eNOS Activity Research Articles

Microvessels-on-chip: Exploring endothelial cells and COVID-19 plasma interaction with nitric oxide metabolites.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), primarily manifests as a flu-like illness with lung injury, often necessitating supplemental oxygen. Elderly individuals and those with pre-existing cardiovascular diseases are at increased risk of mortality. The endothelial barrier disruption observed in patients indicates systemic viral invasion and widespread endotheliitis. Endothelial dysfunction, characterized by impaired nitric oxide (NO) production, contributes to vasoconstriction, inflammation, and coagulation abnormalities seen in COVID-19. In this study, we investigated the impact of COVID-19 patient-derived plasma on the endothelium through NO metabolite analysis using an in vitro 3D micro vessel model. Our experiments revealed alterations in NO metabolites in response to COVID-19 patient plasma perfusion, with BH4+BH2 supplementation improving citrulline levels in severe COVID-19 patient models. Positive correlation between arginase activity and eNOS activity was observed in the severe COVID-19 patient model but not in the mild COVID-19 patient model. These findings underscore the importance of endothelial dysfunction in COVID-19 pathogenesis and highlight potential therapeutic targets for mitigating vascular complications associated with severe infection.

Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation.

Endothelial cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that the application of force to endothelial cells stimulates VE-cadherin to activate liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin-stimulated AMPK increases eNOS (also known as NOS3) activity and localization to the plasma membrane, reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton and cellular alignment. Together, these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic and vasodilatory disturbances arise.

Subcellular Localization Guides eNOS Function.

Nitric oxide synthases (NOS) are enzymes responsible for the cellular production of nitric oxide (NO), a highly reactive signaling molecule involved in important physiological and pathological processes. Given its remarkable capacity to diffuse across membranes, NO cannot be stored inside cells and thus requires multiple controlling mechanisms to regulate its biological functions. In particular, the regulation of endothelial nitric oxide synthase (eNOS) activity has been shown to be crucial in vascular homeostasis, primarily affecting cardiovascular disease and other pathophysiological processes of importance for human health. Among other factors, the subcellular localization of eNOS plays an important role in regulating its enzymatic activity and the bioavailability of NO. The aim of this review is to summarize pioneering studies and more recent publications, unveiling some of the factors that influence the subcellular compartmentalization of eNOS and discussing their functional implications in health and disease.

Microvesicles Derived from Nitric Oxide Synthase-Inhibited Endothelial Cells Promote Cell Dysfunction

Introduction: The aims of this study were to determine (1) whether endothelial nitric oxide synthase (eNOS) inhibition stimulates endothelial microvesicles (EMVs) release and (2) the effect of EMVs derived from eNOS-inhibited cells on endothelial cell eNOS, inflammation, apoptosis, and tissue-type plasminogen activator (t-PA). Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor (NG-nitro-l-arginine methyl ester [L-NAME], 300 µM) for 24 h. EMVs from untreated and L-NAME-treated cells were isolated, quantified, and exposed to HUVECs for 24 h. Results: eNOS-inhibited cells released significantly higher EMVs than untreated cells (81 ± 13 vs. 41 ± 15 EMV/μL; p = 0.005). Expression of total eNOS (97.1 ± 16.4 vs. 157.5 ± 31.2 arbitrary units [AUs]; p = 0.01), p-eNOS (4.9 ± 1.2 vs. 9.1 ± 12.6 AUs; p = 0.02), and NO production (5.0 ± 0.8 vs. 7.0 ± 1.3 µmol/L; p = 0.04) were significantly lower in cells treated with EMVs from L-NAME-treated cells. L-NAME-derived EMVs induced significantly higher IL-6 (38.3 ± 10.3 vs. 21.0 ± 3.8 pg/mL; p = 0.01) and IL-8 (38.9 ± 7.0 vs. 27.2 ± 6.2 pg/mL; p = 0.04) production concurrent with higher expression of p-NF-κB p65 (Ser536) (9.7 ± 1.6 vs. 6.1 ± 1.2 AUs; p = 0.01). Expression of activated caspase-3 was higher (9.5 ± 1.1 vs. 6.4 ± 0.4 AUs) and t-PA lower (24.2 ± 4.3 vs. 36.2 ± 8.4 AUs; p = 0.04) in cells treated with L-NAME-derived EMVs. Conclusion: eNOS inhibition induces an increase in EMV release and an EMV phenotype with adverse cellular effects.

Pasteurized Akkermansia muciniphila Ameliorates Preeclampsia in Mice by Enhancing Gut Barrier Integrity, Improving Endothelial Function, and Modulating Gut Metabolic Dysregulation.

Preeclampsia (PE) is a serious complication of pregnancy linked to endothelial dysfunction and an imbalance in the gut microbiota. While Akkermansia muciniphila (AKK) has shown promise in alleviating PE symptoms, the use of live bacteria raises safety concerns. This study explored the potential of pasteurized A. muciniphila (pAKK) as a safer alternative for treating PE, focusing on its effects on endothelial function and metabolic regulation. A PE mouse model was induced via the nitric oxide synthase inhibitor L-NAME, followed by treatment with either pAKK or live AKK. Fecal metabolomic profiling was performed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in vivo and in vitro experiments were used to assess the effects of pAKK on endothelial function and metabolic pathways. pAKK exhibited therapeutic effects comparable to those of live AKK in improving L-NAME-induced PE-like phenotypes in mice, including enhanced gut barrier function and reduced endotoxemia. pAKK also promoted placental angiogenesis by restoring endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production. The in vitro experiments further confirmed that pAKK alleviated L-NAME-induced NO reduction and endothelial dysfunction in human umbilical vein endothelial cells (HUVECs). Metabolomic analysis revealed that both pAKK and live AKK reversed metabolic disturbances in PE by modulating key metabolites and pathways related to unsaturated fatty acid biosynthesis, folate, and linoleic acid metabolism. As a postbiotic, pAKK may support existing treatments for preeclampsia by improving gut barrier function, restoring endothelial function, and regulating metabolic dysregulation, offering a safer alternative to live bacteria. These findings highlight the potential clinical value of pAKK as an adjunctive therapy in managing PE.

Genetic Manipulation of Caveolin-1 in a Transgenic Mouse Model of Aortic Root Aneurysm: Sex-Dependent Effects on Endothelial and Smooth Muscle Function.

Marfan syndrome (MFS) is a systemic connective tissue disorder stemming from mutations in the gene encoding Fibrillin-1 (Fbn1), a key extracellular matrix glycoprotein. This condition manifests with various clinical features, the most critical of which is the formation of aortic root aneurysms. Reduced nitric oxide (NO) production due to diminished endothelial nitric oxide synthase (eNOS) activity has been linked to MFS aortic aneurysm pathology. Caveolin-1 (Cav1), a structural protein of plasma membrane caveolae, is known to inhibit eNOS activity, suggesting its involvement in MFS aneurysm progression by modulating NO levels. In this study, we examined the role of Cav1 in aortic smooth muscle and endothelial function, aortic wall elasticity, and wall strength in male and female MFS mice (FBN1+/Cys1041Gly) by generating developing Cav1-deficient MFS mice (MFS/Cav1KO). Our findings reveal that Cav1 ablation leads to a pronounced reduction in aortic smooth muscle contraction in response to phenylephrine, attributable to an increase in NO production in the aortic wall. Furthermore, we observed enhanced aortic relaxation responses to acetylcholine in MFS/Cav1KO mice, further underscoring Cav1's inhibitory impact on NO synthesis within the aorta. Notably, van Gieson staining and chamber myography analyses showed improved elastin fiber structure and wall strength in male MFS/Cav1KO mice, whereas these effects were absent in female counterparts. Cav1's regulatory influence on aortic root aneurysm development in MFS through NO-mediated modulation of smooth muscle and endothelial function, with notable sex-dependent variations.

Abstract 4114308: Defective in LYPLA1-Mediated Lysophosphatidylcholine Metabolism Contributes to Diabetes-Induced Atherosclerosis and Subsequent Myocardial Dysfunction

Background and Objective: Diabetes mellitus (DM) is a chronic metabolic condition that can cause serious damage to blood vessels and the heart. While current metabolomics research mainly focused on metabolic profiles during disease onset or at advanced stages, there remains a gap in understanding the molecular mechanisms of metabolites in the progression of diabetic cardiovascular disease. This study aims to explore effective strategies for preventing cardiovascular complications in diabetics by excavating metabolic pathways and mechanisms. Methods: Peripheral blood samples were obtained from 21 healthy individuals, 20 patients diagnosed with DM, 20 patients with coronary heart disease (CHD), and 25 patients with both DM and CHD (DC). Cardiac function, biochemical parameters and serum metabolome were detected. A specialized methodology with rigorous screening criteria was employed to identify differential metabolites associated with diabetes-induced CHD and cardiomyopathy. Mice were injected with LPC16:0 to assess cardiovascular function. Models of diabetes and diabetic atherosclerosis were induced by STZ injection and high-fat diet feeding in WT and ApoE -/- mice. Cardiac function and differential metabolites in aortic and cardiac tissues were assessed. The effect and molecular mechanism of LYPLA1 on vascular function, mitochondrial function, and metabolites-related enzymes were examined in human aortic endothelial cells (HAECs) and AC16 cardiomyocytes. Results: Cardiac function was markedly declined in DM and DC patients, as well as in mice with diabetes and diabetic atherosclerosis. However, CHD patients and atherosclerosis alone did not exhibit evident cardiac dysfunction. Key metabolites associated with diabetic cardiomyopathy and diabetes-induced CHD were identified as LPE18:1 and LPC16:0, respectively. Intraperitoneal administration of LPC16:0 in mice led to decreased LYPLA1, severe myocardial anomalies, and the appearance of plaques in the aortic arch. Either LYPLA1 knockdown or the treatment with high glucose and palmitic acid in HAECs resulted in impaired mitochondrial function, reduced intracellular NO and eNOS activity, which were mitigated by LYPLA1 overexpression. Conclusion: The metabolite LPC16:0 and the regulatory enzyme LYPLA1 are pivotal in driving the advancement of cardiovascular complications triggered by diabetes. Targeting LYPLA1 could represent a promising strategy for preventing diabetes-induced CHD and subsequent cardiomyopathy.

Abstract 4145829: Fatty Acid Binding Protein 4 as Potential Indicator for Gestational Diabetes Mellitus-Induced Fetal Endothelial Dysfunction

Introduction/Background: Gestational diabetes mellitus (GDM) is the most common complication during pregnancy. GDM is leading to an elevated risk for the development of cardiovascular diseases both in the mother and the child in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential role as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. Research Question/Hypothesis: Does FABP4 affect the function of human endothelial cells from patients with GDM? Goals/Aim: Our aim is a better understanding of the role of FABP4 in fetal endothelial dysfunction of patients with GDM and the development of potential therapeutic strategies. Methods/Approach: Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords obtained from normoglycemic and GDM pregnancies. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression pattern. HUVECs were subjected to functional analyses assessing proliferation, migration, NO and insulin signaling. Finally, human endothelial cells were exposed to high laminar flow (30 dyn/cm 2 ) or simvastatin (10 nM) treatment to investigate their impact on FABP4 expression and endothelial function. Results/Data: Bulk RNA sequencing data analysis revealed FABP4 as a gene overexpressed in GDM HUVECs compared to normoglycemic controls. Gene set enrichment analysis showed in addition an enrichment of genes involved in angiogenesis and Notch signaling pathways in human endothelial cells from GDM patients. GDM HUVECs had a significantly higher wound healing capacity compared to normoglycemic controls. The PI3K/AKT/mTOR pathway was enriched in GDM HUVECs. GDM HUVECs displayed impaired activation of AKT and eNOS (expression and phosphorylation) after stimulation with insulin, suggesting a possible insulin resistance. Finally, subjecting HUVECs to high laminar shear stress or simvastatin treatment caused a reduction of FABP4 mRNA expression and an increase of eNOS expression, indicating potential therapeutic strategies to improve endothelial function. Conclusions: We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. High laminar shear stress and medications activating eNOS signaling could protect the endothelium against the negative impact of FABP4 in GDM.

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

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

Moderate physical exercise induces F-actin assembly potentially optimizing eNOS activation.

Moderate physical exercise induces F-actin assembly potentially optimizing eNOS activation.

The effect of CD38 inhibition in age-associated vascular dysfunction

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

Comprehensive analysis of VEGF/VEGFR inhibitor-induced immune-mediated hypertension: integrating pharmacovigilance, clinical data, and preclinical models.

This study aimed to elucidate the differential immunological mechanisms and characteristics of hypertension induced by VEGF inhibitors (VEGFi) and VEGF receptor inhibitors (VEGFRi), with the goal of optimizing monitoring strategies and treatment protocols. We investigated the risk of immune-related adverse events associated with VEGFi/VEGFRi-induced hypertension by analyzing the FDA Adverse Event Reporting System (FAERS) database. Findings were corroborated with blood pressure characteristics observed in clinical patients and preclinical models exposed to various VEGF/VEGFRi. Clinical and preclinical studies were conducted to compare immunological responses and hypertension profiles between inhibitor classes. An integrative analysis across cancer types and species was performed, focusing on key signaling pathways. Analysis of FAERS data, in conjunction with clinical observations, revealed that both VEGFi and VEGFRi significantly elevated the risk of immune-mediated, blood pressure-related adverse events (ROR=7.75, 95% CI: 7.76-7.95). Subsequent clinical and preclinical studies demonstrated differential immunological responses and hypertension profiles between inhibitor classes. VEGFRi exhibited a more rapid onset, greater blood pressure elevation, and higher incidence of immune-mediated adverse events compared to VEGFi (Systolic BP: ROR=0 for VEGFi vs. ROR=12.25, 95% CI: 6.54-22.96 for VEGFRi; Diastolic BP: ROR=5.09, 95% CI: 0.60-43.61 for VEGFi vs. ROR=12.90, 95% CI: 3.73-44.55 for VEGFRi). Integrative analysis across cancer types and species, focusing on key signaling pathways, revealed that VEGF/VEGFRi-induced blood pressure elevation was associated with immunomodulation of the mitogen activated protein kinase (MAPK) pathway (R=-0.379, P=0.0435), alterations in triglyceride metabolism (R=-0.664, P=0.0001), modulation of myo-inositol 1,4,5-trisphosphate-sensitive calcium release channel activity (R=0.389, P=0.0378), and dysregulation of nitric oxide eNOS activation and metabolism (R=-0.439, P=0.0179). The temporal dynamics of these effects demonstrated greater significance than dose-dependent responses. Both VEGFi and VEGFRi significantly augmented the risk of immune-mediated, blood pressure-related adverse events, with VEGFRi inducing a more rapid and pronounced onset of blood pressure elevation and a higher incidence of immune-related, blood pressure-associated adverse events compared to VEGFi.

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

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

Angiopoietin-2 reverses endothelial cell dysfunction in progeria vasculature.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder in children caused by a point mutation in the lamin A gene, resulting in a toxic form of lamin A called progerin. Accelerated atherosclerosis leading to heart attack and stroke are the major causes of death in these patients. Endothelial cell (EC) dysfunction contributes to the pathogenesis of HGPS related cardiovascular diseases (CVD). Endothelial cell-cell communications are important in the development of the vasculature, and their disruptions contribute to cardiovascular pathology. However, it is unclear how progerin interferes with such communications that lead to vascular dysfunction. An antibody array screening of healthy and HGPS patient EC secretomes identified Angiopoietin-2 (Ang2) as a down-regulated signaling molecule in HGPS ECs. A similar down-regulation of Ang2 mRNA and protein was detected in the aortas from an HGPS mouse model. Addition of Ang2 to HGPS ECs rescues vasculogenesis, normalizes endothelial cell migration and gene expression, and restores nitric oxide bioavailability through eNOS activation. Furthermore, Ang2 addition reverses unfavorable paracrine effects of HGPS ECs on vascular smooth muscle cells. Lastly, by utilizing adenine base editor (ABE)-corrected HGPS ECs and progerin-expressing HUVECs, we demonstrated a negative correlation between progerin and Ang2 expression. Lastly, our results indicated that Ang2 exerts its beneficial effect in ECs through Tie2 receptor binding, activating an Akt-mediated pathway. Together, these results provide molecular insights into EC dysfunction in HGPS and suggest that Ang2 treatment has potential therapeutic effects in HGPS-related CVD.

DDAH1 deficiency exacerbates cerebral vascular endothelial dysfunction by aggravating BBB disruption and oxidative stress in thoracic blast-induced brain injury

As terrorist incidents and underground explosion events have become more frequent around the world, brain injury caused by thoracic blast exposure has been more highlighted due to its injured organ, subsequent social and economic burden. It has been reported dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays important roles in regulating vascular endothelial injury repair and angiogenesis, but its role in thoracic blast-induced brain injury remains to be explained. This study seeks to investigate the mechanism of DDAH1 on thoracic blast-induced brain injury. 40 C57BL/6 wild type mice and 40 DDAH1 knockout mice were randomly and equally divided into control group and blast group, respectively. The integrity of blood-brain barrier (BBB) was detected by Evans blue test. The serum inflammatory factors, nitric oxide (NO) contents, and asymmetric dimethylarginine (ADMA) levels were determined through ELISA. HE staining and reactive oxygen species (ROS) detection were performed for histopathological changes. Western blot was used to detect the proteins related to oxidative stress, tight junction, focal adhesion, vascular endothelial injury, and the DDAH1/ADMA/eNOS signaling pathway. DDAH1 deficiency aggravated thoracic blast-induced BBB leakage, inflammatory response, and the increased levels of inflammatory-related factors. Additionally, DDAH1 deficiency also increased ROS generation, MDA and IRE-α expression. Regarding cerebral vascular endothelial dysfunction, DDAH1 deficiency increased the expression of MCAM, FN1, LIMK1, VEGF, MMP9, Vimentin and N-cadherin, while lowering the expression of FMR1, Occludin, claudin-3, claudin-5, Lyn, LIMA1, Glrb, Sez6, Dystrophin, and phosphorylation of VASP. Also, DDAH1 deficiency exacerbated explosion-induced increase of ADMA and decrease of eNOS activity and NO contents. Thus, we conclude that DDAH1 could prevent cerebral vascular endothelial dysfunction and related injury by inhibiting ADMA signaling and increasing eNOS activity in thoracic blast induced brain injury.

Evaluation of anti-hypertensive activity of Quercus leucotrichophora A. camus methanolic extract on DOCA-salt induced hypertensive rats

Quercus leucotrichophora A. camus, commonly known as Banj oak or Ban tree, belongs to the Fagaceae family and is abundantly found in the regions of Shimla, Kullu-Manali, and upper Mandi in Himachal Pradesh. This study explores the medicinal properties of plant seeds, known for their antioxidant and diuretic activity, traditionally utilized in the treatment of hypertension and hypolipidemic, hepatoprotective conditions. PurposeTo investigate the antihypertensive effect of methanolic extract of Quercus leucotrichophora A. camus seeds in DOCA sat induced rats. Material and methodsGround seeds of Quercus leucotrichophora A. camus were stored in an airtight container. A total of 100 gs of dried seed powder were subjected to extraction using 250 ml of methanol and a Soxhlet extractor maintained at 60–65 °C for five hours. The resulting dried extract (5 gs) was stored in an airtight container in a refrigerator at low temperature and administered by rats to test its antihypertensive impact. The assessment of obtained extract impact on hypertension induced by DOCA-salt was conducted through blood pressure measurement and biochemical analysis. ResultsOur study revealed that the methanolic extract of Quercus leucotrichophora A. camus seeds exerted blood pressure-lowering effects, associated with an increase in serum Nitric Oxide (NO) levels. The attenuation of hypertension by extract was further linked to the normalization of serum Na+ and K+concentrations, indicating an improvement in electrolyte imbalance. Additionally, the antioxidant properties of Quercus leucotrichophora A. camus were evident through increased activities of Glutathione (GSH), Glutathione Peroxidase (GHPx), Superoxide Dismutase (SOD), and decrease activity of Thiobarbituric acid reactive substances (TBARS). ConclusionThe findings of our investigation suggest that the antihypertensive action of Quercus leucotrichophora A. camus is correlated with enhanced endothelial nitric oxide synthase (eNOS) activation and an augmented antioxidant defense system. This sheds light on the potential therapeutic benefits of plant under investigation in managing hypertension, emphasizing its role in promoting cardiovascular health. Further research is warranted to elucidate the underlying mechanisms and to explore the full pharmacological potential of Quercus leucotrichophora A. camus in the field of cardiovascular medicine.

Signaling Paradigms of H2S-Induced Vasodilation: A Comprehensive Review.

Hydrogen sulfide (H2S), a gas traditionally considered toxic, is now recognized as a vital endogenous signaling molecule with a complex physiology. This comprehensive study encompasses a systematic literature review that explores the intricate mechanisms underlying H2S-induced vasodilation. The vasodilatory effects of H2S are primarily mediated by activating ATP-sensitive potassium (K_ATP) channels, leading to membrane hyperpolarization and subsequent relaxation of vascular smooth muscle cells (VSMCs). Additionally, H2S inhibits L-type calcium channels, reducing calcium influx and diminishing VSMC contraction. Beyond ion channel modulation, H2S profoundly impacts cyclic nucleotide signaling pathways. It stimulates soluble guanylyl cyclase (sGC), increasing the production of cyclic guanosine monophosphate (cGMP). Elevated cGMP levels activate protein kinase G (PKG), which phosphorylates downstream targets like vasodilator-stimulated phosphoprotein (VASP) and promotes smooth muscle relaxation. The synergy between H2S and nitric oxide (NO) signaling further amplifies vasodilation. H2S enhances NO bioavailability by inhibiting its degradation and stimulating endothelial nitric oxide synthase (eNOS) activity, increasing cGMP levels and potent vasodilatory responses. Protein sulfhydration, a post-translational modification, plays a crucial role in cell signaling. H2S S-sulfurates oxidized cysteine residues, while polysulfides (H2Sn) are responsible for S-sulfurating reduced cysteine residues. Sulfhydration of key proteins like K_ATP channels and sGC enhances their activity, contributing to the overall vasodilatory effect. Furthermore, H2S interaction with endothelium-derived hyperpolarizing factor (EDHF) pathways adds another layer to its vasodilatory mechanism. By enhancing EDHF activity, H2S facilitates the hyperpolarization and relaxation of VSMCs through gap junctions between endothelial cells and VSMCs. Recent findings suggest that H2S can also modulate transient receptor potential (TRP) channels, particularly TRPV4 channels, in endothelial cells. Activating these channels by H2S promotes calcium entry, stimulating the production of vasodilatory agents like NO and prostacyclin, thereby regulating vascular tone. The comprehensive understanding of H2S-induced vasodilation mechanisms highlights its therapeutic potential. The multifaceted approach of H2S in modulating vascular tone presents a promising strategy for developing novel treatments for hypertension, ischemic conditions, and other vascular disorders. The interaction of H2S with ion channels, cyclic nucleotide signaling, NO pathways, ROS (Reactive Oxygen Species) scavenging, protein sulfhydration, and EDHF underscores its complexity and therapeutic relevance. In conclusion, the intricate signaling paradigms of H2S-induced vasodilation offer valuable insights into its physiological role and therapeutic potential, promising innovative approaches for managing various vascular diseases through the modulation of vascular tone.

Circulating extracellular microvesicles associated with electronic cigarette use increase endothelial cell inflammation and reduce nitric oxide production.

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

Vascular endothelial cell morphology and alignment regulate VEGF-induced endothelial nitric oxide synthase activation.

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) inhibits platelet and leukocyte adhesion while promoting vasorelaxation in smooth muscle cells. Dysfunctional regulation of eNOS is a hallmark of various vascular pathologies, notably atherosclerosis, often associated with areas of low shear stress on endothelial cells (ECs). While the link between EC morphology and local hemodynamics is acknowledged, the specific impact of EC morphology on eNOS regulation remains unclear. Morphological differences between elongated, aligned ECs and polygonal, randomly oriented ECs correspond to variations in focal adhesion and cytoskeletal organization, suggesting differing levels of cytoskeletal prestress. However, the functional outcomes of cytoskeletal prestress, particularly in the absence of shear stress, are not extensively studied in ECs. Some evidence suggests that elongated ECs exhibit decreased immunogenicity and enhanced NO production. This study aims to elucidate the signaling pathways governing VEGF-stimulated eNOS regulation in the aligned EC phenotype characterized by elongated and aligned cells within a monolayer. Using anisotropic topographic cues, bovine aortic endothelial cells (BAECs) were elongated and aligned, followed by VEGF treatment in the presence or absence of cytoskeletal tension inhibitors. Phosphorylation of eNOS ser1179, AKT ser437 and FAK Tyr397 in response to VEGF challenge were significantly heightened in aligned ECs compared to unaligned ECs. Moreover this response proved to be robustly tied to cytoskeletal tension as evinced by the abrogation of responses in the presence of the myosin II ATPase inhibitor, blebbistatin. Notably, this work demonstrates for the first time the reliance on FAK phosphorylation in VEGF-mediated eNOS activation and the comparatively greater contribution of the cytoskeletal machinery in propagating VEGF-eNOS signaling in aligned and elongated ECs. This research underscores the importance of utilizing appropriate vascular models in drug development and sheds light on potential mechanisms underlying vascular function and pathology that can help inform vascular graft design.

Polyphenol-rich Aronia melanocarpa juice sustains eNOS activation through phosphorylation and expression via redox-sensitive pathways in endothelial cells.

A sustained formation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is crucial to safeguard the vascular system against the development of cardiovascular diseases. This study investigated the prolonged phosphorylation and expression of eNOS induced by polyphenol-rich Aronia melanocarpa juice (AMJ), along with its underlying mechanisms. The findings revealed that AMJ triggered concentration- and time-dependent increases in eNOS phosphorylation and expression, leading to sustained NO production for 15h. Investigations with various enzymes and inhibitors revealed that the effect of AMJ was associated with redox sensitivity, activating the PI3-kinase/Akt, JNK, and p38 MAPK pathways. These pathways led to the inactivation of transcription factors FoxO1 and FoxO3a through phosphorylation, relieving their repression on eNOS expression. Therefore, the capability of AMJ to consistently trigger prolonged eNOS phosphorylation and expression via complex redox-sensitive pathways highlights its potential for maintaining vascular health and preventing cardiovascular diseases.