Endothelial Nitric Oxide Synthase Research Articles

Blood circulation effect of fermented citrus bioconversion product (FCBP) in EA.hy926 endothelial cells and high-fat diet-fed mouse model

Background: The escalating global burden of cardiovascular diseases, largely driven by unhealthy lifestyle choices and dietary patterns, has intensified the search for effective and safe interventions. With current treatments often marred by significant side effects, the exploration of natural compounds such as flavonoids presents a compelling alternative. Objective: This study investigated the effects of fermented citrus bioconversion product (FCBP), a fermented citrus bioflavonoid, on various markers of cardiovascular health in the context of a high-fat diet. Design: In vivo, a high-fat diet-induced mouse model was used to assess the effects of FCBP on body weight, serum nitric oxide (NO) levels, activated partial thromboplastin time (aPTT), phosphatidylserine (PS) exposure on red blood cells, and the expression of inflammatory markers Intercellular Adhesion Molecule (ICAM)-1 and Vascular Cell Adhesion Molecule (VCAM)-1 in the thoracic aorta. In vitro, EA.hy926 endothelial cells were used to evaluate the compound’s effects on cell viability, NO production, endothelial nitric oxide synthase (eNOS) expression, and cell adhesion molecule (CAM) levels to further understand the mechanisms behind the in vivo findings. Results: In vivo, FCBP supplementation led to a dose-dependent reduction in weight gain, a significant decrease in serum NO levels at 10 mg/kg, and reduced ICAM-1 and VCAM-1 expressions in the thoracic aorta, indicating anti-inflammatory properties. PS exposure on red blood cells was also reduced, suggesting decreased procoagulant activity, while aPTT remained unchanged. In vitro, FCBP was non-cytotoxic to endothelial cells, showed a trend toward increased NO production and eNOS expression, and reduced the expression of ICAM-1 and VCAM-1, supporting its potential anti-inflammatory effects. Conclusions: FCBP demonstrates potential as a bioactive compound for managing cardiovascular health by reducing inflammation, mitigating weight gain, and influencing blood circulation-related parameters under high-fat diet conditions. Further studies, including diverse models and human trials, are warranted to elucidate its mechanisms and compare its efficacy with established cardiovascular therapeutics.

Microvascular insulin resistance with enhanced muscle glucose disposal in CD36 deficiency.

Microvascular dysfunction contributes to insulin resistance. CD36, a fatty acid transporter and modulator of insulin signalling, is abundant in microvascular endothelial cells. Humans carrying the minor allele (G) of CD36 coding variant rs3211938 have 50% reduced CD36 expression and show endothelial dysfunction. We aimed to determine whether G allele carriers have microvascular resistance to insulin and, if so, how this affects glucose disposal. Our multi-disciplinary approach included hyperinsulinaemic-euglycaemic clamps in Cd36-/- and wild-type mice, and in individuals with 50% CD36 deficiency, together with control counterparts, in addition to primary human-derived microvascular endothelial cells with/without CD36 depletion. Insulin clamps showed that Cd36-/- mice have enhanced insulin-stimulated glucose disposal but reduced vascular compliance and capillary perfusion. Intravital microscopy of the gastrocnemius showed unaltered transcapillary insulin flux. CD36-deficient humans had better insulin-stimulated glucose disposal but insulin-unresponsive microvascular blood volume (MBV). Human microvascular cells depleted of CD36 showed impaired insulin activation of Akt, endothelial NO synthase and NO generation. Thus, in CD36 deficiency, microvascular insulin resistance paradoxically associated with enhanced insulin sensitivity of glucose disposal. CD36 deficiency was previously shown to reduce muscle/heart fatty acid uptake, whereas here we showed that it reduced vascular compliance and the ability of insulin to increase MBV for optimising glucose and oxygen delivery. The muscle and heart respond to these energy challenges by transcriptional remodelling priming the tissue for insulin-stimulated glycolytic flux. Reduced oxygen delivery activating hypoxia-induced factors, endothelial release of growth factors or small intracellular vesicles might mediate this adaptation. Targeting NO bioavailability in CD36 deficiency could benefit the microvasculature and muscle/heart metabolism. Clinicaltrials.gov NCT03012386 DATA AVAILABILITY: The RNAseq data generated in this study have been deposited in the NCBI Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo/ ) under accession code GSE235988 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235988 ).

The Adhesion GPCR ADGRL2/LPHN2 Can Protect Against Cellular and Organismal Dysfunction

The most common trigger of sepsis and septic shock is bacterial lipopolysaccharide (LPS). Endothelial cells are among the first to encounter LPS directly. Generally, their function is closely linked to active endothelial NO Synthase (eNOS), which is significantly reduced under septic conditions. LPS treatment of endothelial cells leads to their activation and apoptosis, resulting in loss of integrity and vascular leakage, a hallmark of septic shock. Hence, therapies that prevent endothelial leakage or restore the endothelial barrier would be invaluable for patients. Adhesion GPCRs (aGPCRs) have been largely overlooked in this context, although particularly one of them, ADGRL2/LPHN2, has been implicated in endothelial barrier function. Our study shows that overexpression of ADGRL2 protects endothelial cells from LPS-induced activation, apoptosis, and impaired migration. Mechanistically, ADGRL2 preserves eNOS activity by shifting its binding from Caveolin-1 to Heat Shock Protein 90. Furthermore, ADGRL2 enhances antioxidative responses by increasing NRF2 activity. Notably, we found that this function may be evolutionarily conserved. In the absence of lat-2, a homolog of ADGRL2 in Caenorhabditis elegans, worms show higher ROS levels and altered stress response gene expression. Additionally, lat-2 mutants have a significantly reduced lifespan, altogether indicating a protective role of ADGRL2 against oxidative stress across species.

Peroxisome proliferator-activated receptor agonists as an adjuvant for cancer therapy: A review

Cancer is now one of the leading diseases responsible for the highest mortality rates worldwide. Cancer treatment is extremely intricate and is often associated with less targeted effects and more side effects. In the last few years, two significant revolutions in cancer treatment have altered treatment paradigms: immuno-oncology and the pursuit of actionable changes in oncogene-driven cancers. Significant obstacles continue to exist in both areas of cancer treatment. Next-generation sequencing technologies for molecular prescreening in clinical research are advancing, but their widespread clinical use is hindered by challenges related to the clinical interpretation of large genomic data. Moreover, cancer mono-chemotherapy is associated with issues such as inadequate efficacy, drug resistance, and systemic toxicity. It is not possible to administer cytotoxic drugs limitlessly. Combination therapy was developed in response to this circumstance, with the expectation of achieving high therapeutic efficacy at lower dosages of medication. Peroxisome proliferator-activated receptors (PPARs) are a group of essential lipid sensors and metabolic pathway regulators. In addition, they possess the ability to modulate immunity, inflammation, and endothelial nitric oxide synthase activation. Alongside their well-established functions, new insights into the roles of PPAR agonists in cancer research are emerging. After reviewing current research, it is evident that PPAR modulators have significant potential for managing cancer. This review aims to consolidate the functions of PPAR ligands alongside other cancer therapies. We provide a comprehensive perspective on the applicability of PPAR ligands in cancer treatment as an adjuvant.

Shock wave-pretreated ADMSCs of cell-sheet scaffold (CSS) patched on left ventricular wall (LVW) inhibited LVW remodeling in mini-pig MI ---role CSS on counteracting Laplace's Law of LVW stress: Experimental study.

We investigated whether shock wave (SW)-pretreated autologous adipocyte-derived mesenchymal stem cells (ADMSCs) seeded in the cell-sheet scaffold (CSS) could inhibit left ventricular (LV) remodeling and improve LV ejection fraction (LVEF) in old myocardial infarction (MI). Mini-pigs (n=20) were divided into group 1 (sham-operated control), group 2 (old MI), group 3 (old MI + autologous ADMSCs/1.0×107 in CSS on LV myocardium), and group 4 [old MI + SW (0.12mJ/mm2 for total 140 shots)-pretreated ADMSCs in CSS on LV myocardium]. Treatments started on day 28 after MI induction. In vivo and in vitro studies were conducted. Cell viability/relative mitochondria DNA expression/mitochondrial cytochrome C/adenosine triphosphate concentration in ADMCSs and protein expressions of angiogenesis factors (vascular endothelial growth factor [VEGF]/stromal cell-derived factor-1 [SDF-1])/mitochondrial respiratory chain complexes I-IV/oxygen consumption rate were higher in group 4 than in group 3 (P<0.001). By day 180, LVEF and small vessel numbers in the peri-infarct or infarct area were highest in group 1, lowest in group 2, and significantly lower in group 3 than in group 4. In contrast, the LV dimension was opposite to the pattern of change in LVEF in all groups (P<0.0001). The basal/middle/apical infarct and fibrotic areas were inversely related to LVEF in all groups (all P<0.0001). Protein levels of angiogenetic markers (SDF-1α/C-X-C chemokine receptor type 4/VEGF/angiopoietin-1) were significantly and persistently increased from groups 1 to 4. In contrast, protein levels of endothelial-cell markers (von Willebrand factor or endothelial nitric oxide synthase) showed an identical pattern to LVEF in all groups (all P<0.0001). SW pretreatment of ADMSCs seeded in CSS offered significant benefits in preserving LV performance and ameliorating LV remodeling in mini-pigs with old MI.

β3-adrenergic agonist counters oxidative stress and Na+-K+ pump inhibitory S-glutathionylation of placental cells: Implications for preeclampsia.

Oxidative stress from placental ischemia/reperfusion and hypoxia/reoxygenation (H/R) in preeclampsia is accompanied by Na+-K+ pump inhibition and S-glutathionylation of its β1 subunit (GSS-β1), a modification that inhibits the pump. β3-adrenergic receptor (β3-AR) agonists can reverse GSS-β1. We examined effects of the agonist CL316,243 on GSS-β1 and sources of H/R-induced oxidative stress in immortalized first trimester human trophoblast (HTR-8/SVneo) and freshly isolated placental explants from normal term pregnancies. H/R increased GSS-β1 and, reflecting compromised α1/β1 subunit interaction, it reduced α1/β1 pump subunit co-immunoprecipitation. H/R increased p47phox/p22phox NADPH oxidase subunit co-immunoprecipitation reflecting membrane translocation of cytosolic p47phox that is needed to activate NADPH oxidase. Fluorescence of O2•--sensitive dihydroethidium increased in parallel. H/R increased S-glutathionylation of endothelial nitric oxide synthase (GSS-eNOS) that uncouples NO synthesis towards synthesis of O2•- and reduced trophoblast migration. Oxidative stress induced by tumor necrosis factor α (TNF-α) increased soluble fms-like tyrosine kinase receptor 1 (sFlt-1) trophoblast release, a marker of preeclampsia, and reduced trophoblast integration into endothelial cellular networks. CL316,243 eliminated H/R-induced GSS-β1 and decreases of α1/β1 subunit coimmunoprecipitation, eliminated NADPH oxidase activation and increases in GSS-eNOS, restored trophoblast migration, eliminated increased sFlt-1 release and restored trophoblast integration in endothelial cell networks. H/R induced GSS-β1, α1/β1 subunit co-immunoprecipitation and NADPH oxidase activation of placental explants reflected effects of H/R for trophoblasts and CL316,243 eliminated these changes. We conclude a β3-AR agonist counters key pathophysiological features of preeclampsia in vitro. β3 agonists already in human use for another purpose are potential candidates for re-purposing to treat preeclampsia.

Modulatory potentials of nitric oxide in obesity-induced endothelial dysfunction

Obesity is caused by a significant increase in adipose tissue due to excessive caloric intake that exceeds energy expenditure. Obesity has emerged as a significant public health issue in both poor resource and economic developed nations, owing to its increased incidence and links to chronic diseases, such osteoarthritis, hypertension, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases (CVDs). It is believed that uncoupled eNOS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, lipoxygenase, cyclooxygenase, microsomal P-450 enzymes, and pro-oxidant heme molecules are responsible for the drastic decrease in NO production and the relative increase in reactive oxygen species (ROS) secretion, which are considered as the molecular mechanisms associated with obesity-induced endothelial dysfunction. Vascular endothelium defines a monolayer of cells, which lies between the vascular smooth muscle cells (VSMCs) and the vessel lumen. It performs a number of protective actions facilitated by the release of nitric oxide (NO), a soluble gas that is generated in endothelial cells by the amino acid L-arginine through the action of endothelial nitric oxide synthase (eNOS). The maintenance of vascular homeostasis is facilitated by various biological actions of NO, such as modulation of vascular dilator tone, control of local cell growth, and protection of the endothelium from the harmful effects of cellular components in blood circulation, while maintaining normal endothelial functions. Decreased peripheral arterial endothelium-dependent dilation (EDD) in response to mechanical (intravascular shear) or chemical (acetylcholine) stimuli suggests that decreased NO bioavailability has been recognised as major molecular mechanisms associated with the progress and development of obesity-induced endothelial dysfunction. In this review, the modulatory effects of NO in obesity-induced endothelial dysfunction will be discussed.

Dapagliflozin mitigates cellular stress and inflammation through PI3K/AKT pathway modulation in cardiomyocytes, aortic endothelial cells, and stem cell-derived β cells

Dapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is well-recognized for its therapeutic benefits in type 2 diabetes (T2D) and cardiovascular diseases. In this comprehensive in vitro study, we investigated DAPA’s effects on cardiomyocytes, aortic endothelial cells (AECs), and stem cell-derived beta cells (SC-β), focusing on its impact on hypertrophy, inflammation, and cellular stress. Our results demonstrate that DAPA effectively attenuates isoproterenol (ISO)-induced hypertrophy in cardiomyocytes, reducing cell size and improving cellular structure. Mechanistically, DAPA mitigates reactive oxygen species (ROS) production and inflammation by activating the AKT pathway, which influences downstream markers of fibrosis, hypertrophy, and inflammation. Additionally, DAPA’s modulation of SGLT2, the Na+/H + exchanger 1 (NHE1), and glucose transporter (GLUT 1) type 1 highlights its critical role in maintaining cellular ion balance and glucose metabolism, providing insights into its cardioprotective mechanisms. In aortic endothelial cells (AECs), DAPA exhibited notable anti-inflammatory properties by restoring AKT and phosphoinositide 3-kinase (PI3K) expression, enhancing mitogen-activated protein kinase (MAPK) activation, and downregulating inflammatory cytokines at both the gene and protein levels. Furthermore, DAPA alleviated tumor necrosis factor (TNFα)-induced inflammation and stress responses while enhancing endothelial nitric oxide synthase (eNOS) expression, suggesting its potential to preserve vascular function and improve endothelial health. Investigating SC-β cells, we found that DAPA enhances insulin functionality without altering cell identity, indicating potential benefits for diabetes management. DAPA also upregulated MAFA, PI3K, and NRF2 expression, positively influencing β-cell function and stress response. Additionally, it attenuated NLRP3 activation in inflammation and reduced NHE1 and glucose-regulated protein GRP78 expression, offering novel insights into its anti-inflammatory and stress-modulating effects. Overall, our findings elucidate the multifaceted therapeutic potential of DAPA across various cellular models, emphasizing its role in mitigating hypertrophy, inflammation, and cellular stress through the activation of the AKT pathway and other signaling cascades. These mechanisms may not only contribute to enhanced cardiac and endothelial function but also underscore DAPA’s potential to address metabolic dysregulation in T2D.Graphical abstract

Sirt5 as a mediator of endothelial and vascular dysfunction: potential therapeutic target in aging-related vascular diseases

Abstract Background Aging is one of the most dominant cardiovascular (CV) risk factors. Sirt5 belongs to the lifespan-regulating sirtuin superfamily as a protein deacylase. However, its regulatory effects on vascular function and aging are yet unclear. Purpose We therefore aimed to investigate the roles of Sirt5 on endothelial and vascular function during aging, decipher the underlying mechanisms, and to explore its potential as a therapeutic target through in vivo and in vitro studies. Methods Aortic expression of Sirt5 were studied in C5BL/6 wild type (WT) mice during aging, followed by vascular functional and structural assessment in WT mice and Sirt5-overexpressing mice (Sirt5/Tg) using the established myograph system and histological staining respectively. Furthermore, the regulatory role of Sirt5 on endothelial function was investigated in senescent primary human aortic endothelial cells (HAECs, passages 13-15) by studying its interference with endothelial nitric oxide synthase (eNOS) expression and activity in knock-down experiments using siRNA. The therapeutic potential of Sirt5 inhibition was explored at the tissue level in myograph experiments. Results Aged WT mice had increased level of Sirt5 in aorta compared to the young ones. Overexpression of Sirt5 in vivo led to significantly reduced aortic endothelial-dependent relaxation and displayed thicker collagen deposition in the adventitia compared to aged WT controls. The reduction of vascular relaxing response in Sirt5/Tg mice was prevented by an addition of eNOS inhibitor (L-name), indicating that Sirt5 regulated vascular function through the eNOS pathway. In senescent HAECs, knock-down of Sirt5 markedly induced total eNOS expression and increased its activity (phospho-eNOS Ser1177), further confirming the regulatory role of Sirt5 on eNOS pathway. In line with the above, aorta from aged WT mice displayed improved endothelial function after pharmacological inhibition of Sirt5. Conclusion Vascular Sirt5 expression increases with age and contributes to aging-related endothelial dysfunction by downregulating the eNOS pathway. Thus, Sirt5 inhibition may represent a novel therapeutic approach to maintain vascular function during aging. Ongoing studies will investigate Sirt5 silencing in-vivo using RNA interference for its therapeutic potential to prevent aging-related vascular dysfunction.

Lipoprotein(a) regulates vascular redox state and inflammatory response in patients with coronary artery disease

Abstract Background Circulating Lipoprotein(a) (Lp(a)) is associated with cardiovascular disease, and over 90% of its variation in plasma is genetically determined. However, neither the causal nature of this association nor the underlying mechanisms are fully documented. Purpose We explored the hypothesis that Lp(a) triggers atherogenesis by dysregulating vascular redox-sensitive inflammatory state. Methods 1027 patients with advanced coronary artery disease (CAD) undergoing cardiac surgery were genotyped and a genetic signature (LPA) determining Lp(a) levels was generated. RNA-sequencing and vascular superoxide (O2.-) measurements were performed in internal mammary arteries (IMA), and the contribution of NADPH-oxidases and uncoupled endothelial nitric oxide synthase (eNOS) were determined. Patients were followed up for a median of 5.07 years. Results A genetic screening identified 7 SNPs (LPA) that were associated with increased Lp(a) plasma levels (A). Patients in the alternative LPA variant group, as well as patients with higher plasma Lp(a) levels, had significantly higher basal arterial O2.-. Patients with diabetes had significantly lower plasma Lp(a) compared to non-diabetics and the association between plasma Lp(a) and arterial O2.- was driven by non-diabetic patients. The effect of Lp(a) on vascular redox state was primarily due to eNOS uncoupling, resulting from reduced vascular tetrahydrobiopterin (BH4) bioavailability. Indeed, patients in the alternative LPA variant group or with higher plasma Lp(a) levels had a greater arterial eNOS-derived (LNAME-inhibitable) O2.- production (B,C). Levels of eNOS essential cofactor BH4 (defined by the ratio of plasma BH4 to its oxidation product BH2) were lower in patients with higher plasma Lp(a) levels (D). There was no significant impact of Lp(a) variability on vascular NADPH oxidase-derived O2.-. RNA sequencing of arterial tissue revealed dysregulation of nitrosative and inflammatory signalling in patients with high Lp(a), although there was no association with systemic biomarkers of inflammation (i.e. hsCRP) or oxidative stress (i.e. malondialdehyde). Finally, both LPA and high plasma Lp(a) were associated with elevated risk for cardiovascular mortality (E,F). When the cox regression models were additionally corrected for arterial O2.- production, both the models for the LPA genetic signature (HR[95% CI]=2.053[0.180-23.428], p=0.563) and plasma Lp(a) (HR[95% CI]=1.700[0.325-8.885], p=0.529) lost significance. This implies that redox-sensitive inflammatory signalling may be a link between Lp(a) and cardiovascular risk. All above associations were independent from plasma ApoB. Conclusions This study demonstrates for the first time that a genetically determined increase in plasma Lp(a) results in dysregulated vascular redox/nitrosative signalling in patients with atherosclerosis, revealing new therapeutic opportunities in cardiovascular prevention.

The effect of CD38 inhibition in age-associated vascular dysfunction

Abstract Introduction Aging is characterized by age-related vascular dysfunction, mainly caused by endothelial dysfunction and arterial stiffness. Levels of nicotinamide adenine dinucleotide (NAD) decrease with aging and accordingly, NAD is thought to be involved in the progression of age-related vascular dysfunction. Recent studies reported the involvement of CD38 in NAD regulation; indeed, its inhibition significantly prolongs the lifespan of progeroid and aged mice. However, whether CD38 is involved in the progression of age-related vascular dysfunction remains to be investigated. Methods CD38 expression was investigated in aortic homogenates of young (12-16 weeks) and aged (18-24 months) C57BL/6 wild-type mice, as well as in vitro in young (passage 5-7) and senescent (passage 14-15) primary human aortic endothelial cells (HAECs). Ex vivo tension myograph experiments were performed on thoracic aortas isolated from aged C57BL/6 mice (22-24 months old) pre-incubated for 1 hour with CD38-specific inhibitor, 78c, or DMSO as control. The effect of CD38 inhibition was also observed in vitro in senescent HAECs. Results A significant upregulation of CD38 expression was detected in the aortas of aged mice compared to the young ones as well as in senescent HAECs compared to young cells. Ex vivo tension myograph experiments showed a significant increase in endothelium-dependent relaxation responses to acetylcholine upon CD38 inhibition. The improvement of endothelial function was prevented by the addition of endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME), but not by the COXs inhibitor, indomethacin, indicating that the observed effect achieved by CD38 inhibition is eNOS dependent. The involvement of the eNOS pathway was further supported by the in vitro finding in HAECs of increased eNOS activation upon CD38 inhibition. Conclusion The herein-reported data suggests an involvement of CD38 in the development of age-related endothelial dysfunction. Further studies are ongoing to investigate the underlying molecular mechanisms as well as the effect of long-term CD38 inhibitor supplementation on the progression of age-related vascular dysfunction.

Odoratin balances ROS/NO through EZH2/PPARγ signalling to improve myocardial fibrosis.

Myocardial fibrosis is a critical concern in clinical medicine. This study explores the potential of odoratin as a treatment for myocardial fibrosis and investigates its underlying mechanisms. In vitro experiments involved stimulating primary mouse cardiomyocytes with TGF-β1, followed by odoratin treatment, to assess levels of reactive oxygen species (ROS) and nitric oxide (NO). In vivo, a mouse model of myocardial fibrosis was established using abdominal aortic constriction (AAC) and treated with odoratin. ROS and NO levels in myocardial tissue were then evaluated. Immunofluorescence and Western blotting analysis showed that odoratin reduced excess ROS, enhanced NO production and decreased fibrosis-related protein expression in vitro. In vivo, odoratin significantly improved cardiac function, reduced ROS, increased NO levels and mitigated fibrosis in AAC-induced mice. Both in vitro and in vivo, odoratin inhibited the expression of NADPH oxidase 4 and EZH2, while promoting the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS) and PPARγ. The anti-fibrotic effects of odoratin were reversed by PPARγ antagonism, and EZH2 overexpression diminished PPARγ activation by odoratin. These findings suggest that odoratin may combat myocardial fibrosis by balancing ROS and NO through PPARγ activation, with EZH2 inhibition likely playing a key regulatory role.

Orexin A protects against cerebral ischemia-reperfusion injury by enhancing reperfusion in ischemic cortex via HIF-1α-ET-1/eNOS pathway

The purpose of this study was to investigate the protective effect and underlying mechanism of orexin A on cerebral ischemia-reperfusion injury, specifically through vasodilation mediated by the hypoxia inducible factor-1α (HIF-1α)-Endothelin-1(ET-1)/endothelial nitric oxide synthase (eNOS) pathway. A model of middle cerebral artery occlusion was established in both wild-type SD rats with exogenous orexin A intervention and in orexin A transgenic rats. Neurological deficit scores and cerebral infarction areas were assessed, and ischemic cortical blood flow was monitored. Gene and protein expression levels of HIF-1α, HIF-2α, ET-1, and three types of NOS were detected using real-time RT-qPCR and Western blot analysis, respectively. Additionally, nitric oxide (NO) levels in the cortex were analyzed through biochemical detection methods. Orexin A demonstrated a protective effect by reducing cerebral infarction and improving neurological deficits, which was achieved by increasing cortical blood flow during reperfusion. This protective mechanism was associated with upregulated HIF-1α expression, downregulated ET-1 expression, upregulated eNOS expression, and increased NO production. This study demonstrates the protective effect of orexin A on cerebral ischemia-reperfusion injury, achieved by regulating the release of vasomotor substances to enhance cortical blood flow during reperfusion. These findings suggest that orexin A may represent a potential therapeutic strategy for ischemic stroke.

Association of SIRT1 (rs7069102) Gene polymorphism with premature myocardial infarction in young Egyptian patients

Abstract Background Premature myocardial infarction (PMI) is one of the most pressing global issues in modern cardiology. Recently, the incidence of PMI has gradually increased. Researches found that genetics is a major contributor in its development. SIRT1, an extremely conserved class III NAD-dependent deacetylase, has been linked to numerous cardiovascular disorders and engaged in a number of cellular functions. This work investigated the association between SIRT1 SNP rs7069102 in Egyptian patients ≤ 40 years old with premature ST-elevation Myocardial infarction (STEMI). Methods This cross sectional, single-center study included patients younger than 40 with STEMI (PMI group, n = 140) and a control group (n = 140) of healthy subjects of comparable age. In addition to clinical examination and standard tests, all participants underwent echocardiography, coronary angiography, SIRT1 (rs7069102) genotyping, and nitric oxide assay. Results The risk for PMI was increased in CG or CC genotype carriers of SIRT1 gene rs7069102 (OR: 3.93, 95% Cl: 2.25–6.86), as did carriers of the C allele (OR: 2.26, 95% Cl: 1.65–3.86). In the PMI group, endothelial nitric oxide synthase (eNOS) was significantly decreased; whereas, neuronal nitric oxide synthase (nNOS) was significantly increased. Conclusions SIRT1 single-nucleotide polymorphism (rs7069102) may confer an increased risk for PMI in young Egyptian patients with affecting endothelial nitric oxide synthase protein expressions. Traditional cardiovascular risk factors are prevalent in patients with PMI, with dietary behaviors, obesity, diabetes and dyslipidemia serving as independent risk factors for PMI. Clinical trial registration number: NCT05160844.

Limosilactobacillus reuteri HY7503 and Its Cellular Proteins Alleviate Endothelial Dysfunction by Increasing Nitric Oxide Production and Regulating Cell Adhesion Molecule Levels.

Endothelial dysfunction, which is marked by a reduction in nitric oxide (NO) production or an imbalance in relaxing and contracting factor levels, exacerbates atherosclerosis by promoting the production of cell adhesion molecules and cytokines. This study aimed to investigate the effects of Limosilactobacillus reuteri HY7503, a novel probiotic isolated from raw milk, on endothelial dysfunction. Five lactic acid bacterial strains were screened for their antioxidant, anti-inflammatory, and endothelium-protective properties; L. reuteri HY7503 had the most potent effect. In a mouse model of angiotensin II-induced endothelial dysfunction, L. reuteri HY7503 reduced vascular thickening (19.78%), increased serum NO levels (226.70%), upregulated endothelial NO synthase (eNOS) expression in the aortic tissue, and decreased levels of cell adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and serum cytokines (tumor necrosis factor-alpha [TNF-α] and interleukin-6 [IL-6]). In TNF-α-treated human umbilical vein endothelial cells (HUVECs), L. reuteri HY7503 enhanced NO production and reduced cell adhesion molecule levels. In HUVECs, surface-layer proteins (SLPs) were more effective than extracellular vesicles (exosomes) in increasing NO production and decreasing cell adhesion molecule levels. These findings suggested that L. reuteri HY7503 may serve as a functional probiotic that alleviates endothelial dysfunction.

Lack of AMP-activated protein kinase-α1 reduces nitric oxide synthesis in thoracic aorta perivascular adipose tissue

ObjectivePerivascular adipose tissue (PVAT) releases anti-contractile bioactive molecules including NO. PVAT anti-contractile activity is attenuated in mice lacking AMPKα1 (AMP-activated protein kinase-α1). As AMPK regulates endothelial NO synthase (eNOS) activity in cultured cells, NO synthesis was examined in PVAT from AMPKα1 knockout (KO) mice. Methods and resultsEndothelium-denuded thoracic or abdominal aortic rings were isolated from wild type (WT) and KO mice. NOS inhibition enhanced vasoconstriction in PVAT-intact thoracic aortic rings from mice of either genotype yet had no effect on abdominal rings as assessed by wire myography. Thoracic aorta PVAT exhibited increased NO production, NOS activity and levels of the brown adipose tissue marker uncoupling protein-1 (UCP1) compared to abdominal PVAT. In KO mice, NO production was significantly reduced in thoracic but not abdominal PVAT. Reduced NO production in KO thoracic PVAT was not due to altered levels or phosphorylation of eNOS but was associated with increased caveolin-1:eNOS association and caveolin-1 Tyr14 phosphorylation. A peptide that disrupts eNOS:caveolin-1 association increased NO synthesis and reduced vasoconstriction of PVAT-intact thoracic but not abdominal aortic rings. KO thoracic PVAT also exhibited reduced UCP1 levels. ConclusionsMurine thoracic aorta PVAT exhibits higher NO synthesis and UCP1 levels than abdominal aortic PVAT. Downregulation of AMPK suppresses NO synthesis which may contribute to the reduced anticontractile activity and reduced brown adipose tissue phenotype of KO thoracic PVAT. The mechanism underlying the effect of AMPK downregulation likely results from increased caveolin-1:eNOS association associated with caveolin-1 Tyr14 phosphorylation.

Transient changes in L-arginine, asymmetric and symmetric dimethyl arginine in triathletes following Norseman Xtreme Triathlon.

Arterial vasodilation is dependent on nitric oxide synthesized from L-arginine by endothelial nitric oxide synthase. Triathletes are reported to display altered serum concentrations of nitric oxide metabolites such as L-arginine, asymmetric dimethyl arginine (ADMA) and symmetric dimethyl arginine (SDMA) shortly after completing long-distance triathlon races. In other populations, similar changes to nitric oxide metabolites are established risk markers of cardiovascular disease. The objective of this study was to assess serum concentrations of metabolites for endothelial nitric oxide synthesis in triathletes one week following a long-distance triathlon race. In this prospective observational study, we used high-performance liquid chromatography to measure circulating concentrations of L-arginine, ADMA, and SDMA in triathletes. Venous blood samples were collected before, immediately after, day one, and one week following the triathlon race. Serum concentrations and L-arginine/ADMA ratio were determined for each time-point and compared to baseline. L-arginine/ADMA ratio was reduced on day one (147 ± 32 vs 163 ± 40, p < 0.02). ADMA was reduced immediately after and increased at day one and remained elevated at oneweek (0.29 ± 0.05μM, p < 0.001, 0.44 ± 0.08μM, p < 0.001 and 0.42 ± 0.07μM, p = 0.04, respectively vs 0.40 ± 0.05μM). SDMA was increased at all time-points when compared to baseline (0.48 ± 0.10μM, p < 0.001, 0.53 ± 0.11μM, p < 0.001 and 0.42 ± 0.08μM, p = 0.048 vs 0.38 ± 0.05μM). L-arginine was only decreased immediately after (46.0 ± 9.3μM vs. 64.6 ± 16.1μM, p < 0.001). Long-distance triathlon racing induces altered levels of metabolites for endothelial nitric oxide production that mostly normalizes within one week following racing. The clinical relevance of these transient changes has yet to be elucidated in the athletic population.

Cognitive Impairment and Synaptic Dysfunction in Cardiovascular Disorders: The New Frontiers of the Heart-Brain Axis.

Within the central nervous system, synaptic plasticity, fundamental to processes like learning and memory, is largely driven by activity-dependent changes in synaptic strength. This plasticity often manifests as long-term potentiation (LTP) and long-term depression (LTD), which are bidirectional modulations of synaptic efficacy. Strong epidemiological and experimental evidence show that the heart-brain axis could be severely compromised by both neurological and cardiovascular disorders. Particularly, cardiovascular disorders, such as heart failure, hypertension, obesity, diabetes and insulin resistance, and arrhythmias, may lead to cognitive impairment, a condition known as cardiogenic dementia. Herein, we review the available knowledge on the synaptic and molecular mechanisms by which cardiogenic dementia may arise and describe how LTP and/or LTD induction and maintenance may be compromised in the CA1 region of the hippocampus by heart failure, metabolic syndrome, and arrhythmias. We also discuss the emerging evidence that endothelial dysfunction may contribute to directly altering hippocampal LTP by impairing the synaptically induced activation of the endothelial nitric oxide synthase. A better understanding of how CV disorders impact on the proper function of central synapses will shed novel light on the molecular underpinnings of cardiogenic dementia, thereby providing a new perspective for more specific pharmacological treatments.

Generation and characterization of a conditional eNOS knock out mouse model for cell-specific reactivation of eNOS in gain-of-function studies

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in the vessel wall regulates blood pressure and cardiovascular hemodynamics. In this study, we generated conditional eNOS knock out (KO) mice characterized by a duplicated/inverted exon 2 flanked with two pairs of loxP regions (eNOSinv/inv); a Cre-recombinase activity induces cell-specific reactivation of eNOS, as a result of a flipping of the inverted exon 2 (eNOSfl). This work aimed to test the efficiency of the Cre-mediated cell-specific recombination and the resulting eNOS expression/function. As proof of concept, we crossed eNOSinv/inv mice with DeleterCrepos (DelCrepos) mice, expressing Cre recombinase in all cells. We generated heterozygous eNOSfl/inv or homozygous eNOSfl/fl mice, and eNOSinv/inv littermate mice. We found that both eNOSfl/fl and eNOSfl/inv mice express eNOS and the overall expression level depends on the number of mutated alleles, while eNOSinv/inv mice did not show any eNOS expression. Vascular endothelial function was restored in eNOSfl/fl and eNOSfl/inv mice, as determined by ACh-dependent vasodilation of aortic rings. Cre-dependent reactivation of eNOS in eNOSfl/fl and eNOSfl/inv mice rescued eNOSinv/inv (phenotypically global eNOS KO) mice from hypertension. These findings demonstrate that eNOS expression is restored in eNOSfl/fl mice at comparable physiological levels of WT mice, and its functional activity is independent on the number of the reactivated alleles. Therefore, eNOSinv/inv mice are a useful model for studying the effects of conditional reactivation of eNOS and gene dosage effects in specific cells for gain-of-function studies.

Relationship of cerebrospinal fluid and serum levels of SIL-2R, eNOS, and CD93 with the progression and prognosis of viral encephalitis in children

To investigate the relationship of cerebrospinal fluid and serum levels of soluble interleukin-2 receptor (SIL-2R), endothelial nitric oxide synthase (eNOS), and cluster of differentiation 93 (CD93) with the progression and prognosis of viral encephalitis (VE) in children. Prospectively, 102 children with VE admitted from January 2021 to January 2024 were selected as the VE group. The patients were divided into a mild subgroup (64 patients) and a severe subgroup (38 patients) according to disease progression. The patients were also divided into a good prognosis subgroup (29 patients) and a poor prognosis subgroup (73 patients) according to prognosis. A control group of 102 children with central nervous system diseases who were examined and found not to have VE during the same period was selected. The factors contributing to the poor prognosis of children with VE and the predictive value of SIL-2R, eNOS, and CD93 in cerebrospinal fluid and serum for the poor prognosis of children with VE were evaluated. Cerebrospinal fluid and serum SIL-2R, eNOS, and CD93 levels were significantly increased in the VE group, severe subgroup, and poor prognosis subgroup (P<0.05). Multivariate logistic regression analysis showed that high SIL-2R, eNOS, and CD93 levels in cerebrospinal fluid and serum were risk factors for poor prognosis in children with VE (P<0.05). Receiver operating characteristic curve analysis showed that the combination of cerebrospinal fluid SIL-2R, eNOS, and CD93 was superior to these individual indicators in prediction of poor prognosis in children with VE (P<0.05). Similarly, the combination of serum SIL-2R, eNOS, and CD93 was superior to these individual indicators in prediction of poor prognosis in children with VE (P<0.05). The cerebrospinal fluid and serum levels of SIL-2R, eNOS, and CD93 are significantly elevated in children with VE, and they are associated with VE progression and prognosis.