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Overnutrition-induced endothelial inflammation plays a crucial role in high-fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficientmice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.

Neurofibromatosis type 1 (NF1) is a heritable cancer predisposition syndrome resulting from mutations in the NF1 tumor suppressor gene. Genotype-phenotype correlations for NF1 are rare due to the large number of NF1 mutations and role of modifier genes in manifestations of NF1; however, emerging reports suggest that persons with NF1 display a distinct anthropometric and metabolic phenotype featuring short stature, low body mass index (BMI), increased insulin sensitivity, and protection from diabetes. Nf1 heterozygous (Nf1+/−) mice accurately reflect the dominant inheritance of NF1 and are regularly employed as a model of NF1. Here, we sought to identify whether Nf1+/− mice recapitulate the anthropometric and metabolic features identified in persons with NF1. Littermate 16–20 week-old male wildtype (WT) and Nf1+/− C57B/6J mice underwent nuclear magnetic resonance (NMR), indirect calorimetry, and glucose/insulin/pyruvate tolerance testing. In some experiments, tissues were harvested for NMR and histologic characterization. Nf1+/− mice are leaner with significantly reduced visceral and subcutaneous fat mass, which corresponds with an increased density of small adipocytes and reduced leptin levels. Additionally, Nf1+/− mice are highly reliant on carbohydrates as an energy substrate and display increased glucose clearance and insulin sensitivity, but normal response to pyruvate suggesting enhanced glucose utilization and preserved gluconeogenesis. Finally, WT and Nf1+/− mice subjected to high glucose diet were protected from diet-induced obesity and hyperglycemia. Our data suggest that Nf1+/− mice closely recapitulate the anthropometric and metabolic phenotype identified in persons with NF1, which will impact the interpretation of previous and future translational studies of NF1.

Elevated expression of N-acetyltransferase 1 (NAT1) is associated with invasive and lobular breast carcinomas as well as with bone metastasis following an epithelial-to-mesenchymal transition. We investigated the effect of NAT1 gene deletion in three different human breast cancer cell lines, MDA-MB-231, MCF-7, and ZR-75-1. Human NAT1 was knocked out using CRISPR/Cas9 technology and two different guide RNAs. None of the NAT1 knockout (KO) cell lines exhibited detectable NAT1 activity when measured using its selective substrate p-aminobenzoic acid (PABA). Endogenous acetyl coenzyme A levels (cofactor for acetylation pathways) in NAT1 KO cell lines were significantly elevated in the MDA-MB-231 (p<0.001) and MCF-7 (p=0.0127) but not the ZR-75-1 (p>0.05). Although the effects of NAT1 KO on cell-doubling time were inconsistent across the three breast cancer cell lines, the ability of the NAT1 KO cell lines to form anchorage-independent colonies in soft agar was dramatically and consistently reduced in each of the breast cancer cell lines. The NAT1 KO clones for MDA-MB-231, MCF-7, and ZR-75-1 had a reduction greater than 20-, 6-, and 7- folds in anchorage-independent cell growth, respectively, compared to their parental cell lines (p<0.0001, p<0.0001, and p<0.05, respectively). The results indicate that NAT1 may be an important regulator of cellular acetyl coenzyme A levels and strongly suggest that elevated NAT1 expression in breast cancers contribute to their anchorage-independent growth properties and ultimately metastatic potential.

A defining characteristic of pulmonary hypertension (PH) is the extensive remodeling of pulmonary arteries (PAs), which results in progressive increases in vascular resistance and stiffness and eventual failure of the right ventricle. There is no cure for PH and identification of novel molecular mechanisms that underlie increased proliferation, reduced apoptosis, and excessive extracellular matrix production in pulmonary artery smooth muscle cells (PASMCs) is a vital objective. Galectin-3 (Gal-3) is a chimeric lectin and potent driver of many aspects of fibrosis, but its role in regulating PASMC behavior in PH remains poorly understood. Herein, we evaluated the importance of increased Gal-3 expression and signaling on PA vascular remodeling and cardiopulmonary function in experimental models of PH. Gal-3 expression was quantified by qRT-PCR, immunoblotting, and immunofluorescence imaging, and its functional role was assessed by specific Gal-3 inhibitors and CRISPR/Cas9-mediated knockout of Gal-3 in the rat. In rat models of PH, we observed increased Gal-3 expression in PASMCs, which stimulated migration and resistance to apoptosis, whereas silencing or genetic deletion reduced cellular migration and PA fibrosis and increased apoptosis. Gal-3 inhibitors attenuated and reversed PA remodeling and fibrosis, as well as hemodynamic indices in monocrotaline (MCT)-treated rats in vivo. These results were supported by genetic deletion of Gal-3 in both MCT and Sugen Hypoxia rat models. In conclusion, our results suggest that elevated Gal-3 levels contribute to inappropriate PA remodeling in PH by enhancing multiple profibrotic mechanisms. Therapeutic strategies targeting Gal-3 may be of benefit in the treatment of PH.

BACKGROUND: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca(2+) signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca(2+) signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, TRPV4 ion channels are a major Ca(2+) influx pathway in endothelial cells, and regulatory protein AKAP150 enhances the activity of TRPV4 channels. METHODS: We used endothelium-specific knockout mice and high fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide (NO) and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity, and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from non-obese and obese individuals. RESULTS: We show that Ca(2+) influx through TRPV4 channels at myoendothelial projections (MEPs) to smooth muscle cells decreases resting blood pressure in non-obese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule NO attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of iNOS and NOX1 enzymes at MEPs in obese mice generated higher levels of NO and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at MEPs. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150, and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Importantly, peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. CONCLUSIONS: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension, and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Objective Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. Methods We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). Results Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. Conclusions These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.

Objective— Copper transporter ATP7A (copper-transporting/ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM (type2 diabetes mellitus) mice and human in which insulin–Akt (protein kinase B) pathway is selectively impaired. Approach and Results— Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet–induced or db/db T2DM mice. Akt2 (protein kinase B beta) activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B −/− (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhance insulin–Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2 −/− vessels or VSMCs, which is rescued by ATP7A overexpression. Conclusion— Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.

Rationale: Early vascular changes in metabolic disease that precipitate the development of cardiovascular complications are largely driven by reactive oxygen species accumulation, yet the extent to which excess reactive oxygen species derive from specific NADPH oxidase isoforms remains ill defined. Objective: Identify the role of Nox1 in the development of microvascular dysfunction in metabolic disease. Methods and Results: Four genotypes were generated by breeding Nox1 knockout mice with db/db mice: lean (H db W nox1 ), lean Nox1 knockout (H db K nox1 ), obese (K db W nox1 ), and obese KK (K db K nox1 ). The degree of adiposity, insulin resistance, and dyslipidemia in KW mice was not influenced by Nox1 deletion as determined by nuclear magnetic resonance spectroscopy, glucose tolerance tests, and plasma analyses. Endothelium-dependent responses to acetylcholine in pressurized mesenteric arteries were reduced in KW versus HW ( P P Conclusions: Nox1 deletion reduces oxidant load and restores microvascular health in db/db mice without influencing the degree of metabolic dysfunction. Therefore, targeted Nox1 inhibition may be effective in the prevention of vascular complications.

Traditional aerobic exercise reduces the risk of developing chronic diseases by inducing immune, metabolic, and myokine responses. Following traditional exercise, both the magnitude and time-course of these beneficial responses are different between obese compared to normal weight individuals. Although obesity may affect the ability to engage in traditional exercise, whole body vibration (WBV) has emerged as a more tolerable form of exercise . The impact of WBV on immune, metabolic, and myokine responses as well as differences between normal weight and obese individuals, however, is unknown. Purpose To determine if WBV elicits differential magnitudes and time-courses of immune, metabolic, and myokine responses between obese and normal weight individuals. Methods 21 participants [Obese (OB): n = 11, Age: 33 ± 4 y, percent body fat (%BF): 39.1 ± 2.4% & Normal weight (NW) n = 10, Age: 28 ± 8 y, %BF: 17.4 ± 2.1%] engaged in 10 cycles of WBV exercise [1 cycle = 1 min of vibration followed by 30 s of rest]. Blood samples were collected pre-WBV (PRE), immediately (POST), 3 h (3H), and 24 h (24H) post-WBV and analyzed for leukocytes, insulin, glucose, and myokines (IL-6, decorin, myostatin). Results The peak (3H) percent change in neutrophil counts (OB: 13.9 ± 17.4 vs. NW: 47.2 ± 6.2%Δ; p = 0.007) was different between groups. The percent change in neutrophil percentages was increased in NW (POST: -1.6 ± 2.0 vs. 3H: 13.0 ± 7.2%Δ, p = 0.019) but not OB (p > 0.05). HOMA β-cell function was increased at 24H (PRE: 83.4 ± 5.4 vs. 24H: 131.0 ± 14.1%; p = 0.013) in NW and was not altered in OB (p > 0.05). PRE IL-6 was greater in OB compared to NW (OB: 2.7 ± 0.6 vs. NW: 0.6 ± 0.1 pg/mL; p = 0.011); however, the percent change from PRE to peak (3H) was greater in NW (OB: 148.1 ± 47.9 vs. NW: 1277.9 ± 597.6 %Δ; p = 0.035). Creatine kinase, decorin, and myostatin were not significantly altered in either group (p > 0.05). Conclusion Taken together, these data suggest that acute whole body vibration elicits favorable immune, metabolic, and myokine responses and that these responses differ between obese and normal weight individuals., Highlights • Whole body vibration (WBV) normalizes lymphocytes in obese. • 10 min of WBV facilitates increases in neutrophils in normal weight but not obese. • WBV produces myokine IL-6 in both obese and normal weight. • WBV improves glucose metabolism in obese. • Improvements in glucose metabolism correspond to peak IL-6 concentrations.

Insulin resistance-related disorders are associated with endothelial dysfunction. Accumulating evidence has suggested a role for adenosine signaling in the regulation of endothelial function. Here, we identified a crucial role of endothelial adenosine kinase (ADK) in the regulation of insulin resistance. Feeding mice with a high-fat diet (HFD) markedly enhanced the expression of endothelial Adk. Ablation of endothelial Adk in HFD-fed mice improved glucose tolerance and insulin sensitivity and decreased hepatic steatosis, adipose inflammation and adiposity, which were associated with improved arteriole vasodilation, decreased inflammation and increased adipose angiogenesis. Mechanistically, ADK inhibition or knockdown in human umbilical vein endothelial cells (HUVECs) elevated intracellular adenosine level and increased endothelial nitric oxide synthase (NOS3) activity, resulting in an increase in nitric oxide (NO) production. Antagonism of adenosine receptor A2b abolished ADK-knockdown-enhanced NOS3 expression in HUVECs. Additionally, increased phosphorylation of NOS3 in ADK-knockdown HUVECs was regulated by an adenosine receptor-independent mechanism. These data suggest that Adk-deficiency-elevated intracellular adenosine in endothelial cells ameliorates diet-induced insulin resistance and metabolic disorders, and this is associated with an enhancement of NO production caused by increased NOS3 expression and activation. Therefore, ADK is a potential target for the prevention and treatment of metabolic disorders associated with insulin resistance.

Diabetes is the leading cause of kidney failure, accounting for >45% of new cases of dialysis. Diabetic nephropathy is characterized by inflammation, fibrosis, and oxidant stress, pathologic features that are shared by many other chronic inflammatory diseases. The cytokine IL-17A was initially implicated as a mediator of chronic inflammatory diseases, but recent studies dispute these findings and suggest that IL-17A can favorably modulate inflammation. Here, we examined the role of IL-17A in diabetic nephropathy. We observed that IL-17A levels in plasma and urine were reduced in patients with advanced diabetic nephropathy. Type 1 diabetic mice that are genetically deficient in IL-17A developed more severe nephropathy, whereas administration of low-dose IL-17A prevented diabetic nephropathy in models of type 1 and type 2 diabetes. Moreover, IL-17A administration effectively treated, prevented, and reversed established nephropathy in genetic models of diabetes. Protective effects were also observed after administration of IL-17F but not IL-17C or IL-17E. Notably, tubular epithelial cell-specific overexpression of IL-17A was sufficient to suppress diabetic nephropathy. Mechanistically, IL-17A administration suppressed phosphorylation of signal transducer and activator of transcription 3, a central mediator of fibrosis, upregulated anti-inflammatory microglia/macrophage WAP domain protein in an AMP-activated protein kinase-dependent manner and favorably modulated renal oxidative stress and AMP-activated protein kinase activation. Administration of recombinant microglia/macrophage WAP domain protein suppressed diabetes-induced albuminuria and enhanced M2 marker expression. These observations suggest that the beneficial effects of IL-17 are isoform-specific and identify low-dose IL-17A administration as a promising therapeutic approach in diabetic kidney disease.

Blood pressure exhibits a robust circadian rhythm in health. In hypertension, sleep apnea, and even shift work, this balanced rhythm is perturbed via elevations in night-time blood pressure, inflicting silent damage to the vasculature and body organs. Herein, we examined the influence of circadian dysfunction during experimental hypertension in mice. Using radiotelemetry to measure ambulatory blood pressure and activity, the effects of angiotensin II administration were studied in wild-type (WT) and period isoform knockout (KO) mice (Per2-KO, Per2, 3-KO, and Per1, 2, 3-KO/Per triple KO [TKO] mice). On a normal diet, administration of angiotensin II caused nondipping blood pressure and exacerbated vascular hypertrophy in the Period isoform KO mice relative to WT mice. To study the endogenous effects of angiotensin II stimulation, we then administered a low-salt diet to the mice, which does stimulate endogenous angiotensin II in addition to lowering blood pressure. A low-salt diet decreased blood pressure in wild-type mice. In contrast, Period isoform KO mice lost their circadian rhythm in blood pressure on a low-salt diet, because of an increase in resting blood pressure, which was restorable to rhythmicity by the angiotensin receptor blocker losartan. Chronic administration of low salt caused vascular hypertrophy in Period isoform KO mice, which also exhibited increased renin levels and altered angiotensin 1 receptor expression. These data suggest that circadian clock genes may act to inhibit or control renin/angiotensin signaling. Moreover, circadian disorders such as sleep apnea and shift work may alter the homeostatic responses to sodium restriction to potentially influence nocturnal hypertension.

Background Obesity compromises cardiometabolic function and is associated with hypertension and chronic kidney disease. Exercise ameliorates these conditions, even without weight loss. Although the mechanisms of exercise's benefits remain unclear, augmented lean body mass is a suspected mechanism. Myostatin is a potent negative regulator of skeletal muscle mass that is upregulated in obesity and downregulated with exercise. The current study tested the hypothesis that deletion of myostatin would increase muscle mass and reduce blood pressure and kidney injury in obesity. Methods and Results Myostatin knockout mice were crossed to db/db mice, and metabolic and cardiovascular functions were examined. Deletion of myostatin increased skeletal muscle mass by ≈50% to 60% without concomitant weight loss or reduction in fat mass. Increased blood pressure in obesity was prevented by the deletion of myostatin, but did not confer additional benefit against salt loading. Kidney injury was evident because of increased albuminuria, which was abolished in obese mice lacking myostatin. Glycosuria, total urine volume, and whole kidney NOX‐4 levels were increased in obesity and prevented by myostatin deletion, arguing that increased muscle mass provides a multipronged defense against renal dysfunction in obese mice. Conclusions These experimental observations suggest that loss of muscle mass is a novel risk factor in obesity‐derived cardiovascular dysfunction. Interventions that increase muscle mass, either through exercise or pharmacologically, may help limit cardiovascular disease in obese individuals.

Pneumonia is a leading cause of death in children and the elderly worldwide, accounting for 15% of all deaths of children under 5 years old. Streptococcus pneumoniae is a common and aggressive cause of pneumonia and can also contribute to meningitis and sepsis. Despite the widespread use of antibiotics, mortality rates for pneumonia remain unacceptably high in part due to the release of bacterial toxins. Pneumolysin (PLY), is a cholesterol-dependent toxin that is produced by Streptococcus and it is both necessary and sufficient for the development of the extensive pulmonary permeability edema that underlies acute lung injury (ALI). The mechanisms by which PLY disrupt the pulmonary endothelial barrier are not fully understood. Previously we found that reactive oxygen species (ROS) contribute to the barrier destructive effects of PLY and identified an unexpected but potent role of Hsp70 in suppressing ROS production. The ability of Hsp70 to influence PLY-induced barrier dysfunction is not yet described and the goal of the current study was to identify whether Hsp70 upregulation is an effective strategy to protect the lung microvascular endothelial barrier from G+ bacterial toxins. Overexpression of Hsp70 via adenovirus-mediated gene transfer attenuated PLY-stimulated increases in permeability in human lung microvascular endothelial cells (HLMVEC) with no evidence of cytotoxicity. To adopt a more translational approach, we employed a pharmacological approach using geranylgeranylacetone (GGA) to acutely upregulate endogenous Hsp70 expression. Following acute treatment (6h) with GGA, HLMVECs exposed to PLY displayed improved cell viability and enhanced endothelial barrier function as measured by both Electric Cell-substrate Impedance Sensing (ECIS) and transwell permeability assays compared to control treated cells. PLY promoted increased mitochondrial ROS, decreased mitochondrial oxygen consumption and increased caspase 3 cleavage and cell death, which were collectively improved in cells pre-treated with GGA. In mice, IP pre-treatment with GGA 24h prior to IT administration of PLY resulted in significantly less Evans Blue Dye extravasation compared to vehicle, indicating preserved endothelial barrier integrity and suggesting that the acute upregulation of Hsp70 may be an effective therapeutic approach in the treatment of lung injury associated with pneumonia.

The objective of this study is to test the hypothesis that increased muscle mass has positive effects on cardiovascular function. Specifically, we tested the hypothesis that increases in lean body mass caused by deletion of myostatin improves cardiac performance and vascular function. Echocardiography was used to quantify left ventricular function at baseline and after acute administration of propranolol and isoproterenol to assess β ‐adrenergic reactivity. Additionally, resistance vessels in several beds were removed, cannulated, pressurized to 60 mmHg and reactivity to vasoactive stimuli was assessed. Hemodynamics were measured using in vivo radiotelemetry. Myostatin deletion results in increased fractional shortening at baseline. Additionally, arterioles in the coronary and muscular microcirculations are more sensitive to endothelial‐dependent dilation while nonmuscular beds or the aorta were unaffected. β ‐adrenergic dilation was increased in both coronary and conduit arteries, suggesting a systemic effect of increased muscle mass on vascular function. Overall hemodynamics and physical characteristics (heart weight and size) remained unchanged. Myostatin deletion mimics in part the effects of exercise on cardiovascular function. It significantly increases lean muscle mass and results in muscle‐specific increases in endothelium‐dependent vasodilation. This suggests that increases in muscle mass may serve as a buffer against pathological states that specifically target cardiac function (heart failure), the β ‐adrenergic system (age), and nitric oxide bio‐availability (atherosclerosis). Taken together, pharmacological inhibition of the myostatin pathway could prove an excellent mechanism by which the benefits of exercise can be conferred in patients that are unable to exercise.

Pulmonary Arterial Hypertension (PAH) is characterized by excessive vascular cell proliferation, inward remodeling and increased stiffness and inflammation of the pulmonary blood vessels. We found that galectin-3 (Gal-3) is upregulated in PA from multiple models of PAH including monocrotaline (MCT), MCT + pneumonectomy, and SUGEN/hypoxia rats as well as in human PAH and correlated with severity of disease. Gal-3 is a β-galactoside binding lectin implicated in signaling pathways regulating cell proliferation, inflammation and fibrosis, but its role in PAH is poorly defined. Confocal analysis revealed the majority of Gal-3 expression in the media of PA of both rodent models and humans. Selective inhibitors of Gal-3 attenuated PAH in MCT-treated rats and reduced indices of proliferation, fibrosis and increased apoptosis in PA. Overexpression of Gal-3 in PASMC increased proliferation, migration and expression of profibrotic molecules and protected from apoptosis. Acute exposure of cultured HPASMC with various mitogens and factors important in the development of PAH, failed to increase Gal-3 expression. In contrast, PASMC isolated from rats with PAH exhibited an enduring capacity for increased proliferation and expressed higher levels of Gal-3 suggesting an epigenetic mechanism regulating Gal-3 expression. We found that treatment of PASMC with inhibitors of DNA methylation robustly increased Gal-3 expression in control human and rat PASMC but not in MCT-derived PASMC. Methylation analysis of DNA isolated from PA using MeDIP-qPCR and pyrosequencing revealed hypomethylation of Gal-3 proximal promoter. Analysis of DNA methyltransferase expression in PA revealed a significant loss of only Dnmt3A expression in hypertensive PA. To assess the role of local methylation in the regulation of Gal-3 expression we used CRISPR-dCas9. Targeted Gal-3 promoter methylation using multiple RNA guides and dCas9-Dnmt3A-Dnmt3L effectively reduced Gal-3 expression in SMC isolated from MCT rat PA and reversed the excessive proliferation. These results advance an important role of methylation-dependent mechanisms in Gal-3 signaling and provide a mechanism for the enduring changes in vascular cell behavior observed in PAH.

Objective— Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. Approach and Results— Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. Conclusion— These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. Expression of NAT1 is elevated in several cancers including breast cancer. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. This study explored polar metabolome differences between MDA-MB-231 breast cancer cells expressing varying levels of NAT1 activity using an untargeted approach. Three MDA-MB-231 breast adenocarcinoma cell lines that stably express wild-type, increased, and decreased levels of human NAT1 were investigated for differences in polar metabolic profile using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) system. Increased levels of human NAT1 in the transformed cell lines resulted in a statistically significant decreased abundance of the metabolite palmitoleic acid (q = 0.0006), when compared to normal and decreased levels of human NAT1. The fatty acid synthesis pathway utilizes acetyl coenzyme A (acetyl-CoA) in the first two reactions of the pathway and eventually leads to the synthesis of palmitoleic acid. These data suggest a link between increased levels of NAT1 activity and decreased flux of acetyl-CoA through this portion of the fatty acid synthesis pathway.

Obesity is a major risk factor for hypertension. The copresentation of hypertension and insulin resistance (IR) suggests a role for IR in blood pressure (BP) dysregulation. To test this hypothesis, peripheral IR has been genetically subtracted in a model of obesity by crossing leptin receptor mutant mice (K db H PTP ) with mice lacking protein tyrosine phosphatase 1B (insulin desensitizer, H db K PTP ) to generate obese insulin-sensitive mice (K db K PTP ). BP was recorded in lean (H db H PTP , H db K PTP ) and obese (K db H PTP , K db K PTP ) mice via telemetry, and a frequency analysis of the recording was performed to determine BP variability. Correction of IR in obese mice normalized BP values to baseline levels (H db H PTP : 116±2 mm Hg; K db H PTP : 129±4 mm Hg; K db K PTP : 114±5 mm Hg) and restored BP variability by decreasing its standard deviation and the frequency of BP values over the upper autoregulatory limit of the kidneys. However, although IR-induced increases in proteinuria (versus 53±13 μg/d, H db H PTP ) were corrected in K db K PTP (112±39 versus 422±159 μg/d, K db H PTP ), glomerular hypertrophy was not. IR reduced plasma aldosterone levels ruling out a role for mineralocorticoids in the development of hypertension. Taken together, these data indicate that correction of IR prevents hypertension, BP variability, and microalbuminuria in obese mice. Although the mechanism remains to be fully determined, increases in aldosterone or sympathoactivation of the cardiovascular system seem to be less likely contributors.

Diseases of the coronary circulation remain the leading cause of death in Western society despite impressive advances in diagnosis, pharmacotherapy and post-event management. Part of this statistic likely stems from a parallel increase in the prevalence of obesity and metabolic dysfunction, both significant risk factors for coronary disease. Obesity and diabetes pose unique challenges for the heart and their impact on the coronary vasculature remains incompletely understood. The vascular endothelium is a major interface between arterial function and the physical and chemical components of blood flow. Proper function of the endothelium is necessary to preserve hemostasis, maintain vascular tone and limit the extent of vascular diseases such as atherosclerosis. Given its central role in vascular health, endothelial dysfunction has been the source of considerable research interest in diabetes and obesity. In the current review, we will examine the pathologic impact of obesity and diabetes on coronary function and the extent to which these two factors impact endothelial function. This article is part of a Special Issue entitled “Coronary Blood Flow”.

Arginase can cause vascular dysfunction by competing with nitric oxide synthase for l-arginine and by increasing cell proliferation and collagen formation, which promote vascular fibrosis/stiffening. We have shown that increased arginase expression/activity contribute to vascular endothelial cell (EC) dysfunction. Here, we examined the roles of the two arginase isoforms, arginase I and II (AI and AII, respectively), in this process. Experiments were performed using streptozotocin-induced diabetic mice: wild-type (WT) mice and knockout mice lacking the AII isoform alone (AI+/+AII−/−) or in combination with partial deletion of AI (AI+/−AII −/−). EC-dependent vasorelaxation of aortic rings and arterial fibrosis and stiffness were assessed in relation to arginase activity and expression. Diabetes reduced mean EC-dependent vasorelaxation markedly in diabetic WT and AI+/+AII−/− aortas (53% and 44% vs. controls, respectively) compared with a 27% decrease in AI+/−AII −/− vessels. Coronary fibrosis was also increased in diabetic WT and AI+/+AII−/− mice (1.9- and 1.7-fold vs. controls, respectively) but was not altered in AI+/−AII −/− diabetic mice. Carotid stiffness was increased by 142% in WT diabetic mice compared with 51% in AI+/+AII−/− mice and 19% in AI+/−AII −/− mice. In diabetic WT and AI+/+AII−/− mice, aortic arginase activity and AI expression were significantly increased compared with control mice, but neither parameter was altered in AI+/−AII −/− mice. In summary, AI+/−AII −/− mice exhibit better EC-dependent vasodilation and less vascular stiffness and coronary fibrosis compared with diabetic WT and AI+/+AII−/− mice. These data indicate a major involvement of AI in diabetes-induced vascular dysfunction.

Objective— To determine if elasticity in blood vessels is compromised in circadian clock–mutant mice (Bmal1-knockout [KO] and Per-triple KO) and if matrix metalloproteinases (MMPs) might confer these changes in compliance. Methods and Results— High-resolution ultrasonography in vivo revealed impaired remodeling and increased pulse-wave velocity in the arteries of Bmal1-KO and Per-triple KO mice. In addition, compliance of remodeled arteries and naïve pressurized arterioles ex vivo from Bmal1-KO and Per-triple KO mice was reduced, consistent with stiffening of the vascular bed. The observed vascular stiffness was coincident with dysregulation of MMP-2 and MMP-9 in Bmal1-KO mice. Furthermore, inhibition of MMPs improved indexes of pathological remodeling in wild-type mice, but the effect was abolished in Bmal1-KO mice. Conclusion— Circadian clock dysfunction contributes to hardening of arteries, which may involve impaired control of the extracellular matrix composition.

Insulin resistance is an underlying mechanism of type 2 diabetes and its vascular complications. Recent evidence suggests that crosstalk between angiotensin II (Ang II) and the insulin signaling in vascular smooth muscle cell (VSMC) may contribute to cellular insulin resistance. We hypothesized that Ang II inhibits the anti-mitogenic pathways while enhancing the mitogenic pathways stimulated by insulin via activation of Protein Tyrosine Phosphatase-1B (PTP-1B) in VSMC. We found that Ang II significantly inhibited insulin-induced phosphorylation of tyrosine 608 of IRS-1 and serine 473 of Akt, a downstream member of anti-mitogenic pathway of insulin. In contrast, Ang II increased the serine phosphorylation of IRS-1 which was not affected by the presence of insulin. Activation of p42/p44 MAPK (a mitogenic pathway) induced by insulin was further enhanced by Ang II. Transfection of VSMC with PTP-1B antisense oligonucleotide markedly reduced the effects of Ang II on insulin signaling. Furthermore, an increase in VSMC growth was attenuated by PTP-1B antisense only in the presence of both Ang II and insulin. Finally, we also showed that Ang II -induced activation of PTP-1B in VSMC was PKA/JAK2 dependent. We conclude that Ang II modulates both anti-mitogenic and mitogenic pathways of insulin via the activation of PTP-1B.

Obesity is a risk factor for stroke, but the determinants of increased stroke risk in obesity are unknown. We have previously reported that obese Zucker rats (OZRs) have a worse stroke outcome and display evidence of remodeling of the middle cerebral artery (MCA), in parallel with hypertension, compared with lean controls. This study tested the hypothesis that hypertension is an essential determinant of cerebral vascular remodeling and increased stroke damage in OZRs. Blood pressure was measured by telemetery in lean and obese rats with and without hydrochlorthiazide (HCT; 2 mg·kg−1·day−1) from 8 to 15 wk of age. A separate group of rats was also chronically fed a low-sodium (LS) diet. Vessel structure was assessed in isolated, pressurized MCAs. Cerebral ischemia was induced for 60 min using an intralumenal suture technique, followed by 24 h of reperfusion. HCT treatment effectively prevented the increase in blood pressure in obese rats; however, the LS diet did not lower pressure. Importantly, infarct size was normalized by HCT after ischemia-reperfusion injury. Additionally, HCT improved the changes in MCA structure observed in untreated OZRs. There were no benefits of the LS diet on stroke injury or vessel structure. These results indicate that increased pressure is essential for driving the changes in infarct size in OZRs.

Background— Obesity causes hypertension and sympathoactivation, a process proposed to be mediated by leptin. Protein tyrosine phosphatase 1B (PTP1B), a major new pharmaceutical target in the treatment of obesity and type II diabetes mellitus, constrains the metabolic actions of leptin, but the extent to which PTP1B regulates its cardiovascular effects is unclear. This study examined the hypothesis that PTP1B is a negative regulator of the cardiovascular effects of leptin. Methods and Results— PTP1B knockout mice had lower body fat but higher mean arterial pressure (116±5 versus 105±5 mm Hg, P P P P 1 -adrenergic receptor subtypes, consistent with blunted constriction to phenylephrine. Conclusions— These data indicate that PTP1B is a key regulator of the cardiovascular effects of leptin and that reduced vascular adrenergic reactivity provides a compensatory limit to the effects of leptin on mean arterial pressure.

Impairment of vascular growth is a hallmark of diabetic complications, but the progression and mechanisms are poorly understood. To determine whether obesity and early diabetes impair endothelium-dependent vasodilatation and vascular response to ischemia, microvascular function as well as angiogenic responses to ischemia were assessed in young (C57) and 6-month-old lean mice (old C57), in obese (db-C57) mice, and in mice suffering an early (db-KsJ) and sustained type 2 diabetes (old db-KsJ). Glycemia gradually increased from the db-C57 to the old db-KsJ. Early and established type II diabetes significantly reduced the level of insulin that was significantly increased in obese mice. Endothelial function was assessed in isolated resistance arteries while the angiogenic response induced by unilateral hindlimb ischemia was analyzed, after 28 days, with a laser Doppler flowmeter and angiography. Aging (-21%), obesity (-45%), as well as early (-58%) and sustained type II diabetes (-69%) induced a progressive impairment of the endothelium-dependent relaxation of the gracilis artery. Laser Doppler measurements demonstrated that only early and sustained type II diabetes impaired skin blood flow recovery. Vascular collateralization was reduced with aging and severely impaired in older db-KsJ mice, the two strains of mice in which ischemia reduced eNOS expression. These results demonstrate that endothelial dysfunction induced by obesity is insufficient to alter the angiogenic response to ischemia. Furthermore, the development of frank type II diabetes or increasing age is required to impair the vascular response to hindlimb ischemia. We conclude that additional risk factors or severe endothelial dysfunction may be requisite to impede the angiogenic response to ischemia.

Obesity is a risk factor for stroke, but the mechanisms by which obesity increases stroke risk are unknown. Because microvascular architecture contributes to the outcome of stroke, we hypothesized that middle cerebral arteries (MCAs) from obese Zucker rats (OZRs) undergo inward remodeling and develop increased myogenic tone compared with those in lean Zucker rats (LZRs). We further hypothesized that OZRs have an increased infarct after cerebral ischemia and that changes in vascular structure and function correlate with the development of hypertension in OZRs. Blood pressure was measured by telemetry in LZRs and OZRs from 6 to 17 weeks of age. Vessel structure and function were assessed in isolated MCAs. Stroke damage was assessed after ischemia was induced for 60 minutes followed by 24 hours of reperfusion. Although mean arterial pressure was similar between young rats (6 to 8 weeks old), mean arterial pressure was higher in adult (14 to 17 weeks old) OZRs than in LZRs. MCAs from OZRs had a smaller lumen diameter and increased myogenic vasoconstriction compared with those from LZRs. After ischemia, infarction was 58% larger in OZRs than in LZRs. Before the development of hypertension, MCA myogenic reactivity and lumen diameter, as well as infarct size, were similar between young LZRs and OZRs. Our results indicate that the MCAs of OZRs undergo structural remodeling and that these rats have greater cerebral injury after cerebral ischemia. These cerebrovascular changes correlate with the development of hypertension and suggest that the increased blood pressure may be the major determinant for stroke risk in obese individuals.

High-potassium diets can improve vascular function, yet the effects of potassium supplementation on ischemic stroke have not been studied. We hypothesized that dietary potassium supplementation would reduce ischemic cerebral infarct size by reversing cerebral artery hypertrophy. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were fed diets containing 0.79% potassium (LK) or 2.11% potassium (HK) for 6 wk; Wistar-Kyoto (WKY) rats were fed the LK diet. The HK diet did not reduce blood pressure, as measured by telemetry, in the SHRSP. Cerebral ischemia was induced by middle cerebral artery (MCA) occlusion. The resultant infarct was smaller in the HK-SHRSP than in the LK-SHRSP: 55.1 ± 6.3 vs. 71.4 ± 2.4% of the hemisphere infarcted ( P < 0.05). Infarcts were smaller in WKY rats (33.5 ± 4.8%) than in LK-SHRSP or HK-SHRSP. The vessel wall of MCAs from LK-SHRSP was hypertrophied compared with WKY rats; this was reversed in HK-SHRSP. RT-PCR analysis of the cerebral vessels showed that expression of platelet-derived growth factor receptors-α and -β, epidermal growth factor receptor, and collagen I and III was increased in the vessels from LK-SHRSP compared with WKY rats and reduced in HK-SHRSP. These results suggest that potassium supplementation provides neuroprotection in a model of ischemic stroke independent of blood pressure and possibly through changes in vascular structure.

Obesity is an emerging epidemic worldwide and threatens cardiovascular health due to the complications of accompanying metabolic dysfunction. One potential mechanism by which these complications are advanced is structural alteration in the microcirculation perfusing target organs. In this brief review, the authors focus on the structural adaptations that occur in the microcirculation, with specific focus on changes in basement membrane thickness and vascular structure. By delineating changes in end-stage disease and recent insights from animal studies, they hope to convince the reader (1) that diabetic disease in the limb is associated with profound changes in arteriolar structure and (2) that parallel changes in microvascular architecture precede the onset of frank type II diabetes.

1. Obesity is rapidly increasing in Western populations, driving a parallel increase in hypertension, diabetes and vascular disease. Prior to the development of overt diabetes or hypertension, obese patients spend years in a state of progressive insulin resistance and metabolic disease. Mounting evidence suggests that this insulin-resistant state has deleterious effects on the control of blood flow, thus placing organ systems at a higher risk for end-organ damage and increasing cardiovascular mortality. 2. The purpose of the present review is to examine the current literature on the effects of obesity and insulin resistance on the acute control of vascular tone. Effects on nitric oxide (NO)-mediated control of vascular tone are particularly examined with regard to proximal causes and distal mechanisms of the impaired NO-mediation of vasodilation. 3. Finally, novel pathways of impaired control of perfusion are summarized from the recent literature to identify new avenues of exploring impaired vascular function in patients with metabolic disease.

Obesity causes whole body insulin resistance and impaired vasodilation to nitric oxide (NO). Because NO is a major contributor to the regulation of mesenteric blood flow, the mesenteric circulation of obese animals is faced with reduced capacity to increase flow and increased demand for flow associated with elevated consumption of food. This study hypothesized that insulin resistance impairs NO-mediated dilation but that constrictor reactivity would be reduced to compensate in obese animals. We further hypothesized that elevated superoxide levels caused impaired responses to NO in insulin resistance. Vasodilator reactivity and vasoconstrictor reactivity of mesenteric resistance arteries from lean (LZR) and obese (OZR) Zucker rats were examined in vitro using videomicroscopy. Insulin resistance independent of obesity was induced via fructose feeding in LZR (FF-LZR). Endothelium-dependent NO-mediated dilation was reduced in OZR and FF-LZR compared with LZR. Impairments in NO-mediated dilation were reversed with 1 mM tempol, a SOD mimetic. Constrictor reactivity to norepinephrine was reduced in OZR but not in FF-LZR relative to LZR. Basal mesenteric vascular resistance was similar in LZR and OZR despite impaired NO-dependent dilation in OZR. Mesenteric vascular resistance was increased in FF-LZR relative to LZR. These data indicate that there is reduced constrictor reactivity in OZR that may offset the impaired NO-mediated dilation and preserve mesenteric blood flow in hyperphagic, obese animals.

Fructose feeding has been widely reported to cause hypertension in rats, as assessed indirectly by tail cuff plethysmography. Because there are potentially significant drawbacks associated with plethysmography, we determined whether blood pressure changes could be detected by long-term monitoring with telemetry in age-matched male Sprague-Dawley rats fed either a normal or high-fructose diet for 8 weeks. Fasting plasma glucose (171±10 versus 120±10 mg/dL), plasma insulin (1.8±0.5 versus 0.7±0.1 μg/L), and plasma triglycerides (39±2 versus 30±2 mg/dL) were modestly but significantly elevated in fructose-fed animals. Using the hyperinsulinemic euglycemic clamp technique, the rate of glucose infusion necessary to maintain equivalent plasma glucose was significantly reduced in fructose-fed compared with control animals (22.9±3.6 versus 41.5±2.9 mg/kg per minute; P P

The risk of cerebrovascular disease is four- to sixfold higher in patients with diabetes. Vascular remodeling, characterized by extracellular matrix deposition and an increased media-to-lumen ratio, occurs in diabetes and contributes to the development of complications. However, diabetes-induced changes in the cerebrovascular structure remain unknown. Endothelin-1 (ET-1), a potent vasoconstrictor with profibrotic properties, is chronically elevated in diabetes. To determine diabetes-mediated changes in the cerebrovasculature and the role of ET-1 in this process, type 2 diabetic Goto-Kakizaki (GK) rats were administered an ETA receptor antagonist for 4 weeks. Middle cerebral arteries were harvested and studies were performed to determine vascular structure. Tissue and plasma ET-1 levels were increased in GK rats compared with controls. Significant medial hypertrophy and collagen deposition resulted in an increased wall-to-lumen ratio in diabetic rats that was reduced by ETA receptor antagonism. Vascular matrix metalloproteinase (MMP)-2 activity was higher, but MMP-1 levels were significantly reduced in GK rats, and MMP levels were restored to control levels by ETA receptor antagonism. We conclude that ET-1 promotes cerebrovascular remodeling in type 2 diabetes through differential regulation of MMPs. Augmented cerebrovascular remodeling may contribute to an increased risk of stroke in diabetes, and ETA receptor antagonism may offer a novel therapeutic target.

Excessive cellular proliferation is a major contributor to the pathological changes associated with the secondary complications of diabetes. In particular, hyperglycemia (HG)-induced growth of vascular smooth muscle cells (VSMC) and glomerular mesangial cells (GMC) are characteristic features of the cardiovascular and renal complications of diabetes. VSMC and GMC respond to traditional growth factors, however in diabetes this occurs in the context of an environment, enriched in circulating vasoactive mediators and HG. For example, signaling via the angiotensin II (Ang II) pathway has been implicated in the pathogenesis of diabetic vascular disease. Recent findings indicate that HG and Ang II activate intracellular processes, including the polyol pathway and the generation of reactive oxygen species. These pathways activate the JAK (janus kinase)/STAT (signal transducers and activators of transcription) signaling cascades in both VSMC and GMC. Activation of the latter signaling cascade can stimulate excessive proliferation and growth of these cells, contributing to the accelerated atherosclerosis and nephropathy seen in the diabetic state. This review focuses on key factors in the diabetic microenvironment, in particular the interplay between HG, accumulation of advanced glycation end products and Ang II mediated signaling events both in vitro and in vivo.

Previously, using an animal model of syndrome X, the obese Zucker rat (OZR), we documented impaired endothelium-dependent vasodilation. The aim of this study was to determine whether reduced expression or altered posttranslational regulation of endothelial nitric oxide synthase (eNOS) underlies the vascular dysfunction in OZR rats. There was no significant difference in the relative abundance of eNOS in hearts, aortas, or skeletal muscle between lean Zucker rats (LZR) and OZR regardless of age. There was no difference in eNOS mRNA levels, as determined by real-time PCR, between LZR and OZR. The inability of insulin resistance to modulate eNOS expression was also documented in two additional in vivo models, the ob/ob mouse and the fructose-fed rat, and in vitro via adenoviral expression of protein tyrosine phosphatase 1B in endothelial cells. We next investigated whether changes in the acute posttranslational regulation of eNOS occurs with insulin resistance. Phosphorylation of eNOS at S632 (human S633) and T494 was not different between LZR and OZR; however, phosphorylation of S1176 was significantly enhanced in OZR. Phosphorylation of S1176 was not different in the ob/ob mouse or in fructose-fed rats. The association of heat shock protein 90 with eNOS, a key regulatory step controlling nitric oxide and aberrant O2−production, was not different between OZR and LZR. Taken together, these results suggest that changes in eNOS expression or posttranslation regulation do not underlie the vascular dysfunction seen with insulin resistance and that other mechanisms, such as altered localization, reduced availability of cofactors, substrates, and the elevated production of O2−, may be responsible.

DEY, APARAJITA, ROGER S. WILLIAMS, DAVID M. POLLOCK, DAVID W. STEPP, JOHN W. NEWMAN, BRUCE D. HAMMOCK, AND JOHN D. IMIG. Altered Kidney CYP2C and cyclooxygenase-2 levels are associated with obesity-related albuminuria. Obes Res. 2004;12: 1278 –1289. Objective: To determine cytochrome P450 (CYP450) and cyclooxygenase (COX) expression and metabolite regulation and renal damage in the early stages of obesity-related hypertension and diabetes. Research Methods and Procedures: Obese and lean Zucker rats at 10 to 12 weeks of age were studied. Blood pressure was measured in the conscious state using radiotelemetry. Blood glucose levels and body weight were measured periodically. Protein expression of CYP450 and COX enzymes in the kidney cortex, renal microvessels, and glomeruli was studied. The levels of CYP450 and COX metabolites in urine were measured, and urinary albumin excretion, an indicator of kidney damage, was measured. Results: Body weight and blood glucose averaged 432 20 grams and 105 5 mg/dl, respectively, in obese Zucker rats as compared with 320 8 grams and 91 5 mg/dl, respectively, in age-matched 10- to 12-week-old lean Zucker rats. Renal microvascular CYP4A and COX-2 protein levels were increased 2.3- and 17.0-fold, respectively, in obese Zucker rats. The protein expression of CYP2C11 and CYP2C23 was decreased 2.0-fold in renal microvessels isolated from obese Zucker rats when compared with lean Zucker rats. The urinary excretion rate of thromboxane B2 was increased significantly in obese Zucker as compared with lean Zucker rats (22.0 1.8 vs. 13.4 1.0 ng/d). Urinary albumin excretion, an index of kidney damage, was increased in the obese Zucker rat at this early age. Discussion: These results suggest that increased CYP4A and COX-2 protein levels and decreased CYP2C11 and CYP2C23 protein levels occur in association with microalbuminuria during the onset of obesity-related hypertension and type 2 diabetes.

Peripheral microvascular dysfunction is a common affliction in patients with the metabolic syndrome X. Previous studies have described a number of vascular impairments in vasomotor control in both human patients and animal models of syndrome X, but the net effect of these impairments on microvascular structure has not been examined. The goal of the current study was to test the hypothesis that syndrome X reduces muscle perfusion and induces vascular remodeling. The obese Zucker rat was used as a model of syndrome X, and the microcirculation of the hindlimb and brain were examined. Obese Zucker rats were obese, hyperlipidemic, hyperinsulinemic, and hyperglycemic. Blood flow to the hindlimb was reduced by 59% in obese rats relative to lean rats. Skeletal muscle resistance arteries of the hindlimb microcirculation of obese rats had thinner walls, smaller lumens, and reduced distensibility. Hindlimb microvessels from obese rats also demonstrated reduced expression of vascular smooth muscle cell markers. Each of these traits is consistent with low-flow remodeling. In contrast, the cerebral microcirculation, where flow is vigorously autoregulated, showed no vascular remodeling nor were there changes in microvascular smooth muscle marker expression. Neither physical activity nor muscle mass were significantly different between lean and obese rats. Taken together, these findings suggest that syndrome X, by reducing hindlimb blood flow, induces a marked remodeling of microcirculation to favor smaller, less distensible vessels. This remodeling may result in an architectural limitation of maximum perfusion capacity and may be an important maladaption in the progression of peripheral microvascular disease.

Background A sedentary lifestyle is an independent risk factor for cardiovascular disease and exercise has been shown to ameliorate this risk. Inactivity is associated with a loss of muscle mass, which is also reversed with isometric exercise training. The relationship between muscle mass and vascular function is poorly defined. The aims of the current study were to determine whether increasing muscle mass by genetic deletion of myostatin, a negative regulator of muscle growth, can influence vascular function in mesenteric arteries from obese db/db mice. Methods and Results Myostatin expression was elevated in skeletal muscle of obese mice and associated with reduced muscle mass (30% to 50%). Myostatin deletion increased muscle mass in lean (40% to 60%) and obese (80% to 115%) mice through increased muscle fiber size ( P NO ) synthase inhibitor l ‐ NG ‐nitroarginine methyl ester ( l ‐ NAME) . Prostacyclin ( PGI 2 )‐ and endothelium‐derived hyperpolarizing factor ( EDHF)‐ mediated vasodilation were preserved in obese mice and unaffected by myostatin deletion. Reactive oxygen species) was elevated in the mesenteric endothelium of obese mice and down‐regulated by deletion of myostatin in obese mice. Impaired vasodilation in obese mice was improved by NADPH oxidase inhibitor ( GKT 136901). Treatment with sepiapterin, which increases levels of tetrahydrobiopterin, improved vasodilation in obese mice, an improvement blocked by l ‐ NAME . Conclusions Increasing muscle mass by genetic deletion of myostatin improves NO‐ , but not PGI 2 ‐ or EDHF‐ mediated vasodilation in obese mice; this vasodilation improvement is mediated by down‐regulation of superoxide.

Adipose tissue serves as both a storage site for excess calories and as an endocrine organ, secreting hormones such as adiponectin that promote metabolic homeostasis. In obesity, adipose tissue expands primarily by hypertrophy (enlargement of existing adipocytes) rather than hyperplasia (generation of new adipocytes via adipogenic differentiation of preadipocytes). Progressive adipocyte hypertrophy leads to inflammation, insulin resistance, dyslipidemia, and ectopic lipid deposition, the hallmark characteristics of metabolic disease. We demonstrate that during chronic high fat feeding in mice, adipogenic differentiation is impaired due to the actions of histone deacetylase 9 (HDAC9), a member of the class II family of HDACs. Mechanistically, upregulated HDAC9 expression blocks the adipogenic differentiation program during chronic high fat feeding, leading to accumulation of improperly differentiated adipocytes with diminished expression of adiponectin. These adipocytes are inefficient at storing lipid, resulting in ectopic lipid deposition in the liver. HDAC9 gene deletion prevents the detrimental effects of chronic high fat feeding on adipogenic differentiation, increases adiponectin expression, and enhances energy expenditure by promoting beige adipogenesis, thus leading to reduced body mass and improved metabolic homeostasis. HDAC9 is therefore emerging as a critical regulator of adipose tissue health and a novel therapeutic target for obesity-related disease.

Increases in muscle mass, as observed with exercise, are positive influences of cardiovascular health. Recent studies from our lab have shown the increase muscle mass via deletion of myostatin improves vasodilation but the underlying mechanisms are unknown. Improving insulin resistance has been shown to improve oxidant stress, specifically via expression of NOX1. Hypothesis: Increasing muscle mass by deletion of muscle growth negative regulator myostatin, improves vascular function in mesenteric arteries from obese db/db mice via reducing NOX-mediated oxidant stress in the endothelium. Myostatin deletion increased muscle mass in both lean (gastrocnemius 57.93%, gluteus maximus 60.95%) and obese mice (gastrocnemius 79.64%, gluteus maximus 112.32%). Fasting glucose, HbA1c and glucose tolerance are improved in obese myostatin null mice. Obese mice demonstrate superoxide-mediated impairment of ACh-induced vasodilation compared to lean mice. Deletion of myostatin in obese mice improved ACh-induced vasodilation...

For over 50 years, it has been recognized that coronary blood flow is precisely matched to cardiac metabolism. The interactions which govern this matching remain unknown. In the current review, 3 specific aspects of coronary flow regulation will be discussed: Specialization of function in different microvascular domains, influence of cardiac region on microvascular function and the interactions of vasoactive agents in control of coronary blood flow. Each level of the coronary microcirculation is affected by different physical and chemical forces within the heart. These forces place special demands on these vessels and are in turn met by specialized vasodilator responses, including metabolic and flow-mediated vasodilation. Perfusion of the heart is also profoundly affected by the region perfused. The endocardium is affected by forces, notably cardiac contraction, in a different manner than the epicardium. Thus, the microcirculation has specialized to meet these demands. Finally, the factors determining microvascular tone appear to be coordinated such that the loss of any individual dilator, such as nitric oxide, can be compensated for by the increased contribution of another, such as adenosine. This interplay may serve to protect the heart from ischemia during the early phases of coronary vascular disease when individual dilators may be impaired.

Obesity is a major cause of hypertension, but links between the obese and hypertensive states remain incompletely understood. A major component of cardiovascular function in obese individuals is a state of sympathoactivation. A postulated mechanism of this sympathoactivation is the activation of specific classes of neurons commonly associated with metabolic control, which also affect sympathetic outflow to cardiovascular targets. One class of neurons is characterized by expression of melanocortin-4 receptors (MC4R) which are activated by metabolic signals such as leptin and insulin. In this study, we examined the effects of deletion of MC4R in a novel rat model. MC4R knockout (KO) rats are obese and profoundly insulin resistant without frank diabetes. Despite these conditions, MC4R KO rats are normotensive. Moderate bradycardia and significant increases in peripheral resistance were evident in MC4R KO rats. To determine if the dissociation between hypertension and obesity was associated with changes in vascular function, in vitro reactivity to vasoactive agents and in vivo reactivity to sympathetic blockade were examined. Vasodilator function was not affected by obesity in MC4R KO rats. Reactivity to phenylephrine was reduced, suggesting desensitization of adrenergic signaling. In response to ganglionic blockade with mecamylamine, blood pressure and hindlimb resistance fell more in MC4R KO rats, suggesting that sympathoactivation of the vascular was still evident, despite the absence of hypertension. These findings suggest that obesity causes sympathoactivation of the vasculature despite the absence of MC4R. Dissociation of obesity from hypertension in this model may reflect more renal mechanisms of blood pressure control.

Protein tyrosine phosphatase 1B (PTP1B), a molecular brake on leptin and insulin signaling pathways, is an important regulator of metabolic functions. Due to the close proximity between the signaling pathways regulating the metabolic and the cardiovascular systems, it has been hypothesized that PTP1B might have a role in the control of cardiovascular functions in the context of metabolic disorders. Evidence from population-based studies demonstrates that polymorphisms of the human PTP1B gene, PTPN1, predispose to both type II diabetes and cardiovascular disease, including hypertension and atherosclerosis. Consistent with these findings, genetic deletion of PTP1B in lean and obese animal models has proven to be protective against obesity-induced hypertension and endothelial dysfunction. While both human and animals studies mostly support indirect effects of PTP1B on the cardiovascular system via its control of metabolic function, evidence from in vitro studies suggests direct effects of PTP1B on endothelial and vascular smooth muscle cell integrity. Taken together these data identify PTP1B as a major regulator of cardiovascular function and suggest that manipulation of PTP1B expression and activity could provide a novel therapeutic approach to prevent obesity-related cardiovascular disease.

Insulin resistance is a powerful predictor of cardiovascular disease; however, the mechanistic link remains unclear. This study aims to determine if early cardiovascular changes associated with short‐term fructose feeding in the absence of obesity manifest as abnormal blood pressure control. Metabolic dysfunction was induced in Lean Zucker rats by short‐term high‐fructose feeding. Rats were implanted with telemetry devices for the measurement of mean arterial blood pressure (MAP) and subjected to air jet stress at 5 and 8 weeks after feeding. Additional animals were catheterized under anesthesia for the determination of MAP and blood flow responses in the hind limb and mesenteric vascular beds to intravenous injection of isoproterenol (0.001–0.5 μm), a β‐adrenergic agonist. Metabolic dysfunction in high‐fructose rats was not accompanied by changes in 24‐h MAP. Yet, animals fed a high‐fructose diet for 8 weeks exhibited a marked impairment in blood pressure recovery after air‐jet stress. Dose‐dependent decreases in MAP and peripheral blood flow in response to isoproterenol treatment were significantly attenuated in high‐fructose rats. These data suggest that impaired blood pressure recovery to acute mental stress precedes the onset of hypertension in the early stages of insulin resistance. Further, blunted responses to isoproterenol implicate β 2‐adrenergic sensitivity as a possible mechanism responsible for altered blood pressure control after short‐term high‐fructose feeding.