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.

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.

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.

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.

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.

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.

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.

Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries.

BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.

Amphetamine increases vascular permeability by modulating endothelial actin cytoskeleton and NO synthase via PAR-1 and VEGF-R.

Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction-force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (-75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (-50.8±3.7%, p<0.001) and Threonine495 (-44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse.

Nur77 mitigates endothelial dysfunction through activation of both nitric oxide production and anti-oxidant pathways.

BACKGROUND: Nur77 belongs to the member of orphan nuclear receptor 4A family that plays critical roles in maintaining vascular homeostasis. This study aims to determine whether Nur77 plays a role in attenuating vascular dysfunction, and if so, to determine the molecular mechanisms involved. METHODS: Both Nur77 knockout (Nur77 KO) and Nur77 endothelial specific transgenic mice (Nur77-Tg) were employed to examine the functional significance of Nur77 in vascular endothelium in vivo. Endothelium-dependent vasodilatation to acetylcholine (Ach) and reactive oxygen species (ROS) production was determined under inflammatory and high glucose conditions. Expression of genes was determined by real-time PCR and western blot analysis. RESULTS: In response to tumor necrosis factor alpha (TNF-α) treatment and diabetes, the endothelium-dependent vasodilatation to Ach was significantly impaired in aorta from Nur77 KO as compared with those from the wild-type (WT) mice. Endothelial specific overexpression of Nur77 markedly prevented both TNF-α- and high glucose-induced endothelial dysfunction. Compared with WT mice, after TNF-α and high glucose treatment, ROS production in aorta was significantly increased in Nur77 KO mice, but it was inhibited in Nur77-Tg mice, as determined by dihydroethidium (DHE) staining. Furthermore, we demonstrated that Nur77 overexpression substantially increased the expression of several key enzymes involved in nitric oxide (NO) production and ROS scavenging, including endothelial nitric oxide synthase (eNOS), guanosine triphosphate cyclohydrolase 1 (GCH-1), glutathione peroxidase-1 (GPx-1), and superoxide dismutases (SODs). Mechanistically, we found that Nur77 increased GCH1 mRNA stability by inhibiting the expression of microRNA-133a, while Nur77 upregulated SOD1 expression through directly binding to the human SOD1 promoter in vascular endothelial cells. CONCLUSION: Our results suggest that Nur77 plays an essential role in attenuating endothelial dysfunction through activating NO production and anti-oxidant pathways in vascular endothelium. Targeted activation of Nur77 may provide a novel therapeutic approach for the treatment of cardiovascular diseases associated with endothelial dysfunction.

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Black Soybean Seed Coat Extract Improves Endothelial Function and Upregulates Oxidative Stress Marker Expression in Healthy Volunteers by Stimulating Nitric Oxide Production in Endothelial Cells.

Black soybean seed coat extract (BE) contains multiple bioactive polyphenols, including flavan-3-ols and anthocyanins. BE improves endothelial function; however, it is unclear whether BE protects endothelial cells from senescence. In this study, we examined the effects of BE on endothelial cell senescence and vascular function in healthy individuals. High concentrations of glucose were used to induce senescence in bovine aortic endothelial cells incubated with BE. Senescence, vascular function, and oxidative stress markers were measured. Incubation with BE remarkably inhibited senescence-associated ß-galactosidase and lactate dehydrogenase activities and dose dependently reduced intracellular reactive oxygen species levels in bovine aortic endothelial cells. BE treatment increased the levels of endothelial nitric oxide synthase (eNOS) mRNA and endothelial nitric oxide (NO) metabolites and increased the mRNA expression of klotho, a gene associated with an antiaging phenotype. To examine the effects of BE in humans, we conducted a clinical study using the second derivative of the fingertip photoplethysmogram to investigate vascular function and aging in 24 healthy volunteers. The participants consumed BE supplements (100 mg/day) or a placebo for 2 weeks. When compared with the placebo group, the BE group showed considerably improved vascular function, NO metabolite levels, and oxidative stress. These results suggest that BE supplementation improves endothelial function, possibly through antioxidant activity and NO production, and may consequently reduce the cardiovascular risk associated with aging. BE supplementation may be an effective and safe approach to reduce the risk of atherosclerosis and cardiovascular disease; however, additional studies investigating chronic vascular inflammation are needed.

Danhong formula alleviates endothelial dysfunction and reduces blood pressure in hypertension by regulating MicroRNA 24 - Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential hypertension (EH) is one of the important risk factors of cardio-cerebrovascular diseases, and it can significantly increase the incidence and mortality of acute myocardial infarction, cerebral infarction and hemorrhage. Danhong Formula (DHF) was consisting of Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese) (Plant names have been checked with http://www.the plant list.org on June 28th, 2023) was approved by State Food and Drug Administration of China, that has been used for thousands of years in the treatment of cardiovascular diseases in China with proven safety and efficacy. Though our previous studies have found that DHF improved endothelial dysfunction (ED) and decreased high blood pressure (BP), the underlying mechanisms of its antihypertensive effect still remain unclear. AIM OF THE STUDY: This study investigated whether DHF regulated MicroRNA 24- Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase (miR-24 - PI3K/AKT/eNOS) axis to produce antihypertensive effect and improve endothelial dysfunction. MATERIALS AND METHODS: Firstly, the chemical components of DHF were analyzed by UHPLC-MS. After that, BP was continuously monitored within the 1st, 3rd, and 4th week in SHR to evaluate the antihypertensive effect of DHF intraperitoneal injection. In addition, not only the contents of serum nitric oxide (NO), prostacyclin (PGI2), and angiotensin II (Ang II) were detected, but also the isolated aorta ring experiment was conducted to evaluate the vasomotoricity to evaluate of DHF on improving endothelial dysfunction. Key proteins or mRNA expression associated with miR-24 - PI3K/AKT/eNOS axis in aorta were detected by capillary Western blot, immunohistochemistry or RT-PCR to explore the underlying mechanisms. Index of NO, Ang II PGI2 and key proteins or mRNA expression were also conducted in miR-24-3p over-expression HUVECs model. RESULTS: Compared with SHR control group, DHF (4 mL/kg/day, 2 mL/kg/day, 1 mL/kg/day) treatment significantly reduced high BP in SHR and selectively increased acetylcholine (Ach) induced vasodilation, but not sodium nitroprusside (SNP) in a manner of concentration dependency in isolated aorta ring. DHF (4 mL/kg/day, 1 mL/kg/day) treatment was accompanying an increment of NO and PGI2, and lowering AngII in SHR. Moreover, DHF treatment significantly up-regulated expression of p-PI3K, p-AKT, mTOR, eNOS and p-eNOS, but down-regulated miR-24-3p expression in aorta. Compared with miR-24-3p over-expression HUVECs model group, DHF treatment inhibited miR- 24-3p expression and up-regulated p-PI3K, p-AKT, mTOR and eNOS mRNA expression. Similarly, DHF treatment increased PI3K, AKT, mTOR and eNOS protein expression in HUVECs by Western blot. CONCLUSIONS: These findings suggest that DHF alleviates endothelial dysfunction and reduces high BP in SHR mediated by down-regulating miR-24 via ultimately facilitating up-regulation of PI3K/AKT/eNOS axis. This current study firstly demonstrates a potential direction for antihypertensive mechanism of DHF from microRNA aspect and will promote its clinical applications.

Methylation of eNOS in the rat penile corpus cavernosum under different pathological states and its relationship with erectile function.

BACKGROUND: It has been shown that methylation in the promoter region of eNOS can downregulate eNOS expression resulting in the endothelial dysfunction. However, it is unclear whether low androgen levels and type 1 diabetes cause ED by methylating the promoter region of eNOS in the penile corpus cavernosum. OBJECTIVE: To clarify the effects of type 1 diabetes and hypo-androgen status on the methylation level of the promoter region of the eNOS gene in penile cavernous tissue and their relationship with the erectile function. METHODS: Fifty-eight eight-week-old male Sprague-Dawley rats were randomly divided into six groups (n = 6): sham operation group, castration group, castration+testosterone (cast+T) group, normoglycemia group, diabetic group, and diabetic+methyltransferase inhibitor (5-aza-dc, 1.5 mg/kg) group. The ICPmax/MAP, serum T, the concentration of nitric oxide (NO), the expression of DNMT1, DNMT3a, DNMT3b, and eNOS, and the methylation level of the eNOS promoter region in penile corpus cavernosum of rat were examined 4 weeks after surgery in the sham-operated group, the castration group, and the castration + testosterone replacement group. Those tests were examined after 6 weeks using of methylation inhibitors in the normoglycemic group, the diabetic group, and the diabetic + methylation inhibitor group. RESULTS: ICPmax/MAP, DNMT1, DNMT3a, DNMT3b, eNOS, and NO levels were significantly lower in castrated rats than in sham and cast+T rats (P < 0.05). ICPmax/MAP, eNOS, and NO levels were lower, and DNMT1, DNMT3a, and DNMT3b expression levels were significantly increased in the diabetic group compared with the normoglycemic and diabetic+methyltransferase inhibitor groups (P < 0.05). There was no significant difference in the methylation level of the promoter region of eNOS in penile cavernous tissue of castrated rats compared with the sham group or the testosterone replacement group. The methylation level of the promoter region of eNOS in penile cavernous tissue was significantly higher in the diabetic group than in the normoglycemic group and diabetic+methyltransferase inhibitor group (P < 0.05). CONCLUSION: Although low androgen status inhibited the level of methyltransferase in rat penile cavernous tissue, did not affect the level of methylation in the promoter region of eNOS. Hyperglycemia inhibits the NO level in the penile cavernous tissue and the erectile function of rats by upregulating the methyltransferase level in the penile cavernous tissue and the methylation level in the promoter region of eNOS. Methylation inhibitors can partly improve the erectile function in type 1 diabetic rats.

The hydroxamic acid derivative YPX-C-05 alleviates hypertension and vascular dysfunction through the PI3K/Akt/eNOS pathway.

BACKGROUND: Hypertension is a prevalent cardiovascular disease characterized by elevated blood pressure and increased vascular resistance. HDAC inhibitors have emerged as potential therapeutic agents due to their ability to modulate gene expression and cellular processes. YPX-C-05, a novel hydroxamic acid-based HDAC inhibitor, shows promise in its vasodilatory effects and potential targets for hypertension treatment. In this study, we aimed to elucidate the mechanisms underlying YPX-C-05's vasodilatory effects and explore its therapeutic potential in hypertension. METHODS: To determine the ex vivo vasodilatory effects of YPX-C-05, isolated aortic rings precontracted with phenylephrine were used. We assessed YPX-C-05's inhibitory effects on HDACs and its impact on histone H4 deacetylation levels in endothelial cells. Network pharmacology analysis was employed to predict putative targets of YPX-C-05 for hypertension treatment. To investigate the involvement of the PI3K/Akt/eNOS pathway, we employed enzyme-linked immunosorbent assay and to assess the levels of NO, ET-1, BH2, and BH4 in human umbilical vein endothelial cells. And we also analyzed the mRNA expression of eNOS and ET-1. Furthermore, Western blotting was conducted to quantify the phosphorylated and total Akt and eNOS levels in human umbilical vein endothelial cell lysates following treatment with YPX-C-05. In order to elucidate the vasodilatory mechanism of YPX-C-05, we employed pharmacological inhibitors for evaluation purposes. Furthermore, we evaluated the chronic antihypertensive effects of YPX-C-05 on N-omega-nitro-L-arginine-induced hypertensive mice in an in vivo model. Vascular remodeling was assessed through histological analysis. RESULTS: Our findings demonstrated that YPX-C-05 exerts significant vasodilatory effects in isolated aortic rings precontracted with phenylephrine. Furthermore, YPX-C-05 exhibited inhibitory effects on HDACs and increased histone H4 acetylation in endothelial cells. Network pharmacology analysis predicted YPX-C-05 might activate endothelial eNOS via PI3K/Akt signaling pathway. Inhibition of the PI3K/Akt/eNOS pathway attenuated the vasodilatory effects of YPX-C-05, as evidenced by reduced levels of phosphorylated Akt and eNOS in human umbilical vein endothelial cell lysates. The chronic administration of YPX-C-05 in N-omega-nitro-L-arginine-induced hypertensive mice resulted in significant antihypertensive effects. Histological analysis demonstrated a reduction in vascular remodeling, further supporting the therapeutic potential of YPX-C-05 in hypertension. CONCLUSION: This study demonstrates for the first time that the novel hydroxamic acid-based HDAC inhibitor YPX-C-05 produces significant antihypertensive and vasodilatory effects through the PI3K/Akt/eNOS pathway. Our findings support the developing prospect of YPX-C-05 as a novel antihypertensive drug.

Optimizing irradiation conditions for low-intensity pulsed ultrasound to upregulate endothelial nitric oxide synthase.

PURPOSE: Here we aimed to develop a minimally invasive treatment for ischemic heart disease and demonstrate that low-intensity pulsed ultrasound (LIPUS) therapy improves myocardial ischemia by promoting myocardial angiogenesis in a porcine model of chronic myocardial ischemia. Studies to date determined the optimal treatment conditions within the range of settings available with existing ultrasound equipment and did not investigate a wider range of conditions. METHODS: We investigated a broad range of five parameters associated with ultrasound irradiation conditions that promote expression of endothelial nitric oxide synthase (eNOS), a key molecule that promotes angiogenesis in human coronary artery endothelial cells (HCAEC). RESULTS: Suboptimal irradiation conditions included 1-MHz ultrasound frequency, 500-kPa sound pressure, 20-min total irradiation time, 32-48-[Formula: see text] pulse duration, and 320-[Formula: see text] pulse repetition time. Furthermore, a proposed index, [Formula: see text], calculated as the product of power and the total number of irradiation cycles applied to cells using LIPUS, uniformly revealed the experimental eNOS expression associated with the various values of five parameters under different irradiation conditions. CONCLUSION: We determined the suboptimal ultrasound irradiation conditions for promoting eNOS expression in HCAEC.

PSD-95 inhibitor Tat-NR2B9c (NA-1) protects the integrity of the blood-brain barrier after transient middle artery occlusion in rats by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

BACKGROUND: Protection against ischemic stroke may be most effective when multiple components of the neurovascular unit are protected, yet current treatments target mainly neurons. Here we explored whether the PSD-95 inhibitor Tat-NR2B9c (NA-1) can protect not only neurons but also the blood-brain barrier. METHODS: Adult male Sprague-Dawley rats were randomly divided into three groups, which were subjected to either sham surgery or transient cerebral ischemia-reperfusion, after which some animals were treated with Tat-NR2B9c. The therapeutic efficacy of Tat-NR2B9c was assessed in terms of the degree of neurological deficit and cerebral infarction, integrity of the blood-brain barrier, cerebral water content, as well as expression of PSD-95, nitric oxide synthase, and matrix metalloprotease-9. RESULTS: Tat-NR2B9c (NA-1) ameliorated neurofunctional deficit, reduced cerebral infarction, mitigated blood-brain barrier injury and improved its integrity following ischemia-reperfusion, leading to less cerebral edema. These improvements were associated with upregulation of tight junction proteins in the blood-brain barrier. At the same time, Tat-NR2B9c (NA-1) downregulated neuronal nitric oxide synthase and matrix metalloprotease-9, while reversing the ischemia-induced downregulation of endothelial nitric oxide synthase in brain. We report here the first evidence that PSD-95 is expressed in vascular endothelial cells in the brain. CONCLUSION: Our experiments in a rat model of transient occlusion of the middle cerebral artery suggest that Tat-NR2B9c (NA-1) can mitigate ischemic injury to the blood-brain barrier, and that it may do so by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

Exercise training and vascular heterogeneity in db/db mice: evidence for regional- and duration-dependent effects.

Exercise training (ET) has several health benefits; however, our understanding of regional adaptations to ET is limited. We examined the functional and molecular adaptations to short- and long-term ET in elastic and muscular conduit arteries of db/db mice in relation to changes in cardiovascular risk factors. Diabetic mice and their controls were exercised at moderate intensity for 4 or 8 weeks. The vasodilatory and contractile responses of thoracic aortae and femoral arteries isolated from the same animals were examined. Blood and aortic samples were used to measure hyperglycemia, oxidative stress, inflammation, dyslipidemia, protein expression of SOD isoforms, COX, eNOS, and Akt. Short-term ET improved nitric oxide (NO) mediated vasorelaxation in the aortae and femoral arteries of db/db mice in parallel with increased SOD2 and SOD3 expression, reduced oxidative stress and triglycerides, and independent of weight loss, glycemia, or inflammation. Long-term ET reduced body weight in parallel with reduced systemic inflammation and improved insulin sensitivity along with increased SOD1, Akt, and eNOS expression and improved NO vasorelaxation. Exercise did not restore NOS- and COX-independent vasodilatation in femoral arteries, nor did it mitigate the hypercontractility in the aortae of db/db mice; rather ET transiently increased contractility in association with upregulated COX-2. Long-term ET differentially affected the aortae and femoral arteries contractile responses. ET improved NO-mediated vasodilation in both arteries likely due to collective systemic effects. ET did not mitigate all diabetes-induced vasculopathies. Optimization of the ET regimen can help develop comprehensive management of type 2 diabetes.