Superoxide Dismutase Mimetic Tempol Research Articles

TNFα Induces DNA and Histone Hypomethylation and Pulmonary Artery Smooth Muscle Cell Proliferation Partly via Excessive Superoxide Formation.

Objective: The level of tumor necrosis factor-α (TNF-α) is upregulated during the development of pulmonary vascular remodeling and pulmonary hypertension. A hallmark of pulmonary arterial (PA) remodeling is the excessive proliferation of PA smooth muscle cells (PASMCs). The purpose of this study is to investigate whether TNF-α induces PASMC proliferation and explore the potential mechanisms. Methods: PASMCs were isolated from 8-week-old male Sprague-Dawley rats and treated with 0, 20, or 200 ng/mL TNF-α for 24 or 48 h. After treatment, cell number, superoxide production, histone acetylation, DNA methylation, and histone methylation were assessed. Results: TNF-α treatment increased NADPH oxidase activity, superoxide production, and cell numbers compared to untreated controls. TNF-α-induced PASMC proliferation was rescued by a superoxide dismutase mimetic tempol. TNF-α treatment did not affect histone acetylation at either dose but did significantly decrease DNA methylation. DNA methyltransferase 1 activity was unchanged by TNF-α treatment. Further investigation using QRT-RT-PCR revealed that GADD45-α, a potential mediator of DNA demethylation, was increased after TNF-α treatment. RNAi inhibition of GADD45-α alone increased DNA methylation. TNF-α impaired the epigenetic mechanism leading to DNA hypomethylation, which can be abolished by a superoxide scavenger tempol. TNF-α treatment also decreased H3-K4 methylation. TNF-α-induced PASMC proliferation may involve the H3-K4 demethylase enzyme, lysine-specific demethylase 1 (LSD1). Conclusions: TNF-α-induced PASMC proliferation may be partly associated with excessive superoxide formation and histone and DNA methylation.

Tempol improves aortic mechanics in a mouse model of hypertension

Hypertension-induced arterial remodeling is thought to be a response to increases in both mechanical stress and oxidative stress. The superoxide dismutase mimetic Tempol has been shown to reduce adverse aortic remodeling in multiple murine models of hypertension but in the absence of a detailed assessment of the biaxial biomechanics. We show that concurrent treatment with Tempol in a common mouse model of systemic hypertension results in modest reductions in both wall thickening and circumferential material stiffness that yet work together to achieve a significant reduction in calculated aortic pulse wave velocity. Reducing elevated values of pulse wave velocity engenders multiple benefits to cardiovascular function.

Supraphysiological levels of testosterone induces endothelial injury via increasing generation of reactive oxygen species and ERK1/2 activation

Introduction: High levels of testosterone (Testo) is associated with higher cardiovascular risk by positively modulate the generation of reactive oxygen species (ROS) and inflammatory response. Excessive ROS generation by NADPH oxidases (NOX, major source of ROS in the vasculature) leads to endothelial dysfunction, apoptosis, and inflammation. However, the intracellular mechanisms whereby supraphysiological levels of Testo promote cardiovascular damage are still ill defined. We sought to determine whether supraphysiological levels of Testo induces endothelial cell dysfunction via ROS generation and ERK1/2 activation. Methods: To study whether Testo present the same effects independent on endothelial cell type, we used Primary Human Mesenteric vascular Endothelial Cells (HMEC) and Human Umbilical Vein Endothelial Cells (HUVEC). Cells were stimulated with Testo (10 −7 M, for different time points) in the presence or absence of superoxide dismutase mimetic (Tempol, 10 -4 M, for 30 min). NOX activity, gene expression, and protein expression were determined pharmacological assays and immunoblotting, respectively. Results: Testo increased superoxide anion (O 2 − ) generation in HMEC and HUVEC at short (10 min) stimulation times, which was abolished by superoxide dismutase (SOD) mimetic [(% of control) Ctrl: 100.0 ± 0.0 n=5 vs Testo: 363.2 ± 19.77 n=6 vs. Tempol+Testo: 156.7 ± 12.12 n=6, *P<0.05]. Furthermore, long-term treatment (24 h) of endothelial cells with Testo increased Nox4 expression at gene (2.44-Fold change) and protein levels [(% of control) Ctrl: 100.0 ± 0.0 n=4 vs. Testo: 157.3 ± 4.49 n=4, *P<0.05]. Testo did not affect Nox1, Nox2, and Nox5 expressions. Furthermore, Testo increased ERK1/2 phosphorylation at 10 minutes and promoted endothelial cell inflammation at 8h, characterized by increased ICAM-1(6.49-Fold changes) and VCAM-1 [(1.42-Fold changes) gene expressions. To confirm that Testo-induced ROS production is leading to endothelial injury via ERK1/2, we treated endothelial cells with Tempol prior Testo treatment. Interestingly, superoxide dismutase mimetic prevented ERK1/2 phosphorylation induced by Testo [(% of control) Testo: 161.2 ± 11.39 n=5 vs. Tempol+Testo: 91.45 ± 9.61 n=5, *P<0.05]. Conclusion: These data indicate that supraphysiological levels of testosterone induce endothelial dysfunction via ROS generation and inflammatory response. Our data helps understanding the intracellular mechanisms that justify the cardiovascular damage caused by supraphysiological doses of testosterone and places antioxidant therapy as a possible pharmacological approach to treat patients with dysregulated testosterone levels. 2022/00103-3, São Paulo Research Foundation (FAPESP). NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Initiation of 3,3-dimethyl-1-butanol at midlife prevents endothelial dysfunction and attenuates in vivo aortic stiffening with ageing in mice.

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide-driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome-derived trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3-dimethyl-1-butanol (DMB). We investigated whether DMB supplementation could prevent age-related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium-dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO-mediated EDD and lower superoxide-related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress-strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long-term DMB supplementation prevents/attenuates age-related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. KEY POINTS: Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3-dimethyl-1-butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO-suppressing interventions have potential for translation to humans for reducing CV risk with ageing.

PERMISSIVE ROLE OF ANGIOTENSIN II IN MANTENANCE OF ENDOTHELIUM-DEPENDENT VASODILATION IN CEREBRAL MICROCIRCULATION

Objective: A number of data suggest, rather controversial, permissive role of angiotensin II (ANGII) in maintaining vasodilator responses in microcirculation, via its interaction with AT1 receptors (AT1R). This study aimed to determine the role of ANGII and AT1R in the mechanisms of flow-induced dilation (FID) in middle cerebral arteries (MCA) and incidence of oxidative stress at the functional, cellular and molecular level in the cerebral vasculature and in the serum of Sprague-Dawley (SD) rats fed high salt diet (HS). Design and method: Eleven-week old, male SD rats were randomly assigned to control group (CTRL, 0.4% NaCl in rat chow); Losartan group (rats on a standard diet given the AT1R blocker losartan (1 mg/mL) in drinking water); HS group (7 days 4% NaCl in rat chow) or HS+ANGII group (7 days HS with 3 days ANGII administration via osmotic minipumps (100 ng/kg/min on days 4–7)). FID of MCA was determined in absence/presence of the NOS inhibitor L-NAME, the non-selective cyclooxygenases inhibitor indomethacin, and the superoxide dismutase mimetic TEMPOL. Gene and protein expression of antioxidative enzymes and enzymes involved in FID mechanisms were determined by RT-qPCR and western blot. Vascular NO and superoxide/ROS levels were assessed by direct fluorescence. Serum systemic oxidative stress parameters was measured by spectrophotometry. All experimental procedures conformed to the EU Directive 2010/63/EU. Results: HS or AT1R blockade impaired FID and increased oxidative stress. Supplementation of ANGII restored the mechanisms of FID in MCA of rats fed HS diet to the ones present in CTRL rats without affecting arterial blood pressure by decreasing systemic oxidative stress and decreasing superoxide/ROS levels and increased NO bioavailability in the vascular wall of HS treated rats. The AT1R blockade resulted in significantly attenuated endothelium-dependent dilation, changed mechanisms of FID, increased vascular and systemic oxidative stress and decreased expression/ activity of antioxidative enzymes. Conclusions: ANGII is crucial in the preservation of vascular oxidative stress-antioxidant system balance and via AT1R, in maintenance of physiological vasodilation mechanisms of MCA. Suppression of ANGII is a key link between HS intake and development of endothelial dysfunction, and impaired regulation of cerebral blood flow.

The Impact of Long‐Duration Exposure to Microgravity and Galactic Cosmic Radiation on Corpus Cavernosum Function

During spaceflight missions, astronauts are exposed to an extreme environment with high levels of radiation and microgravity, which negatively influence the cardiovascular system. With an increased development in space exploration and growing interest in manned missions to mars, it is of high importance to evaluate the health risks associated with long‐duration spaceflights. The increased risk of oxidative stress and inflammatory damage post‐spaceflight has been associated with cardiovascular dysfunction, but little is known about the influence of space exposure on erectile function, which is a critical component for quality of life in men. The purpose of this investigation was to determine the influence of long‐duration spaceflight on corpus cavernosum (CC) function. 86 adult male Sprague‐Dawley rats were randomized into 6 different groups with half of them enduring 4‐weeks of hindlimb unloading (HLU), and exposed to sham, 0.75Gy or 1.5Gy of simulated galactic cosmic radiation at the Ground‐based GCR Simulator at the NASA Space Radiation Laboratory (NSRL). Following a 6–9‐month recovery the rats were sacrificed, and CC tissue segments were harvested and mounted into a muscle strip myograph system for ex vivo functional assessment. CC reactivity to six 10‐s,30V‐electric field stimulations with progressive frequencies in the range of 1‐32 Hz targeting the non‐adrenergic non‐cholinergic (NANC) stimulation was assessed. The effects of HLU and radiation were determined by two‐way repeated measures ANOVA. High levels of radiation significantly decreased NANC‐mediated relaxation of the CC. Treatment with nitric oxide synthase (NOS) inhibitor L‐NAME completely inhibited tissue response to electric stimulation, showing that the relaxation was mainly driven by nitric oxide. Incubation of the CC tissue with the xanthine oxidase inhibitor allopurinol, the mitochondria‐targeted antioxidant mito‐TEMPO, and the superoxide dismutase (SOD) mimetic TEMPOL increased the relaxation response of the erectile tissue for the groups exposed to radiation, with little or opposite effect on the groups under no radiation exposure. Upon treatment with the Arginase inhibitor S‐(2‐boronoethyl)‐l‐cysteine (BEC), CC tissue for the groups under radiation exposure showed a significant improvement in relaxation under NANC electric field stimulation. Comparably, western blot analysis showed increased levels of oxidative stress and lipid peroxidation in the corpus cavernosum on the groups under radiation exposure by assessment of 4‐hydroxynonenal (HNE), as well as higher levels of arginase 1 and 2. This information suggests that increased oxidative stress from radiation exposure in long spaceflight trips impairs corpus cavernosum vasoreactivity and exposes new factors to consider with space exploration to ensure that astronauts can return to normal life after long trips in space.

A thrombin-triggered self-regulating anticoagulant strategy combined with anti-inflammatory capacity for blood-contacting implants.

Interrelated coagulation and inflammation are impediments to endothelialization, a prerequisite for the long-term function of cardiovascular materials. Here, we proposed a self-regulating anticoagulant coating strategy combined with anti-inflammatory capacity, which consisted of thrombin-responsive nanogels with anticoagulant and anti-inflammatory components. As an anticoagulant, rivaroxaban was encapsulated in nanogels cross-linked by thrombin-cleavable peptide and released upon the trigger of environmental thrombin, blocking the further coagulation cascade. The superoxide dismutase mimetic Tempol imparted the antioxidant property. Polyphenol epigallocatechin gallate (EGCG), in addition to its anti-inflammatory function in synergy with Tempol, also acted as a weak cross-linker to stabilize the coating. The effectiveness and versatility of this coating were validated using two typical cardiovascular devices as models, biological valves and vascular stents. It was demonstrated that the coating worked as a precise strategy to resist coagulation and inflammation, escorted reendothelialization on the cardiovascular devices, and provided a new perspective for designing endothelium-like functional coatings.

Low-dose angiotensin II supplementation restores flow-induced dilation mechanisms in cerebral arteries of Sprague-Dawley rats on a high salt diet.

Salt-induced suppression of angiotensin II contributes to impaired endothelium-dependent vascular reactivity. The present study investigated the effect of chronic low-dose angiotensin II (ANG II) supplementation on the mechanisms of flow-induced dilation (FID) and oxidative stress at the cellular and molecular level in middle cerebral arteries (MCA) of male Sprague-Dawley rats fed high salt diet. Rats (10 weeks old) were randomly assigned to a low salt diet group (0.4% NaCl in rat chow); high salt diet group (7 days 4% NaCl in rat chow) or HS+ANG II group [7 days high salt diet with 3 days ANG II administration via osmotic minipumps (100 ng/kg per min on days 4-7)]. FID was determined in absence/presence of the NOS inhibitor L-NAME, the non-selective cyclooxygenase (COX-1,2) inhibitor indomethacin, a selective inhibitor of CYP450 epoxygenase activity (MS-PPOH) and the superoxide dismutase mimetic TEMPOL. Gene expression of antioxidative enzymes, and of genes and proteins involved in FID mechanisms were determined by RT-qPCR and western blot. Vascular nitric oxide and superoxide/reactive oxygen species levels were assessed by direct fluorescence. Serum systemic oxidative stress parameters were measured by spectrophotometry. Chronic low-dose ANG II supplementation in high salt fed rats restored FID of MCAs, which was nitric oxide, prostanoid and epoxyeicosatrienoic acid dependent. ANG II changed the protein/gene expression of COXs, HIF-1α and VEGF and significantly increased GPx4 and EC-SOD antioxidative enzyme expression, decreased systemic oxidative stress, decreased superoxide/ROS levels and increased nitric oxide bioavailability in the vascular wall. Physiological levels of circulating ANG II are crucial to maintain the HIF-1α dependent mechanisms of FID and vascular oxidative balance without affecting mean arterial pressure.

Role of Outer Membrane Vesicles From Helicobacter pylori in Atherosclerosis.

Infection is thought to be involved in the pathogenesis of atherosclerosis. Studies have shown the association between helicobacter pylori (H. pylori) and coronary artery disease. It is interesting to find H. pylori DNA and cytotoxin-associated gene A (CagA) protein in atherosclerotic plaque. Outer membrane vesicles (OMVs), secreted by H. pylori, exert effects in the distant organ or tissue. However, whether or not OMVs from H. pylori are involved in the pathogenesis of atherosclerosis remains unknown. Our present study found that treatment with OMVs from CagA-positive H. pylori accelerated atherosclerosis plaque formation in ApoE–/– mice. H. pylori-derived OMVs inhibited proliferation and promoted apoptosis of human umbilical vein endothelial cells (HUVECs), which was also reflected in in vivo studies. These effects were normalized to some degree after treatment with lipopolysaccharide (LPS)-depleted CagA-positive OMVs or CagA-negative OMVs. Treatment with H. pylori-derived OMVs increased reactive oxygen species (ROS) levels and enhanced the activation of nuclear factor-κB (NF-κB) in HUVECs, which were reversed to some degree in the presence of a superoxide dismutase mimetic TEMPOL and a NF-κB inhibitor BAY11-7082. Expressions of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), two inflammatory factors, were augmented after treatment with OMVs from H. pylori. These suggest that H. pylori-derived OMVs accelerate atherosclerosis plaque formation via endothelium injury. CagA and LPS from H. pylori-OMVs, at least in part, participate in these processes, which may be involved with the activation of ROS/NF-κB signaling pathway. These may provide a novel strategy to reduce the incidence and development of atherosclerosis.

Role of the Polyol Pathway in Locomotor Recovery and Wallerian Degeneration after Spinal Cord Contusion Injury.

Spinal cord contusion injury leads to Wallerian degeneration of axonal tracts, resulting in irreversible paralysis. Contusion injury causes perfusion loss by thrombosis and vasospasm, resulting in spinal cord ischemia. In several tissues, including heart and brain, ischemia activates polyol pathway enzymes—aldose reductase (AR) and sorbitol dehydrogenase (SDH)—that convert glucose to sorbitol and fructose in reactions, causing oxidative stress and tissue loss. We sought to determine whether activation of this pathway, which has been termed glucotoxicity, contributes to tissue loss after spinal cord contusion injury. We tested individual treatments with AR inhibitors (sorbinil or ARI-809), SDH inhibitor (CP-470711), superoxide dismutase mimetic (tempol), or combined sorbinil and tempol. Each treatment significantly increased locomotor recovery and reduced loss of spinal cord tissue in a standard model of spinal cord contusion in rats. Tissue levels of sorbitol and axonal AR (AKR1B10) expression were increased after contusion injury, consistent with activation of the polyol pathway. Sorbinil treatment inhibited the above changes and also decreased axonal swelling and loss, characteristic of Wallerian degeneration. Treatment with tempol induced recovery of locomotor function that was similar in magnitude, but non-additive to sorbinil, suggesting a shared mechanism of action by reactive oxygen species (ROS). Exogenous induction of hyperglycemia further increased injury-induced axonal swelling, consistent with glucotoxicity. Unexpectedly, contusion increased spinal cord levels of glucose, the primary polyol pathway substrate. These results support roles for spinal glucose elevation and tissue glucotoxicity by the polyol pathway after spinal cord contusion injury that results in ROS-mediated axonal degeneration.

Combination of Exercise Training and SOD Mimetic Tempol Enhances Upregulation of Nitric Oxide Synthase in the Kidney of Spontaneously Hypertensive Rats.

Both exercise training (Ex) and superoxide dismutase (SOD) mimetic tempol have antihypertensive and renal protective effects in rodent models of several hypertensions. We recently reported that Ex increases nitric oxide (NO) production and the expression levels of endothelial and neuronal NO synthase (eNOS and nNOS) in the kidney and aorta of the spontaneously hypertensive rats (SHR) and normotensive Wistar–Kyoto rats (WKY). We also found that endogenous hydrogen peroxide (H2O2) upregulates the expression levels of eNOS and nNOS in SHR. To elucidate the mechanism of the Ex-upregulated NO system in the kidney, we examined the additive effect of Ex and tempol on the renal NO system in SHR and WKY. Our data showed that, in SHR, both Ex and tempol increase the levels of H2O2 and nitrate/nitrite (NOx) in plasma and urine. We also observed an increased renal NOS activity and upregulated expression levels of eNOS and nNOS with decreased NADPH oxidase activity. The effects of the combination of Ex and tempol on these variables were cumulate in SHR. On the other hand, we found that Ex increases these variables with increased renal NADPH oxidase activity, but tempol did not change these variables or affect the Ex-induced upregulation in the activity and expression of NOS in WKY. The SOD activity in the kidney and aorta was activated by tempol only in SHR, but not in WKY; whereas Ex increased SOD activity only in the aorta in both SHR and WKY. These results indicate that Ex-induced endogenous H2O2 produced in the blood vessel and other organs outside of the kidney may be carried to the kidney by blood flow and stimulates the NO system in the kidney.

Role of Arginase in Selective Impairment of Endothelium-Dependent Nitric Oxide Synthase-Mediated Dilation of Retinal Arterioles during Early Diabetes.

PurposeRetinal vasomotor activity can be regulated by two major endothelial enzymes, nitric oxide synthase (NOS) and cyclooxygenase (COX). The vascular arginase also consumes a NOS substrate and thus impedes NOS-mediated vasodilation. Diabetes mellitus exhibits vascular complications in the retina with elevated oxidative stress and compromised NOS-mediated vasodilation. However, the underlying molecular mechanisms remain unclear, and the effect of diabetes on COX-mediated vasodilation is unknown. Herein, we examined the relative impact of diabetes on retinal arteriolar dilations to COX and NOS activation and the roles of arginase and superoxide in diabetes-induced vasomotor dysfunction.MethodsRetinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks of hyperglycemia, 433 ± 27 mg/dL) or age-matched control pigs (97 ± 4 mg/dL). The vasodilations to bradykinin (NOS activator) and histamine (NOS/COX activator) were examined in vitro.ResultsRetinal arteriolar dilations to histamine and bradykinin were significantly reduced after 2 weeks of diabetes. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated the dilations of control vessels, but not diabetic vessels, to histamine. In the presence of L-NAME and COX inhibitor indomethacin, histamine-induced dilations of control and diabetic vessels were reduced similarly. Treatment of diabetic vessels with arginase inhibitor nor-NOHA, but not superoxide dismutase mimetic TEMPOL, preserved both histamine- and bradykinin-induced dilations in an L-NAME-sensitive manner.ConclusionsArginase, rather than superoxide, impairs endothelium-dependent NOS-mediated dilation of retinal arterioles during diabetes, whereas vasodilation mediated by COX remains intact. Blockade of vascular arginase may improve endothelial function of retinal arterioles during early onset of diabetes.

Deficiency of the oxidative stress–responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy

Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For invitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes invivo and high glucose invitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

Tempol Improves Renal Autoregulation and Kidney Function Following Ischemia‐Reperfusion Injury in Rats

Renal ischemia‐reperfusion (IR) induced acute kidney injury is characterized by inflammation, oxidative stress, increased renal vascular resistance, and reduced glomerular filtration rate. Our previous study revealed that renal autoregulation was impaired in IR, but restored with acute exposure to ROS scavengers or NADPH oxidase inhibitor. Accordingly, we postulated that chronic treatment with the superoxide dismutase mimetic Tempol would improve renal autoregulatory capacity and kidney function following kidney IR. IR was induced by 60‐minute bilateral renal artery occlusion. IR rats were treated with or without Tempol (25 mg/kg/day) in drinking water over a 7‐day period following surgery. Afferent arteriole autoregulatory behavior was assessed in vitro using blood‐perfused juxtamedullary nephrons (n = 6–7/each groups). Afferent arterioles from sham‐operated rats exhibited pressure‐dependent vasoreactivity. Baseline diameter averaged 13.4 ± 0.5 μm at a renal perfusion pressure (RPP) of 100 mmHg and increased to 117 ± 4% of baseline at RPP of 65 mmHg and decreased to 69 ± 2% at RPP of 170 mmHg, respectively. Afferent arterioles from IR rats however, only increased to 104 ± 2% and decreased to just 91 ± 5% of baseline (11.7 ± 0.6 μm) at 65 and 170 mmHg, respectively (P < 0.05 vs sham), indicating persistently impaired autoregulatory capacity. In contrast, Tempol‐treated IR rats exhibited normal autoregulatory function as the pressure‐diameter profile was similar to the shams. Afferent arterioles diameter increased to 109 ± 2% of baseline (13.3 ± 0.9 μm) and decreased to 69 ± 4% at RPP of 65 and 170 mmHg, respectively (P > 0.05 vs. sham). Preglomerular microvessels (PGMV) were collected from one set of sham, IR and IR+Tempol rats (n = 8 rats/group) on day 7 following IR. Steady‐state mRNA of NADPH oxidase subunits, MCP‐1 and TGF‐β in these PGMV were markedly elevated in IR compared to sham rats. The NADPH oxidase subunits, p67phox and p47phox but not p22phox, were increased by 4.5 and 3‐fold respectively in IR vs. sham (P < 0.05). MCP‐1 mRNA increased 6.7‐fold and TGF‐β 4–fold compared to sham (P < 0.05). Tempol treatment significantly suppressed the IR‐induced increase in mRNA expression of all measured markers. Glomerular filtration rate (GFR) was assessed in conscious rats using a transdermal non‐invasive clearance device with intravenous injection of FITC‐sinistrin on day 0 and day 7. GFR remained constant (1.0–1.4 mL/min/100g, n=6) in sham. On day 7 after IR, GFR was 0.27 ± 0.17 mL/min/100g (n=6, P < 0.05 vs sham) in untreated rats, and was 0.60 ± 0.12 mL/min/100g in Tempol‐treated IR rats (n=4, P > 0.05 vs sham). Tempol treatment also significantly reduced albuminuria. These results demonstrated that chronic Tempol treatment ameliorated IR‐induced chronic impairment of autoregulatory capacity and associated increases in expression of NADPH oxidase, MCP‐1 and TGF‐β in PGMV. We conclude that oxidative stress in the renal microvasculature contributed to a persistent lack of functional recovery from severe IR injury.Support or Funding InformationThis study was supported by NIH grants (DK106500 to ZG, and DK044628 and HL095499 to EWI).

Initiation of the Gut Microbiome Targeted Compound 3,3‐Dimethyl‐1‐butanol at Mid‐life Prevents Age‐related Vascular Dysfunction

Aging induces vascular dysfunction (arterial stiffening and endothelial dysfunction) mediated by superoxide‐driven oxidative stress and reduced nitric oxide (NO) bioavailability, which increases risk of cardiovascular (CV) diseases. A potential intervention target is the gut microbiome‐derived metabolite trimethylamine N‐oxide (TMAO), which increases in circulation with advancing age, is associated with higher CV risk, and can be inhibited by the food‐derived compound 3,3‐dimethyl‐1‐butanol (DMB).PurposeTo determine if DMB initiated at mid‐life (i.e., prior to the onset of dysfunction) prevents the development of age‐related vascular dysfunction.MethodsMiddle‐aged (18 mo) male C57BL/6 mice received drinking water with (n=24) or without (control: n=27) 1% DMB until sacrifice at 21, 24, or 27 mo of age (n=7–13/age/treatment). Arterial stiffness was assessed serially in vivo by aortic pulse wave velocity (aPWV). Endothelial function (ex vivo carotid artery endothelium‐dependent dilation [EDD] to acetylcholine), plasma TMAO (LC‐MS) and aortic superoxide (electron paramagnetic resonance spectroscopy) were assessed post‐sacrifice. To determine the prevention of age‐related vascular dysfunction with DMB, functional measures were made in a reference group of young (5 mo) male C57BL/6 mice (YC, n=12).ResultsPlasma TMAO increased with age vs YC (3.1±0.5 μM), but tended to be lower in DMB (5.6±0.5 μM) vs control (6.9±0.8 μM) mice at 24 and 27 mo (p=0.15). In control mice, aPWV was similar to YC (356±14 cm/s) at 18/21 mo (359±12/368±11 cm/s, both p>0.54), but increased with age (24 mo: 386±12 cm/s, p=0.06 vs 18 mo; 27 mo: 432±10 cm/s, p<0.001 vs 18 mo); this increase was attenuated by ~50% in DMB mice (aPWV at 24 mo: 359±9 cm/s; 27 mo: 388±9 cm/s, both p=0.01 vs control). Age‐related declines in peak EDD were apparent in control mice at 24 mo (79±3 vs YC 93±2%, p<0.001) and 27 mo (76±3%, p<0.0001 vs YC) and were fully prevented by DMB (24 mo: 91±3%; 27 mo: 93±1%; both p<0.05 vs control). The effects of DMB at 24 and 27 mo (data combined across these ages) were mediated by reduced aortic superoxide production (control 4.7±0.5 vs DMB 3.1±0.4 AU, p=0.01), which reduced tonic superoxide‐associated suppression of EDD (normalization of EDD with superoxide dismutase mimetic TEMPOL) and restored NO‐mediated dilation (the difference in peak EDD in the absence vs presence of the NO synthase inhibitor L‐NAME: control 45±6 vs DMB 69±4%, p<0.01). There were no differences in smooth muscle sensitivity to NO (peak dilation to sodium nitroprusside; 96±1 vs 98±1%, p=0.12), indicating improvements in EDD were endothelium‐specific.ConclusionSupplementation with DMB from mid‐life through old age in mice prevented the development of arterial stiffening and endothelial dysfunction. DMB may be a promising therapeutic strategy for preventing age‐related vascular dysfunction and thereby reducing CV risk with aging in humans.Support or Funding InformationR01 HL1348870‐02S1, R21 AG060884, F32 HL140875

Impaired Dilator Reactivity of Middle Cerebral Arteries in Goto‐Kakizaki Rats with Type II Diabetes Mellitus

Type II diabetes mellitus (T2DM) is a highly prevalent disease and is associated with the development of cerebrovascular disease. The objective of the study was to determine whether alterations to the regulation of cerebral vascular tone exist in a non‐obese model of T2DM and to determine the mechanistic contributors to changes in this response. Middle cerebral arteries (MCA) from 17–18‐wk‐old male Wistar Kyoto (WKY) and Goto‐Kakizaki (GK) were isolated and cannulated with glass micropipettes. Vascular reactivity was assessed in response to challenge with acetylcholine (ACh), sodium nitroprusside (SNP), reductions in Po2, 5‐HT, and increased intraluminal pressure. Vessels were treated with the competitive nitric oxide synthase (NOS) inhibitor L‐NAME, the cyclooxygenase inhibitor indomethacin, and the cell‐permeable superoxide dismutase (SOD) mimetic TEMPOL to support interpretation of mechanistic contributors to changes in vessel reactivity. Similar measurements of vascular reactivity to methacholine (MCh), phenylephrine, SNP, and reductions in PO2 were measured in aortic ring preparations, along with measurements of vascular NO production and metabolites of arachidonic acid. There was an impairment of vessel dilation within the MCA and aorta of GK in response to ACh/MCh and hypoxia but not SNP. Decreased vascular NO release and ratio of PGI2/TxA2 were measured within the aorta in response to hypoxia, and increasing MCh concentration. No change was seen in the constrictor response to either 5‐HT or phenylephrine within the MCA or aorta respectively or in the myogenic activation of the MCA. Treatment with L‐NAME severely blunted the dilator reactivity of both the MCA and aorta, while TEMPOL showed mild improvements in GK, and indomethacin yielded improved dilator reactivity to MCh in GK but blunted it in WKY. These results suggest that the vasodilator reactivity of the MCA and aorta of GK in response to endothelium‐dependent dilator stimuli is impaired vs. WKY. This may be due to decreased NO bioavailability and altered arachidonic acid metabolism as a result of a pro‐oxidant pro‐inflammatory condition present in T2DM. The lack of change to the constrictor response of MCA and aorta in GK suggests a lack of structural remodeling as a result of T2DM at this age.Support or Funding InformationAmerican Heart Association and National Institutes of Health (USA); Canadian Institutes for Health Research and Natural Sciences and Engineering Research Council (Canada)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prevention of Obesity‐Associated Coronary and Cardiac Diastolic Dysfunction by Deletion of Smooth Muscle Cell Mineralocorticoid Receptor in Females

Coronary microvascular dysfunction is independently predictive of cardiac diastolic dysfunction and both conditions are common in obesity and associated metabolic syndrome. Recent work by us and others indicates that mineralocorticoid receptor (MR) antagonism treats obesity‐associated coronary and cardiac diastolic dysfunction. Vascular cell‐specific MR involvement in these obesity‐related defects remains unclear. Thus, we evaluated the hypothesis that deletion of smooth muscle cell (SMC) MR would prevent obesity‐associated coronary and cardiac diastolic dysfunction in females. Female wild‐type (WT) and SMC MR knockout (KO) mice were fed a standard chow or western diet (WD) to induce obesity for 16 weeks. WD feeding induced obesity, hyperinsulinemia, hypercholesterolemia, and visceral adipose inflammation in both WT and SMC MR KO mice. Assessment of coronary vascular function via wire myography revealed WD‐induced impairment of endothelium‐dependent vasodilation and enhanced vasoconstriction to the thromboxane A2 analog U46619 in WT but not SMC MR KO mice. Vasodilation to the nitric‐oxide donor sodium nitroprusside was unchanged by WD feeding. Global quantitative proteomics of aortas from each group revealed a number of differentially expressed proteins following WD feeding including upregulation of the oxidative stress‐related small GTPase Rac‐1 in WT but not SMC MR KO aortas. Accordingly, pretreatment of coronary arteries with the superoxide dismutase mimetic Tempol restored endothelium‐dependent vasodilation in WD‐fed WT mice with no effect in SMC MR KO vessels. Furthermore, WD feeding induced cardiac diastolic dysfunction assessed by echocardiography (i.e., increased E/E′, reduced E′/A′, increased diastolic stiffness). Coincident with prevention of WD‐induced coronary dysfunction, SMC MR deletion prevented WD‐induced cardiac diastolic dysfunction. Importantly, this cardioprotective effect of SMC MR deletion occurred independent of changes in left ventricular hypertrophy, blood pressure, aortic stiffening, and proteinuria. Taken together, our data demonstrate a central role of SMC MR signaling underlying WD‐induced coronary and cardiac diastolic dysfunction in females. Importantly, this study adds to an emerging body of evidence suggesting a vascular origin of cardiac dysfunction in obesity.Support or Funding InformationSupported by NIH (HL136386 to SBB, HL119290 to IZJ) and the Department of Veterans Affairs BLR&D (CDA‐2 IK2 BX002030). BC is supported by Department of Veterans Affairs (IK6 BX004016 and I01 BX002255).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prenatal hypoxia affected endothelium-dependent vasodilation in mesenteric arteries of aged offspring via increased oxidative stress

Prenatal hypoxia can affect vascular functions in young offspring. However, there is limited knowledge regarding whether and how prenatal hypoxia influences vascular functions in aged offspring. This study compared the effects of prenatal hypoxia on the mesenteric arteries (MA) between a young adult and aged offspring and investigated the underlying mechanisms. Pregnant rats were randomly divided into the control and prenatal hypoxia groups. The vascular functions and molecular levels were assessed in 5-month-old (5 M) or 20-month-old (20 M) offspring. Prenatal hypoxia decreased acetylcholine-mediated vascular relaxations in 20-M but not 5-M offspring. Sodium nitroprusside-mediated relaxation curves were not altered by prenatal hypoxia in 5- and 20-M offspring. Prenatal hypoxia enhanced the contractile responses caused by phenylephrine, phorbol 12,13-dibutyrate, and 5-hydroxytryptamine only in 5-M offspring. The endothelial NO synthase (eNOS) activities were decreased along with downregulated eNOS mRNA expression and phosphorylated eNOS/total eNOS protein expression in 20-M offspring with prenatal hypoxia. The NADPH oxidase (NOX) inhibitor apocynin and superoxide dismutase (SOD) mimetic tempol restored the acetylcholine-mediated weaker relaxations in 20-M offspring with prenatal hypoxia. Enzyme-linked immunosorbent and dihydroethidium assay showed that prenatal hypoxia enhanced oxidative stress in 20-M offspring. Transmission electron microscopy showed that prenatal hypoxia damaged mitochondrial structures in the MA endothelial cells of 20-M offspring. Increased NOX2 protein expression and decreased SOD3 expression were found in 20-M offspring. The results demonstrated that endothelial dysfunction induced by intrauterine hypoxia occurred with aging via enhanced oxidative stress and decreased nitric oxide activities in aged offspring.

Targeted Therapy of Atherosclerosis by a Broad-Spectrum Reactive Oxygen Species Scavenging Nanoparticle with Intrinsic Anti-inflammatory Activity.

Atherosclerosis is a leading cause of vascular diseases worldwide. Whereas antioxidative therapy has been considered promising for the treatment of atherosclerosis in view of a critical role of reactive oxygen species (ROS) in the pathogenesis of atherosclerosis, currently available antioxidants showed considerably limited clinical outcomes. Herein, we hypothesize that a broad-spectrum ROS-scavenging nanoparticle can serve as an effective therapy for atherosclerosis, taking advantage of its antioxidative stress activity and targeting effects. As a proof of concept, a broad-spectrum ROS-eliminating material was synthesized by covalently conjugating a superoxide dismutase mimetic agent Tempol and a hydrogen-peroxide-eliminating compound of phenylboronic acid pinacol ester onto a cyclic polysaccharide β-cyclodextrin (abbreviated as TPCD). TPCD could be easily processed into a nanoparticle (TPCD NP). The obtained nanotherapy TPCD NP could be efficiently and rapidly internalized by macrophages and vascular smooth muscle cells (VSMCs). TPCD NPs significantly attenuated ROS-induced inflammation and cell apoptosis in macrophages, by eliminating overproduced intracellular ROS. Also, TPCD NPs effectively inhibited foam cell formation in macrophages and VSMCs by decreasing internalization of oxidized low-density lipoprotein. After intravenous (i.v.) administration, TPCD NPs accumulated in atherosclerotic lesions of apolipoprotein E-deficient (ApoE-/-) mice by passive targeting through the dysfunctional endothelium and translocation via inflammatory cells. TPCD NPs significantly inhibited the development of atherosclerosis in ApoE-/- mice after i.v. delivery. More importantly, therapy with TPCD NPs afforded stabilized plaques with less cholesterol crystals, a smaller necrotic core, thicker fibrous cap, and lower macrophages and matrix metalloproteinase-9, compared with those treated with control drugs previously developed for antiatherosclerosis. The therapeutic benefits of TPCD NPs mainly resulted from reduced systemic and local oxidative stress and inflammation as well as decreased inflammatory cell infiltration in atherosclerotic plaques. Preliminary in vivo tests implied that TPCD NPs were safe after long-term treatment via i.v. injection. Consequently, TPCD NPs can be developed as a potential antiatherosclerotic nanotherapy.

High Glucose Inhibits Neural Stem Cell Differentiation Through Oxidative Stress and Endoplasmic Reticulum Stress.

Maternal diabetes induces neural tube defects by suppressing neurogenesis in the developing neuroepithelium. Our recent study further revealed that high glucose inhibited embryonic stem cell differentiation into neural lineage cells. However, the mechanism whereby high glucose suppresses neural differentiation is unclear. To investigate whether high glucose-induced oxidative stress and endoplasmic reticulum (ER) stress lead to the inhibition of neural differentiation, the effect of high glucose on neural stem cell (the C17.2 cell line) differentiation was examined. Neural stem cells were cultured in normal glucose (5 mM) or high glucose (25 mM) differentiation medium for 3, 5, and 7 days. High glucose suppressed neural stem cell differentiation by significantly decreasing the expression of the neuron marker Tuj1 and the glial cell marker GFAP and the numbers of Tuj1+ and GFAP+ cells. The antioxidant enzyme superoxide dismutase mimetic Tempol reversed high glucose-decreased Tuj1 and GFAP expression and restored the numbers of neurons and glial cells differentiated from neural stem cells. Hydrogen peroxide treatment imitated the inhibitory effect of high glucose on neural stem cell differentiation. Both high glucose and hydrogen peroxide triggered ER stress, whereas Tempol blocked high glucose-induced ER stress. The ER stress inhibitor, 4-phenylbutyrate, abolished the inhibition of high glucose or hydrogen peroxide on neural stem cell differentiation. Thus, oxidative stress and its resultant ER stress mediate the inhibitory effect of high glucose on neural stem cell differentiation.