Preserved ß-adrenergic-mediated vasodilation in skeletal muscle of young adults with obesity despite shifts in cyclooxygenase and nitric oxide synthase.

Central adiposity is associated with greater sympathetic support of blood pressure. ß-adrenergic receptors (ß-AR) buffer sympathetically mediated vasoconstriction and ß-AR-mediated vasodilation is attenuated in preclinical models of obesity. With this information, we hypothesized ß-AR vasodilation would be lower in obese compared with normal weight adults. Because ß-AR vasodilation in normal weight adults is limited by cyclooxygenase (COX) restraint of nitric oxide synthase (NOS), we further explored the contributions of COX and NOS to ß-AR vasodilation in this cohort. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured and forearm vascular conductance (FVC) was calculated (FVC = FBF/MAP). The rise in FVC from baseline (ΔFVC) was quantified during graded brachial artery infusion of isoproterenol (Iso, 1-12 ng/100 g/min) in normal weight (n = 36) and adults with obesity (n = 22) (18-40 yr old). In a subset of participants, Iso-mediated vasodilation was examined before and during inhibition of NOS [NG-monomethyl-l-arginine (l-NMMA)], COX (ketorolac), and NOS + COX (l-NMMA + ketorolac). Iso-mediated increases in FVC did not differ between groups (P = 0.57). l-NMMA attenuated Iso-mediated ΔFVC in normal weight (P = 0.03) but not adults with obesity (P = 0.27). In normal weight adults, ketorolac increased Iso-mediated ΔFVC (P < 0.01) and this response was lost with concurrent l-NMMA (P = 0.67). In contrast, neither ketorolac (P = 0.81) nor ketorolac + l-NMMA (P = 0.40) altered Iso-mediated ΔFVC in adults with obesity. Despite shifts in COX and NOS, ß-AR vasodilation is preserved in young adults with obesity. These data highlight the presence of a compensatory shift in microvascular control mechanisms in younger humans with obesity.NEW & NOTEWORTHY We examined ß-adrenergic receptor-mediated vasodilation in skeletal muscle of humans with obesity and normal weight. Results show that despite shifts in the contribution of cyclooxygenase and nitric oxide synthase, ß-adrenergic-mediated vasodilation is relatively preserved in young, otherwise healthy adults with obesity. These data highlight the presence of subclinical changes in microvascular control mechanisms early in the obesity process and suggest duration of obesity and/or the addition of primary aging may be necessary for overt dysfunction.

Role of serotonin in regulation of pancreatic and mesenteric arterial function in diabetic mice.

The aim of this study was to investigate the reaction of pancreatic and mesenteric artery to 5-hydroxytryptamine (5-HT, serotonin) and the mechanism of nitric oxide in diabetes. Diabetic mice were induced by streptozotocin through intraperitoneal injection. The vascular tension of the pancreatic, mesenteric and brain basilar arteries in diabetic and control mice were measured by myograph in the applications of angiotensin II, 5-HT, 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI), 5-HT1B/1D receptor agonist sumatriptan, 5-HT2B receptor agonist BW723C86, 5-HT1D receptor antagonist Palonosetron and 5-HT2 receptor antagonist Sarpogrelate. The effect of 5-HT on arteries pretreated with L-NAME and sodium nitroprusside (SNP) on arteries pretreated with norepinephrine were measured. The mRNA expressions of eNOS, 5-HT1B, 5-HT1D, 5-HT2A and 5-HT2B in pancreatic and mesenteric arteries were measured by Real-time PCR. The concentration of 5-HT in plasma and eNOS in pancreatic and mesenteric arteries were tested. Our results showed that the tension of pancreatic and mesenteric arteries in diabetic mice impaired to 5-HT, but not Ang II, and to DOI and sumatriptan, but normalized by incubation with L-NAME. Pancreatic and mesenteric arteries showed no differences to SNP after pretreated with NE between diabetic and control mice. The mRNA of eNOS and 5-HT receptors in pancreatic and mesenteric artery showed no difference between control and diabetic mice. We conclude that the effect of 5-HT on the tension of pancreatic and mesenteric arteries decrease in diabetic mice. It may due to the decreased activity of 5-HT receptors and the activation of eNOS, which causes nitric oxide to release more and makes the tension of vessels decreased.

miR-744/eNOS/NO axis: A novel target to halt triple negative breast cancer progression.

BACKGROUND: Nitric oxide (NO) may have a dual role in cancer. At low concentrations, endogenous NO promotes tumor growth and proliferation. However, at very high concentrations, it mediates cancer cell apoptosis and inhibits cancer growth. High levels of NO have been observed in blood of breast cancer (BC) patients, which increases tumor blood flow and promotes angiogenesis. To date, the regulation of NO-synthesizing enzyme, eNOS, by miRNAs has not been adequately investigated in BC. Therefore, the main aim of this study is to unravel the possible regulation of eNOS by miRNAs in BC and to examine their influence on NO production and BC progression. METHODS: Expression profile of eNOS in Egyptian BC patients and MDA-MB-231 cell lines was investigated using qRT-PCR. In-silico analysis was performed to predict a putative upstream regulator of eNOS. miR-744-5p was selected and its expression was quantified in BC tissues using qRT-PCR. MDA-MB-231 cells were cultured and transfected with miR-744-5p using lipofection method. NO levels were determined using Griess Reagent. Cellular viability and colony-forming ability were assessed using MTT and colony-forming assays; respectively. RESULTS: eNOS and miR-744-5p were significantly up-regulated in BC tissues compared to paired normal tissues. In-silico analysis revealed that miR-744-5p putatively binds to eNOS transcript with high binding scores. Transfection of MDA-MB-231 cells with miR-744-5p mimics resulted in a significant up-regulation of eNOS and consequently NO levels. In addition, miR-744-5p transfection led to an increase in cellular viability and colony-forming ability of the MDA-MB-231. CONCLUSION: miR-744-5p acts as an upstream positive regulator of the NO synthesizing enzyme, eNOS which in turn elevates NO levels. Furthermore, miR-744-5p is a novel oncogenic miRNA in BC. Thus, targeting miR-744/eNOS/NO axis may act as a therapeutic tool in TNBC.

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway.

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O2 ) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.

Molecular dynamics study of in silico mutations in the auto-inhibitory loop of human endothelial nitric oxide synthase FMN sub-domain.

Structural flexibility of the peptide linker connecting two domains is essential for the functioning of multi-domain complex. Nitric oxide synthase (NOS) isoforms contain the oxygenase and the reductase domains connected by calmodulin binding linker (CBL) region. Additionally, the endothelial NOS (eNOS) isoform contain an auto-inhibitory loop (AI) in the FMN reductase sub-domain which represses the inter-domain electron transfer process. Binding of Ca2+-Calmodulin complex on the CBL region relieves the AI loop repression and facilitates electron transfer from FMN in the reductase domain to the heme in the oxygenase domain. Few experimental studies have reported that in vitro mutation of Serine-615 (S615D) and Serine-633 (S633D) in the FMN reductase sub-domain to aspartic acid increased NO production and increased Ca2+ sensitivity. To understand the role of AI loop in eNOS repression and activation in serine mutants (S615D and S633D), we modelled the FMN reductase sub-domain of human eNOS protein with and without the CBL region. Molecular dynamics simulations performed indicated that the mutant protein AI loop structure was stabilized by salt bridge formed between D615 and R602. It was also found that mutation increased the flexibility of C-terminal residues of eNOS CBL region. The hinge-like movement of the AI loop allowed rotation of the FMN sub-domain clockwise which may favour electron-transfer in the mutant protein. This study provides insight on mutation (S615D and S633D) induced changes in AI loop and increased flexibility of CBL region which may lead to the protein activation and may also facilitate Calmodulin binding at physiological Ca2+ concentration. Graphical Abstract Mutation of amino acid residues contribute to structural changes at molecular level leading to alteration in protein dynamics and its function. Serine-615 and Serine-633 in the auto-inhibitory loop of human eNOS reductase model was mutated to aspartic acid in silico and molecular dynamics simulations of the protein showed that steric hindrance due to mutation altered the auto-inhibitory loop rearrangement and the FMN sub-domain movement favouring electron transfer.

Participation of NO-Dependent Mechanisms in the Effects of Increased Systemic Level of Interleukin-1ß on Pial Microvessels under Conditions of Acute Hypoxia.

We studied the effect of increased systemic levels of the proinflammatory cytokine IL-1ß on the vasomotor reactions of pial microvessels in anesthetized rats under conditions of experimentally simulated progressively increasing acute normobaric hypoxia. Vital microscopy showed that more pronounced dilatation of pial vessels in response to IL-1ß under hypoxic conditions was almost completely prevented by pretreatment with non-specific NO synthase blocker L-NAME. These findings indicate the involvement of NO-dependent mechanisms in the vasodilator effect of proinflammatory cytokines under conditions of acute hypoxic exposure.

Cardiomyocyte-derived small extracellular vesicles can signal eNOS activation in cardiac microvascular endothelial cells to protect against Ischemia/Reperfusion injury.

Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods:Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.

Inhibition of eNOS by L-NAME resulting in rat hind limb developmental defects through PFKFB3 mediated angiogenetic pathway.

L-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether L-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that L-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.

Protective Effect of Astragaloside IV on High Glucose-Induced Endothelial Dysfunction via Inhibition of P2X7R Dependent P38 MAPK Signaling Pathway.

Vascular endothelial dysfunction is associated with increased mortality in patients with diabetes. Astragaloside IV (As-IV) is a bioactive saponin with therapeutic potential as an anti-inflammatory and antiendothelial dysfunction. However, the underlying mechanism for how As-IV ameliorated endothelial dysfunction is still unclear. Therefore, in this study, we examined the protective effect of As-IV against endothelial dysfunction and explored potential molecular biology mechanism. In vivo, rats were intraperitoneally injected with streptozotocin (STZ) at a dose of 65 mg/kg body weight to establish a diabetic model. In vitro studies, rat aortic endothelial cells (RAOEC) were pretreated with As-IV, SB203580 (p38 MAPK inhibitor) for 2 h prior to the addition of high glucose (33 mM glucose). Our findings indicated that As-IV improved impaired endothelium-dependent relaxation and increased the levels of endothelial NO synthase (eNOS) and nitric oxide (NO) both in vivo and in vitro. Besides, As-IV treatment inhibited the elevated inflammation and oxidative stress in diabetic model both in vivo and in vitro. Moreover, As-IV administration reversed the upregulated expression of P2X7R and p-p38 MAPK in vivo and in vitro. Additionally, the effects of both P2X7R siRNA and SB203580 on endothelial cells were similar to As-IV. Collectively, our study demonstrated that As-IV rescued endothelial dysfunction induced by high glucose via inhibition of P2X7R dependent p38 MAPK signaling pathway. This provides a theoretical basis for the further study of the vascular endothelial protective effects of As-IV.

Propofol Protects Against TNF-α-induced Blood-brain Barrier Disruption via the PIM-1/eNOS/NO Pathway.

BACKGROUND: The Inflammatory cytokine, tumor necrosis factor-α (TNF-α), disrupts blood-brain barrier (BBB). Propofol reportedly exerts an anti-inflammatory effect in the central nervous system. OBJECTIVE: We hypothesized that propofol could provide a protective effect against TNF-α-induced disruption in human cerebral microvascular endothelial cells (hCMEC/D3 cells) and explored the underlying mechanisms. METHODS: The hCMEC/D3 cell monolayers were pretreated with propofol, followed by TNF-α treatment. The integrity of BBB was reflected by assessing the trans-endothelial electrical resistance (TEER) and determining the expression of proteins within tight junctions (TJs). The effect of propofol on TNF-α-modulated nitric oxide production was measured by a nitrate reductase assay kit. The expression of ZO-1, claudin-5, occludin, TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), proviral-integration site for Moloney murine leukaemia virus (PIM)-1kinase, the phosphorylation of endothelial nitric oxide synthase at ser633 (peNOS-ser633) were detected by western blot. RESULTS: In hCMEC/D3 cells, TNF-α treatment markedly disrupted the integrity of BBB. Further, we found TNF-α treatment could increase the expression of PIM-1, then activate the phosphorylation of eNOS and induce the release of nitric oxide (NO). More importantly, we found that TNF- α-impaired BBB integrity could be reversed by propofol. CONCLUSION: These results suggest that the PIM-1/eNOS/NO pathway plays a vital role, in which Propofol protects against TNF-α-induced blood-brain barrier disruption.

Blood pressure-lowering effects of a Bowman-Birk inhibitor and its derived peptides in normotensive and hypertensive rats.

Bioactive plant peptides have received considerable interest as potential antihypertensive agents with potentially fewer side effects than antihypertensive drugs. Here, the blood pressure-lowering effects of the Bowman-Birk protease inhibitor, BTCI, and its derived peptides, PepChy and PepTry, were investigated using normotensive (Wistar-WR) and spontaneously hypertensive rats (SHR). BTCI inhibited the proteases trypsin and chymotrypsin, respectively, at 6 µM and 40 µM, a 10-fold greater inhibition than observed with PepTry (60 µM) and PepChy (400 µM). These molecules also inhibited angiotensin converting enzyme (ACE) with IC50 values of 54.6 ± 2.9; 24.7 ± 1.1; and 24.4 ± 1.1 µM, respectively, occluding its catalytic site, as indicated by molecular docking simulation, mainly for PepChy and PepTry. Gavage administration of BTCI and the peptides promoted a decrease of systolic and diastolic blood pressure and an increase of renal and aortic vascular conductance. These effects were more expressive in SHR than in WR. Additionally, BTCI, PepChy and PepTry promoted coronary vasodilation and negative inotropic effects in isolated perfused hearts. The nitric oxide synthase inhibitor blunted the BTCI and PepChy, with no cardiac effects on PepTry. The findings of this study indicate a therapeutic potential of BTCI and its related peptides in the treatment of hypertension.

Endothelial ATP-Sensitive Potassium Channel Protects Against the Development of Hypertension and Atherosclerosis.

In the endothelium, ATP-sensitive potassium (KATP) channels are thought to couple cellular metabolism with membrane excitability, calcium entry, and endothelial mediator release. We hypothesized that endothelial KATP channels have a broad role protecting against high blood pressure and atherosclerosis. Endothelial-specific Kir6.1 KO mice (eKO) and eKO mice on an apolipoprotein E KO background were generated (A-eKO) to investigate the role of KATP channels in the endothelium. Basal blood pressure was not elevated in eKO mice. However, when challenged with a high-salt diet and the eNOS inhibitor L-NAME, eKO mice became more hypertensive than their littermate controls. In aorta, NO release at least partly contributes to the endothelium-dependent vasorelaxation induced by pinacidil. In A-eKO mice atherosclerotic plaque density was significantly greater than in their littermate controls when challenged with a high-fat diet, particularly in the aortic arch region. Levels of endothelial dysfunction markers were higher in eKO compared with WT mice; however, these were not significant for A-eKO mice compared with their littermate controls. Furthermore, decreased vascular reactivity was observed in the mesenteric arteries of A-eKO mice, but not in aorta when on a high-fat diet. Our data support a role for endothelial Kir6.1-containing KATP channels in the endothelial protection against environmental stressors: the maintenance of blood pressure homeostasis in response to high salt and endothelial integrity when challenged with a high-fat diet.

Increased Blood Pressure Causes Lymphatic Endothelial Dysfunction via Oxidative Stress in Spontaneously Hypertensive Rats.

The lymphatic system is involved in the pathogenesis of edema, inflammation, and cancer metastasis. Because lymph vessels control fluid electrolytes and volume balance, changes in lymphatic activity can be expected to alter systemic blood pressure. This study examined possible changes in lymphatic contractile properties in spontaneously hypertensive rats (SHR). Thoracic ducts isolated from 10- to 12-week-old SHR exhibited either decreased acetylcholine-induced endothelium-dependent relaxation or sodium nitroprusside-induced endothelium-independent relaxation compared with age-matched Wister-Kyoto rats. The impairment in acetylcholine responsiveness was more pronounced than sodium nitroprusside responsiveness. N-Nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor blunted acetylcholine-induced relaxation in Wister-Kyoto rats, indicating an involvement of endothelial nitric oxide production. Endothelial dysfunction in lymph vessels of SHR was attenuated by tempol (a superoxide dismutase mimetic), apocynin, or VAS-2870 (NADPH oxidase inhibitors). Consistent with these observations, nitrotyrosine levels were significantly elevated in SHR, indicative of increased oxidative stress. In addition, protein expression of NADPH oxidase 2 and phosphorylation of p47phox (Ser345) were significantly increased in SHR. Further, SB203580 (a p38 MAPK inhibitor) restored the acetylcholine-induced relaxation in SHR. It is notable that 4-week-old SHR, which exhibited normal blood pressure, did not show any decreased activity of acetylcholine- or sodium nitroprusside-induced relaxation. Additionally, antihypertensive treatment of 4-week-old SHR with hydrochlorothiazide and reserpine or hydrochlorothiazide and hydralazine for 6 weeks completely restored lymphatic endothelial dysfunction. We conclude that contractile activity of lymphatic vessels is functionally impaired with the development of increasing blood pressure, which is mediated through increased oxidative stress via the p38 MAPK/NADPH oxidase 2 pathway.

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms.

OBJECTIVE: Cardiovascular disease is a leading cause of death worldwide. Obesity-related metabolic disorders including dyslipidemia cause impaired collateralization under ischemic conditions, thereby resulting in exacerbated cardiovascular dysfunction. Pemafibrate is a novel selective PPARα modulator, which has been reported to improve atherogenic dyslipidemia, in particular, hypertriglyceridemia and low HDL-cholesterol. Here, we investigated whether pemafibrate modulates the revascularization process in a mouse model of hindlimb ischemia. METHODS AND RESULTS: Male wild-type (WT) mice were randomly assigned to two groups, normal diet or pemafibrate admixture diet from the ages of 6 weeks. After 4 weeks, mice were subjected to unilateral hindlimb surgery to remove the left femoral artery and vein. Pemafibrate treatment enhanced blood flow recovery and capillary formation in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of endothelial nitric oxide synthase (eNOS). Treatment of cultured endothelial cells with pemafibrate resulted in increased network formation and migratory activity, which were blocked by pretreatment with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Pemafibrate treatment also increased plasma levels of the PPARα-regulated gene, fibroblast growth factor (FGF) 21 in WT mice. Systemic administration of adenoviral vectors expressing FGF21 (Ad-FGF21) to WT mice enhanced blood flow recovery, capillary density and eNOS phosphorylation in ischemic limbs. Treatment of cultured endothelial cells with FGF21 protein led to increases in endothelial cell network formation and migration, which were canceled by pretreatment with L-NAME. Furthermore, administration of pemafibrate or Ad-FGF21 had no effects on blood flow in ischemic limbs in eNOS-deficient mice. CONCLUSION: These data suggest that pemafibrate can promote revascularization in response to ischemia, at least in part, through direct and FGF21-mediated modulation of endothelial cell function. Thus, pemafibrate could be a potentially beneficial drug for ischemic vascular disease.

Endothelial dysfunction induced by hydroxyl radicals - the hidden face of biodegradable Fe-based materials for coronary stents.

Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we show that Fe has a strong potential to generate hydroxyl radicals (HO) during corrosion. This HO generation, but not corrosion, can be inhibited by catalase. Oxidative stress was observed (increased HO-1 expression) in aortic rings after direct exposure to Fe, but not in the presence of catalase or after indirect exposure. This oxidative stress response induced an uncoupling of eNOS in, and a consequent reduced NO production by endothelial cells exposed to Fe. In isolated rat aortic rings NO production was also reduced by HO generated during Fe corrosion, as indicated by the protective role of catalase. Finally, all these mechanisms contributed to impaired endothelium-dependent relaxation in aortic rings caused by HO generated during the direct contact with Fe. This deleterious impact of Fe corrosion on the endothelial function should be integrated when considering the use of biodegradable Fe-based alloys for vascular implants.

Omega-3 and Omega-6 polyunsaturated fatty acids stimulate vascular differentiation of mouse embryonic stem cells.

Polyunsaturated fatty acids (PUFAs) and their metabolites may influence cell fate regulation. Herein, we investigated the effects of linoleic acid (LA) as ω-6 PUFA, eicosapentaenoic acid (EPA) as ω-3 PUFA and palmitic acid (PA) on vasculogenesis of embryonic stem (ES) cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers fetal liver kinase-1, CD31 as well as VE-cadherin, whereas PA was without effect. LA and EPA increased reactive oxygen species (ROS) and nitric oxide (NO), activated endothelial NO synthase (eNOS) and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA, sulfo-N-succinimidyl oleate which is an antagonist of CD36, the scavenger receptor for fatty acid uptake as well as by a CD36 blocking antibody. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 and inhibition of eNOS by L-NAME blunted vasculogenesis. PUFAs stimulated AMP activated protein kinase-α (AMPK-α) as well as peroxisome proliferator-activated receptor-α (PPAR-α). AMPK activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA-induced vasculogenesis was blunted by the PPAR-α inhibitor GW6471. In conclusion, ω-3 and ω-6 PUFAs stimulate vascular differentiation of ES cells via mechanisms involving calcium, ROS and NO, which regulate function of the energy sensors AMPK and PPAR-α and determine the metabolic signature of vascular cell differentiation.

Nicorandil ameliorates bleomycin-induced pulmonary fibrosis in rats through modulating eNOS, iNOS, TXNIP and HIF-1α levels.

Bleomycin (BLM) is one of the most common anti-cancer drugs used to treat numerous types of tumors. However, pulmonary toxicity is considered the most dramatic effect of BLM. Therefore, BLM has been frequently used for induction of pulmonary fibrosis. This study aimed to evaluate the effect of nicorandil on BLM-induced pulmonary fibrosis and explore the possible mechanisms. BLM was instilled intratracheally into male Sprague-Dawley rats as a single dose (5 mg/kg) and oral nicorandil was given (30 mg/kg/day) for 6 weeks after BLM challenge. At the end of experimental period, rats were sacrificed, and lung histopathology and biochemical parameters were evaluated. Nicorandil therapy attenuated lung inflammation and fibrosis elicited by BLM. Nicorandil significantly reduced total protein content, lactate dehydrogenase (LDH) activity and total and differential cell counts. Moreover, nicorandil diminished lung levels of malondialdehyde and total nitrite/nitrate, in addition to increasing lung contents of reduced glutathione and superoxide dismutase activity. Nicorandil reduced lung and bronchoalveolar lavage fluid contents of hypoxia inducible factor-1α (HIF-1α) and lung content of thioredoxin-interacting protein (TXNIP). Besides, nicorandil significantly improved histological lesions and reduced collagen deposition as well as hydroxyproline content. Immunohistochemical examination revealed that nicorandil-treated rats exhibited significant diminutions in protein expression levels of transforming growth factor beta-1(TGF-ß1) and inducible nitric oxide synthase (iNOS) and enhanced pulmonary protein expression of endothelial NOS (eNOS). In conclusion, these results illustrate the possible potential effects of nicorandil for managing pulmonary fibrosis caused by BLM.

VEGF and bFGF induction by nitric oxide is associated with hyperbaric oxygen-induced angiogenesis and muscle regeneration.

Hyperbaric oxygen (HBO) treatment promotes early recovery from muscle injury. Reactive oxygen species (ROS) upregulation is a key mechanism of HBO, which produces high O2 content in tissues through increased dissolution of oxygen at high pressure. Nitric oxide (NO), a type of ROS, generally stabilizes hypoxia-inducible factor (HIF) 1α and stimulates secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from endothelial cells and macrophages, which then induces angiogenesis. The purpose of the present study was to investigate whether HBO could promote angiogenesis via induction of NO and induce muscle regeneration in contused rat skeletal muscles. The HBO protocol consisted of 2.5 atmospheres absolute (ATA) 100% oxygen for 120 minutes, once a day for 5 consecutive days. We also evaluated the effects of a ROS inhibitor (NAC) or NOS-specific inhibitor (L-NAME) on HBO. HBO significantly increased NO3-, VEGF, and bFGF levels and stabilized HIF1α within 1 day. HBO promoted blood vessel formation at 3-7 days and muscle healing at 5-7 days after contusion. Administration of both NAC and L-NAME before HBO suppressed angiogenesis and muscle regeneration even after HBO. HBO thus promoted angiogenesis and muscle regeneration mainly through generation of NO in the early phase after muscle contusion injury.

Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria.

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

Neuronal nitric oxide synthase regulation of calcium cycling in ventricular cardiomyocytes is independent of Cav1.2 channel modulation under basal conditions.

Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that-against the currently prevailing view-basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO.