miR-369-3p ameliorates diabetes-associated atherosclerosis by regulating macrophage succinate-GPR91 signaling.

AIMS: Diabetes leads to dysregulated macrophage immunometabolism, contributing to accelerated atherosclerosis progression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages, yet their therapeutic potential in diabetes-associated atherosclerosis remains unclear. METHODS AND RESULTS: MiRNA profiling revealed significantly lower miR-369-3p expression in aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose containing (HFSC) diet for 12 weeks. miR-369-3p was also reduced in peripheral blood mononuclear cells (PBMCs) from diabetic patients with coronary artery disease (CAD). Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. In vitro, oxLDL treatment reduced miR-369-3p expression in mouse bone marrow-derived macrophages (BMDMs). Metabolic profiling in BMDMs revealed that miR-369-3p overexpression blocked the oxLDL-mediated increase in the cellular metabolite succinate and reduced mitochondrial respiration (OXPHOS) and inflammation (lL-1ß, TNF-a, IL-6). Mechanistically, miR-369-3p targeted the succinate receptor (GPR91) and alleviated the oxLDL-induced activation of inflammasome signaling pathways. Therapeutic administration of miR-369-3p mimics in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and diabetes-accelerated atherosclerosis, evident by a decrease in plaque size and pro-inflammatory Ly6Chi monocytes. RNA-seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p treated mice, consistent with an increase in macrophage efferocytosis in lesions. Finally, a GPR91 antagonist attenuated oxLDL-induced inflammation in primary monocytes from human subjects with diabetes. CONCLUSION: These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism.

Coronary Microvascular Function Following Severe Preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific hypertensive disorder associated with an imbalance in circulating proangiogenic and antiangiogenic proteins. Preclinical evidence implicates microvascular dysfunction as a potential mediator of preeclampsia-associated cardiovascular risk. METHODS: Women with singleton pregnancies complicated by severe antepartum-onset preeclampsia and a comparator group with normotensive deliveries underwent cardiac positron emission tomography within 4 weeks of delivery. A control group of premenopausal, nonpostpartum women was also included. Myocardial flow reserve, myocardial blood flow, and coronary vascular resistance were compared across groups. sFlt-1 (soluble fms-like tyrosine kinase receptor-1) and PlGF (placental growth factor) were measured at imaging. RESULTS: The primary cohort included 19 women with severe preeclampsia (imaged at a mean of 15.3 days postpartum), 5 with normotensive pregnancy (mean, 14.4 days postpartum), and 13 nonpostpartum female controls. Preeclampsia was associated with lower myocardial flow reserve (ß, -0.67 [95% CI, -1.21 to -0.13]; P=0.016), lower stress myocardial blood flow (ß, -0.68 [95% CI, -1.07 to -0.29] mL/min per g; P=0.001), and higher stress coronary vascular resistance (ß, +12.4 [95% CI, 6.0 to 18.7] mm Hg/mL per min/g; P=0.001) versus nonpostpartum controls. Myocardial flow reserve and coronary vascular resistance after normotensive pregnancy were intermediate between preeclamptic and nonpostpartum groups. Following preeclampsia, myocardial flow reserve was positively associated with time following delivery (P=0.008). The sFlt-1/PlGF ratio strongly correlated with rest myocardial blood flow (r=0.71; P<0.001), independent of hemodynamics. CONCLUSIONS: In this exploratory cross-sectional study, we observed reduced coronary microvascular function in the early postpartum period following preeclampsia, suggesting that systemic microvascular dysfunction in preeclampsia involves coronary microcirculation. Further research is needed to establish interventions to mitigate the risk of preeclampsia-associated cardiovascular disease.

Coronary Microvascular Function Following Severe Preeclampsia.

Background: Preeclampsia is a pregnancy-specific hypertensive disorder associated with an imbalance in circulating pro- and anti-angiogenic proteins. Preclinical evidence implicates microvascular dysfunction as a potential mediator of preeclampsia-associated cardiovascular risk. Methods: Women with singleton pregnancies complicated by severe antepartum-onset preeclampsia and a comparator group with normotensive deliveries underwent cardiac positron emission tomography (PET) within 4 weeks of delivery. A control group of pre-menopausal, non-postpartum women was also included. Myocardial flow reserve (MFR), myocardial blood flow (MBF), and coronary vascular resistance (CVR) were compared across groups. Soluble fms-like tyrosine kinase receptor-1 (sFlt-1) and placental growth factor (PlGF) were measured at imaging. Results: The primary cohort included 19 women with severe preeclampsia (imaged at a mean 16.0 days postpartum), 5 with normotensive pregnancy (mean 14.4 days postpartum), and 13 non-postpartum female controls. Preeclampsia was associated with lower MFR (ß=-0.67 [95% CI -1.21 to -0.13]; P=0.016), lower stress MBF (ß=-0.68 [95% CI, -1.07 to -0.29] mL/min/g; P=0.001), and higher stress CVR (ß=+12.4 [95% CI 6.0 to 18.7] mmHg/mL/min/g; P=0.001) vs. non-postpartum controls. MFR and CVR after normotensive pregnancy were intermediate between preeclamptic and non-postpartum groups. Following preeclampsia, MFR was positively associated with time following delivery (P=0.008). The sFlt-1/PlGF ratio strongly correlated with rest MBF (r=0.71; P<0.001), independent of hemodynamics. Conclusions: In this exploratory study, we observed reduced coronary microvascular function in the early postpartum period following severe preeclampsia, suggesting that systemic microvascular dysfunction in preeclampsia involves the coronary microcirculation. Further research is needed to establish interventions to mitigate risk of preeclampsia-associated cardiovascular disease.

Pod-based e-liquids impair human vascular endothelial cell function.

Pod-based electronic (e-) cigarettes more efficiently deliver nicotine using a protonated formulation. The cardiovascular effects associated with these devices are poorly understood. We evaluated whether pod-based e-liquids and their individual components impair endothelial cell function. We isolated endothelial cells from people who are pod users (n = 10), tobacco never users (n = 7), and combustible cigarette users (n = 6). After a structured use, pod users had lower acetylcholine-mediated endothelial nitric oxide synthase (eNOS) activation compared with never users and was similar to levels from combustible cigarette users (overall P = 0.008, P = 0.01 pod vs never; P = 0.96 pod vs combustible cigarette). The effects of pod-based e-cigarettes and their constituents on vascular cell function were further studied in commercially available human aortic endothelial cells (HAECs) incubated with flavored JUUL e-liquids or propylene glycol (PG):vegetable glycerol (VG) at 30:70 ratio with or without 60 mg/mL nicotine salt for 90 min. A progressive increase in cell death with JUUL e-liquid exposure was observed across 0.0001-1% dilutions; PG:VG vehicle with and without nicotine salt induced cell death. A23187-stimulated nitric oxide production was decreased with all JUUL e-liquid flavors, PG:VG and nicotine salt exposures. Aerosols generated by JUUL e-liquid heating similarly decreased stimulated nitric oxide production. Only mint flavored e-liquids increased inflammation and menthol flavored e-liquids enhanced oxidative stress in HAECs. In conclusion, pod e-liquids and their individual components appear to impair endothelial cell function. These findings indicate the potential harm of pod-based devices on endothelial cell function and thus may be relevant to cardiovascular injury in pod type e-cigarette users.

Association of electronic cigarette use with circulating angiogenic cell levels in healthy young adults: Evidence for chronic systemic injury.

BACKGROUND: Circulating angiogenic cells (CACs) are indicative of vascular health and repair capacity; however, their relationship with chronic e-cigarette use is unclear. This study aims to assess the association between e-cigarette use and CAC levels. METHODS: We analyzed CAC levels in 324 healthy participants aged 21-45 years from the cross-sectional Cardiovascular Injury due to Tobacco Use study in four groups: never tobacco users (n = 65), sole e-cigarette users (n = 19), sole combustible cigarette users (n = 212), and dual users (n = 28). A total of 15 CAC subpopulations with four cell surface markers were measured using flow cytometry: CD146 (endothelial), CD34 (stem), CD45 (leukocyte), and AC133 (early progenitor/stem). Generalized linear models with gamma distribution and log-link were generated to assess association between CACs and smoking status. Benjamini-Hochberg were used to adjust p-values for multiple comparisons. RESULTS: The cohort was 47% female, 51% Black/African American, with a mean (± SD) age of 31 ± 7 years. Sole cigarette use was significantly associated with higher levels of two endothelial marker CACs (Q ⩽ 0.05). Dual users had higher levels of four endothelial marker CACs and one early progenitor/stem marker CAC (Q ⩽ 0.05). Sole e-cigarette users had higher levels of one endothelial and one leukocyte marker CAC (Q ⩽ 0.05). CONCLUSION: Dual use of e-cigarettes and combustible cigarettes was associated with higher levels of endothelial origin CACs, indicative of vascular injury. Sole use of e-cigarettes was associated with higher endothelial and inflammatory CACs, suggesting ongoing systemic injury. Distinct patterns of changes in CAC subpopulations suggest that CACs may be informative biomarkers of changes in vascular health due to tobacco product use.

Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis.

AIMS: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. RESULTS: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. CONCLUSIONS: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Lipid profiles in users of combustible and electronic cigarettes.

Electronic cigarette use has especially risen among adolescents and young adults. The aim of this study was to investigate fasting blood glucose and lipid profiles in chronic combustible cigarette and electronic cigarette users. We evaluated participants aged 21 to 45 (n = 525, mean age 31 ± 7 years, 45% women) without established cardiovascular disease or risk factors who were combustible cigarette users (n = 290), electronic cigarette users (n = 131; 65 sole users and 66 dual users), or never users (n = 104). In the first wave of enrollment (2014-2017), electronic cigarette users reported their products as first, second and third generation devices (e-cig users) and were all largely current (i.e., dual) or former (sole) combustible cigarette users, whereas in the second wave of enrollment (2019-2020), electronic cigarette users all reported pod-based device use (pod users) and included more sole users who were never smokers. In multivariable-adjusted analyses comparing to never users, both sole e-cig users and combustible cigarette users had higher glucose and triglycerides and lower high-density lipoprotein (HDL) cholesterol levels. Dual e-cig users showed higher triglycerides and very-low-density lipoprotein cholesterol, and lower HDL cholesterol compared to never users. In contrast, pod users (both sole and dual) had lipid profiles and glucose levels similar to never users. Overall, users of early generation electronic cigarettes display adverse metabolic profiles. In contrast, pod-based electronic cigarette users have similar lipid profiles to never users. Future studies are needed to understand the cumulative effects of electronic cigarette use on cardiometabolic health.

O-GlcNAcylation Mediates Glucose-Induced Alterations in Endothelial Cell Phenotype in Human Diabetes Mellitus.

Background Posttranslational protein modification with O-linked N-acetylglucosamine (O-GlcNAc) is linked to high glucose levels in type 2 diabetes mellitus (T2DM) and may alter cellular function. We sought to elucidate the involvement of O-GlcNAc modification in endothelial dysfunction in patients with T2DM. Methods and Results Freshly isolated endothelial cells obtained by J-wire biopsy from a forearm vein of patients with T2DM (n=18) was compared with controls (n=10). Endothelial O-GlcNAc levels were 1.8-ford higher in T2DM patients than in nondiabetic controls (P=0.003). Higher endothelial O-GlcNAc levels correlated with serum fasting blood glucose level (r=0.433, P=0.024) and hemoglobin A1c (r=0.418, P=0.042). In endothelial cells from patients with T2DM, normal glucose conditions (24 hours at 5 mmol/L) lowered O-GlcNAc levels and restored insulin-mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O-GlcNAc levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O-GlcNAcase inhibitor, increased O-GlcNAc levels and blunted the improvement of insulin-mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O-GlcNAc modification in the dynamic, glucose-induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2DM. O-GlcNAc protein modification may be a treatment target for vascular dysfunction in T2DM.

Alterations in Vascular Function Associated With the Use of Combustible and Electronic Cigarettes.

Background Electronic cigarettes (e-cigarettes) have been proposed as a potential harm reduction tool for combustible cigarette smokers. The majority of adult e-cigarette users continue to smoke combustible cigarettes and are considered dual users. The vascular impact of e-cigarettes remains incompletely defined. Methods and Results We examined the association of e-cigarette use with measures of vascular function and tonometry, preclinical measures of cardiovascular injury. As part of the CITU (Cardiovascular Injury due to Tobacco Use) study, we performed noninvasive vascular function testing in individuals without known cardiovascular disease or cardiovascular disease risk factors who were nonsmokers (n=94), users of combustible cigarettes (n=285), users of e-cigarettes (n=36), or dual users (n=52). In unadjusted analyses, measures of arterial stiffness including carotid-femoral pulse wave velocity, augmentation index, carotid-radial pulse wave velocity, and central blood pressures differed across the use groups. In multivariable models adjusted for age, sex, race, and study site, combustible cigarette smokers had higher augmentation index compared with nonusers (129.8±1.5 versus 118.8±2.7, P=0.003). The augmentation index was similar between combustible cigarette smokers compared with sole e-cigarette users (129.8±1.5 versus 126.2±5.9, P=1.0) and dual users (129.8±1.5 versus 134.9±4.0, P=1.0). Endothelial cells from combustible cigarette smokers and sole e-cigarette users produced less nitric oxide in response to A23187 stimulation compared with nonsmokers, suggestive of impaired endothelial nitric oxide synthase signaling. Conclusions Our findings suggest that e-cigarette use is not associated with a more favorable vascular profile. Future longitudinal studies are needed to evaluate the long-term risks of sustained e-cigarette use.

Liraglutide Treatment Reduces Endothelial Endoplasmic Reticulum Stress and Insulin Resistance in Patients With Diabetes Mellitus.

Background Prior studies have shown that nutrient excess induces endoplasmic reticulum ( ER ) stress in nonvascular tissues from patients with diabetes mellitus ( DM ). ER stress and the subsequent unfolded protein response may be protective, but sustained activation may drive vascular injury. Whether ER stress contributes to endothelial dysfunction in patients with DM remains unknown. Methods and Results To characterize vascular ER stress, we isolated endothelial cells from 42 patients with DM and 37 subjects without DM. Endothelial cells from patients with DM displayed higher levels of ER stress markers compared with controls without DM. Both the early adaptive response, evidenced by higher phosphorylated protein kinase-like ER eukaryotic initiation factor-2a kinase and inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.02, P=0.007, respectively), and the chronic ER stress response evidenced by higher C/ EBP α-homologous protein ( P=0.02), were activated in patients with DM . Higher inositol-requiring ER-to-nucleus signaling protein 1 activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction ( r=0.53, P=0.02). Acute treatment with liraglutide, a glucagon-like peptide 1 receptor agonist, reduced p-inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.01), and the activation of its downstream target c-jun N-terminal kinase ( P=0.025) in endothelial cells from patients with DM . Furthermore, liraglutide restored insulin-stimulated endothelial nitric oxide synthase activation in patients with DM ( P=0.019). Conclusions In summary, our data suggest that ER stress contributes to vascular insulin resistance and endothelial dysfunction in patients with DM . Further, we have demonstrated that liraglutide ameliorates ER stress, decreases c-jun N-terminal kinase activation and restores insulin-mediated endothelial nitric oxide synthase activation in endothelial cells from patients with DM .

Flavorings in Tobacco Products Induce Endothelial Cell Dysfunction.

OBJECTIVE: Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function. APPROACH AND RESULTS: Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1-100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction. CONCLUSIONS: Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity.

Endothelial Nox4-based NADPH oxidase regulates atherosclerosis via soluble epoxide hydrolase.

Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. OBJECTIVE: Our goal was to investigate the mechanisms of endothelial Nox4 in regulating atherosclerosis. APPROACH AND RESULTS: Atherosclerosis-prone conditions (disturbed blood flow, type I diabetes, and Western diet) downregulated endothelial Nox4 mRNA in arteries. To address whether the downregulated endothelial Nox4 was directly involved in the development of atherosclerosis, we generated mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), driven by the endothelial specific promoter Tie-2, on atherosclerosis-prone genetic background (ApoE deficient mice) to mimic the effect of decreased endothelial Nox4. Nox4DN significantly increased type I diabetes-induced aortic stiffness and atherosclerotic lesions. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly upregulated in Nox4DN endothelial cells (EC). Inhibition of sEH activity in Nox4DN EC suppressed inflammation and macrophage adhesion to EC. On the contrary, overexpression of endothelial wild type Nox4 suppressed sEH, ameliorated Western diet-induced atherosclerosis and decreased aortic stiffness. CONCLUSIONS: Atherosclerosis-prone conditions downregulated endothelial Nox4 to accelerate the progress of atherosclerosis, at least in part, by upregulating sEH to enhance inflammation.

The redox mechanism for vascular barrier dysfunction associated with metabolic disorders: Glutathionylation of Rac1 in endothelial cells.

BACKGROUND: Oxidative stress is implicated in increased vascular permeability associated with metabolic disorders, but the underlying redox mechanism is poorly defined. S-glutathionylation, a stable adduct of glutathione with protein sulfhydryl, is a reversible oxidative modification of protein and is emerging as an important redox signaling paradigm in cardiovascular physiopathology. The present study determines the role of protein S-glutathionylation in metabolic stress-induced endothelial cell permeability. METHODS AND RESULTS: In endothelial cells isolated from patients with type-2 diabetes mellitus, protein S-glutathionylation level was increased. This change was also observed in aortic endothelium in ApoE deficient (ApoE-/-) mice fed on Western diet. Metabolic stress-induced protein S-glutathionylation in human aortic endothelial cells (HAEC) was positively correlated with elevated endothelial cell permeability, as reflected by disassembly of cell-cell adherens junctions and cortical actin structures. These impairments were reversed by adenoviral overexpression of a specific de-glutathionylation enzyme, glutaredoxin-1 in cultured HAECs. Consistently, transgenic overexpression of human Glrx-1 in ApoE-/- mice fed the Western diet attenuated endothelial protein S-glutathionylation, actin cytoskeletal disorganization, and vascular permeability in the aorta. Mechanistically, glutathionylation and inactivation of Rac1, a small RhoGPase, were associated with endothelial hyperpermeability caused by metabolic stress. Glutathionylation of Rac1 on cysteine 81 and 157 located adjacent to guanine nucleotide binding site was required for the metabolic stress to inhibit Rac1 activity and promote endothelial hyperpermeability. CONCLUSIONS: Glutathionylation and inactivation of Rac1 in endothelial cells represent a novel redox mechanism of vascular barrier dysfunction associated with metabolic disorders.

Vascular Smooth Muscle Sirtuin-1 Protects Against Diet-Induced Aortic Stiffness.

Arterial stiffness, a major cardiovascular risk factor, develops within 2 months in mice fed a high-fat, high-sucrose (HFHS) diet, serving as a model of human metabolic syndrome, and it is associated with activation of proinflammatory and oxidant pathways in vascular smooth muscle (VSM) cells. Sirtuin-1 (SirT1) is an NAD(+)-dependent deacetylase regulated by the cellular metabolic status. Our goal was to study the effects of VSM SirT1 on arterial stiffness in the context of diet-induced metabolic syndrome. Overnight fasting acutely decreased arterial stiffness, measured in vivo by pulse wave velocity, in mice fed HFHS for 2 or 8 months, but not in mice lacking SirT1 in VSM (SMKO). Similarly, VSM-specific genetic SirT1 overexpression (SMTG) prevented pulse wave velocity increases induced by HFHS feeding, during 8 months. Administration of resveratrol or S17834, 2 polyphenolic compounds known to activate SirT1, prevented HFHS-induced arterial stiffness and were mimicked by global SirT1 overexpression (SirT1 bacterial artificial chromosome overexpressor), without evident metabolic improvements. In addition, HFHS-induced pulse wave velocity increases were reversed by 1-week treatment with a specific, small molecule SirT1 activator (SRT1720). These beneficial effects of pharmacological or genetic SirT1 activation, against HFHS-induced arterial stiffness, were associated with a decrease in nuclear factor kappa light chain enhancer of activated B cells (NFκB) activation and vascular cell adhesion molecule (VCAM-1) and p47phox protein expressions, in aorta and VSM cells. In conclusion, VSM SirT1 activation decreases arterial stiffness in the setting of obesity by stimulating anti-inflammatory and antioxidant pathways in the aorta. SirT1 activators may represent a novel therapeutic approach to prevent arterial stiffness and associated cardiovascular complications in overweight/obese individuals with metabolic syndrome.

Mitochondrial DNA damage and vascular function in patients with diabetes mellitus and atherosclerotic cardiovascular disease.

OBJECTIVE: Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. APPROACH AND RESULTS: We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (ß = 0.14 ± 0.13, P = 0.04 and ß = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. CONCLUSIONS: We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.

Pro-atherogenic role of smooth muscle Nox4-based NADPH oxidase.

UNLABELLED: Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. OBJECTIVE: Our goal was to investigate the role of smooth muscle Nox4 in atherosclerosis. APPROACH AND RESULTS: Atherosclerosis-prone conditions (disturbed blood flow and Western diet) increased Nox4 mRNA level in smooth muscle of arteries. To address whether upregulated smooth muscle Nox4 under atherosclerosis-prone conditions was directly involved in the development of atherosclerosis, mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), specifically in smooth muscle, were generated on a FVB/N ApoE deficient genetic background to counter the effect of increased smooth muscle Nox4. Nox4DN significantly decreased aortic stiffness and atherosclerotic lesions, with no effect on blood pressure. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly downregulated in Nox4DN smooth muscle cells (SMC), at both mRNA and protein levels. Downregulation of sEH by siRNA decreased SMC proliferation and migration, and suppressed inflammation and macrophage adhesion to SMC. CONCLUSIONS: Downregulation of smooth muscle Nox4 inhibits atherosclerosis by suppressing sEH, which, at least in part, accounts for inhibition of SMC proliferation, migration and inflammation.

Endothelial Dysfunction in Human Diabetes Is Mediated by Wnt5a-JNK Signaling.

OBJECTIVE: Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. APPROACH AND RESULTS: We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. CONCLUSIONS: Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus.

Glutathione adducts on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery.

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.

Nox2 mediates high fat high sucrose diet-induced nitric oxide dysfunction and inflammation in aortic smooth muscle cells.

Diet-induced obesity and metabolic syndrome are important contributors to cardiovascular diseases. The decreased nitric oxide (NO) bioactivity in endothelium and the impaired response of smooth muscle cell (SMC) to NO significantly contribute to vascular pathologies, including atherosclerosis and arterial restenosis after angioplasty. Sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is an important mediator of NO function in both endothelial cells and SMCs, and its irreversible oxidation impairs its stimulation by NO. We used C57BL/6J mice fed a high fat high sucrose diet (HFHSD) to study the role of SMC SERCA in diet-induced obesity and metabolic syndrome. We found that HFHSD upregulated Nox2 based NADPH oxidase, induced inflammation, increased irreversible SERCA oxidation, and suppressed the response of aortic SERCA to NO. Cultured aortic SMCs from mice fed HFHSD showed increased reactive oxygen species production, Nox2 upregulation, irreversible SERCA oxidation, inflammation, and a decreased ability of NO to inhibit SMC migration. Overexpression of wild type SERCA2b or downregulation of Nox2 restored NO-mediated inhibition of migration in SMCs isolated from HFHSD-fed mice. In addition, tumor necrosis factor alpha (TNFα) increased Nox2 which induced SERCA oxidation and inflammation. Taken together, Nox2 induced by HFHSD plays significant roles in controlling SMC responses to NO and TNFα-mediated inflammation, which may contribute to the development of cardiovascular diseases in diet-induced obesity and metabolic syndrome.

Arterial stiffening precedes systolic hypertension in diet-induced obesity.

Stiffening of conduit arteries is a risk factor for cardiovascular morbidity. Aortic wall stiffening increases pulsatile hemodynamic forces that are detrimental to the microcirculation in highly perfused organs, such as the heart, brain, and kidney. Arterial stiffness is associated with hypertension but presumed to be due to an adaptive response to increased hemodynamic load. In contrast, a recent clinical study found that stiffness precedes and may contribute to the development of hypertension although the mechanisms underlying hypertension are unknown. Here, we report that in a diet-induced model of obesity, arterial stiffness, measured in vivo, develops within 1 month of the initiation of the diet and precedes the development of hypertension by 5 months. Diet-induced obese mice recapitulate the metabolic syndrome and are characterized by inflammation in visceral fat and aorta. Normalization of the metabolic state by weight loss resulted in return of arterial stiffness and blood pressure to normal. Our findings support the hypothesis that arterial stiffness is a cause rather than a consequence of hypertension.