherogenic protein, thrombospondin-1 (TSP1), in hyperglycemia- or hyperleptinemia-induced atherosclerosis. The goal of the present study was to interrogate sex-specific differences in the role of TSP1 on MetS-induced atherosclerosis. We generated a mouse model of combined MetS and atherosclerosis (KKAy +/- ApoE -/- ) by crossing obese hyperglycemic agouti KKAy +/- mice with atherosclerotic ApoE -/- . Male and female age-matched MetS KKAy +/- ApoE -/- and non-MetS KKAy -/- ApoE -/- mice were placed on standard lab diet from 4-24 wks age. After overnight fasting, mice were harvested; plasma, aorta and heart were collected for various studies. In male MetS KKAy +/- ApoE -/- , increased aortic root ORO-positive lipid burden, reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in aortic vessels associated with augmented TSP1 expression vs. non-MetS KKAy -/- ApoE -/- mice. In contrast, no significant differences in lesion lipid burden, TSP1, LMOD and SRF expression were detected between the female genotypes. To delineate whether TSP1 plays a direct role in SMC de-differentiation in MetS, we next crossed KKAy +/- with TSP1 -/- to generate MetS mice with and without global TSP1 deletion. Male and female KKAy +/- TSP1 +/+ (with intact TSP1) and KKAy +/- TSP1 -/- (lacking TSP1) on standard lab diet from 4-18 wks age were harvested at endpoint for aortic tissue collection. In male KKAy +/- TSP1 -/- aortic vessels, SM contractile marker expression (LMOD, calponin) was markedly increased vs. KKAy +/- TSP1 +/+ , suggesting reduced SMC de-differentiation. In contrast, TSP1 deletion had no effect on SM contractile marker expression in female MetS KKAy +/- . Together, these data suggest a sex-specific role of TSP1 on SMC de-differentiation and atherosclerotic lesion formation in MetS.

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.

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.