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Parallel to the increasing prevalence of obesity in the world, the mortality from cardiovascular disease has also increased. Low-grade chronic inflammation in obesity disrupts vascular homeostasis, and the dysregulation of adipocyte-derived endocrine and paracrine effects contributes to endothelial dysfunction. Besides the adipose tissue inflammation, decreased nitric oxide (NO)-bioavailability, insulin resistance (IR), and oxidized low-density lipoproteins (oxLDLs) are the main factors contributing to endothelial dysfunction in obesity and the development of cardiorenal metabolic syndrome. While normal healthy perivascular adipose tissue (PVAT) ensures the dilation of blood vessels, obesity-associated PVAT leads to a change in the profile of the released adipo-cytokines, resulting in a decreased vasorelaxing effect. Higher stiffness parameter ß, increased oxidative stress, upregulation of pro-inflammatory cytokines, and nicotinamide adenine dinucleotide phosphate (NADP) oxidase in PVAT turn the macrophages into pro-atherogenic phenotypes by oxLDL-induced adipocyte-derived exosome-macrophage crosstalk and contribute to the endothelial dysfunction. In clinical practice, carotid ultrasound, higher leptin levels correlate with irisin over-secretion by human visceral and subcutaneous adipose tissues, and remnant cholesterol (RC) levels predict atherosclerotic disease in obesity. As a novel therapeutic strategy for cardiovascular protection, liraglutide improves vascular dysfunction by modulating a cyclic adenosine monophosphate (cAMP)-independent protein kinase A (PKA)-AMP-activated protein kinase (AMPK) pathway in PVAT in obese individuals. Because the renin-angiotensin-aldosterone system (RAAS) activity, hyperinsulinemia, and the resultant IR play key roles in the progression of cardiovascular disease in obesity, RAAS-targeted therapies contribute to improving endothelial dysfunction. By contrast, arginase reciprocally inhibits NO formation and promotes oxidative stress. Thus, targeting arginase activity as a key mediator in endothelial dysfunction has therapeutic potential in obesity-related vascular comorbidities. Obesity-related endothelial dysfunction plays a pivotal role in the progression of type 2 diabetes (T2D). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (thiazolidinedione), is a popular drug for treating diabetes; however, it leads to increased cardiovascular risk. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin (EMPA) significantly improves endothelial dysfunction and mortality occurring through redox-dependent mechanisms. Although endothelial dysfunction and oxidative stress are alleviated by either metformin or EMPA, currently used drugs to treat obesity-related diabetes neither possess the same anti-inflammatory potential nor simultaneously target endothelial cell dysfunction and obesity equally. While therapeutic interventions with glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide or bariatric surgery reverse regenerative cell exhaustion, support vascular repair mechanisms, and improve cardiometabolic risk in individuals with T2D and obesity, the GLP-1 analog exendin-4 attenuates endothelial endoplasmic reticulum stress.

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High glucose-induced endothelial dysfunction is an important pathological feature of diabetic vasculopathy. While genome-wide studies have identified an association between type 2 diabetes mellitus (T2DM) and increased expression of a C2 calcium-dependent domain containing 4B (C2CD4B), no study has yet explored the possible direct effect of C2CD4B on vascular function. Vascular reactivity studies were conducted using a pressure myograph, and nitric oxide and oxidative stress were assessed through difluorofluorescein diacetate and dihydroethidium, respectively. We demonstrate that high glucose upregulated both mRNA and protein expression of C2CD4B in mice mesenteric arteries in a time-dependent manner. Notably, the inhibition of C2CD4B expression by genetic knockdown efficiently prevented hyperglycemia-induced oxidative stress, endothelial dysfunction, and loss of nitric oxide (NO) bioavailability. Recombinant C2CD4B evoked endothelial dysfunction of mice mesenteric arteries, an effect associated with increased reactive oxygen species (ROS) and decreased NO production. In isolated human umbilical vein endothelial cells (HUVECs), C2CD4B increased phosphorylation of endothelial nitric oxide synthase (eNOS) at the inhibitory site Thr495 and reduced eNOS dimerization. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), Akt, and PKCα effectively attenuated oxidative stress, NO reduction, impairment of endothelial function, and eNOS uncoupling induced by C2CD4B. These data demonstrate, for the first time, that C2CD4B exerts a direct effect on vascular endothelium via a phosphoinositide 3-kinase (PI3K)/Akt/PKCα-signaling pathway, providing a new perspective on C2CD4B as a promising therapeutic target for the prevention of oxidative stress in diabetes-induced endothelial dysfunction.

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Vascular endothelial cells (ECs) senescence plays a crucial role in vascular aging that promotes the initiation and progression of cardiovascular disease. The mutation of Grb10-interacting GYF protein 2 (GIGYF2) is strongly associated with the pathogenesis of aging-related diseases, whereas its role in regulating ECs senescence and dysfunction still remains elusive. In this study, we found aberrant hyperexpression of GIGYF2 in senescent human ECs and aortas of old mice. Silencing GIGYF2 in senescent ECs suppressed eNOS-uncoupling, senescence, and endothelial dysfunction. Conversely, in nonsenescent cells, overexpressing GIGYF2 promoted eNOS-uncoupling, cellular senescence, endothelial dysfunction, and activation of the mTORC1-SK61 pathway, which were ablated by rapamycin or antioxidant N-Acetyl-l-cysteine (NAC). Transcriptome analysis revealed that staufen double-stranded RNA binding protein 1 (STAU1) is remarkably downregulated in the GIGYF2-depleted ECs. STAU1 depletion significantly attenuated GIGYF2-induced cellular senescence, dysfunction, and inflammation in young ECs. Furthermore, we disclosed that GIGYF2 acting as an RNA binding protein (RBP) enhances STAU1 mRNA stability, and that the intron region of the late endosomal/lysosomal adaptor MAPK and mTOR activator 4 (LAMTOR4) could bind to STAU1 protein to upregulate LAMTOR4 expression. Immunofluorescence staining showed that GIGYF2 overexpression promoted the translocation of mTORC1 to lysosome. In the mice model, GIGYF2flox/flox Cdh-Cre+ mice protected aged mice from aging-associated vascular endothelium-dependent relaxation and arterial stiffness. Our work discloses that GIGYF2 serving as an RBP enhances the mRNA stability of STAU1 that upregulates LAMTOR4 expression through binding with its intron region, which activates the mTORC1-S6K1 signaling via recruitment of mTORC1 to the lysosomal membrane, ultimately leading to ECs senescence, dysfunction, and vascular aging. Disrupting the GIGYF2-STAU1-mTORC1 signaling cascade may represent a promising therapeutic approach against vascular aging and aging-related cardiovascular diseases.

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Nitric oxide (NO) is one of the most important molecules released by endothelial cells, and its antiatherogenic properties support cardiovascular homeostasis. Diminished NO bioavailability is a common hallmark of endothelial dysfunction underlying the pathogenesis of the cardiovascular disease. Vascular NO is synthesized by endothelial nitric oxide synthase (eNOS) from the substrate L-arginine (L-Arg), with tetrahydrobiopterin (BH4) as an essential cofactor. Cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, aging, or smoking increase vascular oxidative stress that strongly affects eNOS activity and leads to eNOS uncoupling. Uncoupled eNOS produces superoxide anion (O2-) instead of NO, thus becoming a source of harmful free radicals exacerbating the oxidative stress further. eNOS uncoupling is thought to be one of the major underlying causes of endothelial dysfunction observed in the pathogenesis of vascular diseases. Here, we discuss the main mechanisms of eNOS uncoupling, including oxidative depletion of the critical eNOS cofactor BH4, deficiency of eNOS substrate L-Arg, or accumulation of its analog asymmetrical dimethylarginine (ADMA), and eNOS S-glutathionylation. Moreover, potential therapeutic approaches that prevent eNOS uncoupling by improving cofactor availability, restoration of L-Arg/ADMA ratio, or modulation of eNOS S-glutathionylation are briefly outlined.

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Aortic aneurysms are prevalent and severe vascular diseases with high mortality from unpredicted ruptures, while the only treatment option is surgical correction of large aneurysms with considerable risk. We have shown that folic acid (FA) is highly effective in alleviating development of aneurysms although not sufficient to completely attenuate aneurysm formation. Here, we examined therapeutic effects on aneurysms of combining FA with Nifedipine as novel and potentially more effective oral medication. Oral administration with FA (15 mg/kg/day) significantly reduced incidence of AAA from 85.71% to 18.75% in Ang II-infused apolipoprotein E (apoE) null mice, while combination of FA with Nifedipine (1.5, 5.0 or 20 mg/kg/day) substantially and completely further reduced incidence of AAA to 12.5%, 11.76% and 0.00% respectively in a dose-dependent manner. The combinatory therapy substantially and completely further alleviated enlargement of abdominal aortas defined by ultrasound, vascular remodeling characterized by elastin degradation and adventitial hypertrophy, as well as aortic superoxide production and eNOS uncoupling activity also in a dose-dependent manner, with combination of FA with 20 mg/kg/day Nifedipine attenuating all of these features by 100% to control levels. Aortic NO and H4B bioavailabilities were also dose-dependently further improved by combining FA with Nifedipine. These data establish entirely innovative and robust therapeutic regime of FA combined with Nifedipine for the treatment of aortic aneurysms. The comminatory therapy can serve as a first-in-class and most effective oral medication for aortic aneurysms, which can be rapidly translated into clinical practice to revolutionize management of the devastating vascular diseases of aortic aneurysms known as silent killers.

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We have previously shown that circulating levels of tetrahydrobiopterin (H4B) function as a robust biomarker for aortic aneurysms in several independent animal models. In the present study, we examined diagnostic and predictive values of circulating H4B levels in human patients of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) for the first time, while clinically applicable biomarkers for aortic aneurysms have never been previously available. Ninety-five patients scheduled for TAA repair surgeries and 53 control subjects were recruited at University of California Los Angeles (UCLA) Ronald Regan Medical Center, while 44 control subjects and 29 AAA patients were recruited through National Institute of Health (NIH) National Disease Research Interchange (NDRI) program. We had intriguing observations that circulating H4B levels were substantially lower in TAA and AAA patients, linearly correlated with aortic H4B levels (blood: R = 0.8071, p < 0.0001, n = 75; plasma: R = 0.7983, p < 0.0001, n = 75), and associated with incidence of TAA (blood: adjusted OR 0.495; 95% CI 0.379-0.647; p < 0.001; plasma: adjusted OR 0.501; 95% CI 0.385-0.652; p < 0.001) or AAA (blood: adjusted OR 0.329; 95% CI 0.125-0.868; p = 0.025) after adjustment for other factors. Blood or plasma H4B levels below 0.2 pmol/µg serve as an important threshold for prediction of aortic aneurysms independent of age and gender (for TAA risk - blood: adjusted OR 419.67; 95% CI 59.191-2975.540; p < 0.001; plasma: adjusted OR 206.11; 95% CI 40.956-1037.279; p < 0.001). This threshold was also significantly associated with incidence of AAA (p < 0.001 by Chi-square analysis). In addition, we observed previously unrecognized inverse association of Statin use with TAA, and an association of AAA with arrhythmia. Taken together, our data strongly demonstrate for the first time that circulating H4B levels can serve as a first-in-class, sensitive, robust and independent biomarker for clinical diagnosis and prediction of TAA and AAA in human patients, which can be rapidly translated to bedside to fundamentally improve clinical management of the devastating human disease of aortic aneurysms.

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Limited medical therapies have been implemented for the treatment of the devastating cardiorespiratory disease of pulmonary hypertension (PH) while none of which is sufficiently effective to stop or regress development of PH. We have previously shown that netrin-1, an axon-guiding protein during development, protects against ischemia reperfusion injury induced myocardial infarction via modest and stable production of nitric oxide (NO) and attenuation of oxidative stress. Since NO deficiency and oxidative stress-mediated vascular remodeling play important roles in the pathogenesis of PH, our present study investigated therapeutic effects on PH of netrin-1 and its derived small peptides. Infused into mice for 3 weeks during exposure to hypoxia, netrin-1 and netrin-1 derived small peptides V1, V2 or V3 substantially alleviated pathophysiological and molecular features of PH, as indicated by abrogated increases in mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP), attenuated right ventricular hypertrophy, diminished vascular remodeling of medial thickening and upregulation in smooth muscle alpha-actin (SMA) and proliferative cell nuclear antigen (PCNA), and alleviated perivascular and peribronchial fibrosis reflected by collagen deposition. NO bioavailability was substantially improved by treatment with netrin-1 and netrin-1 derived small peptides, while hypoxia induced increases in total superoxide production and eNOS uncoupling activity were all attenuated. These dual mechanisms of increasing NO bioavailability and decreasing oxidative stress at the same time, underlie robust protective effects on PH of netrin-1 and its derived small peptides, which are different from existing medications that primarily target NO signaling alone. Our data for the first time demonstrate intriguing findings that netrin-1 and netrin-1 derived small peptides can be used as novel and robust therapeutics for the treatment of PH.

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Atrial inflammation and fibrosis have long been considered culprits in the development of atrial fibrillation (AF). Prior clinical studies showed that corticosteroid therapy is beneficial in patients with AF. Here we sought to determine whether prednisone treatment prevents atrial tachypacing (ATP) induced atrial fibrosis.Dogs were randomized into the sham, ATP, ATP + low-dose prednisone (ALP), and ATP + high-dose prednisone (AHP) groups. After 6 days of recovery from surgery, dogs were subjected to ATP at 400 beats per minute for 4 weeks while being treated with prednisone (15 or 40 mg/day) or a placebo. Pacemakers were not activated in the sham group.Compared with the ATP group, the expression of collagen I, collagen III, α-smooth muscle actin, transforming growth factor-ß1 and connective tissue growth factor were significantly reduced in the ALP and AHP groups. Fluorescence assays showed that reactive oxygen species formation in the right atrium was suppressed in the ALP and AHP groups compared with the ATP group. The protein level of NADPH oxidase 2 was reduced in the ALP and AHP groups' versus ATP group, while NOX4 and NOX5 were unchanged. ATP-induced downregulation of BH4 and eNOS uncoupling in the atria was partially restored in the prednisone-treated groups.Our study demonstrated that atrial fibrosis induced by ATP were suppressed by prednisone. Low-dose prednisone was also effective in suppressing the development of atrial fibrosis.

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Cardiovascular disease (CVD) is a major cause of mortality worldwide. A better understanding of the mechanisms underlying CVD is key for better management or prevention. Oxidative stress has been strongly implicated in the pathogenesis of CVD. Indeed, several studies demonstrated that reactive oxygen species (ROS), via different mechanisms, can lead to endothelial cell (EC) dysfunction, a major player in the etiology of several CVDs. ROS appears to modulate a plethora of EC biological processes that are critical for the integrity of the endothelial function. This review seeks to dissect the role of oxidative stress-induced endothelial dysfunction in CVD development, with emphasis on the underlying mechanisms and pathways. Special attention is given to ROS-induced reduction of NO bioavailability, ROS-induced inflammation, and ROS-induced mitochondrial dysfunction. A better understanding and appraisal of these pathways may be essential to attenuate oxidative stress or reverse EC dysfunction, and hence, reduce CVD burden.

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We recently reported a mouse model of chronic electronic cigarette (e-cig) exposure-induced cardiovascular pathology, where long-term exposure to e-cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Here, we delineate the underlying mechanisms of ECV-induced vascular endothelial dysfunction (VED), a central trigger of cardiovascular disease. C57/BL6 male mice were exposed to ECV generated from e-cig liquid containing 0, 6, or 24 mg/mL nicotine for 16 and 60 wk. Time-dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine-induced aortic relaxation in ECV-exposed mice, compared with air-exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3-nitrotyrosine levels in the aorta of ECV-exposed mice were observed, indicating that ECV-induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV-induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt-dependent eNOS phosphorylation occurred in the aorta of ECV-exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV-induced superoxide generation in the aorta of ECV-exposed mice. Together, our data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.NEW & NOTEWORTHY Underlying mechanisms of e-cig-induced vascular endothelial dysfunction are delineated. e-cig exposure activates and increases expression of NADPH oxidase and disrupts activation and coupling of eNOS, leading to a vicious cycle of superoxide generation and peroxynitrite formation, with tetrahydrobiopterin depletion, causing loss of NO that triggers vascular endothelial dysfunction. This process is progressive, increasing with the duration of e-cig exposure, and is more severe in the presence of nicotine, but observed even with nicotine-free vaping.

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Mercury has been shown to be a significant health risk factor and is positively associated with cardiovascular diseases. Evidence reveals that men are more likely to develop cardiovascular diseases than women during reproductive age. However, the effects of mercury in females remain poorly investigated, despite the finding that female hormones demonstrate a cardioprotective role. In the present study, we evaluated whether chronic mercury chloride exposure could alter blood pressure and vascular function of the female rat aorta. Ten-week-old female Wistar rats were divided into two groups: control (vehicle) and mercury treated (first dose of 4.6 µg/kg, subsequent daily doses of 0.07 µg/kg), im. Mercury treatment did not modify systolic blood pressure (SBP) but increased vascular reactivity due to the reduction of nitric oxide bioavailability associated with the increase in reactive oxygen species from endothelial nitric oxide synthase (eNOS) uncoupling. Furthermore, increased participation of the cyclooxygenase-2 pathway occurred through an imbalance in thromboxane 2 and prostacyclin 2. However, the oestrogen signalling pathway was not altered in either group. These results demonstrated that chronic exposure to mercury in females induced endothelial dysfunction and, consequently, increased aortic vascular reactivity, causing vascular damage to the female rat aorta and representing a risk of cardiovascular diseases.

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Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2-•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

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Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The initial stage of CVDs is characterized by endothelial dysfunction, defined as the limited bioavailability of nitric oxide (NO). Thus, any factors that interfere with the synthesis or metabolism of NO in endothelial cells are involved in CVD pathogenesis. It is well established that hypoxia is both the triggering factor as well as the accompanying factor in cardiovascular disease, and diminished tissue oxygen levels have been reported to influence endothelial NO bioavailability. In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS) from L-Arg, with tetrahydrobiopterin (BH4) as an essential cofactor. Here, we discuss the mechanisms by which hypoxia affects NO bioavailability, including regulation of eNOS expression and activity. What is particularly important is the fact that hypoxia contributes to the depletion of cofactor BH4 and deficiency of substrate L-Arg, and thus elicits eNOS uncoupling-a state in which the enzyme produces superoxide instead of NO. eNOS uncoupling and the resulting oxidative stress is the major driver of endothelial dysfunction and atherogenesis. Moreover, hypoxia induces impairment in mitochondrial respiration and endothelial cell activation; thus, oxidative stress and inflammation, along with the hypoxic response, contribute to the development of endothelial dysfunction.

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Acute lung injury (ALI) is a devastating clinical syndrome with no effective therapies. Inflammasome activation has been reported to play a critical role in the initiation and progression of ALI. The molecular mechanisms involved in regulating the activation of inflammasome in ALI remains unresolved, although increases in mitochondrial derived reactive oxygen species (mito-ROS) are involved. Our previous work has shown that the mitochondrial redistribution of uncoupled eNOS impairs mitochondrial bioenergetics and increases mito-ROS generation. Thus, the focus of our study was to determine if lipopolysaccharide (LPS)-mediated inflammasome activation involves the mitochondrial redistribution of uncoupled eNOS. Our data show that the increase in mito-ROS involved in LPS-mediated inflammasome activation is associated with the disruption of mitochondrial bioenergetics in human lung microvascular endothelial cells (HLMVEC) and the mitochondrial redistribution of eNOS. These effects are dependent on RhoA-ROCK signaling and are mediated via increased phosphorylation of eNOS at Threonine (T)-495. A derivative of the mitochondrial targeted Szeto-Schiller peptide (SSP) attached to the antioxidant Tiron (T-SSP), significantly attenuated LPS-mediated mito-ROS generation and inflammasome activation in HLMVEC. Further, T-SSP attenuated mitochondrial superoxide production in a mouse model of sepsis induced ALI. This in turn significantly reduced the inflammatory response and attenuated lung injury. Thus, our findings show that the mitochondrial redistribution of uncoupled eNOS is intimately involved in the activation of the inflammatory response in ALI and implicate attenuating mito-ROS as a therapeutic strategy in humans.

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Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH4) synthesis and biopterin ratio ([BH4]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH3 to BH4, direct scavenging of superoxide (O2•-) and peroxynitrite (ONOO-) and increasing eNOS activity. The model predicted that Asc at 0.1-100 µM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1-10 mM reduced O2•- and ONOO- levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction.

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In the present study we aimed to identify novel mechanisms and therapeutics for thoracic aortic aneurysm (TAA) in Fbn1C1039G/+ Marfan Syndrome (MFS) mice. The expression of mature/active TGFß and its downstream effector NOX4 were upregulated while tetrahydrobiopterin (H4B) salvage enzyme dihydrofolate reductase (DHFR) was downregulated in Fbn1C1039G/+ mice. In vivo treatment with anti-TGFß completely attenuated NOX4 expression, restored DHFR protein abundance, reduced ROS production, recoupled eNOS and attenuated aneurysm formation. Intriguingly, oral administration with folic acid (FA) to recouple eNOS markedly alleviated expansion of aortic roots and abdominal aortas in Fbn1C1039G/+ mice, which was attributed to substantially upregulated DHFR expression and activity in the endothelium to restore tissue and circulating levels of H4B. Notably, circulating H4B levels were accurately predictive of tissue H4B bioavailability, and negatively associated with expansion of aortic roots, indicating a novel biomarker role of circulating H4B for TAA. Furthermore, FA diet abrogated TGFß and NOX4 expression, disrupting the feed-forward loop to inactivate TGFß/NOX4/DHFR/eNOS uncoupling axis in vivo and in vitro, while PTIO, a NO scavenger, reversed this effect in cultured human aortic endothelial cells (HAECs). Besides, expression of the rate limiting H4B synthetic enzyme GTP cyclohydrolase 1 (GTPCHI), was downregulated in Fbn1C1039G/+ mice at baseline. In cultured HAECs, RNAi inhibition of fibrillin resulted in reduced GTPCHI expression, while this response was abrogated by anti-TGFß, indicating TGFß-dependent downregulation of GTPCHI in response to fibrillin deficiency. Taken together, our data for the first time reveal that uncoupled eNOS plays a central role in TAA formation, while anti-TGFß and FA diet robustly abolish aneurysm formation via inactivation of a novel TGFß/NOX4/DHFR/eNOS uncoupling/TGFß feed-forward pathway. Correction of fibrillin deficiency is additionally beneficial via preservation of GTPCHI function.

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Significance: Hypertension has major health consequences, which is associated with endothelial dysfunction. Endothelial nitric oxide synthase (eNOS)-produced nitric oxide (NO) signaling in the vasculature plays an important role in maintaining vascular homeostasis. Considering the importance of NO system, this review aims to provide a brief overview of the biochemistry of members of NO signaling, including GTPCH1 [guanosine 5'-triphosphate (GTP) cyclohydrolase 1], tetrahydrobiopterin (BH4), and eNOS. Recent Advances: Being NO signaling activators and regulators of eNOS signaling, BH4 treatment is getting widespread attention either as potential therapeutic agents or as preventive agents. Recent clinical trials also support that BH4 treatment could be considered a promising therapeutic in hypertension. Critical Issues: Under conditions of BH4 depletion, eNOS-generated superoxides trigger pathological events. Abnormalities in NO availability and BH4 deficiency lead to disturbed redox regulation causing pathological events. This disturbed signaling influences the development of systemic hypertension as well as pulmonary hypertension. Future Directions: Considering the importance of BH4 and NO to improve the translational significance, it is essential to continue research on this field to manipulate BH4 to increase the efficacy for treating hypertension. Thus, this review also examines the current state of knowledge on the effects of eNOS activators on preclinical models and humans to utilize this information for potential therapy.

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Endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (PAH). Ion channelome changes have long been connected to vascular remodeling in PAH, yet only recently has the focus shifted towards Ca2+-activated Cl- channels (CaCC). The most prominent member of the CaCC TMEM16A has been shown to contribute to the pathogenesis of idiopathic PAH (IPAH) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (PAECs) and in the development of endothelial dysfunction remains underrepresented. Here we report enhanced TMEM16A activity in IPAH PAECs by whole-cell patch-clamp recordings. Using adenoviral-mediated TMEM16A increase in healthy primary human PAECs in vitro and in human pulmonary arteries ex vivo, we demonstrate the functional consequences of the augmented TMEM16A activity: alterations of Ca2+ dynamics and eNOS activity as well as decreased NO production, PAECs proliferation, wound healing, tube formation and acetylcholine-mediated relaxation of human pulmonary arteries. We propose that the ERK1/2 pathway is specifically affected by elevated TMEM16A activity, leading to these pathological changes. With this work we introduce increased TMEM16A activity in the cell membrane of human PAECs for the development of endothelial dysfunction in PAH.

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Arginine (L-arginine), is an amino acid involved in a number of biological processes, including the biosynthesis of proteins, host immune response, urea cycle, and nitric oxide production. In this systematic review, we focus on the functional role of arginine in the regulation of endothelial function and vascular tone. Both clinical and preclinical studies are examined, analyzing the effects of arginine supplementation in hypertension, ischemic heart disease, aging, peripheral artery disease, and diabetes mellitus.