Background-In obesity, the excessive synthesis of aldosterone contributes to the development and progression of metabolic and cardiovascular dysfunctions. Obesity-induced hyperaldosteronism is independent of the known regulators of aldosterone secretion, but reliant on unidentified adipocyte-derived factors. We hypothesized that the adipokine leptin is a direct regulator of aldosterone synthase (CYP11B2) expression and aldosterone release and promotes cardiovascular dysfunction via aldosterone-dependent mechanisms. Methods and Results-Immunostaining of human adrenal cross-sections and adrenocortical cells revealed that adrenocortical cells coexpress CYP11B2 and leptin receptors. Measurements of adrenal CYP11B2 expression and plasma aldosterone levels showed that increases in endogenous (obesity) or exogenous (infusion) leptin dose-dependently raised CYP11B2 expression and aldosterone without elevating plasma angiotensin II, potassium or corticosterone. Neither angiotensin II receptors blockade nor α and β adrenergic receptors inhibition blunted leptin-induced aldosterone secretion. Identical results were obtained in cultured adrenocortical cells. Enhanced leptin signaling elevated CYP11B2 expression and plasma aldosterone, whereas deficiency in leptin or leptin receptors blunted obesity-induced increases in CYP11B2 and aldosterone, ruling out a role for obesity per se. Leptin increased intracellular calcium, elevated calmodulin and calmodulinkinase II expression, whereas calcium chelation blunted leptin-mediated increases in CYP11B2, in adrenocortical cells. Mineralocorticoid receptor blockade blunted leptin-induced endothelial dysfunction and increases in cardiac fibrotic markers. Conclusions-Leptin is a newly described regulator of aldosterone synthesis that acts directly on adrenal glomerulosa cells to increase CYP11B2 expression and enhance aldosterone production via calcium-dependent mechanisms. Furthermore, leptin-mediated aldosterone secretion contributes to cardiovascular disease by promoting endothelial dysfunction and the expression of profibrotic markers in the heart.

Abstract-Chronic increases in blood flow increase arterial diameter and NO-dependent dilation in resistance arteries.Because endothelial dysfunction accompanies metabolic syndrome, we hypothesized that flow-mediated remodeling might be impaired in obese rat resistance arteries. Obese and lean Zucker rat mesenteric resistance arteries were exposed to chronic flow increases through arterial ligation in vivo: arteries exposed to high flow were compared with normal flow arteries. Diameter was measured in vitro in cannulated arteries using pressure arteriography. After 7 days, outward remodeling (diameter increased from 346Ϯ9 to 412Ϯ11 m at 100 mm Hg) occurred in lean high-flow arteries. Endothelium-dependent tone was reduced in high-flow arteries from obese rats by contrast with lean animals. On the other hand, diameter enlargement occurred similarly in the 2 strains. The involvement of NO in endothelium-dependent dilation (evidenced by NO blockade) and endothelial NO synthase phosphorylation was smaller in obese than in lean rats. Superoxide anion and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression (p67phox and gp91phox) increased in obese rats and were higher in high-flow than in control arteries. Acute Tempol (a catalase mimetic), catalase plus superoxide dismutase, and L-arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat normal and high-flow arteries to the level found in lean control arteries. Thus, flow-induced remodeling in obese resistance arteries was associated with a reduced endothelium-mediated dilation because of a decreased NO bioavailability and an excessive superoxide production. This dysfunction might have negative consequences in ischemic diseases in patients with obesity or metabolic syndrome. Key Words: resistance arteries Ⅲ shear stress Ⅲ NO Ⅲ reactive oxygen species Ⅲ mechanotransduction Ⅲ obesity Ⅲ metabolic syndrome T he metabolic syndrome is a common health problem. Its incidence and prevalence are increasing in parallel with the enhanced prevalence and incidence of obesity and type 2 diabetes. 1,2 This syndrome is not only a metabolic disorder; it is also associated with endothelial dysfunction 3 and vascular remodeling leading to a reduction in arterial diameter. 4 Patients with metabolic syndrome demonstrate an increasing risk of overall mortality, as well as cardiovascular morbidity and mortality. 5 Basically, an increase in blood pressure or a decrease in blood flow induces a transient adjustment in vessel diameter mediated by changes in myogenic tone and by the release of vasoactive substances. On the other hand, increasing blood flow induces a vasodilation mediated by endothelium-derived NO. A long-term exposition to altered mechanical forces induces vascular remodeling to restore tensile and shear stresses. 6,7 Long-term increase in shear stress enhances NO production by endothelial cells, and we have shown previously that NO has a key role in vascular remodeling of large 8 and resistance arteries in response to an in...

Obesity is a major risk factor for cardiovascular disease in males and females. Whether obesity triggers cardiovascular disease via similar mechanisms in both the sexes is, however, unknown. In males, the adipokine leptin highly contributes to obesity-related cardiovascular disease by increasing sympathetic activity. Females secrete 3× to 4× more leptin than males, but do not exhibit high sympathetic tone with obesity. Nevertheless, females show inappropriately high aldosterone levels that positively correlate with adiposity and blood pressure (BP). We hypothesized that leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in females. Leptin control of the cardiovascular function was analyzed in female mice sensitized to leptin via the deletion of protein tyrosine phosphatase 1b (knockout) and in agouti yellow obese hyperleptinemic mice (Ay). Hypersensitivity to leptin (wild-type, 115±2; protein tyrosine phosphatase 1b knockout, 124±2 mm Hg; P<0.05) and obesity elevated BP (a/a, 113±1; Ay, 128±7 mm Hg; P<0.05) and impaired endothelial function. Chronic leptin receptor antagonism restored BP and endothelial function in protein tyrosine phosphatase 1b knockout and Ay mice. Hypersensitivity to leptin and obesity reduced BP response to ganglionic blockade in both strains and plasma catecholamine levels in protein tyrosine phosphatase 1b knockout mice. Hypersensitivity to leptin and obesity significantly increased plasma aldosterone levels and adrenal CYP11B2 expression. Chronic leptin receptor antagonism reduced aldosterone levels. Furthermore, chronic leptin and mineralocorticoid receptor blockade reduced BP and improved endothelial function in both leptin-sensitized and obese hyperleptinemic female mice. Together, these data demonstrate that leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in female mice and suggest that obesity leads to cardiovascular disease via sex-specific mechanisms.

Objective-Notch3, a member of the evolutionary conserved Notch receptor family, is primarily expressed in vascular smooth muscle cells. Genetic studies in human and mice revealed a critical role for Notch3 in the structural integrity of distal resistance arteries by regulating arterial differentiation and postnatal maturation. Methods and Results-We investigated the role of Notch3 in vascular tone in small resistance vessels (tail and cerebral arteries) and large (carotid) arteries isolated from Notch3-deficient mice using arteriography. Passive diameter and compliance were unaltered in mutant arteries. Similarly, contractions to phenylephrine, KCl, angiotensin II, and thromboxane A2 as well as dilation to acetylcholine or sodium nitroprusside were unaffected. However, Notch3 deficiency induced a dramatic reduction in pressure-induced myogenic tone associated with a higher flow (shear stress)-mediated dilation in tail and cerebral resistance arteries only. Furthermore, RhoA activity and myosin light chain phosphorylation, measured in pressurized tail arteries, were significantly reduced in Notch3KO mice. Additionally, myogenic tone inhibition by the Rho kinase inhibitor Y27632 was attenuated in mutant tail arteries. Conclusions-Notch3 plays an important role in the control of vascular mechano-transduction, by modulating the RhoA/Rho kinase pathway, with opposite effects on myogenic tone and flow-mediated dilation in the resistance circulation. (Arterioscler Thromb Vasc

Although the anorexic effects of leptin are lost in obesity, leptin-mediated sympatho-activation is preserved. The cardiovascular consequences of leptin-mediated sympatho-activation in obesity are poorly understood. We tested the hypothesis that 32 weeks of high fat diet (HFD) induces metabolic leptin resistance but preserves leptin-mediated sympatho-activation of the cardiovascular system. HFD in mice significantly increased body weight and plasma leptin concentrations but significantly reduced the anorexic effects of leptin. HFD increased heart rate (HR), stroke volume, cardiac output and plasma aldosterone levels but not blood pressure (BP). As reflected by the contractile response to phenylephrine measured both in vivo and ex vivo, vascular adrenergic reactivity was reduced by HFD suggesting that reductions in sympathetic tone to the periphery vasculature may mitigate sympatho-activation of the heart and the renin angiotensin aldosterone system (RAAS). Tachyphlyaxis was partially restored by symptho-inhibition and not present in ob/ob and db/db mice, despite obesity, arguing for a sympatho-mediated and leptin-specific mechanism. While infusion of leptin in HFD mice had no effect on HR or BP, it further increased aldosterone levels and further reduced vascular adrenergic tone in the absence of weight loss, indicating persistent leptin-mediated stimulation of the cardiovascular system in obesity. In conclusion, these data indicate that despite metabolic leptin resistance, leptin-mediated stimulation of the heart, the vasculature and aldosterone production persists in obesity. BP effects in response to leptin may be limited by a tachyphylactic response in the circulation suggesting that failure of adrenergic desensitization may be a requisite step for hypertension in the context of obesity.

Peroxynitrite (ONOO−) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO− interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO− scavenger, iron-(III)-tetrakis(N-methyl-4'pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO− reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO−-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose–exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO− reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO− selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients.

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 (116Ϯ5 versus 105Ϯ5 mm Hg, PϽ0.05) than controls. Leptin infusion produced a greater anorexic effect in PTP1B knockout mice and a marked increase in mean arterial pressure (135Ϯ5 mm Hg) in PTP1B knockout mice only. The decrease in mean arterial pressure in response to ganglionic blockade was higher in PTP1B knockout mice (Ϫ38Ϯ3% versus Ϫ29Ϯ3%, PϽ0.05), which suggests increased sympathetic tone. PTP1B deletion blunted mean arterial pressure responses to phenylephrine injection (55Ϯ10% versus 93Ϯ7%, PϽ0.05). Phenylephrine-induced aortic contraction was reduced in PTP1B knockout mice (57.7Ϯ9% versus 96.3Ϯ12% of KCl, PϽ0.05), consistent with desensitization to chronically elevated sympathetic tone. Furthermore, PTP1B deletion significantly reduced gene expression of 3 ␣ 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.

Objectives-Increased formation of reactive oxygen species (ROS) has been identified as a causative factor in endothelial dysfunction by reducing NO bioavailability and uncoupling endothelial nitric oxide synthase (eNOS). However, the specific contribution of ROS to endothelial function is not well understood. Methods and Results-A major source of intracellular ROS is the NADPH oxidase (Nox) family of enzymes. The goal of the current study was to directly assess the contribution of NADPH oxidase derived superoxide to eNOS function by expressing Nox5, a single gene product that constitutively produces superoxide within cells. Paradoxically, we found that instead of inhibiting eNOS, coexpression of Nox5 increased NO release from both bovine and human endothelial cells. To establish the functional significance of this observation in intact blood vessels, the endothelium of mouse aorta was transduced with Nox5 or control adenoviruses. Nox5 potently inhibited Ach-induced relaxation and potentiated contractile responses to phenylephrine. In precontracted aortae, acute exposure to superoxide dismutase induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. (EC) has been identified as a causative factor in this process by reducing NO bioavailability, uncoupling eNOS via BH4 depletion or homodimer disruption, and also by altering redox-sensitive signaling cascades. However, elevated superoxide production is frequently accompanied by changes in blood pressure, cellular signaling, hormones, and the composition of the extracellular milieu, making it difficult to ascertain the independent effects of intracellular ROS. For example, endothelial function is reduced in animal models of type II diabetes, angiotensin-dependent hypertension, and atherosclerosis, and this deficit is accompanied by significant increases in superoxide formation. 2-4 However, whether increased superoxide is the causative factor, a participant, or requires the cooperation of other factors present in the extracellular milieu is not yet known. Conclusions-These