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Graphical abstract, Highlights • Subchronic HCB-administration in male rats generates arterial hypertension. • AT1 activity is involved in HCB- induced hypertension. • AT1 activity is involved in arterial morphological changes caused by HCB. • AT1 activity is involved in HCB- produced alterations in arterial relaxation. • Losartan prevents HCB-produced alterations in TGF-β1, eNOS and AT1 expressions., Environmental hexachlorobenzene (HCB) increases blood pressure (BP) in female rats, causing alterations in arterial structure and function. Here we study the role of Angiotensin II receptor type 1 (AT1) in HCB-induced hypertension through the use of AT1 antagonist losartan. HCB-treated male rats showed a 22.7% increase in BP which was prevented by losartan. Losartan blocked HCB-induced changes in arterial morphology (decreased aorta cell number and increased wall thickness). Losartan also prevented HCB-induced alterations in artery relaxation by acetylcholine and nitroprusside but not the reduction in the maximum contraction by phenylephrine. Losartan rescued arterial molecular alterations caused by HCB (i.e. an increase in TGF-β1 and AT1 expression and a decrease in eNOS expression and nitrite levels) and reduced hydrogen sulfide plasma concentration. In conclusion: in this work we demonstrate that AT1 activity is involved in HCB effects on the vascular system leading to hypertension.

Graphical abstract, Highlights • Naringin inhibits and/or scavenge in vitro free radicals. • Exposure to cyclophosphamide (CYCP) invoked erythrocytotoxicity. • Erythrocytotoxicity was characterized by oxidative stress and dyslipidaemia. • LDH (a marker of anaerobic ATP generation) was also depleted. • Naringin pre-administered reversed the CYCP-induced erythrocytotoxicity., Earlier reports have shown that Cyclophosphamide (CYCP), an anti-malignant drug, elicited cytotoxicity; and that naringin has several beneficial potentials against oxidative stress and dyslipidaemias. We investigated the influence of naringin on free radical scavenging, cellular integrity, cellular ATP, antioxidants, oxidative stress, and lipid profiles in the CYCP-induced erythrocytotoxicity rat model. Rats were pretreated orally by gavage for fourteen consecutive days with three doses (50, 100, and 200 mg/kg) naringin before single CYCP (200 mg/kg, i.p.) administration. Afterwards, the rats were sacrificed. Naringin concentrations required for 50 % scavenging hydrogen peroxide and nitric oxide radical were 0.27 mg/mL and 0.28 mg/mL, respectively. Naringin pretreatment significantly (p < 0.05) protected erythrocytes plasma membrane architecture and integrity by abolishing CYCP-induced decrease in the activity of erythrocyte LDH (a marker of ATP). Pretreatment with naringin remarkably (p < 0.05) reversed CYCP-induced decreases in the erythrocytes glutathione levels, activities of glutathione-S-transferase, catalase, glutathione peroxidase, and glutathione reductase; attenuated CYCP-mediated increases in erythrocytes levels of malondialdehyde, nitric oxide, and major lipids (cholesterol, triacylglycerol, phospholipids, and non-esterified fatty acids). Taken together, different acute pretreatment doses of naringin might avert CYCP-mediated erythrocytes dysfunctions via its antioxidant, free-radical scavenging, and anti-dyslipidaemia properties.

Background and aim Renal ischemia-reperfusion is associated with inflammation and oxidative stress. As a major compound in black pepper, piperine has anti-inflammatory and anti-oxidative properties. In present study, the protective effects of oral administration of piperine in renal ischemia-reperfusion (IR) induced acute kidney injuries (AKI) were investigated. Experimental procedure Male Wistar rats received piperine (10 or 20 mg/kg.bw) or vehicle for 10 days. The artery and vein of both kidneys were then clamped for 30 min, followed by a 24-h reperfusion period. Concentrations of creatinine and urea-nitrogen in descending aorta blood were measured, and malondialdehyde (MDA) and ferric reducing/antioxidant power (FRAP) levels were measured in kidney tissue to evaluate the oxidative stress. Inflammation was evaluated by measuring the TNF-α and ICAM-1 mRNA expression levels in renal cortical tissue using Real Time PCR method and counting leukocytes infiltration to interstitium. Further measured were tissue damages in H & E stained sections. Results Renal IR reduced FRAP, while increasing the plasma concentrations of creatinine and urea-nitrogen, tissue MDA level, TNF-α and ICAM-1 mRNA expressions, leukocyte infiltration and histopathologic injuries. Piperine administration significantly reduced the plasma concentrations of creatinine and urea-nitrogen, expression of pro-inflammatory factors, oxidative stress and renal histopathologic injuries. It is to be noted that 20 mg/kg dose was more effective. Conclusion Our results suggest piperine protects the kidney against ischemia-reperfusion induced acute kidney injuries by its anti-inflammatory and anti-oxidative properties., Graphical abstract Image 102975, Highlights • Renal ischemia-reperfusion increased the inflammation and oxidative stress parameters. • Ischemia-reperfusion increased histopathological damages and functional parameters. • Piperine pretreatment significantly reduced the inflammation and oxidative stress. • Piperine administration ameliorated renal function and histopathologic damages.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease, which is accompanied by progressive joint damage and disability. The intolerability of conventional antirheumatic drugs by some patients necessitates the search for effective antirheumatic agents having better tolerability. In the current work, we aimed to investigate the efficacy of cinnamaldehyde, tadalafil, and aliskiren as potential antirheumatic candidates and to explore their modulatory effects on joint destruction, inflammatory response, and intracellular signaling. Arthritis was induced in female Wistar rats by complete Freund's adjuvant (CFA) 0.4 ml s.c. on days 1, 4, and 7. Treated groups received their respective drugs, starting from day 13, daily for 3 weeks. Methotrexate and prednisolone were the standard antirheumatic drugs, while cinnamaldehyde, tadalafil, and aliskiren were the test agents. Treatment with cinnamaldehyde, tadalafil, or aliskiren reduced serum levels of rheumatoid factor, and pro-inflammatory cytokines; tumor necrosis factor-alpha and interleukin-6 (IL-6), along with elevated level of IL-10 which is an anti-inflammatory cytokine. Besides, cartilage and bone destruction biomarkers; matrix metalloproteinase-3 (MMP-3) and receptor activator of nuclear factor-kappa B ligand (RANKL); were significantly reduced after treatment with the test agents, which was further confirmed by histopathological investigation. The elevated protein expressions of phosphorylated-Janus kinase 2 (p-JAK2), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), and inducible nitric oxide synthase (iNOS) in articular tissue were markedly attenuated after treatment with cinnamaldehyde, tadalafil, or aliskiren, while that of endothelial nitric oxide synthase (eNOS) was greatly enhanced. In addition, oxidative stress and inflammatory markers such as malondialdehyde, nitric oxide, and myeloperoxidase were reduced in joint tissue after treatment with the test agents, while glutathione content was elevated. Furthermore, the renin inhibitor aliskiren produced effects close to those of the normal and methotrexate, the gold standard antirheumatic drug, in most of the measured parameters. Collectively, these findings led to the assumption that the downregulation of IL-6/JAK2/STAT3 signaling by cinnamaldehyde, tadalafil, and aliskiren could alleviate joint destruction by MMP-3 and RANKL, reduce iNOS, and enhance eNOS expressions. Moreover, aliskiren could be a promising therapeutic agent for RA, because of its ability to normalize most of the measured parameters after CFA-induced arthritis.

Graphical abstract, Highlights • Methanol extract of Psiadia punctulata (MAPP) produced a significant vasodilation. • Chloroform fraction and its methylated flavonoids were responsible for this effect. • Vasodilation is referred to endothelial nitric oxide and, Ca2+ dependent eNOS. • Interference with calcium entrance is another possible mechanism of vasodilation., Vasodilators are important pharmacologic agents for managing and/or treating hypertension. Medicinal plants are considered as valuable source of bioactive compounds. We used a bioguided approach to isolate, identify, and investigate the possible vasodilation activities and mechanism(s) of the prepared methanol extract from aerial parts of Psiadia punctulata (MAPP), its bioactive fraction and active compounds. Vascular effects of MAPP were studied using isolated artery technique in the presence or absence of specific candidate pathways inhibitors, and found to produce a significant vasodilation of phenylephrine preconstricted rat aortae. The bioactive chloroform fraction yielded five methoxylated flavonoids: umuhengerin (1), gardenin A (2), gardenin B (3), luteolin-3′,4′ -dimethyl ether (4), and 5,3′-dihydroxy-6,7,4′,5′-tetramethoxyflavone (5). Metabolites 1, 4, and 5 produced a significant vasodilation. Removal of the endothelium significantly inhibited MAPP vasodilation. Nitric oxide synthase inhibition and not prostacycline inhibition or K+ channel blocking, was found to cause the observed vasodilation inhibition. Both guanylate cyclase and adenylate cyclase inhibitions markedly inhibited MAPP vasodilation. In conclusion MAPP possesses vasodilation activities that is mediated through endothelial nitric oxide pathway, calcium dependent endothelial nitric oxide synthase activation, and interference with the depolarization process through calcium channel blocking activity.

Cardiovascular diseases are a rapidly growing epidemic with high morbidity and mortality. There is an urgent need to develop nutraceutical-based therapy with minimum side effects to reduce cardiovascular risk. Panax ginseng occupies a prominent status in herbal medicine for its various therapeutic effects against inflammation, allergy, diabetes, cardiovascular diseases, and even cancer, with positive, beneficial, and restorative effects. The active components found in most P. ginseng varieties are known to include ginsenosides, polysaccharides, peptides, alkaloids, polyacetylene, and phenolic compounds, which are considered to be the main pharmacologically active constituents in ginseng. P. ginseng is an adaptogen. That is, it supports living organisms to maintain optimal homeostasis by exerting effects that counteract physiological changes caused by physical, chemical, or biological stressors. P. ginseng possesses immunomodulatory (including both immunostimulatory and immunosuppressive), neuromodulatory, and cardioprotective effects; suppresses anxiety; and balances vascular tone. P. ginseng has an antihypertensive effect that has been explained by its vasorelaxant action, and paradoxically, it is also known to increase blood pressure by vasoconstriction and help maintain cardiovascular health. Here, we discuss the potential adaptogenic effects of P. ginseng on the cardiovascular system and outline a future research perspective in this area.

Visual Abstract, Highlights • The role of ERα non-nuclear signaling in female heart failure was investigated, using a novel mouse line in which ERα non-nuclear signaling was selectively disrupted by inhibiting the interaction between ERα and striatin, a scaffold protein residing at caveolae. • ERα non-nuclear signaling was linked to myocardial PKG activity in female hearts and ameliorated cardiac maladaptive remodeling induced by pressure overload. • ERα non-nuclear signaling was indispensable to the therapeutic efficacy of cGMP-PDE5 inhibition in heart failure but not to that of sGC stimulation. • sGC stimulation potently ameliorated cardiac remodeling, regardless of estrogen conditions, in sharp contrast to PDE5 inhibition. • The study provided the first in vivo evidence for the role of ERα non-nuclear signaling in heart failure, linking it to cGMP-PKG pathways. The data also supported the advantage of sGC stimulation over PDE5 inhibition as a potential therapeutic strategy in treating heart failure in post-menopausal women, highlighting the need for female-specific therapeutic strategies., Summary Using genetically engineered mice lacking estrogen receptor-α non-nuclear signaling, this study demonstrated that estrogen receptor−α non-nuclear signaling activated myocardial cyclic guanosine monophosphate-dependent protein kinase G and conferred protection against cardiac remodeling induced by pressure overload. This pathway was indispensable to the therapeutic efficacy of cyclic guanosine monophosphate−phosphodiesterase 5 inhibition but not to that of soluble guanylate cyclase stimulation. These results might partially explain the equivocal results of phosphodiesterase 5 inhibitor efficacy and also provide the molecular basis for the advantage of using a soluble guanylate cyclase simulator as a new therapeutic option in post-menopausal women. This study also highlighted the need for female-specific therapeutic strategies for heart failure.

JHEP reports 3(4), 100316 (2021). doi:10.1016/j.jhepr.2021.100316, Published by Elsevier, Amsterdam

Visual Abstract, Highlights • CMECs exert a direct positive effect on cardiomyocyte contraction and relaxation, which is mainly mediated by endothelial-derived NO. • Pro-inflammatory stimulation of CMECs by pre-incubation with TNF-α or interleukin-1β abrogates the positive regulatory function of these cells on cardiomyocyte contractile property. • Mechanistically, pro-inflammatory activation of CMECs leads to mitochondrial and cytoplasmic ROS accumulation that results in the scavenging of NO. • Empagliflozin directly restores the beneficial effect of CMECs on cardiomyocyte contraction and relaxation by reducing TNF-α-induced mitochondrial and cytoplasmic ROS accumulation, which leads to reinstatement of CMEC-derived NO delivery., Summary The positive findings of the EMPA-REG OUTCOME trial (Randomized, Placebo-Controlled Cardiovascular Outcome Trial of Empagliflozin) on heart failure (HF) outcome in patients with type 2 diabetes mellitus suggest a direct effect of empagliflozin on the heart. These patients frequently have HF with preserved ejection fraction (HFpEF), in which a metabolic risk-related pro-inflammatory state induces cardiac microvascular endothelial cell (CMEC) dysfunction with subsequent cardiomyocyte (CM) contractility impairment. This study showed that CMECs confer a direct positive effect on contraction and relaxation of CMs, an effect that requires nitric oxide, is diminished after CMEC stimulation with tumor necrosis factor-α, and is restored by empagliflozin. Our findings on the effect of empagliflozin on CMEC-mediated preservation of CM function suggests that empagliflozin can be used to treat the cardiac mechanical implications of microvascular dysfunction in HFpEF.

Hemodialysis vascular access dysfunction is a common and intractable problem in clinical practice with no definitive therapy yet available. As a key mediator of vascular and cardiac maladaptive remodeling, mineralocorticoid receptor (MR) plays a pivotal role in vascular fibrosis and intimal hyperplasia (IH) and is potentiated locally in hemodialysis vascular access following diverse injuries, like barotrauma, cannulation and shear stress. MR-related genomic and non-genomic pathways are responsible for triggering vascular smooth muscle cell activation, proliferation, migration and extracellular matrix overproduction. In endothelial cells, MR signaling diminishes nitric oxide production and its bioavailability, but amplifies reactive oxygen species, leading to an inflammatory state. Moreover, MR favors macrophage polarization towards a pro-inflammatory phenotype. In clinical settings like post-angioplasty or stenting restenosis, the beneficial effect of MR antagonists on vascular fibrosis and IH has been validated. In aggregate, therapeutic targeting of MR may provide a new avenue to prevent hemodialysis vascular access dysfunction., Highlights • MR signaling is instrumental in both insufficient outward remodeling and exuberant inward remodeling of AVF. • The effects of MR in VSMC, endothelial cell, and macrophage act synergistically to promote IH and vascular fibrosis in AVF. • Pharmacological targeting of MR represents a novel therapeutic strategy to prevent hemodialysis vascular access dysfunction.

Graphical abstract, Highlights • The coexistence of diabetes and hypertension represents a major risk factor for the onset of cardiovascular problems. • The purinergic signaling pathway constitutes a ubiquitous system of cell–cell communication. • Purinergic system plays a pivotal role in physiopathological conditions. • Several alterations in purine metabolism have been demonstrated in diabetes and hypertension. • Purinergic system can be an important target related to therapeutic approaches in diabetes and hypertension., Diabetes mellitus (DM) and hypertension are highly prevalent worldwide health problems and frequently associated with severe clinical complications, such as diabetic cardiomyopathy, nephropathy, retinopathy, neuropathy, stroke, and cardiac arrhythmia, among others. Despite all existing research results and reasonable speculations, knowledge about the role of purinergic system in individuals with DM and hypertension remains restricted. Purinergic signaling accounts for a complex network of receptors and extracellular enzymes responsible for the recognition and degradation of extracellular nucleotides and adenosine. The main components of this system that will be presented in this review are: P1 and P2 receptors and the enzymatic cascade composed by CD39 (NTPDase; with ATP and ADP as a substrate), CD73 (5′-nucleotidase; with AMP as a substrate), and adenosine deaminase (ADA; with adenosine as a substrate). The purinergic system has recently emerged as a central player in several physiopathological conditions, particularly those linked to inflammatory responses such as diabetes and hypertension. Therefore, the present review focuses on changes in both purinergic P1 and P2 receptor expression as well as the activities of CD39, CD73, and ADA in diabetes and hypertension conditions. It can be postulated that the manipulation of the purinergic axis at different levels can prevent or exacerbate the insurgency and evolution of diabetes and hypertension working as a compensatory mechanism.

In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 μM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 μM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that ∼78% of NO metabolism in SMCs is Cygb-dependent. Of this, ∼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis–Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.

Graphical abstract, COVID-19 is an ongoing viral pandemic disease that is caused by SARS-CoV2, inducing severe pneumonia in humans. However, several classes of repurposed drugs have been recommended, no specific vaccines or effective therapeutic interventions for COVID-19 are developed till now. Viral dependence on ACE-2, as entry receptors, drove the researchers into RAS impact on COVID-19 pathogenesis. Several evidences have pointed at Neprilysin (NEP) as one of pulmonary RAS components. Considering the protective effect of NEP against pulmonary inflammatory reactions and fibrosis, it is suggested to direct the future efforts towards its potential role in COVID-19 pathophysiology. Thus, the review aimed to shed light on the potential beneficial effects of NEP pathways as a novel target for COVID-19 therapy by summarizing its possible molecular mechanisms. Additional experimental and clinical studies explaining more the relationships between NEP and COVID-19 will greatly benefit in designing the future treatment approaches.

Background Dysfunction of liver sinusoidal endothelial cells (LSECs) is permissive for the progression of liver fibrosis and cirrhosis and responsible for its clinical complications. Here, we have mapped the spatial distribution of heterogeneous liver ECs in normal vs cirrhotic mouse livers and identified zone-specific transcriptomic changes of LSECs associated with liver cirrhosis using scRNA-seq technology. Approach & Results Cirrhosis was generated in endothelial specific green fluorescent protein (GFP) reporter mice through carbon tetrachloride inhalation for 12 weeks. GFP-positive liver EC populations were isolated from control and cirrhotic mice by FACS. We identified 6 clusters of liver EC populations including 3 clusters of LSECs, 2 clusters of vascular ECs and 1 cluster of lymphatic ECs. Based on previously reported LSEC-landmarks, we mapped the 3 clusters of LSECs in zones 1, 2, and 3, and determined phenotypic changes in each zone between control and cirrhotic mice. We found genes representing capillarization of LSECs (eg, CD34) as well as extracellular matrix genes were most upregulated in LSECs of zone 3 in cirrhotic mice, which may contribute to the development of basement membranes. LSECs in cirrhotic mice also demonstrated decreased expression of endocytic receptors, most remarkably in zone 3. Transcription factors (Klf2 [Kruppel-like factor-2], Klf4 [Kruppel-like factor-4], and AP-1) that induce nitric oxide production in response to shear stress were downregulated in LSECs of all zones in cirrhotic mice, implying increased intrahepatic vascular resistance. Conclusion This study deepens our knowledge of the pathogenesis of liver cirrhosis at a spatial, cell-specific level, which is indispensable for the development of novel therapeutic strategies to target the most dysfunctional liver ECs., Graphical abstract

International audience; Thermoregulation is a homeostatic mechanism that is disrupted in some neurological diseases. Patients with multiple sclerosis (MS) are susceptible to increases in body temperature, especially with more severe neurological signs. This condition can become intolerable when these patients suffer febrile infections such as coronavirus disease-2019 (COVID-19). We review the mechanisms of hyperthermia in patients with MS, and they may encounter when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Finally, the thermoregulatory role and relevant adaptation to regular physical exercise are summarized.

Rapid and efficient digestion and absorption of dietary triglycerides and other lipids by the intestine, the packaging of those lipids into lipoprotein chylomicron (CM) particles, and their secretion via the lymphatic duct into the blood circulation are essential in maintaining whole-body lipid and energy homeostasis. Biosynthesis and assembly of CMs in enterocytes is a complex multistep process that is subject to regulation by intracellular signaling pathways as well as by hormones, nutrients, and neural factors extrinsic to the enterocyte. Dysregulation of this process has implications for health and disease, contributing to dyslipidemia and a potentially increased risk of atherosclerotic cardiovascular disease. There is increasing recognition that, besides intracellular regulation of CM assembly and secretion, regulation of postassembly pathways also plays important roles in CM secretion. This review examines recent advances in our understanding of the regulation of CM secretion in relation to mobilization of intestinal lipid stores, drawing particular attention to post-assembly regulatory mechanisms, including intracellular trafficking of triglycerides in enterocytes, CM mobilization from the lamina propria, and regulated transport of CM by intestinal lymphatics. Keywords: Intestine, Chylomicron, Triglycerides, Lymphatics

Visual Abstract, Highlights • Autonomic imbalance has been documented in patients with PAH. • Electrical VNS is known to restore autonomic balance and improve heart failure. • This study aimed to elucidate the therapeutic effects of VNS on severe PAH in a rat model. • VNS significantly restored autonomic balance, decreased mean pulmonary arterial pressure, attenuated pulmonary vascular remodeling, and preserved right ventricular function. In addition, VNS markedly improved the survival of rats with PAH. • Our findings may contribute greatly to the development of device therapy for PAH and widen the clinical applicability of VNS., Summary This study aimed to elucidate the therapeutic effects of electrical vagal nerve stimulation (VNS) on severe pulmonary arterial hypertension in a rat model. In a pathophysiological study, VNS significantly restored autonomic balance, decreased mean pulmonary arterial pressure, attenuated pulmonary vascular remodeling, and preserved right ventricular function. In a survival study, VNS significantly improved the survival rate in both the prevention (VNS from 0 to 5 weeks after a SU5416 injection) and treatment (VNS from 5 to 10 weeks) protocols. Thus, VNS may serve as a novel therapeutic strategy for pulmonary arterial hypertension.

The role of NADPH oxidase (Nox)-derived reactive oxygen species in kidney vascular function has extensively been investigated in the harmful context of oxidative stress in diabetes and obesity-associated kidney disease. Since hydrogen peroxide (H2O2) has recently been involved in the non-nitric oxide (NO) non-prostanoid relaxations of intrarenal arteries, the present study was sought to investigate whether NADPH oxidases may be functional sources of vasodilator H2O2 in the kidney and to assess their role in the endothelium-dependent relaxations of human and rat intrarenal arteries. Renal interlobar arteries isolated from the kidney of renal tumor patients who underwent nephrectomy, and from the kidney of Wistar rats, were mounted in microvascular myographs to assess function. Superoxide (O2.-) and H2O2 production was measured by chemiluminescence and Amplex Red fluorescence, and Nox2 and Nox4 enzymes were detected by Western blotting and by double inmunolabeling along with eNOS. Nox2 and Nox4 proteins were expressed in the endothelium of renal arterioles and glomeruli co-localized with eNOS, levels of expression of both enzymes being higher in the cortex than in isolated arteries. Pharmacological inhibition of Nox with apocynin and of CYP 2C epoxygenases with sulfaphenazol, but not of the NO synthase (NOS), reduced renal NADPH-stimulated O2.- and H2O2 production. Under conditions of cyclooxygenase and NOS blockade, acetylcholine induced endothelium-dependent relaxations that were blunted by the non-selective Nox inhibitor apocynin and by the Nox2 or the Nox1/4 inhibitors gp91ds-tat and GKT136901, respectively. Acetylcholine stimulated H2O2 production that was reduced by gp91ds-tat and by GKT136901. These results suggest the specific involvement of Nox4 and Nox2 subunits as physiologically relevant endothelial sources of H2O2 generation that contribute to the endothelium-dependent vasodilatation of renal arteries and therefore have a protective role in kidney vasculature., Graphical abstract fx1

Cumulus cells play a crucial role as essential mediators in the maturation of ova. Ginger contains 10-gingerol, which induces apoptosis in colon cancer cells. Based on this hypothesis, this study aimed to determine whether 10-gingerol is able to induce apoptosis in normal cells, namely, cumulus cells.This study used anTheThese findings indicate that further examination is warranted for 10-gingerol as a contraception agent.تلعب الخلايا المحيطة بالبويضة دورا حاسما كوسيط أساسي في نضج البويضات. ويحتوي الزنجبيل على ١٠-جينجيرول، الذي يمكن أن يحفز موت الخلايا المبرمج في خلايا سرطان القولون. واستنادا إلى هذه الفرضية، هدفت هذه الدراسة إلى تحديد ما إذا كان ١٠ جينجيرول قادرا على تحريض موت الخلايا المبرمج في خلايا طبيعية، وتحديدا الخلايا المحيطة بالبويضة.استخدمت هذه الدراسة التحليل المختبري للخلايا المحيطة بالبويضة المستزرعة في محلول م ١٩٩ والمحتوي على١٠ جينجيرول في تركيزات مختلفة (١٢، ١٦، و٢٠ ميكرومول) ومن ثم الكشف عن نشاط قاتل للخلايا مبكر باستخدام عدة أنيكسين - فلورسين أيسو ثايو سايانيت للكشف.تُظهر البيانات في التجارب المختبرية أن عدد خلايا الموت المبرمج زادت مع زيادة فترة الحضانة: ١٢ ميكرومول (٦٣.٧١٪ ± ٢.١٩٢٪)، ١٦ ميكرومول (٧٤.٥١٪ ± ٤.٥٩٦٪)، و ٢٠ ميكرومول (٧٨.٧٩٥٪ ± ١.٤٣٥٪). تستطيع المادة ١٠ جينجيرول أن تحفز الموت المبرمج للخلايا في الخلايا المحيطة بالبويضة، إلا أنها تثبط وظائف الجين المسؤول عن برمجة مستَقبِل 5- هيدروكسي تريبتامين وتبطل عمل كلا من جلايكوجين سنثايز كاينايز ٣ بيتا والجين المبرمج لـ ” أي كي تي - ١ “.تشير نتائج هذه الدراسة إلى أن هناك حاجة لمزيد من الفحص لـ ١٠-جينجيرول كعامل منع للحمل.

Vascular peroxidase 1 (VPO1) is a member of the peroxidase family which aggravates oxidative stress by producing hypochlorous acid (HOCl). Our previous study demonstrated that VPO1 plays a critical role in endothelial dysfunction through dimethylarginine dimethylaminohydrolase2 (DDAH2)/asymmetric Dimethylarginine (ADMA) pathway. Hereby we describe the regulatory role of VPO1 on endothelial nitric oxide synthase (eNOS) expression and activity in human umbilical vein endothelial cells (HUVECs). In HUVECs AngiotensinII (100 nM) treatment reduced Nitric Oxide (NO) production, decreased eNOS expression and activity, which were reversed by VPO1 siRNA. Knockdown of VPO1 also attenuated ADMA production and eNOS uncoupling while enhancing phosphorylated ser1177 eNOS expression level. Furthermore, HOCl stimulation was shown to directly induce ADMA production and eNOS uncoupling, decrease phosphorylated ser1177 eNOS expression. It also significantly suppressed eNOS expression and activity together with NO production. Therefore, VPO1 plays a vital role in regulating eNOS expression and activity via hydrogen peroxide (H2O2)-VPO1-HOCl pathway., Highlights • Angiotensin II decreased eNOS expression and activity in HUVECs. • VPO1 plays an important role in regulating eNOS expression and activity in HUVECs. • VPO1 regulates eNOS expression and activity through VPO1/H2O2/HOCl pathway.

Aldosterone, with pro-oxidation and pro-autophagy capabilities, plays a key role in liver fibrosis. However, the mechanisms underlying aldosterone-promoted liver sinusoidal endothelial cells (LSECs) defenestration remain unknown. Caveolin 1 (Cav1) displays close links with autophagy and fenestration. Hence, we aim to investigate the role of Cav1-related autophagy in LSECs defenestration. We found the increase of aldosterone/MR (mineralocorticoid receptor) level, oxidation, autophagy, and defenestration in LSECs in the human fibrotic liver, BDL or hyperaldosteronism models; while antagonizing aldosterone or inhibiting autophagy relieved LSECs defenestration in BDL-induced fibrosis or hyperaldosteronism models. In vitro, fenestrae of primary LSECs gradually shrank, along with the down-regulation of the NO-dependent pathway and the augment of the AMPK-dependent autophagy; these effects were aggravated by rapamycin (an autophagy activator) or aldosterone treatment. Additionally, aldosterone increased oxidation mediated by Cav1, reduced ATP generation, and subsequently induced the AMPK-dependent autophagy, leading to the down-regulation of the NO-dependent pathway and LSECs defenestration. These effects were reversed by MR antagonist spironolactone, antioxidants or autophagy inhibitors. Besides, aldosterone enhanced the co-immunoprecipitation of Cav1 with p62 and ubiquitin, and induced Cav1 co-immunofluorescence staining with LC3, ubiquitin, and F-actin in the perinuclear area of LSECs. Furthermore, aldosterone treatment increased the membrane protein level of Cav1, whereas decrease the cytoplasmic protein level of Cav1, indicating that aldosterone induced Cav1-related selective autophagy and F-actin remodeling to promote defenestration. Consequently, Cav1-related selective autophagy initiated by aldosterone-induced oxidation promotes LSECs defenestration via activating the AMPK-ULK1 pathway and inhibiting the NO-dependent pathway.

Decreased nitric oxide (NO) bioavailability plays a critical role in the pathophysiology of preeclampsia (PE). Recent evidence indicates that S-glutathionylation may occur on the endothelial nitric oxide synthase (eNOS), leading to eNOS uncoupling, characterized by a decreased NO production and an increased generation of superoxide anion (O2•–). We hypothesized that eNOS glutathionylation may occur in PE placentas and participate in eNOS dysfunction. The glutathionylation of eNOS was investigated in thirteen PE-affected patients and in nine normal pregnancies. Immunofluorescence, confocal microscopy and western-blot experiments carried out on eNOS immunoprecipitates, revealed a high level of eNOS glutathionylation in PE placentas, mostly reversed by dithiotreitol (DTT), thus indicative of S-glutathionylation. In order to investigate whether eNOS glutathionylation may alter trophoblast migration, an important event occurring during early placentation, cultured HTR-8/SVneo human trophoblasts (HTR8) were exposed either to low pO2 (O2 1%) or to pO2 changes (O2 1–20%), in order to generate oxidative stress. Trophoblasts exposed to low pO2, did not undergo oxidative stress nor eNOS S-glutathionylation, and were able to generate NO and migrate in a wound closure model. In contrast, trophoblasts submitted to low/high pO2 changes, exhibited oxidative stress and a (DTT reversible) S-glutathionylation of eNOS, associated with reduced NO production and migration. The autonomous production of NO seemed necessary for the migratory potential of HTR8, as suggested by the inhibitory effect of eNOS silencing by small interfering RNAs, and the eNOS inhibitor L-NAME, in low pO2 conditions. Finally, the addition of the NO donor, NOC-18 (5 µM), restored in part the migration of HTR8, thereby emphasizing the role of NO in trophoblast homeostasis. In conclusion, the high level of eNOS S-glutathionylation in PE placentas provides new insights in the mechanism of eNOS dysfunction in this disease., Graphical abstract fx1, Highlights • eNOS is S-glutathionylated in preeclamptic placentas. • Oxidative stress evoked by pO2 changes, triggers eNOS S-glutathionylation in HTR8. • pO2 changes alter the invasive potential of HTR8 in the wound closure model. • The NO donor NOC-18 restores the invasive potential of HTR8.

Endothelial dysfunction is characterized by nitric oxide dysregulation and an altered redox state. Oxidative stress and inflammatory markers prevail, thus promoting atherogenesis and hypertension, important risk factors for the development and progression of heart failure. There has been a reemerging interest in the role that endothelial dysfunction plays in the failing circulation. Accordingly, patients with heart failure are being clinically assessed for endothelial dysfunction via various methods, including flow-mediated vasodilation, peripheral arterial tonometry, quantification of circulating endothelial progenitor cells, and early and late endothelial progenitor cell outgrowth measurements. Although the mechanisms underlying endothelial dysfunction are intimately related to cardiovascular disease and heart failure, it remains unclear whether targeting endothelial dysfunction is a feasible strategy for ameliorating heart failure progression. This review focuses on the pathophysiology of endothelial dysfunction, the mechanisms linking endothelial dysfunction and heart failure, and the various diagnostic methods currently used to measure endothelial function, ultimately highlighting the therapeutic implications of targeting endothelial dysfunction for the treatment of heart failure.

Highlights • Patchouli alcohol (PA) is a bioactive component in essential oil extracted from Pogostemon cablin. • The present review provides the scientific mechanisms for health beneficial activities of PA in diverse disease models. • PA possesses diverse health beneficial activities., Patchouli alcohol (PA), a tricyclic sesquiterpene, is a dominant bioactive component in oil extracted from the aerial parts of Pogostemon cablin (patchouli). Diverse beneficial activities have been reported, including anti-influenza virus, anti-depressant, anti-nociceptive, vasorelaxation, lung protection, brain protection, anti-ulcerogenic, anti-colitis, pre-biotic-like, anti-inflammatory, anti-cancer and protective activities against metabolic diseases. However, detailed mechanistic studies are required to explore the possibility of developing PA as a functional food material or promising drug for the prevention and treatment of human diseases. This review highlights multiple molecular targets and working mechanisms by which PA mediates health benefits.

Cells that are not irradiated but are affected by “stress signal factors” released from irradiated cells are called bystander cells. These cells, as well as directly irradiated ones, express DNA damage-related proteins and display excess DNA damage, chromosome aberrations, mutations, and malignant transformation. This phenomenon has been studied widely in the past 20 years, since its first description by Nagasawa and Little in 1992, and is known as the radiation-induced bystander effect (RIBE). Several factors have been identified as playing a role in the bystander response. This review will focus on one of them, nitric oxide (NO), and its role in the stimulation and propagation of RIBE. The hydrophobic properties of NO, which permit its diffusion through the cytoplasm and plasma membranes, allow this signaling molecule to easily spread from irradiated cells to bystander cells without the involvement of gap junction intercellular communication. NO produced in irradiated tissues mediates cellular regulation through posttranslational modification of a number of regulatory proteins. The best studied of these modifications are S-nitrosylation (reversible oxidation of cysteine) and tyrosine nitration. These modifications can up- or down-regulate the functions of many proteins modulating different NO-dependent effects. These NO-dependent effects include the stimulation of genomic instability (GI) and the accumulation of DNA errors in bystander cells without direct DNA damage., Graphical abstract, Highlights • Ionizing radiation stimulates generation of nitric oxide (NO). • NO stimulates genomic instability by inhibiting BRCA1 protein expression. • NO can diffuse and stimulate genomic instability in the bystander cells. • Propagation of NO from cell-to-cell creates a “mutator fields”. • Definition of the “mutator filed” is proposed.

Critical limb ischemia (CLI) is a potentially life-threatening condition that involves severely reduced blood flow to the peripheral arteries due to arteriosclerosis obliterans (ASO) of the limbs or a similar condition. CLI patients must undergo revascularization to avoid amputation of the lower limbs and improve their survival prognosis. However, the outcomes of conventional surgical revascularization or endovascular therapy are inadequate; therefore, establishing further effective treatment methods is an urgent task. We perform therapeutic angiogenesis using autologous bone marrow-derived mononuclear cells in clinical practice and demonstrated its safety and efficacy for CLI patients for whom conventional treatments failed or are not indicated. Exercise therapies must be devised for CLI patients who have undergone therapeutic angiogenesis to save their limbs and improve survival. Because evidence regarding the efficacy and safety of exercise therapy for CLI patients is lacking, we plan to perform a prospective trial of the efficacy and safety of optimal exercise therapy following therapeutic angiogenesis for CLI patients.The trial will enroll 30 patients between 20 and 79 years with Rutherford category 4 or 5 CLI caused by ASO who will undergo therapeutic angiogenesis. Participants will be randomly allocated to receive either optimal exercise therapy or fixed exercise therapy. Those receiving optimal exercise therapy will undergo tissue muscle oxygen saturation monitoring using near-infrared spectroscopy while performing exercises and will be prescribed optimal exercise therapy. The optimal amount of exercise will be determined on day 8, 31, 61, 91 and 181 after therapeutic angiogenesis. Ethics and dissemination: This protocol was approved by the Institutional Review Boards of Kyoto Prefectural University of Medicine. In accordance with the Helsinki Declaration, written informed consent has been obtained from all participants prior to enrollment. The results of this trial will be disseminated by publication in a peer-reviewed journal. Trial registration: This trial is registered at http://www.umin.ac.jp/ctr/index.htm (identifier: UMIN000035288). Keywords: Critical limb ischemia, Arteriosclerosis obliterans, Therapeutic angiogenesis, Optimal exercise therapy, Tissue muscle oxygen saturation, Near-infrared spectroscopy

Studies revealed that the use of renin-angiotensin-aldosterone system antagonism is not associated with a statistically significant reduction in the risk of cardiovascular events in patients with chronic kidney disease (CKD) compared with that in the general population. We tested the hypothesis that indoxyl sulfate (IS) can interfere with the protective effect of valsartan-mediated on endothelial function in vitro and neovascularization in mice underwent subtotal nephrectomy. In human aortic endothelial cells, we first demonstrated that IS impaired the valsartan-mediated phosphorylation of eNOSThr495, nitric oxide production and tube formation via NADPH oxidase (NOX) and protein kinase C (PKC) phosphorylation, but this effect was suppressed by cotreatment with apocynin and calphostin C. In vivo, IS attenuated valsartan-induced angiogenesis in Matrigel plugs in mice. Moreover, in subtotal nephrectomy mice who underwent hindlimb ischemic surgery, valsartan significantly increased the mobilization of endothelial progenitor cells in circulation as well as the reperfusion of blood flow and density of CD31+ capillaries in ischemic limbs. However, IS attenuated the protective effect of valsartan-induced neovascularization and increased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Cotreatment of apocynin and calphostin C reversed the IS impaired-neovascularization and decreased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Our study suggests that the NOX/PKC/eNOS signaling pathway plays a pivotal role in the IS-mediated inhibition of valsartan-conferred beneficial effects on endothelial function in vitro and neovascularization in subtotal nephrectomy mice. We proposed a novel causative role for IS in cardiovascular complications in CKD patients., Highlights • The use of renin-angiotensin-aldosterone system antagonism fails to reduce in the risk cardiovascular events in patients with CKD. • Indoxyl sulfate interferes with the protective effect of angiotensin II receptor blocker-mediated neovascularization in CKD mice. • Indoxyl sulfate interferes with the beneficial effect of of valsartan on endothelial function by activating the NOX/PKC signaling pathway. • This article proposed a novel causative role for indoxyl sulfate in cardiovascular complications in CKD patients.

Visual Abstract, Highlights • RBCs from mice and patients with type 2 diabetes have increased arginase activity and production of reactive oxygen species. • RBCs from mice and patients with type 2 diabetes aggravate myocardial ischemia-reperfusion injury. • Inhibition of arginase in RBCs from mice and patients with type 2 diabetes improves post-ischemic myocardial recovery via reduced oxidative stress. • Inhibition of nitric oxide synthase in RBC reduces oxidative stress and restores post-ischemic myocardial functional recovery. • These data demonstrate a novel disease mechanism by which RBC drive post-ischemic cardiac dysfunction in type 2 diabetes., Summary This study tested the hypothesis that red blood cell (RBC) arginase represents a potential therapeutic target in ischemia-reperfusion in type 2 diabetes. Post-ischemic cardiac recovery was impaired in hearts from db/db mice compared with wild-type hearts. RBCs from mice and patients with type 2 diabetes attenuated post-ischemic cardiac recovery of nondiabetic hearts. This impaired cardiac recovery was reversed by inhibition of RBCs arginase or nitric oxide synthase. The results suggest that RBCs from type 2 diabetics impair cardiac tolerance to ischemia-reperfusion via a pathway involving arginase activity and nitric oxide synthase-dependent oxidative stress.

Reperfusion injury, the paradoxical tissue response that is manifested by blood flow-deprived and oxygen-starved organs following the restoration of blood flow and tissue oxygenation, has been a focus of basic and clinical research for over 4-decades. While a variety of molecular mechanisms have been proposed to explain this phenomenon, excess production of reactive oxygen species (ROS) continues to receive much attention as a critical factor in the genesis of reperfusion injury. As a consequence, considerable effort has been devoted to identifying the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Of the potential ROS sources described to date, xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase have gained a status as the most likely contributors to reperfusion-induced oxidative stress and represent priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. Although all four enzymatic sources are present in most tissues and are likely to play some role in reperfusion injury, priority and emphasis has been given to specific ROS sources that are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The possibility that multiple ROS sources contribute to reperfusion injury in most tissues is supported by evidence demonstrating that redox-signaling enables ROS produced by one enzymatic source (e.g., Nox) to activate and enhance ROS production by a second source (e.g., mitochondria). This review provides a synopsis of the evidence implicating ROS in reperfusion injury, the clinical implications of this phenomenon, and summarizes current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue., Graphical abstract fx1, Highlights • Reperfusion injury is implicated in a variety of human diseases and disorders. • Evidence implicating ROS in reperfusion injury continues to grow. • Several enzymes are candidate sources of ROS in post-ischemic tissue. • Inter-enzymatic ROS-dependent signaling enhances the oxidative stress caused by I/R. .

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer deaths throughout the world. This study was aimed to analyze oxidative stress and cell damage in a multistage model of liver carcinogenesis induced by diethylnitrosamine (DEN) in rats. Male Wistar rats weighing 145–150g were divided into three groups: control, precancerous lesions (PL) (which received 100mg DEN once a week every 6 weeks up to 28 weeks), and advanced HCC (50mg DEN once/twice per week up to 19 weeks). Lipid peroxidation (TBARS), superoxide dismutase (SOD) activity, and expression of transforming growth factor-1 beta (TGF)-1β, endothelial and inducible nitric oxide syntahese (eNOS, iNOS), NADPH quinone oxireductase (NQO)-1, nuclear factor erythroid 2-related factor (NrF)2, kelch-like ECH-associated protein (Keap)1 and heat shock protein (HSP)70 were measured. TBARS concentration was augmented in the PL and advanced HCC groups. SOD activity, TGF-1β and Nrf2 expression were higher in animals with precancerous lesions. In advanced HCC, expression of NQO1 and iNOS increased while there was a decrease in HPS70 expression. Data obtained provide evidence for the differential activation of proteins involved in oxidative stress and cell damage during progression of carcinogenesis in an animal model of HCC.

Visual Abstract, Highlights • Tetrahydrobiopterin is an essential cofactor for NO production. • Limitation of endogenous tetrahydrobiopterin reduces NO bioavailability, enhances oxidative stress, and impairs vascular function. • Orally supplemented tetrahydrobiopterin has therapeutic challenges because it is rapidly oxidized in vivo. • Here, the authors demonstrate that l-phenylalanine, when administered orally, raises vascular tetrahydrobiopterin, restores NO, reduces superoxide, and enhances vascular function in spontaneously hypertensive rats. • This effect is achieved by activation of a protein complex (GCH1-GFRP) involved in the biosynthesis of tetrahydrobiopterin. • Activation of this protein complex by l-phenylalanine or its analogues represents a novel therapeutic target for vascular disorders underpinned by reduced NO bioavailability., Summary Reduced nitric oxide (NO) bioavailability correlates with impaired cardiovascular function. NO is extremely labile and has been challenging to develop as a therapeutic agent. However, NO bioavailability could be enhanced by pharmacologically targeting endogenous NO regulatory pathways. Tetrahydrobiopterin, an essential cofactor for NO production, is synthesized by GTP cyclohydrolase-1 (GCH1), which complexes with GCH1 feedback regulatory protein (GFRP). The dietary amino acid l-phenylalanine activates this complex, elevating vascular BH4. Here, the authors demonstrate that l-phenylalanine administration restores vascular function in a rodent model of hypertension, suggesting the GCH1-GFRP complex represents a rational therapeutic target for diseases underpinned by endothelial dysfunction.

Atherosclerosis is a multifocal alteration of the vascular wall of medium and large arteries characterized by a local accumulation of cholesterol and non-resolving inflammation. Atherothrombotic complications are the leading cause of disability and mortality in western countries. Neovascularization in atherosclerotic lesions plays a major role in plaque growth and instability. The angiogenic process is mediated by classical angiogenic factors and by additional factors specific to atherosclerotic angiogenesis. In addition to its role in plaque progression, neovascularization may take part in plaque destabilization and thromboembolic events. Anti-angiogenic agents are effective to reduce atherosclerosis progression in various animal models. However, clinical trials with anti-angiogenic drugs, mainly anti-VEGF/VEGFR, used in anti-cancer therapy show cardiovascular adverse effects, and require additional investigations., Graphical abstract fx1, Highlights • Intimal neovascularization in plaque growth and destabilization. • Role of classical and specific angiogenic factors in atherosclerotic angiogenesis. • Inflammation and oxidative stress promote angiogenesis and plaque destabilization. • Angiogenesis promote intraplaque hemorrhages, afflux of blood cells and lipids. • Anti-angiogenic therapy for atherosclerotic angiogenesis.

Insulin resistance promotes vascular endothelial dysfunction and subsequent development of cardiovascular disease. Previously we found that skeletal muscle arteriolar flow-induced dilation (FID) was reduced following a hyperinsulinemic clamp in healthy adults. Therefore, we hypothesized that hyperinsulinemia, a hallmark of insulin resistance, contributes to microvascular endothelial cell dysfunction via inducing oxidative stress that is mediated by NADPH oxidase (Nox) system. We examined the effect of insulin, at levels that are comparable with human hyperinsulinemia on 1) FID of isolated arterioles from human skeletal muscle tissue in the presence and absence of Nox inhibitors and 2) human adipose microvascular endothelial cell (HAMECs) expression of nitric oxide (NO), endothelial NO synthase (eNOS), and Nox-mediated oxidative stress. In six lean healthy participants (mean age 25.5±1.6 y, BMI 21.8±0.9), reactive oxygen species (ROS) were increased while NO and arteriolar FID were reduced following 60 min of ex vivo insulin incubation. These changes were reversed after co-incubation with the Nox isoform 2 (Nox2) inhibitor, VAS2870. In HAMECs, insulin-induced time-dependent increases in Nox2 expression and P47phox phosphorylation were echoed by elevations of superoxide production. In contrast, phosphorylation of eNOS and expression of superoxide dismutase (SOD2 and SOD3) isoforms showed a biphasic response with an increased expression at earlier time points followed by a steep reduction phase. Insulin induced eNOS uncoupling that was synchronized with a drop of NO and a surge of ROS production. These effects were reversed by Tempol (SOD mimetic), Tetrahydrobiopterin (BH4; eNOS cofactor), and VAS2870. Finally, insulin induced nitrotyrosine formation which was reversed by inhibiting NO or superoxide generation. In conclusions, hyperinsulinemia may reduce FID via inducing Nox2-mediated superoxide production in microvascular endothelial cells which reduce the availability of NO and enhances peroxynitrite formation. Therefore, the Nox2 pathway should be considered as a target for the prevention of oxidative stress-associated endothelial dysfunction during hyperinsulinemia., Graphical abstract fx1, Highlights • Hyperinsulinemia impairs FID and induces ROS production in human muscle arterioles. • Insulin-induced ROS production in endotelial cells is mediated by NADPH oxidase. • Long exposure to high insulin levels reduces eNOS phosphorylation and NO production.