Integrated metabolomics analysis reveals mechanistic insights into variability in blood pressure response to thiazide diuretics and beta blockers.

Hypertensive patients with a higher proportion of genetic West African ancestry (%GWAA) have better blood pressure (BP) response to thiazide diuretics (TDs) and worse response to ß-blockers (BBs) than those with lower %GWAA, associated with their lower plasma renin activity (PRA). TDs and BBs are suggested to reduce BP in the long term through vasodilation via incompletely understood mechanisms. This study aimed at identifying pathways underlying ancestral differences in PRA, which might reflect pathways underlying BP-lowering mechanisms of TDs and BBs. Among hypertensive participants enrolled in the Pharmacogenomics Evaluation of Antihypertensive Responses (PEAR) and PEAR-2 trials, we previously identified 8 metabolites associated with baseline PRA and 4 metabolic clusters (including 39 metabolites) that are different between those with GWAA <45% versus ≥45%. In the current study, using Ingenuity Pathway Analysis (IPA), we integrated these signals. Three overlapping metabolic signals within three significantly enriched pathways were identified as associated with both PRA and %GWAA: ceramide signaling, sphingosine 1- phosphate signaling, and endothelial nitric oxide synthase signaling. Literature indicates that the identified pathways are involved in the regulation of the Rho kinase cascade, production of the vasoactive agents nitric oxide, prostacyclin, thromboxane A2, and endothelin 1; the pathways proposed to underlie TD- and BB-induced vasodilatation. These findings may improve our understanding of the BP-lowering mechanisms of TDs and BBs. This might provide a possible step forward in personalizing antihypertensive therapy by identifying patients expected to have robust BP-lowering effects from these drugs.

Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment.

BACKGROUND: Vasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear. METHODS: Vascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation. RESULTS: Our study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1. CONCLUSION: In summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Combined cardiovascular effects of ovariectomy and high-intensity interval training in female spontaneously hypertensive rats.

Hypertensive postmenopausal women are more likely to develop adverse cardiac remodeling and respond less effectively to drug treatment than men. High-intensity interval exercise (HIIE) is a nonpharmacological strategy for the treatment of hypertension; however, the effectiveness in women remains uncertain. This study was designed to evaluate 1) the effects of HIIE training upon morphological and functional markers of cardiovascular health in female SHR and 2) to determine whether the hormonal shift induced by ovariectomy could influence cardiovascular responses to HIIE. Thirty-six SHR were randomly assigned to four groups: ovariectomized sedentary, ovariectomized trained, sham-operated sedentary, and sham-operated trained. The trained rats performed HIIE 5 days/wk for 8 wk. Blood pressure and echocardiographic measurements were performed before and after training in animals. Cardiac response to ß-adrenergic stimulation and the expression of calcium regulatory proteins and estrogen receptors in heart samples were assessed. Endothelium-dependent vasorelaxation in response to acetylcholine was evaluated in aortic rings as well as the expression of nitric oxide synthase isoforms (eNOS and P-eNOS) by Western blotting. In both groups of trained SHR, HIIE induced eccentric cardiac remodeling with greater inotropic and chronotropic effects, as well as an increase in SERCA and ß1AR expression. However, although the trained rats showed improved endothelial function and expression of eNOS and P-eNOS in the aorta, there was no demonstrated effect on blood pressure. In addition, the responses to HIIE training were not affected by ovariectomy. This work highlights the importance of assessing the cardiovascular efficacy and safety of different exercise modalities in women.NEW & NOTEWORTHY This study reports the effects of high-intensity interval exercise (HIIE) training on cardiac and endothelial function in female hypertensive rats. Despite a lack of effect on blood pressure (BP), HIIE training induces eccentric cardiac remodeling with greater functionals effects. Furthermore, training has beneficial effects on endothelial function. However, ovarian hormones do not seem to modulate cardiac and aortic adaptations to this training modality. All this underlines the need to consider training modalities on the cardiovascular system in women.

Zymosan and lipopolysaccharide decrease gene expression of neuronal nitric oxide synthase in peripheral organs in chicks.

Nitric oxide (NO) is gaseous bioactive molecule that is synthesized by NO synthase (NOS). Inducible NOS (iNOS) expression occurs in response to pathogenic challenges, resulting in the production of large amounts of NO. However, there is a lack of knowledge regarding neuronal NOS (nNOS) and endothelial NOS (eNOS) in birds during pathogenic challenge. Therefore, the present study was conducted to determine the influence of intraperitoneal (IP) injection of zymosan (cell wall component of yeast) and lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria) on NOS expression in chicks (Gallus gallus). Furthermore, the effect of NOS inhibitors on the corresponding behavioral and physiological parameters was investigated. Zymosan and LPS injections induced iNOS mRNA expression in several organs. Zymosan had no effect on eNOS mRNA expression in the organs investigated, whereas LPS increased its expression in the pancreas. Zymosan and LPS decreased nNOS mRNA expression in the lung, heart, kidney, and pancreas. The decreased nNOS mRNA expression in pancreas was probably associated with the NO from iNOS provided that such effect was reproduced by IP injection of sodium nitroprusside, which is a NO donor. Furthermore, pancreatic nNOS mRNA expression decreased following subcutaneous injection of corticosterone. Furthermore, IP injections of a nonspecific NOS inhibitor, NG-nitro-L-arginine methyl ester, and an nNOS-specific inhibitor, 7-nitroindazole, resulted in the significant decreases in food intake, cloacal temperature, and feed passage via the digestive tract in chicks. Collectively, the current findings imply the decreased nNOS expression because of fungal and bacterial infections, which affects food intake, body temperature, and the digestive function in birds.

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice.

BACKGROUND: Neutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored. METHODS: The expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured. RESULTS: Results showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1. CONCLUSIONS: NCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

Exposure to emissions generated by 3-dimensional printing with polycarbonate: effects on peripheral vascular function, cardiac vascular morphology and expression of markers of oxidative stress in male rat cardiac tissue.

Three-dimensional (3D) printing with polycarbonate (PC) plastic occurs in manufacturing settings, homes, and schools. Emissions generated during printing with PC stock and bisphenol-A (BPA), an endocrine disrupter in PC, may induce adverse health effects. Inhalation of 3D printer emissions, and changes in endocrine function may lead to cardiovascular dysfunction. The goal of this study was to determine whether there were any changes in markers of peripheral or cardiovascular dysfunction in animals exposed to PC-emissions. Male Sprague Dawley rats were exposed to PC-emissions generated by 3D printing for 1, 4, 8, 15 or 30 d. Exposure induced a reduction in the expression of the antioxidant catalase (Cat) and endothelial nitric oxide synthase (eNos). Endothelin and hypoxia-induced factor 1α transcripts increased after 30 d. Alterations in transcription were associated with elevations in immunostaining for estrogen and androgen receptors, nitrotyrosine, and vascular endothelial growth factor in cardiac arteries of PC-emission exposed animals. There was also a reduction eNOS immunostaining in cardiac arteries from rats exposed to PC-emissions. Histological analyses of heart sections revealed that exposure to PC-emissions resulted in vasoconstriction of cardiac arteries and thickening of the vascular smooth muscle wall, suggesting there was a prolonged vasoconstriction. These findings are consistent with studies showing that inhalation 3D-printer emissions affect cardiovascular function. Although BPA levels in animals were relatively low, exposure-induced changes in immunostaining for estrogen and androgen receptors in cardiac arteries suggest that changes in the action of steroid hormones may have contributed to the alterations in morphology and markers of cardiac function.

Association between nitric oxide synthase (NOS3) gene polymorphisms and diabetic nephropathy: An updated meta-analysis.

Polymorphisms located within NOS3 gene have been investigated as susceptibility variants for diabetic nephropathy (DN) in type 2 diabetes mellitus (T2DM) in a large number of studies. However, these previous articles yielded inconsistent results and we aimed at elucidating the impact of NOS3 variants on DN risk in T2DM by conducting an updated systematic data synthesis. A total of 36 studies (12,807 participants) were selected for qualitative data synthesis, while 33 records with 11,649 subjects were included in the meta-analysis. The pooled analysis demonstrated the association of minor alleles of rs2070744 and rs1799983 with an increased susceptibility to DN (P < 0.001 and P = 0.015 for allelic model, respectively). For both of these variants, a significant effect of subgrouping according to ethnicity was found. Rs869109213 displayed an association with DN susceptibility, with pooled effect measures indicating a predisposing effect of the minor allele a (Prec = 0.002, ORrec = 1.960, 95%CI 1.288-2.983; Paavs. bb = 0.001, ORaavs. bb = 2.014, 95%CI 1.316-3.083). These findings support the effects of NOS3 variants on the risk of developing DN in T2DM.

Intrinsic endothelial hyperresponsiveness to inflammatory mediators drives acute episodes in models of Clarkson disease.

Clarkson disease, or monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS), is a rare, relapsing-remitting disorder featuring the abrupt extravasation of fluids and proteins into peripheral tissues, which in turn leads to hypotensive shock, severe hemoconcentration, and hypoalbuminemia. The specific leakage factor(s) and pathways in ISCLS are unknown, and there is no effective treatment for acute flares. Here, we characterize an autonomous vascular endothelial defect in ISCLS that was recapitulated in patient-derived endothelial cells (ECs) in culture and in a mouse model of disease. ISCLS-derived ECs were functionally hyperresponsive to permeability-inducing factors like VEGF and histamine, in part due to increased endothelial nitric oxide synthase (eNOS) activity. eNOS blockade by administration of N(γ)-nitro-l-arginine methyl ester (l-NAME) ameliorated vascular leakage in an SJL/J mouse model of ISCLS induced by histamine or VEGF challenge. eNOS mislocalization and decreased protein phosphatase 2A (PP2A) expression may contribute to eNOS hyperactivation in ISCLS-derived ECs. Our findings provide mechanistic insights into microvascular barrier dysfunction in ISCLS and highlight a potential therapeutic approach.

Low HDL cholesterol and the eNOS Glu298Asp polymorphism are associated with inducible myocardial ischemia in patients with suspected stable coronary artery disease.

BACKGROUND: The endothelial nitric oxide synthase (eNOS) gene deficiency is known to cause impaired coronary vasodilating capability in animal models. In the general clinical population, the eNOS gene polymorphisms, able to affect eNOS activity, were associated with cardiometabolic risk features and prevalence of coronary artery disease (CAD). AIM: To investigate the association of eNOS Glu298Asp gene polymorphism, cardiometabolic profile, obstructive CAD and inducible myocardial ischemia in patients with suspected stable CAD. METHODS: A total of 506 patients (314 males; mean age 62 ± 9 years) referred for suspected CAD was enrolled. Among these, 325 patients underwent stress ECG or cardiac imaging to assess the presence of inducible myocardial ischemia and 436 patients underwent non-invasive computerized tomography or invasive coronary angiography to assess the presence of obstructive CAD. Clinical characteristics and blood samples were collected for each patient. RESULTS: In the whole population, 49.6% of patients were homozygous for the Glu298 genotype (Glu/Glu), 40.9% heterozygotes (Glu/Asp) and 9.5% homozygous for the 298Asp genotype (Asp/Asp). Obstructive CAD was documented in 178/436 (40.8%) patients undergoing coronary angiography while myocardial ischemia in 160/325 (49.2%) patients undergoing stress testing. Patients with eNOS Asp genotype (Glu/Asp + Asp/Asp) had no significant differences in clinical risk factors and in circulating markers. Independent predictors of obstructive CAD were age, gender, obesity, and low HDL-C. Independent predictors of myocardial ischemia were gender, obesity, low HDL-C and Asp genotype. In the subpopulation in which both stress tests and coronary angiography were performed, the Asp genotype remained associated with increased myocardial ischemia risk after adjustment for obstructive CAD. CONCLUSION: In this population, low-HDL cholesterol was the only cardiometabolic risk determinant of obstructive CAD. The eNOS Glu298Asp gene polymorphism was significantly associated with inducible myocardial ischemia independently of other risk factors and presence of obstructive CAD.

Indole-3-Propionic Acid, a Gut Microbiota-Derived Tryptophan Metabolite, Promotes Endothelial Dysfunction Impairing Purinergic-Induced Nitric Oxide Release in Endothelial Cells.

Different gut microbiota-derived metabolites influence cardiovascular function, and, among all, the role of indole-3-propionic acid (IPA), from tryptophan metabolism, shows controversial effects. The aim of this study was to evaluate its role in endothelial dysfunction. IPA effects were studied on bovine aortic endothelial cells (BAE-1). First, IPA cytotoxicity was evaluated by an MTS assay. Then, the levels of intracellular reactive oxygen species (ROS) were evaluated by a microplate reader or fluorescence microscopy with the CellROX® Green probe, and nitric oxide (NO) production was studied by fluorescence microscopy with the DAR4M-AM probe after acute or chronic treatment. Finally, immunoblotting analysis for endothelial nitric oxide synthase (eNOS) phosphorylation (p-eNOS) was performed. In BAE-1, IPA was not cytotoxic, except for the highest concentration (5 mM) after 48 h of treatment, and it showed neither oxidant nor antioxidant activity. However, the physiological concentration of IPA (1 µM) significantly reduced NO released by adenosine triphosphate (ATP)-stimulated BAE-1. These last data were confirmed by Western blot analysis, where IPA induced a significant reduction in p-eNOS in purinergic-stimulated BAE-1. Given these data, we can speculate that IPA negatively affects the physiological control of vascular tone by impairing the endothelial NO release induced by purinergic stimulation. These results represent a starting point for understanding the mechanisms underlying the relationship between gut microbiota metabolites and cardiometabolic health.

Vascular dysfunction programmed in male rats by topiramate during peripubertal period.

AIM: The present study evaluated whether topiramate (TPM) treatment during the peripubertal period affects vascular parameters of male rats and whether oxidative stress plays a role in these changes. MAIN METHODS: Rats were treated with TPM (41 mg/kg/day, gavage) or vehicle (CTR group) from the postnatal day (PND) 28 to 50. At PND 51 and 120 the rats were evaluated for: thoracic aorta reactivity to phenylephrine, in the presence (Endo+) or absence of endothelium (Endo-), to acetylcholine and to sodium nitroprusside (SNP), aortic thickness and endothelial nitric oxide synthase (eNOS) expression. In serum were analyzed: the antioxidant capacity by ferric reducing antioxidant power assay; endogenous antioxidant reduced glutathione, and superoxide anion. Results were expressed as mean ± s.e.m., differences when p < 0.05. STATISTICS: Two-way ANOVA (and Tukey's) or Student t-test. KEY FINDINGS: At PND 51, the contraction induced by phenylephrine in Endo+ ring was higher in TPM when compared to CTR. At PND 120, the aortic sensitivity to acetylcholine in TPM rats was reduced in comparison with CTR. The aortic eNOs expression and the aortic thickness were similar between the groups. At PND 51 and 120, TPM group presented a decrease in antioxidants when compared to CTR groups and at PND 120, in TPM group the superoxide anion was increased. SIGNIFICANCE: Taken together, the treatment of rats with TPM during peripubertal period promoted permanent impairment of endothelial function probably mediated by oxidative stress.

Fenugreek extract improves diabetes-induced endothelial dysfunction via the arginase 1 pathway.

Endothelial dysfunction (ED) is an initiating trigger and key factor in vascular complications, leading to disability and mortality in individuals with diabetes. The research concerning therapeutic interventions for ED has gained considerable interest. Fenugreek, a commonly used edible plant in dietary consumption, has attracted significant attention due to its management of diabetes and its associated complications. The research presented in this study examines the potential therapeutic benefits of fenugreek in treating ED and investigates the underlying mechanism associated with its effects. The analysis on fenugreek was performed using 70% ethanol extract, and its chemical composition was analyzed using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). In total, we identified 49 compounds present in the fenugreek extract. These compounds encompass flavonoids, saponins, and phospholipids. Then, the models of ED in streptozotocin-induced diabetic mice and high glucose-induced isolated rat aortas were established for research. Through vascular function testing, it was observed that fenugreek extract effectively improved ED induced by diabetes or high glucose. By analyzing the protein expression of arginase 1 (Arg1), Arg activity, Arg1 immunohistochemistry, nitric oxide (NO) level, and the protein expression of endothelial nitric oxide synthase (eNOS), p38 mitogen-activated protein kinase (p38 MAPK), and p-p38 MAPK in aortas, this study revealed that the potential mechanism of fenugreek extract in anti-ED involves the downregulation of Arg1, leading to enhanced NO production. Furthermore, analysis of serum exosomes carrying Arg activity indicates that fenugreek may decrease the activity of Arg transported by serum exosomes, potentially preventing the increase in Arg levels triggered by the uptake of serum exosomes by vascular endothelial cells. In general, this investigation offers valuable observations regarding the curative impact of fenugreek extract on anti-ED in diabetes, revealing the involvement of the Arg1 pathway in its mechanism.

Protein phosphatase 4 mediates palmitic acid-induced endothelial dysfunction by decreasing eNOS phosphorylation at serine 633 in HUVECs.

Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.

Protective effect of alizarin on vascular endothelial dysfunction via inhibiting the type 2 diabetes-induced synthesis of THBS1 and activating the AMPK signaling pathway.

BACKGROUND: In this study, we investigated the protective effects of alizarin (AZ) on endothelial dysfunction (ED). AZ has inhibition of the type 2 diabetes mellitus (T2DM)-induced synthesis of thrombospondin 1 (THBS1). Adenosine 5'-monophosphate- activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. PURPOSE: The aim of this study was to investigate the ameliorative effect of AZ on vascular injury caused by T2DM and to reveal the potential mechanism of AZ in high glucose (HG)-stimulated human umbilical vein endothelial cells (HUVECs) and diabetic model rats. STUDY DESIGN: HUVECs, rats and AMPK-/- transgenic mice were used to investigate the mitigating effects of AZ on vascular endothelial dysfunction caused by T2DM and its in vitro and in vivo molecular mechanisms. METHODS: In type 2 diabetes mellitus rats and HUVECs, the inhibitory effect of alizarin on THBS1 synthesis was verified by immunohistochemistry (IHC), immunofluorescence (IF) and Western blot (WB) so that increase endothelial nitric oxide synthase (eNOS) content in vitro and in vivo. In addition, we verified protein interactions with immunoprecipitation (IP). To probe the mechanism, we also performed AMPKα2 transfection. AMPK's pivotal role in AZ-mediated prevention against T2DM-induced vascular endothelial dysfunction was tested using AMPKα2-/- mice. RESULTS: We first demonstrated that THBS1 and AMPK are targets of AZ. In T2DM, THBS1 was robustly induced by high glucose and inhibited by AZ. Furthermore, AZ activates the AMPK signaling pathway, and recoupled eNOS in stressed endothelial cells which plays a protective role in vascular endothelial dysfunction. CONCLUSIONS: The main finding of this study is that AZ can play a role in different pathways of vascular injury due to T2DM. Mechanistically, alizarin inhibits the increase in THBS1 protein synthesis after high glucose induction and activates AMPKα2, which increases NO release from eNOS, which is essential in the prevention of vascular endothelial dysfunction caused by T2DM.

Hydrogen sulfide dysfunction in metabolic syndrome-associated vascular complications involves cGMP regulation through soluble guanylyl cyclase persulfidation.

Here, by using in vitro and ex vivo approaches, we elucidate the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with metabolic syndrome (MetS). In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, we observe significant hallmarks of endothelial dysfunction, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, a genetic MetS model, we identify a downregulation of CBS and CSE expression in aorta, coupled with a diminished L-cysteine-induced vasorelaxation. Molecular mechanisms of eNOS/NO signaling impairment, dissected using pharmacological and molecular approaches, indicate an altered eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation and increased L-NIO-induced contraction. In vivo treatment with the H2S donor Erucin ameliorates vascular dysfunction observed in db/db mice without impacting eNOS, further highlighting a specific action on smooth muscle component rather than the endothelium. Analyzing the NO signaling pathway in db/db mice aortas, reduced cGMP levels were detected, implicating a defective sGC/cGMP signaling. In vivo Erucin administration restores cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation observed in sGC overexpressed cells, coupled with PDE5 inhibition. In conclusion, our study demonstrates a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MetS involving an impairment of both H2S and NO signaling. Exogenous H2S supplementation through Erucin represents a promising alternative in MetS therapy, targeting smooth muscle cells and supporting the importance of lifestyle and nutrition in managing MetS.

Flavonoid Compounds in Hippophae rhamnoides L. Protect Endothelial Cells from Oxidative Damage Through the PI3K/AKT-eNOS Pathway.

Sea buckthorn, a traditional medicinal plant, has been used for several years in China for the prevention and treatment of various diseases, a practice closely associated with its significant antioxidant activity. The aim of this study was to investigate the protective effects of sea buckthorn flavonoids on vascular endothelial cells in an oxidative stress environment. We isolated and extracted active compounds from sea buckthorn and investigated their impact on endothelial nitric oxide synthase (eNOS) activity through the PI3K/AKT-eNOS signaling pathway through a combination of network pharmacology and cellular experiments, elucidating the regulatory effects of these compounds on endothelial cell functions. Three flavonoids, named Fr.4-2-1, Fr.4-2-2 and Fr.4-2-3, were obtained from sea buckthorn. The results of network pharmacology indicated that they might exert their effects by regulating the PI3K-AKT signaling pathway. In vitro results showed that all three flavonoids were effective in alleviating the degree of oxidative stress in cells, among which Fr.4-2-1 exerted its antioxidant effects by modulating the PI3K/AKT-eNOS pathway. Flavonoids in sea buckthorn can effectively inhibit oxidative stress-induced cellular damage, preserving the integrity and functionality of endothelial cells, which is crucial for maintaining vascular health and function.

Scaffolds and the scaffolding domain: an alternative paradigm for caveolin-1 signaling.

Caveolin-1 (Cav1) is a 22 kDa intracellular protein that is the main protein constituent of bulb-shaped membrane invaginations known as caveolae. Cav1 can be also found in functional non-caveolar structures at the plasma membrane called scaffolds. Scaffolds were originally described as SDS-resistant oligomers composed of 10-15 Cav1 monomers observable as 8S complexes by sucrose velocity gradient centrifugation. Recently, cryoelectron microscopy (cryoEM) and super-resolution microscopy have shown that 8S complexes are interlocking structures composed of 11 Cav1 monomers each, which further assemble modularly to form higher-order scaffolds and caveolae. In addition, Cav1 can act as a critical signaling regulator capable of direct interactions with multiple client proteins, in particular, the endothelial nitric oxide (NO) synthase (eNOS), a role believed by many to be attributable to the highly conserved and versatile scaffolding domain (CSD). However, as the CSD is a hydrophobic domain located by cryoEM to the periphery of the 8S complex, it is predicted to be enmeshed in membrane lipids. This has led some to challenge its ability to interact directly with client proteins and argue that it impacts signaling only indirectly via local alteration of membrane lipids. Here, based on recent advances in our understanding of higher-order Cav1 structure formation, we discuss how the Cav1 CSD may function through both lipid and protein interaction and propose an alternate view in which structural modifications to Cav1 oligomers may impact exposure of the CSD to cytoplasmic client proteins, such as eNOS.

C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

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.

Crystallographic and Computational Insights into Isoform-Selective Dynamics in Nitric Oxide Synthase.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

Absorbed Bioactive Compounds Replicate Guanxin II-Induced Endothelium-Associated in/ex vivo Vasodilation.

OBJECTIVE: To develop an interference-free and rapid method to elucidate Guanxin II (GX II)'s representative vasodilator absorbed bioactive compounds (ABCs) among enormous phytochemicals. METHODS: The contents of ferulic acid, tanshinol, and hydroxysafflor yellow A (FTA) in GX II/rat serum after the oral administration of GX II (30 g/kg) were detected using ultra-performance liquid chromatography-mass spectrometry. Totally 18 rats were randomly assigned to the control group (0.9% normal saline), GX II (30 g/kg) and FTA (5, 28 and 77 mg/kg) by random number table method. Diastolic coronary flow velocity-time integral (VTI), i.e., coronary flow or coronary flow-mediated dilation (CFMD), and endothelium-intact vascular tension of isolated aortic rings were measured. After 12 h of exposure to blank medium or 0.5 mmol/L H2O2, endothelial cells (ECs) were treated with post-dose GX II of supernatant from deproteinized serum (PGSDS, 300 µL PGSDS per 1 mL of culture medium) or FTA (237, 1539, and 1510 mg/mL) for 10 min as control, H2O2, PGSDS and FTA groups. Nitric oxide (NO), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), superoxide dismutase (SOD), malondialdehyde (MDA) and phosphorylated phosphoinositide 3 kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS) were analyzed. PGSDS was developed as a GX II proxy of ex vivo herbal crude extracts. RESULTS: PGSDS effectively eliminates false responses caused by crude GX II preparations. When doses equaled the contents in GX II/its post-dose serum, FTA accounted for 98.17% of GX II -added CFMD and 92.99% of PGSDS-reduced vascular tension. In ECs, FTA/PGSDS was found to have significant antioxidant (lower MDA and higher SOD, P<0.01) and endothelial function-protective (lower VEGF, ET-1, P<0.01) effects. The increases in aortic relaxation, endothelial NO levels and phosphorylated PI3K/Akt/eNOS protein induced by FTA/PGSDS were markedly abolished by NG-nitro-L-arginine methyl ester (L-NA, eNOS inhibitor) and wortmannin (PI3K/AKT inhibitor), respectively, indicating an endothelium-dependent vasodilation via the PI3K/AKT-eNOS pathway (P<0.01). CONCLUSION: This study provides a strategy for rapidly and precisely elucidating GX II's representative in/ex vivo cardioprotective absorbed bioactive compounds (ABCs)-FTA, suggesting its potential in advancing precision ethnomedicine.