eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity.

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

The impact of cardiomotor rehabilitation on endothelial function in elderly patients with chronic heart failure.

BACKGROUND: To explore the effects of cardiac exercise rehabilitation on peripheral blood endothelial progenitor cells (EPC) in elderly patients with chronic heart failure. METHODS: 80 elderly patients with chronic heart failure were selected from March 2017 to March 2019 and randomly divided into two groups (N = 40). The control group was treated routinely and walked freely for 30-60 min every day. The patients in the exercise rehabilitation group developed a cardiac exercise rehabilitation plan. Then, cardiac function and peripheral blood B-natriuretic peptide (BNP) levels in the two groups were compared. The cell viability, proliferation, apoptosis, and invasion ability of EPCs were detected. The levels of the PI3K/AKT pathway and eNOS and VEGF were compared. RESULTS: There were no significant differences in all indexes between the two groups before treatment (P > 0.05), and both improved significantly after treatment (P < 0.05). After treatment, LVEF and LVFS in the exercise rehabilitation group were significantly higher than those in the control group (P < 0.05), and LVEDD and LVESD were significantly lower than those in the control group (P < 0.05). The BNP level in the exercise rehabilitation group was significantly lower than that in the control group (P < 0.05). The cell viability, proliferation, invasion ability of EPC, and the levels of PI3K, AKT, eNOS, and VEGF mRNA and protein in the exercise rehabilitation group were significantly higher than those in the control group. Apoptosis rate was significantly lower than those in the control group (P < 0.05). CONCLUSIONS: Visceral exercise rehabilitation can improve cardiac ejection and myocardial function in elderly patients with chronic heart failure, and can promote the vitality, proliferation, and invasion of peripheral blood EPC, and promote the expression of eNOS and VEGF by upregulating the PI3K/AKT pathway to promote angiogenesis and endothelial function.

Ventilatory responses during and following hypercapnic gas challenge are impaired in male but not female endothelial NOS knock-out mice.

The roles of endothelial nitric oxide synthase (eNOS) in the ventilatory responses during and after a hypercapnic gas challenge (HCC, 5% CO2, 21% O2, 74% N2) were assessed in freely-moving female and male wild-type (WT) C57BL6 mice and eNOS knock-out (eNOS-/-) mice of C57BL6 background using whole body plethysmography. HCC elicited an array of ventilatory responses that were similar in male and female WT mice, such as increases in breathing frequency (with falls in inspiratory and expiratory times), and increases in tidal volume, minute ventilation, peak inspiratory and expiratory flows, and inspiratory and expiratory drives. eNOS-/- male mice had smaller increases in minute ventilation, peak inspiratory flow and inspiratory drive, and smaller decreases in inspiratory time than WT males. Ventilatory responses in female eNOS-/- mice were similar to those in female WT mice. The ventilatory excitatory phase upon return to room-air was similar in both male and female WT mice. However, the post-HCC increases in frequency of breathing (with decreases in inspiratory times), and increases in tidal volume, minute ventilation, inspiratory drive (i.e., tidal volume/inspiratory time) and expiratory drive (i.e., tidal volume/expiratory time), and peak inspiratory and expiratory flows in male eNOS-/- mice were smaller than in male WT mice. In contrast, the post-HCC responses in female eNOS-/- mice were equal to those of the female WT mice. These findings provide the first evidence that the loss of eNOS affects the ventilatory responses during and after HCC in male C57BL6 mice, whereas female C57BL6 mice can compensate for the loss of eNOS, at least in respect to triggering ventilatory responses to HCC.

Noncanonical Thyroid Hormone Receptor α Action Mediates Arterial Vasodilation.

CONTEXT: Hypothyroidism impairs cardiovascular health and contributes to endothelial dysfunction with reduced vasodilation. How 3,5,3'-triiodothyronine (T3) and its receptors are involved in the regulation of vasomotion is not yet fully understood. In general, thyroid hormone receptors (TRs) either influence gene expression (canonical action) or rapidly activate intracellular signaling pathways (noncanonical action). OBJECTIVE: Here we aimed to characterize the T3 action underlying the mechanism of arterial vasodilation and blood pressure (BP) regulation. METHODS: Mesenteric arteries were isolated from male rats, wild-type (WT) mice, TRα knockout (TRα 0) mice, and from knockin mice with a mutation in the DNA-binding domain (TRα GS). In this mutant, DNA binding and thus canonical action is abrogated while noncanonical signaling is preserved. In a wire myograph system, the isolated vessels were preconstricted with norepinephrine. The response to T3 was measured, and the resulting vasodilation (Δ force [mN]) was normalized to maximum contraction with norepinephrine and expressed as percentage vasodilation after maximal preconstriction with norepinephrine (%NE). Isolated vessels were treated with T3 (1 × 10-15 to 1 × 10-5 mol/L) alone and in combination with the endothelial nitric oxide-synthase (eNOS) inhibitor L-NG-nitroarginine methyl ester (L-NAME) or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The endothelium was removed to determine the contribution of T3 to endothelium-dependent vasodilation. The physiological relevance of T3-induced vasodilation was determined by in vivo arterial BP measurements in male and female mice. RESULTS: T3 treatment induced vasodilation of mesenteric arteries from WT mice within 2 minutes (by 21.5 ±â€…1.7%NE). This effect was absent in arteries from TRα 0 mice (by 5.3 ±â€…0.6%NE, P < .001 vs WT) but preserved in TRα GS arteries (by 17.2 ±â€…1.1%NE, not significant vs WT). Inhibition of either eNOS or PI3K reduced T3-mediated vasodilation from 52.7 ±â€…4.5%NE to 28.5 ±â€…4.1%NE and 22.7 ±â€…2.9%NE, respectively. Removal of the endothelium abolished the T3-mediated vasodilation in rat mesenteric arteries (by 36.7 ±â€…5.4%NE vs 3.5 ±â€…6.2%NE). In vivo, T3 injection led to a rapid decrease of arterial BP in WT (by 13.9 ±â€…1.9 mm Hg) and TRα GS mice (by 12.4 ±â€…1.9 mm Hg), but not in TRα 0 mice (by 4.1 ±â€…1.9 mm Hg). CONCLUSION: These results demonstrate that T3 acting through noncanonical TRα action affects cardiovascular physiology by inducing endothelium-dependent vasodilation within minutes via PI3K and eNOS activation.

Apoptosis signal-regulating kinase 1 (ASK1) inhibition reduces endothelial cytokine production without improving permeability after toll-like receptor 4 (TLR4) challenge.

Sepsis represents a life-threatening event often mediated by the host's response to pathogens such as gram-negative organisms, which release the proinflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.

Glu298Asp (rs1799983) Polymorphism Influences Postprandial Vascular Reactivity and the Insulin Response to Meals of Varying Fat Composition in Postmenopausal Women: Findings from the Randomized, Controlled Dietary Intervention and VAScular function (DIVAS)-2 Study.

BACKGROUND: Previous acute studies suggest the Glu298Asp polymorphism (rs1799983) may influence vascular reactivity in response to long-chain n-3 PUFA intake. However, the effects of this genotype on postprandial vascular function after meals rich in SFAs, n-6 PUFAs, and MUFAs are unclear. OBJECTIVES: This study determined the impact of the Glu298Asp polymorphism on changes in vascular function and cardiometabolic risk biomarkers in response to sequential meals of varying fat composition. METHODS: In a randomized, double-blind, crossover, acute study, 32 postmenopausal women (mean ± SD age: 58 ± 5 y; BMI: 25.9 ± 4.1 kg/m2) consumed mixed meals (breakfast: 0 min, 50 g fat; lunch: 330 min, 30 g fat) containing SFAs, n-6 PUFAs, or MUFAs on 3 occasions. Blood samples for cardiometabolic disease risk markers and real-time measures of vascular reactivity [including flow-mediated dilatation (FMD; primary outcome)] were collected/performed before and regularly for 480 min after breakfast. Participants were retrospectively genotyped for the Glu298Asp (rs1799983) polymorphism. Data were analyzed using linear mixed models. RESULTS: For the postprandial %FMD response, a test fat × genotype interaction was observed for the AUC (P = 0.019) but not incremental AUC (IAUC), with the AUC being ∼24% greater after MUFA- than after SFA- and n-6 PUFA-rich meals in the Glu298 homozygotes (P ≤ 0.026). Test fat × genotype interactions were also evident for postprandial insulin (P ≤ 0.005), with the MUFA-rich meals demonstrating significantly higher AUC (12.8%/14.9%), IAUC (14.6%/20.0%), and maximum concentration (20.0%/34.5%) than the SFA- and n-6 PUFA-rich meals, respectively, in Asp298 carriers (P < 0.05). Genotype did not influence other study outcome measures in response to the test fats. CONCLUSIONS: Our findings suggest the Glu298Asp polymorphism may represent a potential determinant of the inter-individual variability in postprandial responsiveness of %FMD and insulin to acute meal fat composition in postmenopausal women. Further studies are required to confirm these observations.This trial was registered at clinicaltrials.gov as NCT02144454.

Gene expression profiles of diabetic kidney disease and neuropathy in eNOS knockout mice: Predictors of pathology and RAS blockade effects.

Diabetic kidney disease (DKD) and diabetic peripheral neuropathy (DPN) are two common diabetic complications. However, their pathogenesis remains elusive and current therapies are only modestly effective. We evaluated genome-wide expression to identify pathways involved in DKD and DPN progression in db/db eNOS-/- mice receiving renin-angiotensin-aldosterone system (RAS)-blocking drugs to mimic the current standard of care for DKD patients. Diabetes and eNOS deletion worsened DKD, which improved with RAS treatment. Diabetes also induced DPN, which was not affected by eNOS deletion or RAS blockade. Given the multiple factors affecting DKD and the graded differences in disease severity across mouse groups, an automatic data analysis method, SOM, or self-organizing map was used to elucidate glomerular transcriptional changes associated with DKD, whereas pairwise bioinformatic analysis was used for DPN. These analyses revealed that enhanced gene expression in several pro-inflammatory networks and reduced expression of development genes correlated with worsening DKD. Although RAS treatment ameliorated the nephropathy phenotype, it did not alter the more abnormal gene expression changes in kidney. Moreover, RAS exacerbated expression of genes related to inflammation and oxidant generation in peripheral nerves. The graded increase in inflammatory gene expression and decrease in development gene expression with DKD progression underline the potentially important role of these pathways in DKD pathogenesis. Since RAS blockers worsened this gene expression pattern in both DKD and DPN, it may partly explain the inadequate therapeutic efficacy of such blockers.

eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects.

Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

Role of endothelial nitric oxide synthase for early brain injury after subarachnoid hemorrhage in mice.

The first few hours and days after subarachnoid hemorrhage (SAH) are characterized by cerebral ischemia, spasms of pial arterioles, and a significant reduction of cerebral microperfusion, however, the mechanisms of this early microcirculatory dysfunction are still unknown. Endothelial nitric oxide production is reduced after SAH and exogenous application of NO reduces post-hemorrhagic microvasospasm. Therefore, we hypothesize that the endothelial NO-synthase (eNOS) may be involved in the formation of microvasospasms, microcirculatory dysfunction, and unfavorable outcome after SAH. SAH was induced in male eNOS deficient (eNOS-/-) mice by endovascular MCA perforation. Three hours later, the cerebral microcirculation was visualized using in vivo 2-photon-microscopy. eNOS-/- mice had more severe SAHs, more severe ischemia, three time more rebleedings, and a massively increased mortality (50 vs. 0%) as compared to wild type (WT) littermate controls. Three hours after SAH eNOS-/- mice had fewer perfused microvessels and 40% more microvasospasms than WT mice. The current study indicates that a proper function of eNOS plays a key role for a favorable outcome after SAH and helps to explain why patients suffering from hypertension or other conditions associated with impaired eNOS function, have a higher risk of unfavorable outcome after SAH.

Endothelial nitric oxide synthase-derived nitric oxide in the regulation of metabolism.

Nitric oxide is an endogenously formed gas that acts as a signaling molecule in the human body. The signaling functions of nitric oxide are accomplished through two primer mechanisms: cGMP-mediated phosphorylation and the formation of S-nitrosocysteine on proteins. This review presents and discusses previous and more recent findings documenting that nitric oxide signaling regulates metabolic activity. These discussions primarily focus on endothelial nitric oxide synthase (eNOS) as the source of nitric oxide.

Dysregulation of Endothelial Nitric Oxide Synthase Does Not Depend on Hemodynamic Alterations in Bicuspid Aortic Valve Aortopathy.

Background Bicuspid aortic valves (BAVs) predispose to ascending aortic aneurysm. Turbulent blood flow and genetic factors have been proposed as underlying mechanisms. Endothelial nitric oxide synthase (eNOS) has been implicated in BAV aortopathy, and its expression is regulated by wall shear stress. We hypothesized that if turbulent flow induces aneurysm formation in patients with a BAV, regional differences in eNOS expression would be observed in BAVs. Methods and Results Ascending aortic specimens were harvested intraoperatively from 48 patients with tricuspid aortic valve (19 dilated, 29 nondilated) and 38 with BAV (28 dilated, 10 nondilated) undergoing cardiac surgery. eNOS mRNA and protein concentration were analyzed at the convex and concave aortic wall. In nondilated aortas, eNOS mRNA and protein concentration were decreased in BAV compared with tricuspid aortic valve (all P<0.05). eNOS expression was increased in association with dilation in BAV aortas (P=0.03), but not in tricuspid aortic valve aortas (P=0.63). There were no regional differences in eNOS mRNA or protein concentration in BAV aortas (all P>0.05). However, eNOS expression was increased at the concave wall (versus convexity) in tricuspid aortic valve dilated aortas (all P<0.05). Conclusions Dysregulated eNOS occurs independent of dilation in BAV aortas, suggesting a potential role for aberrantly regulated eNOS expression in the development of BAV-associated aneurysms. The absence of regional variations of eNOS expression suggests that eNOS dysregulation in BAV aortas is the result of underlying genetic factors associated with BAV disease, rather than changes stimulated by hemodynamic alterations. These findings provide insight into the underlying mechanisms of aortic dilation in patients with a BAV.

Inhibitory effects of Paris saponin I, II, ⠥ and ⠦ on HUVEC cells through regulation of VEGFR2, PI3K/AKT/mTOR, Src/eNOS, PLCγ/ERK/MERK, and JAK2-STAT3 pathways.

Rhizoma Paris is a popular Chinese medicine in clinics. It contains four main saponins which are its major bioactive compounds. These saponins are Paris saponin I, II, VI and VII (PSI, PSII, PSVI and PSVII, respectively). Up to now, the research using HUVEC cells to evaluate the anti-angiogenic activity of four saponins is blank. The purpose of this study was to evaluate the anti-angiogenic properties (also known as angiotoxicity) of the four saponins in Rhizoma Paris on vascular endothelial cells-HUVEC cells, and to investigate the underlying mechanism, which has not been studied before. In this study, MTT assay, Lactate dehydrogenase (LDH) assay, wound healing experiments, transwell cell invasion assay, tubule formation experiment, DAPI staining, AV-PI double staining, and cell cycle analysis were used to determine the effects of Paris saponins. The results showed that, with increases in concentrations of PSI, PSII, PSVI and PSVII, the viability of HUVEC cells decreased significantly. In addition, four saponins dose-dependent enhanced LDH release and inhibited HUVEC cell migration, invasion, and angiogenesis. In terms of mechanism, PSI significantly inhibited protein expression in multiple signaling pathways. In particular, with the VEGF2 as the target, it activate the downstream PI3K / AKT / mTOR, SRC / eNOS, P38, PLCγ / ERK / MERK and JAK2/STAT3 signaling pathways. In conclusion, PSI, PSII, PSVI and PSVII can inhibit endothelial cell proliferation, migration and invasion, block endothelial cell cycle, induce endothelial cell apoptosis, act on protein expression in several anti-angiogenic signaling pathways, and finally inhibit angiogenesis in vitro. This study provides further data support for the clinical application of Paris saponins as antiangiogenic drugs.

Fatty acid transport protein-2 regulates glycemic control and diabetic kidney disease progression.

Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end-stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, after filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2-dependent (FATP2-dependent) mechanism. To address this possibility, genetic (Leprdb/db eNOS-/-) and induced (high-fat diet plus low-dose streptozotocin) mouse models of obesity and DKD were bred with global FATP2 gene-deleted mice (Slc27a2) and then phenotyped. DKD-prone mice with the Slc27a2-/- genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer life span compared with diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2-/- mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2-/- mice was accompanied by ß cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.

Aortic Valve Stenosis and Mitochondrial Dysfunctions: Clinical and Molecular Perspectives.

Calcific aortic stenosis is a disorder that impacts the physiology of heart valves. Fibrocalcific events progress in conjunction with thickening of the valve leaflets. Over the years, these events promote stenosis and obstruction of blood flow. Known and common risk factors are congenital defects, aging and metabolic syndromes linked to high plasma levels of lipoproteins. Inflammation and oxidative stress are the main molecular mediators of the evolution of aortic stenosis in patients and these mediators regulate both the degradation and remodeling processes. Mitochondrial dysfunction and dysregulation of autophagy also contribute to the disease. A better understanding of these cellular impairments might help to develop new ways to treat patients since, at the moment, there is no effective medical treatment to diminish neither the advancement of valve stenosis nor the left ventricular function impairments, and the current approaches are surgical treatment or transcatheter aortic valve replacement with prosthesis.

Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms with Susceptibility to Prostate Cancer: a Comprehensive Systematic Review and Meta-Analysis.

PURPOSE: A variety of studies have evaluated the association of polymorphisms at endothelial nitric oxide synthase (eNOS) gene with risk of prostate cancer. However, the results remain inconclusive. This meta-analysis was performed to derive a more precise estimation between eNOS polymorphisms and prostate cancer risk. MATERIALS AND METHODS: A comprehensive literature search was conducted using PubMed, EMBASE, Wed of Science, Elsevier, Cochrane Library, SciELO, SID, WanFang, VIP, CBD and CNKI database up to March 20, 2020. Odds ratios with 95% confidence intervals were used to assess the strength of the associations. RESULTS: A total of 22 case-control studies including 12 studies with 4,464 cases and 4,347 controls on +894G>T, five studies with 589 cases and 789 controls on VNTR 4a/b, and five studies with 588 cases and 692 controls on -786T > C were selected. Overall, pooled data showed a significant association between eNOS 894G>T, VNTR 4a/b, and -786T > C polymorphisms and an increased risk of prostate cancer in the global population. When stratified by ethnicity, a significant association was found between eNOS +894G>T and -786T>C polymorphisms and risk of prostate cancer in Caucasians. CONCLUSION: Our results indicated that eNOS 894G>T, VNTR 4a/b, and -786T>C polymorphisms were associated with risk of prostate cancer in the global population as well as Caucasian population.

A Role of Inflammation and Immunity in Essential Hypertension-Modeled and Analyzed Using Petri Nets.

Recent studies have shown that the innate and adaptive immune system, together with low-grade inflammation, may play an important role in essential hypertension. In this work, to verify the importance of selected factors for the development of essential hypertension, we created a Petri net-based model and analyzed it. The analysis was based mainly on t-invariants, knockouts of selected fragments of the net and its simulations. The blockade of the renin-angiotensin (RAA) system revealed that the most significant effect on the emergence of essential hypertension has RAA activation. This blockade affects: (1) the formation of angiotensin II, (2) inflammatory process (by influencing C-reactive protein (CRP)), (3) the initiation of blood coagulation, (4) bradykinin generation via the kallikrein-kinin system, (5) activation of lymphocytes in hypertension, (6) the participation of TNF alpha in the activation of the acute phase response, and (7) activation of NADPH oxidase-a key enzyme of oxidative stress. On the other hand, we found that the blockade of the activation of the RAA system may not eliminate hypertension that can occur due to disturbances associated with the osmotically independent binding of Na in the interstitium. Moreover, we revealed that inflammation alone is not enough to trigger primary hypertension, but it can coexist with it. We believe that our research may contribute to a better understanding of the pathology of hypertension. It can help identify potential subprocesses, which blocking will allow better control of essential hypertension.

Ultrasound-Targeted Microbubble Cavitation with Sodium Nitrite Synergistically Enhances Nitric Oxide Production and Microvascular Perfusion.

Microvascular obstruction is a common repercussion of percutaneous coronary intervention for distal microembolization, ischemia-reperfusion injury and inflammation, which increases post-myocardial infarction heart failure and mortality. Ultrasound-targeted microbubble cavitation (UTMC) may resolve microvascular obstruction while activating endothelial nitric oxide synthase (eNOS) and increasing endothelium-derived nitric oxide (NO) bioavailability. Nitrite, a cardioprotective agent, offers an additional source of NO and potential synergy with UTMC. UTMC and nitrite co-therapy increased microvascular perfusion and NO concentration in a rat hindlimb model. Using N-nitro-L-arginine methyl ester for eNOS blockade, we found a three-way interaction effect between nitrite, UTMC and eNOS on microvascular perfusion and NO production. Modulating ultrasound peak negative acoustic pressure (0.33-1.5 MPa) significantly affected outcomes, while microbubble dosage (2 × 108 bubbles/mL, 1.5 mL/h to 1 × 109 bubbles/mL, 3 mL/h) did not. Nitrite co-therapy also protected against oxidative stress. Comparison of nitrite to sodium nitroprusside with UTMC revealed synergistic effects were specific to nitrite. Synergy between UTMC and nitrite holds therapeutic potential for cardiovascular disease.

Apelin directs endothelial cell differentiation and vascular repair following immune-mediated injury.

Sustained, indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized, maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes, associated with expression of apelin. In the adult, the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo, loss of donor heart expression of apelin facilitated graft immune cell infiltration, blunted vascular repair, and worsened occlusive vasculopathy in mice. In vitro, an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus, apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together, these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature.

Acanthopanax senticosus Induces Vasorelaxation via Endothelial Nitric Oxide-Dependent and -Independent Pathways.

Although Acanthopanax senticosus root extract (ASRE), a functional food used in Japan, improves peripheral blood circulation and exerts vasorelaxant effects in rats under healthy conditions, the underlying mechanisms currently remain unclear. Therefore, we investigated the mechanisms responsible for ASRE-induced relaxation in isolated thoracic aortas using organ bath techniques and examined whether ASRE affects systemic and peripheral circulation using a photoplethysmographic tail-cuff system and noncontact laser tissue blood flow meter in Wistar rats. Similar to acetylcholine (ACh), ASRE induced dose-dependent relaxation in aortas pre-contracted with phenylephrine; however, in contrast to ACh, ASRE-induced relaxation was partially inhibited by treatments with antagonists of nitric oxide (NO) synthase and soluble guanylyl cyclase as well as by endothelium removal. Contractile responses to phenylephrine or potassium chloride were observed in the presence of ASRE. The oral administration of ASRE (900 mg/kg/d for 1 wk) decreased systolic blood pressure in rats 3 h after the treatment and did not affect heart rate, tail blood flow, mass, or velocity; this decreasing effect was not observed on day 2. A 1-wk treatment with ASRE did not affect vasorelaxation in response to ASRE. These results demonstrate that ASRE induces vasorelaxation via endothelial NO production and an NO-independent pathway in rats. Based on these findings, positive impacts of ASRE on blood pressure and peripheral blood circulation cannot be expected under healthy conditions as the systemic effects of ASRE are temporary. Instead, caution is needed to prevent the occurrence of side effects (i.e., orthostatic dizziness) at the beginning of ASRE dosing.

MicroRNA-615-5p Regulates Angiogenesis and Tissue Repair by Targeting AKT/eNOS (Protein Kinase B/Endothelial Nitric Oxide Synthase) Signaling in Endothelial Cells.

Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.