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Aims: Anxiety often coexists with migraine, and both conditions share a commonality in oxidative/nitrosative stress and mitochondrial dysfunction contributing to their pathogenesis. β-Sitosterol, a plant sterol, has shown promise in mitigating oxidative/nitrosative stress, enhancing mitochondrial function, and exerting neuroprotective effects. In this study, we investigated the impact of β-sitosterol on migraine-associated anxiety and whether this effect was associated with alleviation of oxidative/nitrosative stress and improvement in mitochondrial function., Methods: Nitroglycerin was used to induce migraine in adult male Wistar rats. β-Sitosterol treatment consisted of daily intraperitoneal injections (10 mg/kg) for 10 days following migraine induction. Anxiety levels were evaluated using open-field test (OFT) and hole-board test (HBT). Frontal cortex samples were analyzed for malondialdehyde (MDA), glutathione (GSH), reactive oxygen/nitrogen species, nitric oxide (NO) (markers of oxidative/nitrosative stress), and ATP (indicator of mitochondrial function)., Results: Migraine induction led to impaired performance in both the OFT and the HBT. Concurrently, it elevated MDA, reactive oxygen/nitrogen species, and NO levels while diminishing GSH levels in the frontal cortex, signifying heightened oxidative/nitrosative stress. Moreover, ATP levels decreased, indicating mitochondrial dysfunction. Treatment with β-sitosterol significantly restored performance in both behavioral assays and normalized the levels of MDA, GSH, reactive oxygen/nitrogen species, NO, and ATP., Conclusion: β-Sitosterol exerted anxiolytic effects in migraine, which can be attributed to its ability to ameliorate oxidative/nitrosative stress and enhance mitochondrial function., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

The purpose of this study was to determine the effect of circulating microvesicles isolated from chronic electronic (e-)cigarette users on cultured human umbilical vein endothelial cell (HUVEC) expression of nuclear factor-κB (NF-κB), cellular cytokine release, phosphorylation of endothelial nitric oxide synthase (eNOS) and NO production. The HUVECs were treated with microvesicles isolated via flow cytometry from nine non-tobacco users (five male and four female; 22 ± 2 years of age) and 10 e-cigarette users (six male and four female; 22 ± 2 years of age). Microvesicles from e-cigarette users induced significantly greater release of interleukin-6 (183.4 ± 23.6 vs. 150.6 ± 15.4 pg/mL; P = 0.002) and interleukin-8 (160.0 ± 31.6 vs. 129.4 ± 11.2 pg/mL; P = 0.01), in addition to expression of p-NF-κB p65 (Ser536) (18.8 ± 3.4 vs. 15.6 ± 1.5 a.u.; P = 0.02) from HUVECs compared with microvesicles from non-tobacco users. Nuclear factor-κB p65 was not significantly different between microvesicles from the non-tobacco users and from the e-cigarette users (87.6 ± 8.7 vs. 90.4 ± 24.6 a.u.; P = 0.701). Neither total eNOS (71.4 ± 21.8 vs. 80.4 ± 24.5 a.u.; P = 0.413) nor p-eNOS (Thr495) (229.2 ± 26.5 vs. 222.1 ± 22.7 a.u.; P = 0.542) was significantly different between microvesicle-treated HUVECs from non-tobacco users and e-cigarette users. However, p-eNOS (Ser1177) (28.9 ± 6.2 vs. 45.8 ± 9.0 a.u.; P < 0.001) expression was significantly lower from e-cigarette users compared with non-tobacco users. Nitric oxide production was significantly lower (8.2 ± 0.6 vs. 9.7 ± 0.9 μmol/L; P = 0.001) in HUVECs treated with microvesicles from e-cigarette users compared with microvesicles from non-tobacco users. This study demonstrated increased NF-κB activation and inflammatory cytokine production, in addition to diminished eNOS activity and NO production resulting from e-cigarette use. HIGHLIGHTS: What is the central question of this study? Circulating microvesicles contribute to cardiovascular health and disease via their effects on the vascular endothelium. The impact of electronic (e-)cigarette use on circulating microvesicle phenotype is not well understood. What is the main finding and its importance? Circulating microvesicles from e-cigarette users increase endothelial cell inflammation and impair endothelial nitric oxide production. Endothelial inflammation and diminished nitric oxide bioavailability are central factors underlying endothelial dysfunction and, in turn, cardiovascular disease risk. Deleterious changes in the functional phenotype of circulating microvesicles might contribute to the reported adverse effects of e-cigarette use on cardiovascular health., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Aim: We aimed to test the hypothesis that a high-salt diet (HS) impairs NO signaling in kidney microvascular endothelial cells through a histone deacetylase 1 (HDAC1)-dependent mechanism., Methods: Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or HS (4% NaCl) for 2 weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured., Results: We found HS-induced impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS-fed rats to be sufficient to induce impaired NO signaling. This indicates the presence of a humoral factor we termed plasma-derived endothelial dysfunction mediator (PDEM). Pretreatment with the antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling., Conclusion: We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling., (© 2024 The Author(s). Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Background: Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor availability. Loss of endothelial function and NO bioavailability are associated with inflammation, including leukocyte activation. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced cardiovascular events in REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) in relation to on-treatment EPA blood levels. The mechanisms of cardiovascular protection for EPA remain incompletely elucidated but likely involve direct effects on the endothelium., Methods and Results: In this study, human ECs were treated with EPA and challenged with the cytokine IL-6 (interleukin-6). Proinflammatory responses in the ECs were confirmed by ELISA capture of sICAM-1 (soluble intercellular adhesion molecule-1) and TNF-α (tumor necrosis factor-α). Global protein expression was determined using liquid chromatography-mass spectrometry tandem mass tag. Release kinetics of NO and peroxynitrite were monitored using porphyrinic nanosensors. IL-6 challenge induced proinflammatory responses from the ECs as evidenced by increased release of sICAM-1 and TNF-α, which correlated with a loss of NO bioavailability. ECs pretreated with EPA modulated expression of 327 proteins by >1-fold ( P <0.05), compared with IL-6 alone. EPA augmented expression of proteins involved in NO production, including heme oxygenase-1 and dimethylarginine dimethylaminohydrolase-1, and 34 proteins annotated as associated with neutrophil degranulation. EPA reversed the endothelial NO synthase uncoupling induced by IL-6 as evidenced by an increased [NO]/[peroxynitrite] release ratio ( P <0.05)., Conclusions: These direct actions of EPA on EC functions during inflammation may contribute to its distinct cardiovascular benefits.

This study aimed to investigate the effects of the n-hexane fraction of the ethanolic seed extract of PG (NFESEPG) on hypertension induced by Nω-nitro-L-arginine methyl ester (L-NAME) in rats. Specifically, the study examined the impact of NFESEPG on blood pressure, oxidative stress markers, NO concentration, angiotensin-converting enzyme (ACE) and arginase activities, and cardiac biomarkers in hypertensive rats. The study involved collecting, identifying, and processing the PG plant to obtain the ethanolic seed extract. The extract was then partitioned with solvents to isolate the n-hexane fraction. Hypertension was induced in rats by oral administration of L-NAME for 10 days, while concurrent treatment with NFESEPG at two doses (200 and 400 mg/kg/day) was administered orally. Blood pressure was measured using a noninvasive tail-cuff method, and various biochemical parameters were assessed. Treatment with both doses of NFESEPG significantly reduced systolic and diastolic blood pressure in L-NAME-induced hypertensive rats. Additionally, NFESEPG administration increased NO concentration and decreased ACE and arginase activities, malondialdehyde (MDA) levels, and cardiac biomarkers in hypertensive rats. The findings indicate that NFESEPG effectively lowered blood pressure in hypertensive rats induced by L-NAME, potentially through mechanisms involving the modulation of oxidative stress, NO bioavailability, and cardiac biomarkers. These results suggest the therapeutic potential of NFESEPG in managing hypertension and related cardiovascular complications., (© 2024 The Author(s). Cell Biochemistry and Function published by John Wiley & Sons Ltd.)

Safe chemicals for drug withdrawal can be extracted from natural sources. This study investigates the effects of clonidine and Thymbra spicata extract (TSE) on mice suffering from morphine withdrawal syndrome. Thymol, which is the active constituent in TSE, was also tested. A total of 90 mice were divided into nine groups. Group 1 was the control group, while Group 2 was given only morphine, and Group 3 received morphine and 0.2 mg/kg of clonidine. Groups 4-6 were given morphine along with 100, 200, and 300 mg/kg of TSE, respectively. Groups 7-9 received morphine plus 30, 60, and 90 mg/kg of Thymol, respectively, for 7 days. An oral naloxone challenge of 3 mg/kg was used to induce withdrawal syndrome in all groups. Improvement of liver enzyme levels (aspartate aminotransferase, alkaline phosphatase, and alanine transaminase) (p < .01) and behavioral responses (frequencies of jumping, frequencies of two-legged standing, Straub tail reaction) (p < .01) were significantly observed in the groups receiving TSE and Thymol (Groups 4-9) compared to Group 2. Additionally, antioxidant activity in these groups was improved compared to Group 2. Nitric oxide significantly decreased in Groups 4 and 6 compared to Groups 2 and 3 (p < .01). Superoxide dismutase increased dramatically in Groups 5, 8, and 9 compared to Groups 2 and 3 (p < .01). Groups 5-9 were significantly different from Group 2 in terms of malondialdehyde levels (p < .01). Certain doses of TSE and Thymol were found to alleviate the narcotics withdrawal symptoms. This similar effect to clonidine can pave the way for their administration in humans., (© 2024 John Wiley & Sons Ltd.)

Cisplatin (CIS) is a platinum-derived chemotherapeutic agent commonly utilized in the treatment of various malignant tumours. However, anticancer doses of the drug cause serious damage to the brain. This study aimed to determine the potential protective effects of tangeretin, which has antioxidant and anti-inflammatory properties, in cisplatin-induced neurotoxicity on BALB/c mice brains. Male BALB/c mice were randomized and separated into four groups. Tangeretin was given for 10 days by gavage. CIS was injected as a single dose of 10 mg/kg intraperitoneally (ip) on the 10th day. Brain tissues, malondialdehyde (MDA), total glutathione (tGSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and nitric oxide (NO) levels were measured to determine oxidative damage and myeloperoxidase, tumour necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), IL-6 and IL-10 were measured to determine inflammatory activity. In addition, 8-OHdG and caspase-3 were analysed by immunofluorescence methods. While CIS administration remarkably elevated reactive oxygen species, MDA, and NO levels in brain tissue compared to the control, tGSH, GPx, SOD and CAT levels were significantly decreased. Also, it has been detected that TNF-α, IL-1β and IL-6 obtained in CIS-treated groups increased as well as IL-10 decreased, thereby elevating the inflammatory response. In addition, 8-OHdG and caspase-3 immunoreactivity in neurons increased with CIS administration. Treatment with tangeretin ameliorated the deterioration in oxidant/antioxidant status, overpowered neuroinflammation and ameliorated neurotoxicity-induced apoptosis. This study shows that tangeretin has beneficial effects on CIS-induced neurodegeneration. Possible mechanisms underlying these beneficial effects include the antioxidant and anti-inflammatory properties of tangeretin., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

In this in vitro study, for the first time, we evaluate the effects of simvastatin-loaded liposome nanoparticles (SIM-LipoNPs) treatment on fibrosis-induced liver microtissues, as simvastatin (SIM) has shown potential benefits in the non-alcoholic fatty liver disease process. We developed multicellular liver microtissues composed of hepatic stellate cells, hepatoblastoma cells and human umbilical vein endothelial cells. The microtissues were supplemented with a combination of palmitic acid and oleic acid to develop fibrosis models. Subsequently, various groups of microtissues were exposed to SIM and SIM-LipoNPs at doses of 5 and 10 mg/mL. The effectiveness of the treatments was evaluated by analysing cell viability, production of reactive oxygen species (ROS) and nitric oxide (NO), the expression of Kruppel-like factor (KLF) 2, and pro-inflammatory cytokines (interleukin(IL)-1 α, IL-1 β, IL-6 and tumour necrosis factor-α), and the expression of collagen I. Our results indicated that SIM-LipoNPs application showed promising results. SIM-LipoNPs effectively amplified the SIM-klf2-NO pathway at a lower dosage compatible with a high dosage of free SIM, which also led to reduced oxidative stress by decreasing ROS levels. SIM-LipoNPs administration also resulted in a significant reduction in pro-inflammatory cytokines and Collagen I mRNA levels, as a marker of fibrosis. In conclusion, our study highlights the considerable therapeutic potential of using SIM-LipoNPs to prevent liver fibrosis progress, underscoring the remarkable properties of SIM-LipoNPs in activating the KLF2-NO pathway and anti-oxidative and anti-inflammatory response., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Objective: The sympathetic-parasympathetic (or axo-axonal) interaction mechanism mediated that neurogenic relaxation, which was dependent on norepinephrine (NE) releases from sympathetic nerve terminal and acts on β 2 -adrenoceptor of parasympathetic nerve terminal, has been reported. As NE is a weak β 2 -adrenoceptor agonist, there is a possibility that synaptic NE is converted to epinephrine by phenylethanolamine-N-methyltransferase (PNMT) and then acts on the β 2 -adrenoceptors to induce neurogenic vasodilation., Methods: Blood vessel myography technique was used to measure relaxation and contraction responses of isolated basilar arterial rings of rats., Results: Nicotine-induced relaxation was sensitive to propranolol, guanethidine (an adrenergic neuronal blocker), and N ω -nitro-l-arginine. Nicotine- and exogenous NE-induced vasorelaxation was partially inhibited by LY-78335 (a PNMT inhibitor), and transmural nerve stimulation depolarized the nitrergic nerve terminal directly and was not inhibited by LY-78335; it then induced the release of nitric oxide (NO). Epinephrine-induced vasorelaxation was not affected by LY-78335. However, these vasorelaxations were completely inhibited by atenolol (a β 1 -adrenoceptor antagonist) combined with ICI-118,551 (a β 2 -adrenoceptor antagonist)., Conclusions: These results suggest that NE may be methylated by PNMT to form epinephrine and cause the release of NO and vasodilation. These results provide further evidence supporting the physiological significance of the axo-axonal interaction mechanism in regulating brainstem vascular tone., (© 2024 John Wiley & Sons Ltd.)

Background: The vasoconstrictor effects of angiotensin II via type 1 angiotensin II receptors in vascular smooth muscle cells are well established, but the direct effects of angiotensin II on vascular endothelial cells (VECs) in vivo and the mechanisms how VECs may mitigate angiotensin II-mediated vasoconstriction are not fully understood. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of angiotensin II on VECs in kidney and brain microvessels in vivo., Methods and Results: Changes in VEC intracellular calcium ([Ca 2+ ] i ) and nitric oxide (NO) production were visualized by intravital multiphoton microscopy of cadherin 5-Salsa6f mice or the endothelial uptake of NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, respectively. Kidney fibrosis by unilateral ureteral obstruction and Ready-to-use adeno-associated virus expressing Mouse Renin 1 gene (Ren1-AAV) hypertension were used as disease models. Acute systemic angiotensin II injections triggered >4-fold increases in VEC [Ca 2+ ] i in brain and kidney resistance arterioles and capillaries that were blocked by pretreatment with the type 1 angiotensin II receptor inhibitor losartan, but not by the type 2 angiotensin II receptor inhibitor PD123319. VEC responded to acute angiotensin II by increased NO production as indicated by >1.5-fold increase in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence intensity. In mice with kidney fibrosis or hypertension, the angiotensin II-induced VEC [Ca 2+ ] i and NO responses were significantly reduced, which was associated with more robust vasoconstrictions, VEC shedding, and microthrombi formation., Conclusions: The present study directly visualized angiotensin II-induced increases in VEC [Ca 2+ ] i and NO production that serve to counterbalance agonist-induced vasoconstriction and maintain residual organ blood flow. These direct and endothelium-specific angiotensin II effects were blunted in disease conditions and linked to endothelial dysfunction and the development of vascular pathologies.

Many patients exhibit persistently reduced pulmonary diffusing capacity after coronavirus disease 2019 (COVID-19). In this study, dual test gas diffusing capacity for carbon monoxide and nitric oxide (D L,CO,NO ) metrics and their relationship to disease severity and physical performance were examined in patients who previously had COVID-19. An initial cohort of 148 patients diagnosed with COVID-19 of all severities between March 2020 and March 2021 had a D L,CO,NO measurement performed using the single-breath method at 5.7 months follow-up. All patients with at least one abnormal D L,CO,NO metric (n = 87) were revaluated at 12.5 months follow-up. The D L,CO,NO was used to provide the pulmonary diffusing capacity for nitric oxide (D L,NO ), the pulmonary diffusing capacity for carbon monoxide (D L,CO,5s ), the alveolar-capillary membrane diffusing capacity and the pulmonary capillary blood volume. At both 5.7 and 12.5 months, physical performance was assessed using a 30 s sit-to-stand test and the 6 min walk test. Approximately 60% of patients exhibited a severity-dependent decline in at least one D L,CO,NO metric at 5.7 months follow-up. At 12.5 months, both D L,NO and D L,CO,5s had returned towards normal but still remained abnormal in two-thirds of the patients. Concurrently, improvements in physical performance were observed, but with no apparent relationship to any D L,CO,NO metric. The severity-dependent decline in D L,NO and D L,CO observed at 5.7 months after COVID-19 appears to be reduced consistently at 12.5 months follow-up in the majority of patients, despite marked improvements in physical performance., (© 2024 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH., (© 2024 John Wiley & Sons Australia, Ltd.)

Aim: Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice., Methods: Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO 2 - and NO 3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated., Results: The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity., Conclusion: In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Levodopa (L-DOPA) is the classical gold standard treatment for Parkinson's disease. However, its chronic administration can lead to the development of L-DOPA-induced dyskinesias (LIDs). Dysregulation of the nitric oxide-cyclic guanosine monophosphate pathway in striatal networks has been linked to deficits in corticostriatal transmission in LIDs. This study investigated the effects of the nitric oxide (NO) donor sodium nitroprusside (SNP) on behavioural and electrophysiological outcomes in sham-operated and 6-hydroxydopamine-lesioned rats chronically treated with vehicle or L-DOPA, respectively. In sham-operated animals, systemic administration of SNP increased the spike probability of putative striatal medium spiny neurons (MSNs) in response to electrical stimulation of the primary motor cortex. In 6-hydroxydopamine-lesioned animals, SNP improved the stepping test performance without exacerbating abnormal involuntary movements. Additionally, SNP significantly increased the responsiveness of putative striatal MSNs in the dyskinetic striatum. These findings highlight the critical role of the NO signalling pathway in facilitating the responsiveness of striatal MSNs in both the intact and dyskinetic striata. The study suggests that SNP has the potential to enhance L-DOPA's effects in the stepping test without exacerbating abnormal involuntary movements, thereby offering new possibilities for optimizing Parkinson's disease therapy. In conclusion, this study highlights the involvement of the NO signalling pathway in the pathophysiology of LIDs., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Endometritis is an inflammatory reaction of the lining of uterus, leading to the occurrence of infertility. Platelet rich plasma (PRP) has been proven to exhibit extremely effective for the treatment of endometrium-associated infertility, but the mechanism of its prevention for endometritis remains unclear., Objective: The present study aimed to investigate the protective effect of PRP against endometritis induced by lipopolysaccharide (LPS) and elucidate the mechanism underlying these effects., Methods: Mouse model of endometritis was established by intrauterine perfusion of LPS. PRP intrauterine infusion was administered at 24 h after LPS induction. After another 24 h, the uterine tissues were harvested to observe histopathological changes, production of proinflammatory cytokines, variation of the Toll-like receptor 4/nuclear factor κB (TLR4/NF-κB) signaling pathways, and validated the anti-inflammatory effect of PRP. The myeloperoxidase (MPO) activity and concentration of nitric oxide (NO) were determined using assay kit. Proinflammatory chemokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6)) were measured by ELISA and Real-Time PCR. The activity of TLR4/NF-κB pathway in uterine tissues was measured by Western blotting., Results: Hematoxylin-eosin staining (H&E) appeared that PRP remarkably relieved the impairment of uterine tissues. Detection of MPO activity and concentration of NO revealed that PRP treatment distinctly mitigated infiltration of inflammatory cells in mice with endometritis induced by LPS. PRP treatment significantly affected the expression of TNF-α, IL-1β, and IL-6. PRP was also found to suppress LPS-induced activation of TLR4/NF-κB pathway., Conclusion: PRP effectively alleviates LPS-induced endometritis via restraining the signal pathway of TLR4/NF-κB. These findings provide a solid foundation for PRP as a potential therapeutic agent for endometritis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: Magnesium (Mg 2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca 2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg 2+ -dependent vessel relaxation., Methods: To uncover these mechanisms, force development was measured ex vivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg 2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused ex vivo using pressure myography. Effects of Mg 2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine., Results: Mg 2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg 2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg 2+ augmented acetylcholine-induced relaxation. SK Ca and IK Ca channel blockers apamin and TRAM34 inhibited Mg 2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca 2+ . Combined blockade of NO, SK Ca , and IK Ca channels significantly reduced Mg 2+ -dependent dilatation in mesenteric resistance vessels., Conclusions: In mouse conductance and resistance arteries Mg 2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca 2+ in smooth muscle cells, and additional unidentified mechanisms., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Background: This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS)., Methods and Results: EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. In vivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions., Conclusions: CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

Background: Nitric oxide (NO) is the most important vasodilator secreted by vascular endothelial cells, and its abnormal synthesis is involved in the development of cardiovascular disease. The prenatal period is a critical time for development and largely determines lifelong vascular health in offspring. Given the high incidence and severity of gestational hypoxia in mid-late pregnancy, it is urgent to further explore whether it affects the long-term synthesis of NO in offspring vascular endothelial cells., Methods and Results: Pregnant Sprague-Dawley rats were housed in a normoxic or hypoxic (10.5% O 2 ) chamber from gestation days 10 to 20. The thoracic aortas of fetal and adult male offspring were isolated for experiments. Gestational hypoxia significantly reduces the NO-dependent vasodilation mediated by acetylcholine in both the fetal and adult offspring thoracic aorta rings. Meanwhile, acetylcholine-induced NO synthesis is impaired in vascular endothelial cells from hypoxic offspring thoracic aortas. We demonstrate that gestational hypoxic offspring exhibit a reduced endothelial NO synthesis capacity, primarily due to increased expression of NADPH oxidase 2 and enhanced reactive oxygen species. Additionally, gestational hypoxic offspring show elevated levels of miR-155-5p in vascular endothelial cells, which is associated with increased expression of NADPH oxidase 2 and reactive oxygen species generation, as well as impaired NO synthesis., Conclusions: The present study is the first to demonstrate that gestational hypoxia impairs endothelial NO synthesis via the miR-155-5p/NADPH oxidase 2/reactive oxygen species axis in offspring vessels. These novel findings indicate that the detrimental effects of gestational hypoxia on fetal vascular function can persist into adulthood, providing new insights into the development of vascular diseases.

Objectives: The objective of our study is to evaluate the involvement of the transient receptor potential vanilloid 4 (TRPV4) in the alteration of lymphatic pumping in response to flow and determine the signaling pathways involved., Methods: We used immunofluorescence imaging and western blotting to assess TRPV4 expression in rat mesenteric lymphatic vessels. We examined inhibition of TRPV4 with HC067047, nitric oxide synthase (NOS) with L-NNA and cyclooxygenases (COXs) with indomethacin on the contractile response of pressurized lymphatic vessels to flow changes induced by a stepwise increase in pressure gradients, and the functionality of endothelial TRPV4 channels by measuring the intracellular Ca 2+ response of primary lymphatic endothelial cell cultures to the selective agonist GSK1016790A., Results: TRPV4 protein was expressed in both the endothelial and the smooth muscle layer of rat mesenteric lymphatics with high endothelial expression around the valve sites. When maintained under constant transmural pressure, most lymphatic vessels displayed a decrease in contraction frequency under conditions of flow and this effect was ablated through inhibition of NOS, COX or TRPV4., Conclusions: Our findings demonstrate a critical role for TRPV4 in the decrease in contraction frequency induced in lymphatic vessels by increases in flow rate via the production and action of nitric oxide and dilatory prostanoids., (© 2023 John Wiley & Sons Ltd.)

Background: Skeletal muscles are postulated to be a potent regulator of systemic nitric oxide homeostasis. In this study, we aimed to evaluate the impact of physical training on the heart and skeletal muscle nitric oxide bioavailability (judged on the basis of intramuscular nitrite and nitrate) in rats., Methods and Results: Rats were trained on a treadmill for 8 weeks, performing mainly endurance running sessions with some sprinting runs. Muscle nitrite (NO 2 - ) and nitrate (NO 3 - ) concentrations were measured using a high-performance liquid chromatography-based method, while amino acids, pyruvate, lactate, and reduced and oxidized glutathione were determined using a liquid chromatography coupled with tandem mass spectrometry technique. The content of muscle nitrite reductases (electron transport chain proteins, myoglobin, and xanthine oxidase) was assessed by western immunoblotting. We found that 8 weeks of endurance training decreased basal NO 2 - in the locomotory muscles and in the heart, without changes in the basal NO 3 - . In the slow-twitch oxidative soleus muscle, the decrease in NO 2 - was already present after the first week of training, and the content of nitrite reductases remained unchanged throughout the entire period of training, except for the electron transport chain protein content, which increased no sooner than after 8 weeks of training., Conclusions: Muscle NO 2 - level, opposed to NO 3 - , decreases in the time course of training. This effect is rapid and already visible in the slow-oxidative soleus after the first week of training. The underlying mechanisms of training-induced muscle NO 2 - decrease may involve an increase in the oxidative stress, as well as metabolite changes related to an increased muscle anaerobic glycolytic activity contributing to (1) direct chemical reduction of NO 2 - or (2) activation of muscle nitrite reductases.

The adverse effects caused by nonselective and selective cyclooxygenase-2 (COX-2) inhibitors remain a challenge for current anti-inflammatory medications. A balanced inhibition of COX-1/-2 represents a promising strategy for the development of novel COX-2 inhibitors. In this study, we present the design and synthesis of a novel series of firocoxib analogues incorporating an amide bond to facilitate essential hydrogen bonding with amino residues in COX-2. The synthesized analogs were evaluated for their inhibitory activity against both COX-1 and COX-2 enzymes. Among them, compound 9d demonstrated potent and balanced inhibition. Inhibition of COX enzymes by 9d in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages resulted in the suppression of the NF-κB signaling pathway to reduced expression of pro-inflammatory factors such as inducible nitric oxide synthase (iNOS), COX-2, nitric oxide (NO), and reactive oxygen species (ROS). The remarkable in vitro anti-inflammatory activity exhibited by 9d positions it as a promising candidate for further development as a novel lead compound for inflammation treatment., (© 2024 John Wiley & Sons Ltd.)

Aims: To mediate its pharmacodynamic effects, glyceryl trinitrate (GTN) requires bioactivation, by which it releases nitric oxide or a nitric oxide moiety. The exact mechanism of GTN bioactivation remains uncertain. Mitochondrial aldehyde dehydrogenase (ALDH-2) has been proposed as the primary enzyme responsible for this bioactivation process. Evidence for the importance of ALDH-2 in GTN bioactivation has been inconsistent, particularly in human models. An alternative hypothesis suggests that decreased ALDH-2 activity leads to accumulation of reactive cytotoxic aldehydes, which either inhibit the vasoactive product(s) of GTN or impair other enzymatic pathways involved in the bioactivation of GTN. We investigated the effect of supplemental vitamin C on vascular responses to GTN in healthy volunteers of East Asian descent, of whom 12 with and 12 without the ALDH-2 polymorphism participated., Methods: Subjects underwent 2 sequential brachial artery infusions of GTN at rates of 5, 11 and 22 nmol/min, separated by a 30-min washout period. The GTN infusions were carried out in the presence and absence of vitamin C using a randomized, crossover design. Venous occlusion plethysmography was used to measure forearm blood flow responses to GTN., Results: Compared to subjects with functional ALDH-2, the variant group exhibited blunted hemodynamic responses to intra-arterial GTN infusions, although this reduction in response was not statically significant. Contrary to our hypothesis, vitamin C had an inhibitory effect on GTN mediated vasodilation as compared to GTN during saline in both groups., Conclusion: We conclude that vitamin C did not augment the acute vascular response to GTN in those with the ALDH-2 polymorphism., (© 2023 British Pharmacological Society.)

Background The mechanisms determining vascular tone are still not completely understood, even though it is a significant factor in blood pressure management. Many circulating proteins have a significant impact on controlling vascular tone. Progranulin displays anti-inflammatory effects and has been extensively studied in neurodegenerative illnesses. We investigated whether progranulin sustains the vascular tone that helps regulate blood pressure. Methods and Results We used male and female C57BL6/J wild type (progranulin +/+ ) and B6(Cg)-Grn tm1.1Aidi /J (progranulin -/- ) to understand the impact of progranulin on vascular contractility and blood pressure. We found that progranulin -/- mice display elevated blood pressure followed by hypercontractility to noradrenaline in mesenteric arteries, which is restored by supplementing the mice with recombinant progranulin. In ex vivo experiments, recombinant progranulin attenuated the vascular contractility to noradrenaline in male and female progranulin +/+ arteries, which was blunted by blocking EphrinA2 or Sortilin1. To understand the mechanisms whereby progranulin evokes anticontractile effects, we inhibited endothelial factors. N(gamma)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) prevented the progranulin effects, whereas indomethacin (cyclooxygenase inhibitor) affected only the contractility in arteries incubated with vehicle, indicating that progranulin increases nitric oxide and decreases contractile prostanoids. Finally, recombinant progranulin induced endothelial nitric oxide synthase phosphorylation and nitric oxide production in isolated mesenteric endothelial cells. Conclusions Circulating progranulin regulates vascular tone and blood pressure via EphrinA2 and Sortilin1 receptors and endothelial nitric oxide synthase activation. Collectively, our data suggest that deficiency in progranulin is a cardiovascular risk factor and that progranulin might be a new therapeutic avenue to treat high blood pressure.

Mephedrone is a representative of synthetic cathinones that is known from its rewarding and psychostimulant effects. It exerts behavioural sensitization after repeated and then interrupted administration. In our study, we investigated a role of the L-arginine-NO-cGMP-dependent signalling in the expression of sensitization to hyperlocomotion evoked by mephedrone. The study was carried out in male albino Swiss mice. The tested mice received mephedrone (2.5 mg/kg) for 5 consecutive days and on the 20th day of the experiment (the 'challenge' day) animals received both mephedrone (2.5 mg/kg) and a given substance that affects the L-arginine-NO-cGMP signalling, that is, L-arginine hydrochloride (125 or 250 mg/kg), 7-nitroindazole (10 or 20 mg/kg), L-NAME (25 or 50 mg/kg) or methylene blue (5 or 10 mg/kg). We observed that 7-nitroindazole, L-NAME and methylene blue inhibited the expression of sensitization to the mephedrone-induced hyperlocomotion. Moreover, we demonstrated that the mephedrone-induced sensitization is accompanied by lowered levels of D 1 receptors and NR2B subunits in the hippocampus, whereas a concurrent administration of L-arginine hydrochloride, 7-nitroindazole and L-NAME with the mephedrone challenge dose reversed these effects. Methylene blue only reversed the mephedrone-induced effects on hippocampal levels of the NR2B subunit. Our study confirms that the L-arginine-NO-cGMP pathway contributes to mechanisms underlying the expression of sensitization to the mephedrone-evoked hyperlocomotion., (© 2023 John Wiley & Sons Australia, Ltd.)

Background: Nitric oxide is a chemical agent produced by endothelial cells in a healthy blood vessel, inhibiting the overgrowth of vascular smooth muscle cells and regulating vessel tone. Liposomes are biocompatible and biodegradable drug carriers with a similar structure to cell bilayer phospholipid membrane that can be used as useful nitric oxide carriers in vascular grafts., Method: Using a custom-designed apparatus, the sheep carotid arteries were decellularized while still maintaining important components of the vascular extracellular matrix (ECM), allowing them to be used as small-diameter vascular grafts. A chemical signal of sodium nitrite was applied to control smooth muscle cells' behavior under static and dynamic cell culture conditions. The thin film hydration approach was used to create nano-liposomes, which were then used as sodium nitrite carriers to control the drug release rate and enhance the amount of drug loaded into the liposomes., Results: The ratio of 80:20:2 for DPPC: Cholesterol: PEG was determined as the optimum formulation of the liposome structure with high drug encapsulation efficiency (98%) and optimum drug release rate (the drug release rate was 40%, 65%, and 83% after 24, 48, and 72 h, respectively). MTT assay results showed an improvement in endothelial cell proliferation in the presence of nano-liposomal sodium nitrite (LNS) at the concentration of 0.5 μg/mL. Using a suitable concentration of liposomal sodium nitrite (0.5 μg/mL) put onto the constructed scaffold resulted in the controllable development of smooth muscle cells in the experiment. The culture of smooth muscle cells in a pulsatile perfusion bioreactor indicated that in the presence of synthesized liposomal sodium nitrite, the overgrowth of smooth muscle cells was inhibited in dynamic cell culture conditions. The mechanical properties of ECM graft were measured, and a multi-scale model with an accuracy of 83% was proposed to predict mechanical properties successfully., Conclusion: The liposomal drug-loaded small-diameter vascular graft can prevent the overgrowth of SMCs and the formation of intimal hyperplasia in the graft. Aside from that, the effect of LNS on endothelial has the potential to stimulate endothelial cell proliferation and re-endothelialization., (© 2023 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

Aim: Dietary nitrate (NO 3 - ) supplementation increases nitric oxide bioavailability and can enhance exercise performance. We investigated the distribution and metabolic fate of ingested NO 3 - at rest and during exercise with a focus on skeletal muscle., Methods: In a randomized, crossover study, 10 healthy volunteers consumed 12.8 mmol 15 N-labeled potassium nitrate (K 15 NO 3 ; NIT) or potassium chloride placebo (PLA). Muscle biopsies were taken at baseline, at 1- and 3-h post-supplement ingestion, and immediately following the completion of 60 maximal intermittent contractions of the knee extensors. Muscle, plasma, saliva, and urine samples were analyzed using chemiluminescence to determine absolute [NO 3 - ] and [NO 2 - ], and by mass spectrometry to determine the proportion of NO 3 - and NO 2 - that was 15 N-labeled., Results: Neither muscle [NO 3 - ] nor [NO 2 - ] were altered by PLA. Following NIT, muscle [NO 3 - ] (but not [NO 2 - ]) was elevated at 1-h (from ~35 to 147 nmol/g, p < 0.001) and 3-h, with almost all of the increase being 15 N-labeled. There was a significant reduction in 15 N-labeled muscle [NO 3 - ] from pre- to post-exercise. Relative to PLA, mean muscle torque production was ~7% greater during the first 18 contractions following NIT. This improvement in torque was correlated with the pre-exercise 15 N-labeled muscle [NO 3 - ] and the magnitude of decline in 15 N-labeled muscle [NO 3 - ] during exercise (r = 0.66 and r = 0.62, respectively; p < 0.01)., Conclusion: This study shows, for the first time, that skeletal muscle rapidly takes up dietary NO 3 - , the elevated muscle [NO 3 - ] following NO 3 - ingestion declines during exercise, and muscle NO 3 - dynamics are associated with enhanced torque production during maximal intermittent muscle contractions., (© 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Background Hypertension and vascular toxicity are major unwanted side effects of antiangiogenic drugs, such as vascular endothelial growth factor inhibitors (VEGFis), which are effective anticancer drugs but have unwanted side effects, including vascular toxicity and hypertension. Poly (ADP-ribose) polymerase (PARP) inhibitors, used to treat ovarian and other cancers, have also been associated with elevated blood pressure. However, when patients with cancer receive both olaparib, a PARP inhibitor, and VEGFi, the risk of blood pressure elevation is reduced. Underlying molecular mechanisms are unclear, but PARP-regulated transient receptor potential cation channel, subfamily M, member 2 (TRPM2), a redox-sensitive calcium channel, may be important. We investigated whether PARP/TRPM2 plays a role in VEGFi-induced vascular dysfunction and whether PARP inhibition ameliorates the vasculopathy associated with VEGF inhibition. Methods and Results Human vascular smooth muscle cells (VSMCs), human aortic endothelial cells, and wild-type mouse mesenteric arteries were studied. Cells/arteries were exposed to axitinib (VEGFi) alone and in combination with olaparib. Reactive oxygen species production, Ca 2+ influx, protein/gene analysis, PARP activity, and TRPM2 signaling were assessed in VSMCs, and nitric oxide levels were determined in endothelial cells. Vascular function was assessed by myography. Axitinib increased PARP activity in VSMCs in a reactive oxygen species-dependent manner. Endothelial dysfunction and hypercontractile responses were ameliorated by olaparib and a TRPM2 blocker (8-Br-cADPR). VSMC reactive oxygen species production, Ca 2+ influx, and phosphorylation of myosin light chain 20 and endothelial nitric oxide synthase (Thr 495 ) were augmented by axitinib and attenuated by olaparib and TRPM2 inhibition. Proinflammatory markers were upregulated in axitinib-stimulated VSMCs, which was reduced by reactive oxygen species scavengers and PARP-TRPM2 inhibition. Human aortic endothelial cells exposed to combined olaparib and axitinib showed nitric oxide levels similar to VEGF-stimulated cells. Conclusions Axitinib-mediated vascular dysfunction involves PARP and TRPM2, which, when inhibited, ameliorate the injurious effects of VEGFi. Our findings define a potential mechanism whereby PARP inhibitor may attenuate vascular toxicity in VEGFi-treated patients with cancer.

The nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway is an effective mechanism involved in the treatment of hypertension. In our search for potential antihypertensive agents, a series of novel NO-donor derivatives of the 4-chromanone skeleton were designed and synthesized by coupling furoxans or nitrooxy NO-donor moieties. All derivatives showed enhanced nitric oxide releasing capacity and vasodilator activity with EC 50 values ranging from 0.0215 μM to 1.46 μM, obviously superior to those of precursor 3. These biological evaluations indicated that all compounds displayed an important vasorelaxant effect, and several compounds (9c, 14b, 14c, 14d) presented good vasodilator activity, with 14c being the best. Furthermore, molecular modeling studies revealed that compound 14c occupied the pocket well with the phosphodiesterase 5 domain in a favorable conformation. In conclusion, we observed that these novel compounds can act as structural templates for the design and subsequent development of new vasodilators and antihypertensive drugs., (© 2022 John Wiley & Sons Ltd.)

Background: We investigated the effect of Erbu Zhuyu decoction (EBZY) on angiogenesis via uterine natural killer (uNK) cells and the PI3K/Akt/eNOS pathway in embryo implantation dysfunction (EID) mice., Methods: Pregnant mice were randomly divided into blank, model, EBZY, progynova, and aspirin groups. Uteri were excised on the 5th day of pregnancy for analysis., Results: Mice in the model group showed pale uteri, a reduced implantation rate, and lower expression levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS) and nitric oxide (NO). Compared to the model group, implantation rates in the medium-dose and high-dose groups of EBZY were significantly higher (P < .05), PI3K and Akt mRNA expression levels in the low-dose group were significantly higher (P < .05, P < .01), and the expression of p-PI3K, p-Akt, and p-eNOS proteins in all treatment groups were significantly increased (P < .01, P < .05). The expression of NO was significantly increased in the low-dose and high-dose groups (P < .01, P < .05, respectively). The level of p-Akt protein in the high-dose group was significantly higher than those in the other treatment groups (P < .01, P < .05). There was no significant difference in the density of uNK cells (P > .05)., Conclusions: EBZY facilitated embryo implantation in EID mice by enhancing endometrial angiogenesis via activation of the PI3K/Akt/eNOS pathway, at least in part. There was no evidence to indicate that EBZY could adjust the expression of uNK., (© 2022 The Authors. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

Nitrate, a nitric oxide (NO) donor, has antiobesity effect in female rats. This study hypothesized that the antiobesity effect of nitrate in female rats is due to the browning of white adipose tissue (WAT). Female Wistar rats (aged 8 months) were divided into two groups (n = 10/group): the control group received tap water and the nitrate group received water containing 100 mg/L of sodium nitrate for 9 months. At months 0, 3, 6, and 9, obesity indices were measured. At month 9, gonadal adipose tissue was used to measure messenger RNA (mRNA) and protein levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), PPAR-γ coactivator 1-α (PGC1-α), uncoupling protein 1 (UCP1), and adipocyte density and area. After the 9-month intervention, nitrate-treated rats had lower body weight, body mass index, thoracic circumference, and abdominal circumference by 6.4% (p = .012), 9.1% (p = .029), 6.0% (p = .056), and 5.7% (p = .098), respectively. In addition, nitrate-treated rats had higher PPAR-γ (mRNA: 1.78-fold, p = .016 and protein: 19%, p = .076), PGC1-α (mRNA: 1.69-fold, p = .012 and protein: 68%, p = .001), and UCP1 (mRNA: 2.50-fold, p = .001 and protein: 81%, p = .001) in gonadal adipose tissue. Nitrate also reduced adipocyte area by 35% (p = .054) and increased adipocyte density by 31% (p = .086). In conclusion, antiobesity effect of nitrate in female rats is associated with increased browning of gonadal adipose tissue as indicated by higher expression of PPAR-γ, PGC1-α, and UCP1 and reduced adipocyte area and increased adipocyte density., (© 2022 John Wiley & Sons Ltd.)

Aim: To study the effect of metformin on plasma levels of arginine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), indicators of the nitric oxide pathway., Materials and Methods: In this post hoc analysis of the HOME trial, we analysed plasma levels of arginine, ADMA and SDMA during the 4.3-year follow-up (comparing the effects of metformin versus placebo on top of insulin therapy). Statistical analysis was performed with a mixed model approach, in which simultaneously constant treatment effects were estimated, as well as time-dependent treatment effects., Results: We found that metformin compared with placebo did not affect ADMA or SDMA plasma levels but rapidly decreased arginine plasma levels and hence the arginine to ADMA ratio. The constant treatment effect on ADMA was 0.99 (95% CI 0.97, 1.00) relative to placebo and the time-dependent treatment effect was 1.00 (95% CI 1.00, 1.01). By contrast, the constant treatment effect on arginine was 0.86 (95% CI 0.84, 0.88), with only a minimal time-dependent change of 1.01 (95% CI 1.00, 1.01)., Conclusions: The potential benefits of metformin on endothelial function cannot be explained by a decrease in ADMA or by improved global arginine availability. The clinical significance of the decreased arginine plasma levels is not clear and can be harmful or beneficial, depending on the mechanism involved. However, a potential effect of metformin on the nitric oxide pathway is not restricted to the studied metabolites., (© 2022 John Wiley & Sons Ltd.)

Hypertension is common in elderly population. We designed to search comprehensively for genes that are chronologically shifted in their expressions and to define their contributions to vascular aging and hypertension. RNA sequencing was conducted to search for senescence-shifted transcripts in human umbilical vein endothelial cells (HUVECs). Small interfering RNA (siRNA), small-molecule drugs, CRISPR/Cas9 techniques, and imaging were used to determine genes' function and contributions to age-related phenotypes of the endothelial cell and blood vessel. Of 25 genes enriched in the term of "regulation of blood pressure," NPRA was changed most significantly. The decreased NPRA expression was replicated in aortas of aged mice. The knockdown of NPRA promoted HUVEC senescence and it decreased expressions of protein kinase cGMP-dependent 1 (PKG), sirtuin 1 (SIRT1), and endothelial nitric oxide synthase (eNOS). Suppression of NPRA also decreased the phosphorylation of AMP-activated protein kinase (AMPK) as well as the ratio of oxidized nicotinamide adenine dinucleotide (NAD + )/reduced nicotinamide adenine dinucleotide (NADH) but increased the production of reactive oxygen species (ROS). 8-Br-cGMP (analog of cGMP), or AICAR (AMPK activator), counteracted the observed changes in HUVECs. The Npr1 +/- mice presented an elevated systolic blood pressure and their vessels became insensitive to endothelial-dependent vasodilators. Further, vessels from Npr1 +/- mice increased Cdkn1a but decreased eNos expressions. These phenotypes were rescued by intravenously administrated 8-Br-cGMP and viral overexpression of human PKG, respectively. In conclusion, we demonstrate NPRA/PKG/AMPK as a novel and critical signaling axis in the modulation of endothelial cell senescence, vascular aging, and hypertension., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Age-related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age-dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. We generated a list of candidate genes that act as AMI regulators by performing a comprehensive analysis of RNAsequencing data from young and aged fly heads and genome-wide RNAi screening data to identify memory-regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. We identified the guanylyl cyclase β-subunit at 100B (gycβ) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycβ in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate-term memory (ITM) without apparent effects on memory acquisition. We also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age-related enhancement of the NO-sGC pathway causes memory impairment., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

DNA damage is a causative factor in ageing of the vasculature and other organs. One of the most important vascular ageing features is reduced nitric oxide (NO)soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling. We hypothesized that the restoration of NO-sGC-cGMP signaling with an sGC activator (BAY 54-6544) may have beneficial effects on vascular ageing and premature death in DNA repair-defective mice undergoing accelerated ageing. Eight weeks of treatment with a non-pressor dosage of BAY 54-6544 restored the decreased in vivo microvascular cutaneous perfusion in progeroid Ercc1 ∆/- mice to the level of wild-type mice. In addition, BAY 54-6544 increased survival of Ercc1 ∆/- mice. In isolated Ercc1 ∆/- aorta, the decreased endothelium-independent vasodilation was restored after chronic BAY 54-6544 treatment. Senescence markers p16 and p21, and markers of inflammation, including Ccl2, Il6 in aorta and liver, and circulating IL-6 and TNF-α were increased in Ercc1 ∆/- , which was lowered by the treatment. Expression of antioxidant genes, including Cyb5r3 and Nqo1, was favorably changed by chronic BAY 54-6544 treatment. In summary, BAY 54-6544 treatment improved the vascular function and survival rates in mice with accelerated ageing, which may have implication in prolonging health span in progeria and normal ageing., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a ruthenium complex nitric oxide (NO) donor that presents a nitrite in its moiety and has been shown to induce vasodilation in various arteries, as well as arterial pressure reduction with no changes in heart rate. Because vascular tone is highly dependent on the cytosolic calcium concentration ([Ca 2+ ]c), the current study aimed to investigate the effects of RuBPY on the intracellular mobilization of calcium stores of rat aortic vascular smooth muscle cells. Vascular reactivity experiments were performed in isolated aortic rings that were contracted with a high concentration of KCl or phenylephrine (Phe). Moreover, primary cultured vascular smooth muscle cells were used to measure [Ca 2+ ]c by confocal microscopy. The NO donor RuBPY decreased the [Ca 2+ ]c and reduced KCl and Phe-induced contractile responses. The selective inhibitor of sarco-endoplasmic Ca-ATPase (SERCA) with thapsigargin impaired the effect of RuBPY on Phe-induced contractile response. RuBPY also reduced caffeine-induced contraction, and the contraction dependent on the capacitive Ca 2+ influx. Therefore, our results suggest that NO released from RuBPY decreased [Ca 2+ ]c by calcium influx blockade and activation of guanylyl-cyclase-cGMP-GK pathway. These results indicate that RuBPY increases Ca 2+ storage in the sarcoplasmic reticulum by SERCA activation and also by capacitive Ca 2+ influx inhibition, which is dependent on the intracellular release of nitric oxide from this compound., (© 2022 John Wiley & Sons Australia, Ltd.)

Background The enterosalivary nitrate-nitrite-nitric oxide (NO 3 -NO 2 -NO) pathway generates NO following oral microbiota-mediated production of salivary nitrite, potentially linking the oral microbiota to reduced cardiometabolic risk. Nitrite depletion by oral bacteria may also be important for determining the net nitrite available systemically. We examine if higher abundance of oral microbial genes favoring increased oral nitrite generation and decreased nitrite depletion is associated with a better cardiometabolic profile cross-sectionally. Methods and Results This study includes 764 adults (mean [SD] age 32 [9] years, 71% women) enrolled in ORIGINS (Oral Infections, Glucose Intolerance, and Insulin Resistance Study). Microbial DNA from subgingival dental plaques underwent 16S rRNA gene sequencing; PICRUSt2 was used to estimate functional gene profiles. To represent the different components and pathways of nitrogen metabolism in bacteria, predicted gene abundances were operationalized to create summary scores by (1) bacterial nitrogen metabolic pathway or (2) biochemical product (NO 2 , NO, or ammonia [NH 3 ]) formed by the action of the bacterial reductases encoded. Finally, nitrite generation-to-depletion ratios of gene abundances were created from the above summary scores. A composite cardiometabolic Z score was created from cardiometabolic risk variables, with higher scores associated with worse cardiometabolic health. We performed multivariable linear regression analysis with cardiometabolic Z score as the outcome and the gene abundance summary scores and ratios as predictor variables, adjusting for sex, age, race, and ethnicity in the simple adjusted model. A 1 SD higher NO versus NH 3 summary ratio was inversely associated with a -0.10 (false discovery rate q =0.003) lower composite cardiometabolic Z score in simple adjusted models. Higher NH 3 summary score (suggestive of nitrite depletion) was associated with higher cardiometabolic risk, with a 0.06 (false discovery rate q =0.04) higher composite cardiometabolic Z score. Conclusions Increased net capacity for nitrite generation versus depletion by oral bacteria, assessed through a metagenome estimation approach, is associated with lower levels of cardiometabolic risk.

It has been postulated that the activation of NMDA receptors (NMDAr) and nitric oxide (NO) production in the hippocampus is involved in the behavioral consequences of stress. Stress triggers NMDAr-induced calcium influx in limbic areas, such as the hippocampus, which in turn activates neuronal NO synthase (nNOS). Inhibition of nNOS or NMDAr activity can prevent stress-induced effects in animal models, but the molecular mechanisms behind this effect are still unclear. In this study, cultured hippocampal neurons treated with NMDA or dexamethasone showed an increased of DNA methyltransferase 3b (DNMT3b) mRNA expression, which was blocked by pre-treatment with nNOS inhibitor n ω -propyl-l-arginine (NPA). In rats submitted to the Learned Helplessness paradigm (LH), we observed that inescapable stress increased DNMT3b mRNA expression at 1h and 24h in the hippocampus. The NOS inhibitors 7-NI and aminoguanidine (AMG) decreased the number of escape failures in LH and counteracted the changes in hippocampal DNMT3b mRNA induced in this behavioral paradigm. Altogether, our data suggest that NO produced in response to NMDAr activation following stress upregulates DNMT3b in the hippocampus., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Aim Alpha-thalassaemia is known to reduce intra-erythrocyte Hb S (sickle haemoglobin) concentration in sickle cell trait ( SCT) subjects. Because Hb S was shown to increase oxidative stress, the purpose of this study was to assess the effects of the coexistence of α-thalassaemia and SCT on oxidative stress markers and nitric oxide ( NO) metabolism after an acute physical exercise. Methods Forty subjects (age: 23.5 ± 2.21 years), SCT carriers ( Hb AS) or healthy subjects ( Hb AA), with (-α T) or without (- Nα T) an associated α-thalassaemia took part in the study. Plasma markers of oxidative stress [advanced oxidation protein products ( AOPP), protein carbonyl, malondialdehyde ( MDA) and nitrotyrosine], anti-oxidant defences and NO metabolism ( NOx) were measured at rest ( Trest), immediately following an incremental maximal exercise test ( Tex) and during recovery ( T1h, T2h and T24h). Results Malondialdehyde expressed as the percentage of changes from baseline was significantly higher in the Hb AS- Nα T compared with Hb AS-α T during recovery (+36.3 ± 14.1% vs. −1.8 ± 13.2% at T1h, P = 0.02; +36.6 ± 13.4% vs. −11.4 ± 12.5% at T2h, P = 0.004 and +24.1 ± 12.3% vs. −14.4 ± 11.5% at T24h, P = 0.02 in Hb AS- Nα T vs. Hb AS-α T). Compared with Hb AS- Nα T, Hb AS-α T had a higher NOx change from baseline at Tex (−23.4 ± 20.6% vs. +57.7 ± 19.3%, respectively; P = 0.005) and lower nitrotyrosine change from baseline at T1h (+7.2 ± 22.2% vs. +93.5%±29.3%, respectively; P = 0.04). Conclusion All these data suggest that the presence of α-thalassaemia may blunt the higher level of oxidative stress and the impaired bioavailability of NO observed in the SCT carriers. [ABSTRACT FROM AUTHOR]

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase ( GSNOR -⁄- ), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR -⁄- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR -⁄- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

Background Saphenous vein grafts (SVGs) are broadly used in coronary artery bypass grafting despite their inferior patency compared with arterial grafts. Recently, the no-touch technique (NT), in which an SVG is harvested with a pedicle of perivascular adipose tissue (PVAT) without conduit distension, was shown to improve long-term patency compared with conventional preparation (CV), wherein outer tissue is removed with distension. The NT was also reportedly associated with reduced atherosclerosis. Although endothelial damage provoked by conventional distension may underlie poor patency when CV is performed, the precise mechanisms underlying the salutary effects of the NT have been unclear. Methods and Results Residual SVGs prepared with CV (CV-SVGs) or NT (NT-SVGs) were obtained during coronary artery bypass grafting. Nitric oxide (NO 2 - /NO 3 - (NO x )) levels after 24 hours of tissue culture were quantified. The protein expression and localization were analyzed. The isometric force of SVG strips was measured. NT-SVGs showed superior NO x production to CV-SVGs. PVAT generated the majority of NO x in NT-SVGs. PVAT highly expressed arginosuccinate synthase 1, a rate-limiting enzyme in the molecular circuit for NO synthesis, thereby continuously providing the substrate for NO. A substantial level of endothelial NO synthase was also expressed in PVAT. Pharmacological inhibition of arginosuccinate synthase 1 or endothelial NO synthase significantly suppressed the NO x production in NT-SVGs. PVAT induced vasorelaxation through NO production, even in the endothelium-denuded SVG strips. Conclusions Preserving PVAT was predominantly involved in the superior NO x production in NT-SVGs. Since NO plays crucial roles in suppressing atherosclerosis, this mechanism may greatly contribute to the excellent patency in NT-SVGs.

Changes in intestinal nitric oxide metabolism are discussed to contribute for the development of intestinal barrier dysfunction in non-alcoholic fatty liver disease (NAFLD). To induce steatosis, female C57BL/6J mice were pair-fed with a liquid control diet (C) or a fat-, fructose- and cholesterol-rich diet (FFC) for 8 weeks. Mice received the diets ± 2.49 g L-arginine/kg bw/day for additional 5 weeks. Furthermore, mice fed C or FFC ± L-arginine/kg bw/day for 8 weeks were concomitantly treated with the arginase inhibitor N ω -hydroxy-nor-L-arginine (nor-NOHA, 0.01 g/kg bw). Liver damage, intestinal barrier function, nitric oxide levels and arginase activity in small intestine were assessed. Also, arginase activity was measured in serum from 13 patients with steatosis (NAFL) and 14 controls. The development of steatosis with beginning inflammation was associated with impaired intestinal barrier function, increased nitric oxide levels and a loss of arginase activity in small intestine in mice. L-arginine supplementation abolished the latter along with an improvement of intestinal barrier dysfunction; nor-NOHA attenuated these effects. In patients with NAFL, arginase activity in serum was significantly lower than in healthy controls. Our data suggest that increased formation of nitric oxide and a loss of intestinal arginase activity is critical in NAFLD-associated intestinal barrier dysfunction., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

In the central nervous system, the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling cascade has an established role in fine-tuning of synaptic transmission. In the present study, we asked which isoform of NO-sensitive guanylyl cyclase, NO-GC1 or NO-GC2, is responsible for generation of N-methyl-d-aspartate (NMDA)- and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-induced cGMP signals and which of the phosphodiesterases (PDEs) is responsible for degradation. To this end, we performed live cell fluorescence measurements of primary hippocampal neurons isolated from NO-GC isoform-deficient mice. Although both isoforms contributed to the NMDA- and AMPA-induced cGMP signals, NO-GC2 clearly played the predominant role. Whereas under PDE-inhibiting conditions the cGMP levels elicited by both glutamatergic ligands were comparable, NMDA-induced cGMP signals were clearly higher than the AMPA-induced ones in the absence of PDE inhibitors. Thus, AMPA-induced cGMP signals are more tightly controlled by PDE-mediated degradation than NMDA-induced signals. In addition, these findings are compatible with the existence of at least two different pools of cGMP in both of which PDE1 and PDE2-known to be highly expressed in the hippocampus-are mainly responsible for cGMP degradation. The finding that distinct pools of cGMP are equipped with different amounts of PDEs highlights the importance of PDEs for the shape of NO-induced cGMP signals in the central nervous system., (© 2021 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Tetragonisca angustula honey was fractioned in a SiO 2 column to furnish three fractions (A-C) in which four hydroxycinnamic acid-Spermidine amides (HCAAs), known as N', N″, N‴-tris-p-coumaroyl spermidine, N', N″-dicaffeoyl, N‴-coumaroyl spermidine, N', N″, N‴-tris-caffeoyl spermidine and N', N″-dicaffeoyl and N‴-feruloyl spermidine were identified in the fractions B and C by electrospray ionization tandem mass spectrometry. A primary culture model previously infected with Neospora caninum (72 h) was used to evaluate the honey fractions (A-C) for two-time intervals: 24 and 72 h. Parasitic reduction ranged from 38% on fraction C (12.5 µg/ml), after 24 h, to 54% and 41% with fractions B and C (25 µg/ml) after 72 h of treatment, respectively. Additionally, HCAAs did not show any cell toxicity for 24 and 72 h. For infected cultures (72 h), the active fractions B (12.5 µg/ml) and C (25 µg/ml) decreased their NO content. In silico studies suggest that HCAAs may affect the parasite's redox pathway and improve the oxidative effect of NO released from infected cells. Here, we presented for the first time, that HCAAs from T. angustula honey have the potential to inhibit the growth of N. caninum protozoa., (© 2021 John Wiley & Sons Ltd.)

The balance between endothelial nitric oxide (NO) synthase (eNOS) activation and production of reactive oxygen species (ROS) is very important for NO homeostasis in liver sinusoidal endothelial cells (LSECs). Overexpression of cyclooxygenase-2 (COX-2), a major intravascular source of ROS production, has been observed in LSECs of cirrhotic liver. However, the links between low NO bioavailability and COX-2 overexpression in LSECs are unknown. This study has confirmed the link between low NO bioavailability and COX-2 overexpression by COX-2-dependent PGE2-EP2-ERK1/2-NOX1/NOX4 signalling pathway in LSECs in vivo and in vitro. In addition, the regulation of COX-2-independent LKB1-AMPK-NRF2-HO-1 signalling pathway on NO homeostasis in LSECs was also elucidated. The combinative effects of celecoxib on diminishment of ROS via COX-2-dependent and COX-2-independent signalling pathways greatly decreased NO scavenging. As a result, LSECs capillarisation was reduced, and endothelial dysfunction was corrected. Furthermore, portal hypertension of cirrhotic liver was ameliorated with substantial decreasing hepatic vascular resistance and great increase of portal blood flow. With the advance understanding of the mechanisms of LSECs protection, celecoxib may serve as a potential therapeutic candidate for patients with cirrhotic portal hypertension., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Aim: The optimal exercise intensity to improve endothelial function remains unclear, as well as whether the addition of hypoxia could potentiate this function. Therefore, the aim of this study was to compare the effects of different exercise intensities in normoxia and hypoxia on vascular reactivity and nitric oxide (NO) bioavailability in mice., Methods: C57BL/6 mice underwent treadmill running three times per week, for 4 weeks at either low, maximal or supramaximal intensity in normoxia or hypoxia (inspire oxygen fraction = 0.13). Vascular reactivity and expression of genes and proteins involved in NO production/bioavailability were assessed in aorta using isolated vessel tension experiments, RT-qPCR and western blot, respectively. Circulating NO metabolites and pro-/antioxidant markers were measured., Results: Hypoxic exercise improved both acetylcholine-induced vasorelaxation and phenylephrine-induced vasoconstriction compared to normoxic exercise, independently of intensity. In hypoxia, a higher acetylcholine-induced vasorelaxation was observed with high intensities (supramaximal and maximal) compared to low intensity. Exercise protocols modulated endothelial nitric oxide synthase (eNOS) and α1-adrenergic receptor (α 1 -AR) mRNA level, but not superoxide dismutase 3 (SOD3) and p47phox. No significant differences were observed for protein expression of α 1 -AR, total eNOS, phosphorylated eNOS, SOD isoforms and p47phox. However, plasma SOD and catalase activities were significantly increased in hypoxic supramaximal compared to hypoxic low intensity, while concentration of nitrotyrosine significantly decreased. The latter was also observed in hypoxic maximal and supramaximal compared to the same intensities in normoxia., Conclusion: Hypoxic high-intensity exercise increases NO bioavailability and improves vascular function, opening promising clinical perspectives for cardiovascular disease prevention., (© 2021 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

In the present study, we aim to investigate the effects of aerobic physical training on perivascular adipose tissue (PVAT)-induced microvascular dysfunction of the femoral artery in obese mice. Microvascular reactivity was evaluated in control sedentary (c-SD), obese sedentary (o-SD) and obese trained (o-TR) male mice (C57BL6/JUnib), in the absence (PVAT-) or the presence (PVAT+) of femoral artery PVAT. We also analyzed protein expression, vascular nitric oxide (NO) production and reactive oxygen species (ROS) generation in PVAT. The blood glucose, triglycerides and total cholesterol levels were increased in the o-SD group, when compared with the c-SD group. The maximal responses and the potency to acetylcholine (ACh) were decreased in PVAT+ compared with PVAT- rings in the o-SD group, accompanied by a decrease in vascular protein expression of peNOS Ser1177 , Cu/Zn-SOD, leptin receptor (Ob-R) and adiponectin receptor (AdipoR1). The protein expression of leptin increased and that of adiponectin decreased in PVAT. Additionally, vascular NO production was reduced and ROS generation was enhanced in PVAT in the o-SD group. Aerobic exercise training was effective for normalizing ACh relaxation response, vascular NO production and ROS generation in the o-TR group. It partially re-established the vascular protein expression of peNOS Ser1177 and the PVAT leptin; normalized the vascular Cu/Zn-SOD and AdipoR1 protein expressions. In obese sedentary mice, the presence of PVAT is involved in the process of microvascular dysfunction of the femoral artery in a pathway associated with increased inflammation and ROS generation. The aerobic exercise training normalized the vascular response, the NO production and/or bioavailability and oxidative stress, with improved vascular expressions of Cu/Zn-SOD, peNOS ser1177 , and AdipoR1., (© 2021 John Wiley & Sons Australia, Ltd.)

Vascular diseases are caused by endothelial dysfunction due to inflammation. On endothelial injury, the expression of extracellular matrix (ECM) is enhanced and nitric oxide (NO) bioavailability becomes deficient. This condition affects endothelial metabolism and leads to vascular destruction. The aim of this investigation was to determine whether substance P (SP) is able to protect the endothelium against inflammatory stress. To this end, aortic endothelial cells were pre-treated with SP, followed by tumour necrosis factor α (TNF-α), and cellular responses were evaluated using a combination of cell biology and quantification assays, as well as western blot analyses. Our results show that TNF-α enhanced ECM expression and reduced NO production within 4 hours, promoting immune cell adhesion to the endothelium and monocyte chemoattractant protein-1 (MCP-1) secretion from aortic smooth muscle cells. However, SP treatment ameliorated TNF-α-induced endothelial impairment by maintaining low ECM levels. Our data suggest that this protective effect is mediated by Akt activation and NO-enriched conditions. The inhibition of aortic endothelial cell injury by SP also reduced MCP-1 production in aortic smooth muscle cells. Together, our data indicate that SP can protect aortic endothelial and smooth muscle cells from inflammatory injury, which suggests that SP may prevent cardiovascular disease., (© 2021 John Wiley & Sons Australia, Ltd.)

Background The vascular pharmacodynamics of anthocyanins is only partially understood. To examine whether the anthocyanin-induced vasorelaxation is related to membrane estrogen receptor activity, the role of ERα or GPER antagonism was ascertained on anthocyanins or 17-β estradiol-(E2) induced vasodilatations and NO production. Methods and Results The rat arterial mesenteric bed was perfused with either anthocyanins or corresponding 3-O-glycosides, or E2, to examine rapid concentration-dependent vasorelaxations. The luminally accessible fraction of NO in mesenteric perfusates before and after anthocyanins or E2 administration was quantified. Likewise, NO-DAF signal detected NO production in primary endothelial cells cultures incubated with anthocyanins or E2 in the absence and presence of ERα (ICI 182,780) or GPER (G-36) selective antagonists. Anthocyanins or corresponding glycosides elicited, within minutes, vasodilation with nanomolar potencies; half maximal anthocyanin response reached 50% to 60% efficacy, in contrast to acetylcholine. The vasorelaxation is of rapid onset and exclusively endothelium-dependent; NOS inhibition annulled the vasorelaxation. The delphinidin vascular response was not modified by 100 nmol/L atropine but significantly attenuated by joint application of ICI plus G-36 (52±4.6 versus 8.5±1.5%), revealing the role of membrane estrogen receptors. Moreover, the anthocyanin or E2-induced NO production was antagonized up to 70% by these antagonists. NO-DAF signal elicited by anthocyanins was annulled by NOS inhibition or by ICI plus G-36 addition. Conclusions The biomedical effect of anthocyanins or 3-O-glycosylates derivatives contained in naturally purple-colored foods or berries is due to increased NO production, and not to the phytochemical's antioxidant potential, highlighting the nutraceutical role of natural products in cardiovascular diseases.

The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca 2+ -permeable nonselective cation channel, is widely distributed in the circulatory system, particularly in vascular endothelial cells (ECs) and smooth muscle cells (SMCs). The TRPV4 channel is activated by various endogenous and exogenous stimuli, including shear stress, low intravascular pressure, and arachidonic acid. TRPV4 has a role in mediating vascular tone and arterial blood pressure. The activation of the TRPV4 channel induces Ca 2+ influx, thereby resulting in endothelium-dependent hyperpolarization and SMC relaxation through SK Ca and IK Ca activation on ECs or through BK Ca activation on SMCs. Ca 2+ binds to calmodulin, which leads to the production of nitric oxide, causing vasodilation. Furthermore, the TRPV4 channel plays an important role in angiogenesis and arteriogenesis and is critical for tumor angiogenesis and growth, since it promotes or inhibits the development of various types of cancer. The TRPV4 channel is involved in the active growth of collateral arteries induced by flow shear stress, which makes it a promising therapeutic target in the occlusion or stenosis of the main arteries. In this review, we explore the role and the potential mechanism of action of the TRPV4 channel in the regulation of vascular tone and in the induction of neovascularization to provide a reference for future research., (© 2021 John Wiley & Sons Ltd.)