Sex differences in the vascular function and related mechanisms: role of 17ß-estradiol.

The incidence of cardiovascular disease (CVD) is lower in premenopausal women but increases with age and menopause compared with similarly aged men. Based on the prevalence of CVD in postmenopausal women, sex hormone-dependent mechanisms have been postulated to be the primary factors responsible for the protection from CVD in premenopausal women. Recent Women's Health Initiative studies, Cochrane Review studies, the Early Versus Late Intervention Trial with Estradiol Study, and the Kronos Early Estrogen Prevention Study have suggested that beneficial effects of hormone replacement therapy (HRT) are seen in women of <60 yr of age and if initiated within <10 yr of menopause. In contrast, the beneficial effects of HRT are not seen in women of >60 yr of age and if commenced after 10 yr of menopause. The higher incidence of CVD and the failure of HRT in postmenopausal aged women could be partly associated with fundamental differences in the vascular structure and function between men and women and in between pre- and postmenopausal women, respectively. In this regard, previous studies from human and animal studies have identified several sex differences in vascular function and associated mechanisms. The female sex hormone 17ß-estradiol regulates the majority of these mechanisms. In this review, we summarize the sex differences in vascular structure, myogenic properties, endothelium-dependent and -independent mechanisms, and the role of 17ß-estradiol in the regulation of vascular function.

Endothelial specific SIRT3 deletion impairs glycolysis and angiogenesis and causes diastolic dysfunction.

Endothelial glycolysis plays a critical role in the regulation of angiogenesis. We investigated the role of Sirtuin 3 (SIRT3) on endothelial cell (EC) glycolytic metabolism, angiogenesis, and diastolic function. Our aim was to test the hypothesis that loss of SIRT3 in ECs impairs endothelial glycolytic metabolism and angiogenesis and contributes to myocardial capillary rarefaction and the development of diastolic dysfunction. Using SIRT3 deficient ECs, SIRT3 was found to regulate a metabolic switch between mitochondrial respiration and glycolysis. SIRT3 knockout (KO)-ECs exhibited higher mitochondrial respiration and reactive oxygen species (ROS) formation. SIRT3 knockout (KO)-ECs exhibited a reduction in the expression of glycolytic enzyme, PFKFB3, and a fall in glycolysis and angiogenesis. Blockade of PFKFB3 reduced glycolysis and downregulated expression of VEGF and Angiopoietin-1 (Ang-1) in ECs. Deletion of SIRT3 in ECs also impaired hypoxia-induced expression of HIF-2α, VEGF, and Ang-1, as well as reduced angiogenesis. In vivo, endothelial-specific SIRT3 KO (ECKO) mice exhibited a myocardial capillary rarefaction together with a reduced coronary flow reserve (CFR) and diastolic dysfunction. Histologic study further demonstrated that knockout of SIRT3 in ECs significantly increased perivascular fibrosis in the coronary artery. These results implicate a role of SIRT3 in modulating endothelial function and cardiac function. Ablation of SIRT3 leads to impairment of EC glycolytic metabolism and angiogenic signaling, which may contribute to coronary microvascular rarefaction and diastolic dysfunction in SIRT3 ECKO mice.

Cellular and Oxidative Mechanisms Associated with Interleukin-6 Signaling in the Vasculature.

Reactive oxygen species, particularly superoxide, promote endothelial dysfunction and alterations in vascular structure. It is increasingly recognized that inflammatory cytokines, such as interleukin-6 (IL-6), contribute to endothelial dysfunction and vascular hypertrophy and fibrosis. IL-6 is increased in a number of cardiovascular diseases, including hypertension. IL-6 is also associated with a higher incidence of future cardiovascular events and all-cause mortality. Both immune and vascular cells produce IL-6 in response to a number of stimuli, such as angiotensin II. The vasculature is responsive to IL-6 produced from vascular and non-vascular sources via classical IL-6 signaling involving a membrane-bound IL-6 receptor (IL-6R) and membrane-bound gp130 via Jak/STAT as well as SHP2-dependent signaling pathways. IL-6 signaling is unique because it can also occur via a soluble IL-6 receptor (sIL-6R) which allows for IL-6 signaling in tissues that do not normally express IL-6R through a process referred to as IL-6 trans-signaling. IL-6 signaling mediates a vast array of effects in the vascular wall, including endothelial activation, vascular permeability, immune cell recruitment, endothelial dysfunction, as well as vascular hypertrophy and fibrosis. Many of the effects of IL-6 on vascular function and structure are representative of loss or reductions in nitric oxide (NO) bioavailability. IL-6 has direct effects on endothelial nitric oxide synthase activity and expression as well as increasing vascular superoxide, which rapidly inactivates NO thereby limiting NO bioavailability. The goal of this review is to highlight both the cellular and oxidative mechanisms associated with IL-6-signaling in the vascular wall in general, in hypertension, and in response to angiotensin II.

Early development of podocyte injury independently of hyperglycemia and elevations in arterial pressure in nondiabetic obese Dahl SS leptin receptor mutant rats.

The current study examined the effect of obesity on the development of renal injury within the genetic background of the Dahl salt-sensitive rat with a dysfunctional leptin receptor derived from zinc-finger nucleases (SSLepRmutant strain). At 6 wk of age, body weight was 35% higher in the SSLepRmutant strain compared with SSWT rats and remained elevated throughout the entire study. The SSLepRmutant strain exhibited impaired glucose tolerance and increased plasma insulin levels at 6 wk of age, suggesting insulin resistance while SSWT rats did not. However, blood glucose levels were normal throughout the course of the study. Systolic arterial pressure (SAP) was similar between the two strains from 6 to 10 wk of age. However, by 18 wk of age, the development of hypertension was more severe in the SSLepRmutant strain compared with SSWT rats (201 ± 10 vs. 155 ± 3 mmHg, respectively). Interestingly, proteinuria was substantially higher at 6 wk of age in the SSLepRmutant strain vs. SSWT rats (241 ± 27 vs. 24 ± 2 mg/day, respectively) and remained elevated until the end of the study. The kidneys from the SSLepRmutant strain displayed significant glomerular injury, including podocyte foot process effacement and lipid droplets compared with SSWT rats as early as 6 wk of age. By 18 wk of age, plasma creatinine levels were twofold higher in the SSLepRmutant strain vs. SSWT rats, suggesting the presence of chronic kidney disease (CKD). Overall, these results indicate that the SSLepRmutant strain develops podocyte injury and proteinuria independently of hyperglycemia and elevated arterial pressure that later progresses to CKD.

Nox2-derived superoxide contributes to cerebral vascular dysfunction in diet-induced obesity.

BACKGROUND AND PURPOSE: Obesity is an increasing epidemic worldwide; however, little is known about effects of obesity produced by high-fat diet (HFD) on the cerebral circulation. The purpose of this study was to examine the functional and temporal effects of a HFD on carotid and cerebral vascular function and to identify mechanisms that contribute to such functional alterations. METHODS: Responses of cerebral arterioles (in vivo) and carotid arteries (in vitro) were examined in C57Bl/6 (wild-type) and Nox2-deficient (Nox2(-/-)) mice fed a control (10%) or a HFD (45% or 60% kcal of fat) for 8, 12, 30, or 36 weeks. RESULTS: In wild-type mice, a HFD produced obesity and endothelial dysfunction by 12 and 36 weeks in cerebral arterioles and carotid arteries, respectively. Endothelial function could be significantly improved with Tempol (a superoxide scavenger) treatment in wild-type mice fed a HFD. Despite producing a similar degree of obesity in both wild-type and Nox2(-/-) mice, endothelial dysfunction was observed only in wild-type, but not in Nox2(-/-), mice fed a HFD. CONCLUSIONS: Endothelial dysfunction produced by a HFD occurs in a temporal manner and appears much earlier in cerebral arterioles than in carotid arteries. Genetic studies revealed that Nox2-derived superoxide plays a major role in endothelial dysfunction produced by a HFD. Such functional changes may serve to predispose blood vessels to reduced vasodilator responses and thus may contribute to alterations in cerebral blood flow associated with obesity.

Early effects of high-fat diet on neurovascular function and focal ischemic brain injury.

Obesity is a risk factor for stroke, but the early effects of high-fat diet (HFD) on neurovascular function and ischemic stroke outcomes remain unclear. The goal of this study was to test the hypotheses that HFD beginning early in life 1) impairs neurovascular coupling, 2) causes cerebrovascular dysfunction, and 3) worsens short-term outcomes after cerebral ischemia. Functional hyperemia and parenchymal arteriole (PA) reactivity were measured in rats after 8 wk of HFD. The effect of HFD on basilar artery function after middle cerebral artery occlusion (MCAO) and associated O-GlcNAcylation were assessed. Neuronal cell death, infarct size, hemorrhagic transformation (HT) frequency/severity, and neurological deficit were evaluated after global ischemia and transient MCAO. HFD caused a 10% increase in body weight and doubled adiposity without a change in lipid profile, blood glucose, and blood pressure. Functional hyperemia and PA relaxation were decreased with HFD. Basilar arteries from stroked HFD rats were more sensitive to contractile factors, and acetylcholine-mediated relaxation was impaired. Vascular O-GlcNAcylated protein content was increased with HFD. This group also showed greater mortality rate, infarct volume, HT occurrence rate, and HT severity and poor functional outcome compared with the control diet group. These results indicate that HFD negatively affects neurovascular coupling and cerebrovascular function even in the absence of dyslipidemia. These early cerebrovascular changes may be the cause of greater cerebral injury and poor outcomes of stroke in these animals.

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method.

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Superimposed Preeclampsia Exacerbates Postpartum Renal Injury Despite Lack of Long-Term Blood Pressure Difference in the Dahl Salt-Sensitive Rat.

Preeclampsia results in increased susceptibility to hypertension and chronic kidney disease postpartum; however, the mechanisms responsible for disease progression in these women remain unknown. The purpose of this study was to test the hypothesis that 2 mechanisms contribute to the link between the maternal syndrome of preeclampsia and the increased postpartum risk of cardiovascular and renal disease: (1) increased T cells in the kidney and (2) a decreased NO:ET-1 (endothelin-1) ratio. Dahl S rats (a previously characterized model of preeclampsia superimposed on chronic hypertension) who experienced 2 pregnancies and virgin littermate controls were studied at 6 months of age. Mean arterial pressure was measured via telemetry, and renal injury was assessed through both histological analysis and measurement of urinary markers including nephrin, podocalyxin, and KIM-1 (kidney injury marker 1). Contributing mechanisms were assessed through flow cytometric analysis of renal T cells, quantification of plasma TNF-α (tumor necrosis factor-α) and IL-10 (interleukin-10), and quantification of urinary concentrations of NO metabolites and ET-1. Although prior pregnancy did not exacerbate the hypertension at 6 months, this group showed greater renal injury compared with virgin littermates. Flow cytometric analyses revealed an increase in renal T cells after pregnancy, and cytokine analysis revealed a systemic proinflammatory shift. Finally, the NO:ET-1 ratio was reduced. These results demonstrate that the link between the maternal syndrome of superimposed preeclampsia and postpartum risk of chronic kidney disease could involve both immune system activation and dysregulation of the NO:ET-1 balance.

Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency.

OBJECTIVE: The goal of this study was to compare vascular function, superoxide levels, and MnSOD protein expression in young (4 to 7 months) and old (22 to 24 months) MnSOD+/+ and MnSOD-deficient (MnSOD+/-) mice. METHODS AND RESULTS: Relaxation of aorta in vitro to the endothelium-dependent dilator acetylcholine (ACh) was similar in young MnSOD+/+ (n=9) and young MnSOD+/- (n=6) mice. This response was impaired in old MnSOD+/+ (n=8) mice and old MnSOD+/- mice (n=14), with dysfunction being greater in old MnSOD-deficient mice (eg, 100 micromol/L ACh produced 77+/-3% [mean+/-SE], 77+/-3%, 70+/-4%, and 57+/-4% relaxation in young MnSOD+/+, young MnSOD+/-, old MnSOD+/+, and old MnSOD+/- mice, respectively). The endothelial dysfunction was similar in mice on both C57BL/6 and CD-1 genetic backgrounds. In contrast to ACh, responses to the endothelium-independent dilator sodium nitroprusside were enhanced in old MnSOD+/+ and MnSOD+/- mice compared with both groups of young mice (P<0.05). Superoxide levels, as measured using lucigenin-enhanced chemiluminescence, were increased more than 2-fold in old MnSOD+/- mice compared with old MnSOD+/+ and young mice (P<0.05). CONCLUSIONS: These data provide the first direct evidence that MnSOD haploinsufficiency results in increased vascular oxidative stress and endothelial dysfunction with aging.

IL-6 deficiency protects against angiotensin II induced endothelial dysfunction and hypertrophy.

OBJECTIVE: The goal of this study was to test the hypothesis that IL-6 mediates the increases in superoxide, vascular hypertrophy, and endothelial dysfunction in response to angiotensin II (Ang II). METHODS AND RESULTS: Responses of carotid arteries from control and IL-6-deficient mice were examined after acute (22-hour) incubation with Ang II (10 nmol/L) or chronic infusion of Ang II (1.4 mg/kg/d for 14 days). The hypertrophic response and endothelial dysfunction produced by Ang II infusion was markedly less in carotid arteries from IL-6-deficient mice than that in control mice. IL-6 deficiency also protected against endothelial dysfunction in response to acute (local) Ang II treatment (eg, 100 mumol/L acetylcholine produced 100+/-4 and 98+/-4% relaxation in vehicle-treated and 51+/-4 and 99+/-4% relaxation in Ang II-treated, control, and IL-6-deficient vessels, respectively). Endothelial dysfunction could be reproduced in vessels from IL-6-deficient mice with combined Ang II plus IL-6 (0.1 nmol/L) treatment. Increases in vascular superoxide and IL-6, as well as reductions in endothelial nitric oxide synthase mRNA expression, produced by Ang II were absent in IL-6-deficient mice. CONCLUSIONS: These data demonstrate that IL-6 is essential for Ang II-induced increases in superoxide, endothelial dysfunction, and vascular hypertrophy.

Spheroid Culture System Confers Differentiated Transcriptome Profile and Functional Advantage to 3T3-L1 Adipocytes.

This study highlights functional differences between 2-D monolayer and 3-D spheroid 3T3-L1 adipocyte culture models and explores the underlying genomic mechanisms responsible for the different phenotypes present. The spheroids showed higher triglyceride accumulation than the monolayer culture and further increase with larger spheroid size. Whole transcriptome analysis indicated significant differential expression of genes related to adipogenesis, including adipocytokine signaling, fatty acid metabolism, and PPAR-γ signaling. Spheroids also showed downregulation of matrix metalloproteinases (MMPs), integrin, actin-cytoskeleton associated genes, and Rho/GTPase3 expression relative to 2-D monolayer, indicating suppression of the Rho-ROCK pathway and thereby promoting adipogenic differentiation. When exposed to linoleic acid (500 µM) and TNF-α (125 ng/mL) to promote chronic adiposity, linoleic acid treatment resulted in increased intracellular triglycerides and subsequent TNF-α treatment resulted in significantly altered adipocytokine signaling, fatty acid metabolism, and PPAR signaling, in addition to upregulation of multiple MMPs in spheroids vs. monolayer. Overall, 3-D spheroids showed enhanced adipogenic phenotype as indicated by triglyceride synthesis and transcriptome changes while retaining sensitivity to a pro-inflammatory stimulus. The 3-D spheroid culture thus may provide a simple, convenient, and sensitive in vitro model to study adipocyte response to metabolic stresses relevant to clinical pathologies.

Heterozygous eNOS Deficient Mice as a Model to Examine the Effects of eNOS Haploinsufficiency on the Cerebral Circulation.

Nitric oxide derived from endothelial nitric oxide synthase (eNOS) has been shown to be a major mediator of endothelium-dependent responses in cerebral blood vessels. Loss of a single eNOS gene is not associated with any apparent negative consequences on endothelial function in most blood vessels. In contrast, we have recently demonstrated that heterozygous eNOS gene deficiency in combination with a high fat diet is associated with marked impairment of endothelial function. These findings provide an important example of eNOS haploinsufficiency and one that directly impacts the cerebral vasculature. A major mechanism associated with the impairment of endothelial function with eNOS deficiency and a high fat diet appears to be related to increases in plasma IL-6 that serves to further reduce the bioavailability of NO either directly or indirectly via reductions in eNOS expression or activity and via increases in vascular superoxide. Taken together, these findings provide important insights into genetic and molecular mechanisms that promote endothelial dysfunction in response to a high fat diet in cerebral blood vessels with inherent reductions in eNOS gene expression, such as those due to eNOS gene polymorphisms. These findings also highlight the importance of eNOS+/- mice to study the effects of eNOS haploinsufficiency on cerebral blood vessels.

A novel genetic model to explore the Brenner hypothesis: Linking nephron endowment and number with hypertension.

Nephron endowment, the total number of nephrons an individual is born with, is determined by both genetic and environmental factors during embryonic development. In 1988, Brenner hypothesized that there was an inverse relationship between nephron number and hypertension. Over the course of one's lifetime it is predicted that even healthy individuals will lose a significant percentage of nephrons as part of normal aging. Thus, a low nephron endowment at birth or in combination with age- or disease-related nephron loss could pre-dispose individuals to the development of hypertension. Currently, it is not clear what minimal number (ie, threshold) of nephrons is associated with susceptibility to glomeruli injury or hypertension, due in part to the lack of relevant animal models. The BPH2 mouse is a unique genetic model of hypertension that has a normotensive line (BPN3 mice) as well as a hypotensive line (BPL1 mice) derived from the original breeding of eight common inbred strains of mice. Thus, we hypothesize that the differences in blood pressure observed in BPH2, BPN3, and BPL1 mice will correlate inversely with nephron number as predicted by the Brenner hypothesis. If our hypothesis is true, then the BPH2 mouse model will provide a unique experimental model to study the impact of nephron endowment and nephron number on susceptibility to renal injury and hypertension.

Impaired myogenic response of the afferent arteriole contributes to the increased susceptibility to renal disease in Milan normotensive rats.

Milan normotensive (MNS) rats are more susceptible to the development of renal disease than Milan hypertensive (MHS) rats, but the genes and pathways involved are unknown. This study compared the myogenic response of isolated perfused afferent arterioles (Af-Art) and autoregulation of renal blood flow (RBF) and glomerular capillary pressure (Pgc) in 6-9-week-old MNS and MHS rats. The diameter of the Af-Art of MHS rats decreased significantly from 14.3 ± 0.5 to 11.5 ± 0.6 µm when perfusion pressure was elevated from 60 to 120 mmHg. In contrast, the diameter of Af-Art of MNS rats did not decrease. RBF was well autoregulated in MHS rats, but it increased by 26% in MNS rats. Pgc rose by 11 mmHg when renal perfusion pressure (RPP) was increased from 100 to 140 mmHg in MNS but not in MHS rats. Protein excretion increased from 10 ± 1 to 245 ± 36 mg/day in MNS rats as they aged from 3 to 11 months but it did not increase in MHS rats. We also compared the development of proteinuria in MNS and MHS rats following the induction of diabetes with streptozotocin. Protein excretion rose from 16 ± 3 to 234 ± 43 mg/day in MNS rats, but it remained unaltered in MHS rats. These data indicate that the myogenic response of the Af-art is impaired in MNS rats and increased transmission of pressure to the glomerulus may contribute to renal injury in MNS rats similar to what is seen in fawn-hooded hypertensive and Dahl salt-sensitive rats.

Hypertrophy of cerebral arterioles in mice deficient in expression of the gene for CuZn superoxide dismutase.

BACKGROUND AND PURPOSE: Reactive oxygen species are believed to be an important determinant of vascular growth. We examined effects of genetic deficiency of copper-zinc superoxide dismutase (CuZnSOD; SOD1) on structure and function of cerebral arterioles. METHODS: Systemic arterial pressure (SAP) and cross-sectional area of the vessel wall (CSA) and superoxide (O2-) levels (relative fluorescence of ethidium [ETH]) were examined in maximally dilated cerebral arterioles in mice with targeted disruption of one (+/-) or both (-/-) genes encoding CuZnSOD. Wild-type littermates served as controls. Vasodilator responses were tested in separate groups of mice. RESULTS: CSA and ETH were significantly increased (P<0.05) in both CuZnSOD+/- and CuZnSOD-/- mice (CSA=435+/-24 and 541+/-48 microm2; ETH=18+/-1 and 34+/-2%) compared with wild-type mice (CSA=327+/-28 microm2; ETH=6%). Furthermore, the increases in CSA and ETH relative to wild-type mice were significantly greater (P<0.05) in CuZnSOD-/- mice than in CuZnSOD+/- mice (CSA=108 versus 214 microm2; ETH=12 versus 28%). In addition, dilatation of cerebral arterioles in response to acetylcholine, but not nitroprusside, was reduced by approximately 25% in CuZnSOD+/- (P<0.075) and 50% in CuZnSOD-/- mice (P<0.05) compared with wild-type mice. CONCLUSIONS: Cerebral arterioles in CuZnSOD+/- and CuZnSOD-/- mice undergo marked hypertrophy. These findings provide the first direct evidence in any blood vessel that CuZnSOD normally inhibits vascular hypertrophy suggesting that CuZnSOD plays a major role in regulation of cerebral vascular growth. The findings also suggest a gene dosing effect of CuZnSOD for increases in O2-, induction of cerebral vascular hypertrophy and impaired endothelium-dependent dilatation.

Cerebral vascular effects of angiotensin II: new insights from genetic models.

Very little is known regarding the mechanisms of action of angiotensin II (Ang II) or the consequences of Ang II-dependent hypertension in the cerebral circulation. We tested the hypothesis that Ang II produces constriction of cerebral arteries that is mediated by activation of AT1A receptors and Rho-kinase. Basilar arteries (baseline diameter approximately 130 microm) from mice were isolated, cannulated and pressurized to measure the vessel diameter. Angiotensin II was a potent constrictor in arteries from male, but not female, mice. Vasoconstriction in response to Ang II was prevented by an inhibitor of Rho-kinase (Y-27632) in control mice, and was reduced by approximately 85% in mice deficient in expression of AT1A receptors. We also examined the chronic effects of Ang II using a model of Ang II-dependent hypertension, mice which overexpress human renin (R+) and angiotensinogen (A+). Responses to the endothelium-dependent agonist acetylcholine were markedly impaired in R+A+ mice (P<0.01) compared with controls, but were restored to normal by a superoxide scavenger (PEG-SOD). A-23187 (another endothelium-dependent agonist) produced vasodilation in control mice, but no response or vasoconstriction in R+A+ mice. In contrast, dilation of the basilar artery in response to a NO donor (NONOate) was similar in R+A+ mice and controls. Thus, Ang II produces potent constriction of cerebral arteries via activation of AT1A receptors and Rho-kinase. There are marked gender differences in cerebral vascular responses to Ang II. Endothelial function is greatly impaired in a genetic model of Ang II-dependent hypertension via a mechanism that involves superoxide.

Increased superoxide and vascular dysfunction in CuZnSOD-deficient mice.

Increased superoxide is thought to play a major role in vascular dysfunction in a variety of disease states. Superoxide dismutase (SOD) limits increases in superoxide; however, the functional significance of selected isoforms of SOD within the vessel wall are unknown. We tested the hypothesis that selective loss of CuZnSOD results in increased superoxide and altered vascular responsiveness in CuZnSOD-deficient (CuZnSOD(-/-)) mice compared with wild-type (CuZnSOD(+/+)) littermates. Total SOD activity was reduced (P<0.05) by approximately 60% and CuZnSOD protein was absent in aorta from CuZnSOD(-/-) as compared with wild-type mice. Vascular superoxide levels, measured using lucigenin (5 micro mol/L)-enhanced chemiluminescence and hydroethidine (2 micro mol/L)-based confocal microscopy, were increased (approximately 2-fold; P<0.05) in CuZnSOD(-/-) mice as compared with wild-type mice. Relaxation of the carotid artery in response to acetylcholine and authentic nitric oxide was impaired (P<0.05) in CuZnSOD(-/-) mice. For example, maximal relaxation to acetylcholine (100 micro mol/L) was 50+/-6% and 69+/-5% in CuZnSOD(-/-) and wild-type mice, respectively. Contractile responses of the carotid artery were enhanced (P<0.05) in CuZnSOD(-/-) mice in response to phenylephrine and serotonin, but not to potassium chloride or U46619. In vivo, dilatation of cerebral arterioles (baseline diameter=31+/-1 micro m) to acetylcholine was reduced by approximately 50% in CuZnSOD(-/-) mice as compared with wild-type mice (P<0.05). These findings provide the first direct insight into the functional importance of CuZnSOD in blood vessels and indicate that this specific isoform of SOD limits increases in superoxide under basal conditions. CuZnSOD-deficiency results in altered responsiveness in both large arteries and microvessels.

Ceramide-induced impairment of endothelial function is prevented by CuZn superoxide dismutase overexpression.

OBJECTIVE: Ceramide is an important intracellular second messenger that may also increase superoxide. The goal of this study was to determine whether overexpression of CuZn superoxide dismutase (SOD) protects against ceramide-induced increases in vascular superoxide and endothelial dysfunction. METHODS AND RESULTS: Carotid arteries from CuZnSOD-transgenic (CuZnSOD-Tg) and nontransgenic littermates were examined in vitro. Immunohistochemistry confirmed that CuZnSOD protein was greater in carotid artery from CuZnSOD-Tg compared with nontransgenic mice. Ceramide (N-acetyl-d-sphingosine; 1 and 10 micromol/L) produced concentration-dependent impairment (P<0.05) of vasorelaxation in response to the endothelium-dependent agonist acetylcholine (ACh) in nontransgenic mice. For example, 100 micromol/L ACh relaxed arteries from nontransgenic mice by 96+/-4% and 52+/-5% in the presence of vehicle and 10 micromol/L ceramide, respectively. In contrast, ceramide (1 or 10 micromol/L) had no effect (P>0.05) on responses of carotid artery to ACh in CuZnSOD-Tg mice. Ceramide had no effect on nitroprusside- or papaverine-induced relaxation in CuZnSOD-Tg or nontransgenic mice. Ceramide increased superoxide in arteries from nontransgenic vessels, and this effect was prevented by polyethyleneglycol-SOD (50 U/mL) or overexpression of CuZnSOD. CONCLUSIONS: These results suggest that ceramide-induced increases in superoxide impair endothelium-dependent relaxation, and that select overexpression of the CuZn isoform of SOD prevents ceramide-induced oxidative stress in vessels.

Cardiac STAT3 Deficiency Impairs Contractility and Metabolic Homeostasis in Hypertension.

Signal transducer and activator of transcription 3 (STAT3) protects the heart from acute ischemic stress. However, the importance of STAT3 to the heart in chronic stress, such as hypertension, is not known. To study this, we used cardiomyocyte-targeted STAT3 knockout (KO) mice and Angiotensin II (ANG II) infusion by osmotic minipumps. After 4 weeks, ANG II induced similar cardiac hypertrophy in wild type (WT) and cardiac Cre-expressing control (CTRL) mice with no impairment of cardiac function. In contrast, STAT3 KO mice exhibited reduced contractile function but similar hypertrophy to CTRL mice. Ejection fraction and fractional shortening decreased by 22.5 and 27.3%, respectively. Since STAT3 has direct protective effects on mitochondrial function, we examined rates of glucose and oleate oxidation by isolated perfused hearts using a Langendorff system. Hearts of ANG II-treated STAT3 KO and CTRL mice had similar rates of oleate oxidation as saline-infused WT mice. Rates of glucose oxidation were similar between hearts of WT plus saline and CTRL plus ANG II mice; however, glucose oxidation was increased by 66% in hearts of ANG II-treated STAT3 KO mice. The ratio of maximal ATP yield from glucose to fatty acid oxidation was 21.1 ± 3.1 in hearts of ANG II-treated STAT3 KO mice vs. 12.6 ± 2.2 in hearts of ANG II-treated CTRL mice. Lactate production was also elevated in hearts of ANG II-treated STAT3 KO mice by 162% compared to ANG II-treated CTRL mice. Our findings indicate that STAT3 is important for maintaining contractile function and metabolic homeostasis in the hypertensive heart, and STAT3 deficiency promotes a switch toward glucose utilization.

Impaired endothelium-dependent responses and enhanced influence of Rho-kinase in cerebral arterioles in type II diabetes.

BACKGROUND AND PURPOSE: Although the incidence of type II diabetes is increasing, very little is known regarding vascular responses in the cerebral circulation in this disease. The goals of this study were to examine the role of superoxide in impaired endothelium-dependent responses and to examine the influence of Rho-kinase on vascular tone in the cerebral microcirculation in type II diabetes. METHODS: Diameter of cerebral arterioles (29+/-1 microm; mean+/-SE) was measured in vivo using a cranial window in anesthetized db/db and control mice. RESULTS: Dilatation of cerebral arterioles in response to acetylcholine (ACh; 1 and 10 micromol/L), but not to nitroprusside, was markedly reduced in db/db mice (eg, 10 micromol/L ACh produced 29+/-1% and 9+/-1% in control and db/db mice, respectively). Superoxide levels were increased (P<0.05) in cerebral arterioles from db/db mice (n=6) compared with controls (n=6). Vasodilatation to ACh in db/db mice was restored to normal by polyethylene glycol-superoxide dismutase (100 U/mL). Y-27632 (1 to 100 micromol/L; a Rho-kinase inhibitor) produced modest vasodilatation in control mice but much greater responses in db/db mice. N(G)-nitro-L-arginine (100 micromol/L; an inhibitor of NO synthase) significantly enhanced Y-27632-induced dilatation in control mice to similar levels as observed in db/db mice. CONCLUSIONS: These findings provide the first evidence for superoxide-mediated impairment of endothelium-dependent responses of cerebral vessels in any model of type II diabetes. In addition, the influence of Rho-kinase on resting tone appears to be selectively enhanced in the cerebral microcirculation in this genetic model of type II diabetes.