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The endothelium plays a pivotal role in the regulation of vascular tone by synthesizing and liberating endothelium-derived relaxing factors inclusive of vasodilator prostaglandins (eg, prostacyclin), nitric oxide (NO), and endothelium-dependent hyperpolarization factors in a distinct blood vessel size–dependent manner. Large conduit arteries are predominantly regulated by NO and small resistance arteries by endothelium-dependent hyperpolarization factors. Accumulating evidence over the past few decades has demonstrated that endothelial dysfunction and coronary vasomotion abnormalities play crucial roles in the pathogenesis of various cardiovascular diseases. Structural and functional alterations of the coronary microvasculature have been coined as coronary microvascular dysfunction (CMD), which is highly prevalent and associated with adverse clinical outcomes in many clinical settings. The major mechanisms of coronary vasomotion abnormalities include enhanced coronary vasoconstrictive reactivity at epicardial and microvascular levels, impaired endothelium-dependent and endothelium-independent coronary vasodilator capacities, and elevated coronary microvascular resistance caused by structural factors. Recent experimental and clinical research has highlighted CMD as the systemic small artery disease beyond the heart, emerging modulators of vascular functions, novel insights into the pathogenesis of cardiovascular diseases associated with CMD, and potential therapeutic interventions to CMD with major clinical implications. In this article, we will summarize the current knowledge on the endothelial modulation of vascular tone and the pathogenesis of coronary macrovascular and microvascular diseases from bench to bedside, with a special emphasis placed on the mechanisms and clinical implications of CMD.

Endothelium-derived hyperpolarizing factor (EDHF) was envisaged as a chemical entity causing vasodilation by hyperpolarizing vascular smooth muscle (VSM) cells and distinct from nitric oxide (NO) ([aka endothelium-derived relaxing factor (EDRF)]) and prostacyclin. The search for an identity for EDHF unraveled the complexity of signaling within small arteries. Hyperpolarization originates within endothelial cells (ECs), spreading to the VSM by 2 branches, 1 chemical and 1 electrical, with the relative contribution varying with artery location, branch order, and prevailing profile of VSM activation. Chemical signals vary likewise and can involve potassium ion, lipid mediators, and hydrogen peroxide, whereas electrical signaling depends on physical contacts formed by homocellular and heterocellular (myoendothelial; MEJ) gap junctions, both able to conduct hyperpolarizing current. The discovery that chemical and electrical signals each arise within ECs resulted in an evolution of the single EDHF concept into the more inclusive, EDH signaling. Recognition of the importance of MEJs and particularly the fact they can support bidirectional signaling also informed the discovery that Ca2+ signals can pass from VSM to ECs during vasoconstriction. This signaling activates negative feedback mediated by NO and EDH forming a myoendothelial feedback circuit, which may also be responsible for basal or constitutive release of NO and EDH activity. The MEJs are housed in endothelial projections, and another spin-off from investigating EDH signaling was the discovery these fine structures contain clusters of signaling proteins to regulate both hyperpolarization and NO release. So, these tiny membrane bridges serve as a signaling superhighway or infobahn, which controls vasoreactivity by responding to signals flowing back and forth between the endothelium and VSM. By allowing bidirectional signaling, MEJs enable sinusoidal vasomotion, co-ordinated cycles of widespread vasoconstriction/vasodilation that optimize time-averaged blood flow. Cardiovascular disease disrupts EC signaling and as a result vasomotion changes to vasospasm.

Background Although high doses of proton pump inhibitors can elicit an anticancer effect, this strategy may impair vascular biology. In particular, their effects on endothelial Ca2⁺ signaling and production of endothelium-derived relaxing factor (EDRF) are unknown. To this end, we investigated the effects of high dosages of omeprazole on endothelial Ca2⁺ responses and EDRF production in primary cultured porcine aortic endothelial cells.Methods and Results Omeprazole (10–1000μM) suppressed both bradykinin (BK)- and thapsigargin-induced endothelial Ca2⁺ response in a dose-dependent manner. Furthermore, omeprazole slightly attenuated Ca2⁺ mobilization from the endoplasmic reticulum, whereas no inhibitory effects on endoplasmic reticulum Ca2⁺-ATPase were observed. Omeprazole decreased BK-induced phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and tended to decrease BK-induced nitric oxide production. Production of prostaglandin I₂ metabolites, especially 6-keto-prostaglandin 1α, also tended to be reduced by omeprazole.Conclusion Our results are the first to indicate that high doses of omeprazole may suppress both store-operated Ca2⁺ channels and partially the G protein-coupled receptor/phospholipase C/inositol 1,4,5-triphosphate pathway, and decreased BKinduced, Ca2⁺-dependent phosphorylation of eNOS(Ser1177). Thus, high dosages of omeprazole impaired EDRF production by attenuating intracellular Ca2⁺ signaling.

We aimed to investigate whether the change in methemoglobin levels (ΔMHb) predicts oxygenation response to inhaled nitric oxide (iNO) in persistent pulmonary hypertension of the newborn (PPHN) with lung disease, with or without pulmonary hypoplasia.In this prospective observational study, infants were categorized based on ΔMHb and oxygenation response (ΔPaO2/FiO2) following iNO: ΔMHb ≤0 or ΔMHb0, and ΔPaO2/FiO2 20 mmHg (Non-responder) or≥20 mmHg (Responder). ΔMHb levels were compared among infants with or without pulmonary hypoplasia.Among infants with pulmonary hypoplasia (n = 28), ΔMHb was not associated with an oxygenation response to iNO or survival without ECMO. Among infants without hypoplasia (n = 29), subjects with ΔMHb0 following iNO (n = 21) had a greater ΔPaO2/FiO2 (median, 64 mmHg; IQR, 127; p 0.01) and 100% survival without extracorporeal membrane oxygenation (ECMO) when compared to infants with ΔMHb ≤0 (n = 8; median 10 mmHg; IQR, 33).PPHN secondary to lung disease without hypoplasia with increased ΔMHb following iNO was associated with better oxygenation response and survival without ECMO compared to subjects without an increase in MHb.

An electrical communication between the endothelial and smooth muscle cells via gap junctions, which provides the signaling pathway known as endothelium-dependent hyperpolarization (EDH), plays a crucial role in controlling the vascular tone. In this study, we investigated the role of gap junctions in the acetylcholine (ACh)-induced EDH-type dilation of rat retinal arterioles in vivo. The dilator response was evaluated by measuring the diameter of retinal arterioles. Intravitreal injection of gap junction blockers (18β-glycyrrhetinic acid and carbenoxolone) reduced the ACh-induced dilation of retinal arterioles. Moreover, the retinal arteriolar response to ACh was attenuated by 18β-glycyrrhetinic acid under treatment with a combination of NG-nitro-L-arginine methyl ester (a nitric oxide (NO) synthase inhibitor; 30 mg/kg) and indomethacin (a cyclooxygenase inhibitor; 5 mg/kg). The NO- and prostaglandin-independent, EDH-related component of ACh-induced dilation of retinal arterioles was prevented by intravitreal injection of iberiotoxin, which inhibits large-conductance Ca2+-activated K+ channels. Furthermore, the combination of 18β-glycyrrhetinic acid and iberiotoxin produced greater attenuation in the EDH-related response than that by the individual agent. Treatment with 18β-glycyrrhetinic acid revealed no significant effect on NOR3 (an NO donor)-induced retinal vasodilator response. These results suggest that gap junctions contribute to the ACh-induced, EDH-type dilation of rat retinal arterioles in vivo.

Perivascular adipose tissue (PVAT) is an additional special type of adipose tissue surrounding blood vessels. Under physiological conditions, PVAT plays a significant role in regulation of vascular tone, intravascular thermoregulation, and vascular smooth muscle cell (VSMC) proliferation. PVAT is responsible for releasing adipocytes-derived relaxing factors (ADRF) and perivascular-derived relaxing factors (PDRF), which have anticontractile properties. Obesity induces increased oxidative stress, an inflammatory state, and hypoxia, which contribute to PVAT dysfunction. The exact mechanism of vascular dysfunction in obesity is still not well clarified; however, there are some pathways such as renin–angiotensin–aldosterone system (RAAS) disorders and PVAT-derived factor dysregulation, which are involved in hypertension and endothelial dysfunction development. Physical activity has a beneficial effect on PVAT function among obese patients by reducing the oxidative stress and inflammatory state. Diet, which is the second most beneficial non-invasive strategy in obesity treatment, may have a positive impact on PVAT-derived factors and may restore the balance in their concentration.

Aneurysmal subarachnoid hemorrhage (aSAH) patients develop delayed cerebral ischemia and delayed deficits (DCI) within 2 weeks of aneurysm rupture at a rate of approximately 30%. DCI is a major contributor to morbidity and mortality after SAH. The cause of DCI is multi-factorial with contributions from microthrombi, blood vessel constriction, inflammation, and cortical spreading depolarizations. Platelets play central roles in hemostasis, inflammation, and vascular function. Within this review, we examine the potential roles of platelets in microthrombi formation, large artery vasospasm, microvessel constriction, inflammation, and cortical spreading depolarization. Evidence from experimental and clinical studies is provided to support the role(s) of platelets in each pathophysiology which contributes to DCI. The review concludes with a suggestion for future therapeutic targets to prevent DCI after aSAH.

Congenital diaphragmatic hernia (CDH) is a rare disease, which affects 1 in 2,500 newborns. Congenital diaphragmatic hernia can interfere with the normal development of the pulmonary parenchyma and vascular bed, and in severe cases, it can lead to the development of severe pulmonary arterial hypertension (PAH) and right ventricular failure. We present a neonate with CDH who developed severe PAH and right ventricular dysfunction and was managed with a unique strategy combining venoarterial extracorporeal membrane oxygenation, prostaglandin E1, and a variety of PAH therapies.

Aims: The aim of this study is to study the colonization of subgingival biofilm (SGB) with periodontopathogenic bacteria species and endothelium-dependent vasodilation in patients with coronary heart disease and concomitant periodontitis. Subjects and Methods: Forty-five patients with cardiovascular diseases (CVDs) were examined – 28 women (62%) and 17 men (38%) aged 53–76 years, including 15 patients with acute myocardial infarction (AMI), 15 patients with exertional angina (pectoris), and 15 patients with chronic periodontitis (CP) without CVD. Dental and cardiological health conditions were determined, a biochemical blood test was conducted, endothelium-dependent vasodilation in the brachial artery was measured, and DNA of periodontopathogenic bacteria in SGB was detected. Results: A reliable interrelation between the colonization of SGB with periodontopathogenic bacteria and development of AMI was established. In AMI patients, the frequency of Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans detection was significantly higher than in the group of participants without cardiovascular disease. The presence of P. gingivalis and A. actinomycetemcomitans in patients with CP directly correlated with severity of periodontal tissue destruction. Endothelium-dependent vasodilation in the brachial artery moderately correlated with patient's cardiological condition (r = 0.3284), biochemical markers of atherosclerosis development (r = 0.6465), and frequency of P. intermedia detection in periodontal pockets (r = 0.3828). Conclusions: Periodontal status in patients with AMI is characterized by unsatisfactory and poor hygiene, increased indices of bleeding on probing, and periodontal pocket depth in comparison to groups of patients with angina pectoris and CP without cardiovascular pathology.

4-hydroxybenzaldehyde (4HB), known as ρ-hydroxybenzaldehyde, is commonly present in traditional Chinese medicine herb, most frequently used for hypertension treatment. This research aims to determine the potency of 4HB's vasorelaxant action. In the study, the vasodilation effect of 4HB was evaluated using in vitro isolated rat aortic rings assay. The aortic rings were pre-incubated with respective antagonists before being pre-contracted with phenylephrine (PE) and challenged with various concentrations of 4HB for mechanistic action studies. R

Background Pulmonary arterial hypertension (PAH) manifests with progressive right ventricular (RV) dysfunction, which eventually impairs the left ventricular function. We hypothesized that 4‐dimensional–flow magnetic resonance imaging can detect flow hemodynamic changes associated with efficient intracardiac flow during noninvasive inhaled nitric oxide (iNO) challenge in children with PAH. Methods and Results Children with PAH (n=10) underwent 2 same‐day separate iNO challenge tests using: (1) 4‐dimensional–flow magnetic resonance imaging and (2) standard catheterization hemodynamics. Intracardiac flow was evaluated using the particle tracking 4‐flow component analysis technique evaluating the direct flow , retained inflow , delayed ejection flow , and residual volume . Respective flow hemodynamic changes were compared with the corresponding catheterization iNO challenge results. The RV analysis revealed decreased direct flow in patients with PAH when compared with controls ( P P P =0.004) and increased proportion of the residual volume ( P =0.014). There was an increase in the RV direct flow during iNO delivery ( P =0.009), with parallel decrease in the residual volume ( P =0.008). Conclusions Children with PAH have abnormal biventricular flow associated with impaired diastolic filling. The flow efficiency is significantly improved in the RV on iNO administration with no change in the left ventricle. The changes in the RV flow have occurred despite the minimal change in catheterization hemodynamics, suggesting that flow hemodynamic evaluation might provide more quantitative insights into vasoreactivity testing in PAH.

In this study, we evaluated the effect of eight weeks of administration of 10% fructose solution to adult Wistar Kyoto (WKY) rats on systolic blood pressure (SBP), plasma and biometric parameters, vasoactive properties of the thoracic aorta (TA), NO synthase (NOS) activity, and the expression of enzymes producing NO and H2S. Eight weeks of fructose administration did not affect SBP, glycaemia, or the plasma levels of total cholesterol or low-density and high-density lipoprotein, however, it significantly increased the plasma levels of γ-glutamyl transferase and alanine transaminase. Chronic fructose intake deteriorated endothelium-dependent vasorelaxation (EDVR) and increased the sensitivity of adrenergic receptors to noradrenaline. Acute NOS inhibition evoked a reduction in EDVR that was similar between groups, however, it increased adrenergic contraction more in fructose-fed rats. CSE inhibition decreased EDVR in WKY but not in fructose-fed rats. The application of a H2S scavenger evoked a reduction in the EDVR in WKY rats and normalized the sensitivity of adrenergic receptors in rats treated with fructose. Fructose intake did not change NOS activity but reduced the expression of eNOS and CBS in the TA and CSE and CBS in the left ventricle. Based on our results, we could assume that the impaired vascular function induced by increased fructose intake was probably not directly associated with a decreased production of NO, but rather with impairment of the NO–H2S interaction and its manifestation in vasoactive responses.

The core pathology of coronavirus disease 2019 (COVID-19) is infection of airway cells by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that results in excessive inflammation and respiratory disease, with cytokine storm and acute respiratory distress syndrome implicated in the most severe cases. Thrombotic complications are a major cause of morbidity and mortality in patients with COVID-19. Patients with pre-existing cardiovascular disease and/or traditional cardiovascular risk factors, including obesity, diabetes mellitus, hypertension and advanced age, are at the highest risk of death from COVID-19. In this Review, we summarize new lines of evidence that point to both platelet and endothelial dysfunction as essential components of COVID-19 pathology and describe the mechanisms that might account for the contribution of cardiovascular risk factors to the most severe outcomes in COVID-19. We highlight the distinct contributions of coagulopathy, thrombocytopathy and endotheliopathy to the pathogenesis of COVID-19 and discuss potential therapeutic strategies in the management of patients with COVD-19. Harnessing the expertise of the biomedical and clinical communities is imperative to expand the available therapeutics beyond anticoagulants and to target both thrombocytopathy and endotheliopathy. Only with such collaborative efforts can we better prepare for further waves and for future coronavirus-related pandemics., This Review summarizes the latest evidence indicating that platelet and endothelial dysfunction are essential components of COVID-19 pathology, describes the potential mechanisms underlying the contribution of cardiovascular risk factors to the most severe outcomes in COVID-19, and highlights the roles of coagulopathy, thrombocytopathy and endotheliopathy in COVID-19 pathogenesis., Key points Venous thromboembolism, arterial thrombosis and thrombotic microangiopathy substantially contribute to increased morbidity and mortality in patients with COVID-19.A complex interaction between coagulopathy, thrombocytopathy and endotheliopathy contributes to COVID-19-associated thromboinflammation.Coagulopathy, thrombocytopathy and endotheliopathy are characteristic features associated with cardiovascular risk factors such as diabetes mellitus, obesity and ageing.The combination of cardiovascular risk factors and infection with SARS-CoV-2 leads to exacerbated thrombosis and increased mortality.Age has an important role in COVID-19 pathogenesis; young patients without a predisposition to coagulopathy, thrombocytopathy and endotheliopathy can have a distinct multisystem inflammatory syndrome that includes a Kawasaki disease-like syndrome in very young individuals.Combination therapies targeting inflammation, coagulopathy, thrombocytopathy and endotheliopathy are likely to be more successful than a single agent in tackling the COVID-19-associated thrombotic complications.

Diabetes mellitus (DM)-related morbidity and mortality are steadily rising worldwide, affecting about half a billion people worldwide. A significant proportion of diabetic cases are in the elderly, which is concerning given the increasing aging population. Proper nutrition is an important component in the effective management of diabetes in the elderly. A plethora of active substances of plant origin exhibit potency to target the pathogenesis of diabetes mellitus. The nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. In this study, the effect of Hungarian sour cherry, which is rich in anthocyanins, on hyperglycemia-induced endothelial dysfunction was tested using human umbilical cord vein endothelial cells (HUVECs). HUVECs were maintained under both normoglycemic (5 mM) and hyperglycemic (30 mM) conditions with or without two concentrations (1.50 ng/&micro, L) of anthocyanin-rich sour cherry extract. Hyperglycemia-induced oxidative stress and inflammatory response and damaged vasorelaxation processes were investigated by evaluating the level of reactive oxygen species (ROS) and gene expression of four proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF- &alpha, ), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1&alpha, (IL-1&alpha, ), as well as the gene expression of nitric oxide synthase (NOS) endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1). It was found that hyperglycemia-induced oxidative stress was significantly suppressed by anthocyanin-rich sour cherry extract in a concentration-dependent manner. The gene expression of the tested proinflammatory cytokines increased under hyperglycemic conditions but was significantly reduced by both 1 and 50 ng/&micro, L anthocyanin-rich sour cherry extract. Further, although increased ET-1 and ECE-1 expression due to hyperglycemia was reduced by anthocyanin-rich sour cherry extract, NOS expression was increased by the extract. Collectively, these data suggest that anthocyanin-rich sour cherry extract could alleviate hyperglycemia-induced endothelial dysfunction due to its antioxidant, anti-inflammatory, and vasorelaxant effects.

Although vascular dysfunction is a key event in the development of diabetic complications, and abnormal toll-like receptor 4 (TLR4) may contribute to the pathophysiology of vascular diseases, the direct relationships between TLR4 and vascular function in diabetic arteries are still poorly understood. Thus, to investigate whether pharmacological blockade of TLR4 affects vascular function in the superior mesenteric artery (SMA) of streptozotocin (STZ)-induced diabetic rats, the SMA was isolated from male Wistar rat injected once with STZ (65 mg/kg, 27-34 weeks) which was treated with TAK-242 (10-6 M), a TLR4 inhibitor, for approximately 1 d using organ culture techniques. After incubation, functional and biochemical studies were performed. In the functional study, treatment with TAK-242 increased acetylcholine (ACh)-induced relaxation of the diabetic SMA in the intact condition. Sodium nitroprusside (SNP)-induced relaxation was also increased in the TAK-242-treated group compared with the vehicle-treated group. Under cyclooxygenase (COX) blockade by indomethacin (10-5 M), ACh-induced relaxation was similar in the vehicle- and TAK-242-treated groups. In addition, ACh-induced relaxation in the combined presence of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NNA) (10-4 M), and indomethacin (10-5 M) was similar in the vehicle- and TAK-242-treated groups. The productions of thromboxane (TX) B2 in cultured medium in the presence of ACh (10-5 M) were lower in the TAK-242-treated group than in the vehicle-treated group. These data suggested that TAK-242 could augment endothelium-dependent relaxation by partly suppressing vasoconstrictor TXA2 or increasing NO signaling. TLR4 inhibition may be a novel therapeutic strategy to assist in the management of diabetes-associated vascular complications.

Objective: Nitric oxide (NO) plays several protective roles in ischemia/reperfusion (I/R) injury. Neonates undergoing the Norwood procedure are subject to develop I/R injury due to the immaturity of their organs and the potential need to interrupt or decrease systemic flow during surgery. We hypothesized that NO administration during cardiopulmonary bypass (CPB) ameliorates the I/R and could help the postoperative recovery after the Norwood procedure. Methods: Twenty-four neonates who underwent a Norwood procedure were enrolled in a prospective randomized blinded controlled trial to receive NO (12 patients) or placebo (12 patients) into the oxygenator of the CPB circuit during the Norwood procedure. Markers of I/R injury were collected at baseline (T0), after weaning from CPB before modified ultrafiltration (T1), after modified ultrafiltration (T2), and at 12 hours (T3) and 24 hours (T4) after surgery, and they were compared between both groups, as well as other postoperative clinical variables. Results: There was no difference in age, weight, anatomical diagnosis, CPB, and aortic cross-clamp time between both groups. Troponin levels were lower in the study group at T1 (0.62 ± 58 ng/mL vs 0.87 ± 0.58 ng/mL, P = .31) and became significantly lower at T2 (0.36 ± 0.32 ng/mL vs 0.97 ± 0.48 ng/mL, P = .009).There were no significant differences between both groups for all other markers. Despite a lower troponin level, there was no difference in inotropic scores or ventricular function between both groups. Time to start diuresis, time to sternal closure and extubation, and intensive care unit and hospital stay were not different between both groups. Conclusion: Systemic administration of NO during the Norwood procedure has myocardial protective effects (lower Troponin levels) but we observed no effect on postoperative recovery. Larger sample size may be needed to show clinical differences.

Background: Pulmonary vasodilators as add-on to current treatment strategies in acute pulmonary embolism may improve right ventricular unloading and hence improve patient outcome. We aimed to investigate whether stimulation of the nitric oxide (NO)–soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) pathway with riociguat, sildenafil or inhaled NO causes pulmonary vasodilation and improves right ventricular function in a porcine model of acute intermediate risk pulmonary embolism. Methods: Two large autologous blood clots were administered to the pulmonary circulation of 28 pigs (60 kg). Animals were randomized to four increasing, clinically equivalent doses of riociguat ( n=6), sildenafil ( n=6), inhaled NO ( n=6) or vehicle ( n=6). Sham animals ( n=4) did not receive pulmonary embolism or treatment. Haemodynamic responses were evaluated at baseline, after pulmonary embolism and after each dose using invasive pressure measurements, transoesophageal echocardiography, respiratory parameters and blood analysis. Results: Pulmonary embolism caused a three-fold increase in pulmonary vascular resistance compared with baseline (pulmonary embolism: 352±29 vs. baseline: 107±6 dynes, pConclusion: Stimulation of the NO–sGC–cGMP pathway by riociguat, sildenafil and inhaled NO reduces pulmonary vascular resistance in a porcine model of acute pulmonary embolism without lowering systemic blood pressure.

Purpose: To determine whether the absence of transglutaminase 2 enzyme (TG2) in TG2 knockout mice (TG2-/-) protect them against early age-related functional and histological arterial changes. Methods: Pulse wave velocity (PWV) was measured using non-invasive Doppler and mean arterial pressure (MAP) was measured in awake mice using tail-cuff system. Thoracic aortas were excised for evaluation of endothelial dependent vasodilation (EDV) by wire myography, as well as histological analyses. Results: PWV and MAP were similar in TG2-/-mice to age-matched wild type (WT) control mice. Old WT mice exhibited a markedly attenuated EDV as compared to young WT animals. The TG2-/-young and old mice had enhanced EDV responses (p

Nitric oxide is a wound mediator that promotes wound healing. We hypothesized that topical application of nitric oxide would speed reepithelialization, enhance angiogenesis, and reduce scar thickness in a partial thickness porcine burn model.While under general anesthesia, 20 partial thickness burns were created on the backs of four female Yorkshire swine using a 2.5cm×2.5cm×7.5cm, 150-g aluminum bar, preheated to 80°C and applied for 20s. The necrotic epidermis was removed and the burns were randomized to low, medium, and high concentrations of a novel nitric-oxide (NO) releasing drug or its ointment vehicle applied 3 times weekly for 28 days. Full thickness punch biopsies were performed at 8, 11, 14 and 28 days after injury to determine percentage wound reepithelialization and scar thickness using HE staining and blood vessel density using CD31 staining.At day 11, the percentages (SD) wound reepithelialization were: control, 26.3 (34.6); low NO, 23.9 (36.9); medium NO, 43.3 (42.9); and high NO, 59.9 (43.6); ANOVA, P=0.02. The number of CD31 stained blood vessels at days 8 and 11 were greater in wounds treated with high dose NO vs. controls (48.1 vs. 22.9 [P0.001] and 44.0 vs. 33.5 [P=0.05] per 1mmTreatment of partial thickness porcine burns with high concentrations of topical NO resulted in earlier reepithelization and increased angiogenesis but not reduced scar thickness compared with its control vehicle in a partial thickness porcine burn model.

Purpose To correlate outflow function and outflow tract vessel diameter changes induced by nitric oxide (NO). Methods In a porcine anterior segment perfusion model, the effects of a nitric oxide donor (100 μM DETA-NO) on outflow facility were compared with controls (n = 8 per group) with trabecular meshwork (TM) and after circumferential ab interno trabeculectomy (AIT). Outflow structures were assessed with spectral-domain optical coherence tomography (SD-OCT) before and after NO, or an NO synthase inhibitor (100 μM L-NAME) and the vasoconstrictor, endothelin-1 (100 pg/mL ET-1). Scans were processed with a custom macroscript and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA). Results The facility increased after DETA-NO (Δ of 0.189 ± 0.081 μL/min·mm Hg, P = 0.034) and AIT (Δ of 0.251 ± 0.094 μL/min·mm Hg, P = 0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (Δ of 0.190 ± 0.074 μL/min·mm Hg, P = 0.023) and CSA by 13.9% (P < 0.001). L-NAME + ET-1 decreased CSA by -8.6% (P < 0.001). NO increased the diameter of focal constrictions 5.0 ± 3.8-fold. Conclusions NO can dilate vessels of the distal outflow tract and increase outflow facility in a TM-independent fashion. There are short, focally constricting vessel sections that display large diameter changes and may have a substantial impact on outflow.

Arterial wall viscosity (AWV) is a major cause of energy dissipation along the arterial tree. Its determinants remain controversial but an active endothelial regulation has been suggested. Our objective was to assess in humans the physiological role of endothelium-derived nitric oxide (NO), epoxyeicosatrienoic acids and the effect of modulating smooth muscle tone in the regulation of AWV. We simultaneously measured radial artery diameter, wall thickness, and arterial pressure in healthy volunteers during the local infusion of inhibitors of NO-synthase ( N G -monomethyl- l -arginine), epoxyeicosatrienoic acids synthesis by cytochrome P450 (fluconazole), the epoxyeicosatrienoic acids cellular targets calcium-activated potassium channels (tetraethylammonium), alone and in combination. AWV was estimated from the relative viscosity expressed as the ratio of the area of the hysteresis loop of the pressure–diameter relationship to the area under the loading phase. Arterial tone was assessed by measuring change in wall stiffness and midwall stress. N G -monomethyl- l -arginine paradoxically reduced relative viscosity (34.9±8.9%–28.9±8.6%). Conversely, relative viscosity was not modified by fluconazole (33.5±15.5%–32.0±13.6%) but increased by tetraethylammonium (31.7±6.6%–35.7±8.0%). This increase was more marked with N G -monomethyl- l -arginine+fluconazole (31.1±10.7%–43.3±13.2%) and N G -monomethyl- l -arginine+tetraethylammonium (29.5±2.3%–41.5±11.1%) compared with inhibitors alone. Sodium nitroprusside decreased AWV (35.4±2.9%–28.7±2.0%). These effects were associated with parallel change in tone but of different magnitude for similar variations in viscosity, suggesting tone-dependent and independent mechanisms. In conclusion, this is the first demonstration that the endothelial factors, NO and epoxyeicosatrienoic acids, regulate AWV in humans and support the role of arterial tone in this regulation. Clinical Trial Registration— URL: https://eudract.ema.europa.eu . Unique identifier: RCB2007-A001-10-53.

Peripheral artery disease (PAD) is a morbid condition whereby ischemic peripheral muscle causes pain and tissue breakdown. Interestingly, PAD risk factors, e.g. diabetes mellitus, cause endothelial dysfunction secondary to decreased nitric oxide (NO) levels, which could explain treatment failures. Previously, we demonstrated 670nm light (R/NIR) increased NO from nitrosyl-heme stores, therefore we hypothesized R/NIR can stimulate vasodilation in healthy and diabetic blood vessels. Vasodilation was tested by ex vivo pressure myography in wild type C57Bl/6, endothelial nitric oxide synthase (eNOS) knockout, and db/db mice (10 mW/cm2 for 5min with 10 min dark period). NOS inhibition with N-Nitroarginine methyl ester (L-NAME) or the NO scavenger Carboxy-PTIO (c-PTIO) tested the specificity of NO production. 4,5-Diaminofluorescein diacetate (DAF-2) measured NO in human dermal microvascular endothelial cells (HMVEC-d). R/NIR significantly increased vasodilation in wild type and NOS inhibited groups, however R/NIR dilation was totally abolished with c-PTIO and blood vessel denudation. Interestingly, the bath solution from intact R/NIR stimulated vessels could dilate light naïve vessels in a NO dependent manner. Characterization of the bath identified a NO generating substance suggestive of S-nitrosothiols or non heme iron nitrosyl complexes. Consistent with the finding of an endothelial source of NO, intracellular NO increased with R/NIR in HMVEC-d treated with and without L-NAME (1mM), yet c-PTIO (100μm) reduced NO production. R/NIR significantly dilated db/db blood vessels. In conclusion, R/NIR stimulates vasodilation by release of NO bound substances from the endothelium. In a diabetes model of endothelial dysfunction, R/NIR restores vasodilation, which lends the potential for new treatments for diabetic vascular disease.

While the biological role of nitric oxide (NO) synthase (NOS) is appreciated, several fundamental aspects of the NOS/NO-related signaling pathway(s) remain incompletely understood. Canonically, the NOS-derived NO diffuses through the (inter)cellular milieu to bind the prosthetic ferro(Fe2+)-heme group of the soluble guanylyl cyclase (sGC). The formation of ternary NO-ferroheme-sGC complex results in the enzyme activation and accelerated production of the second messenger, cyclic GMP. This paper argues that cells dynamically generate mobile/exchangeable NO-ferroheme species, which activate sGC and regulate the function of some other biomolecules. In contrast to free NO, the mobile NO-ferroheme may ensure safe, efficient and coordinated delivery of the signal within and between cells. The NO-heme signaling may contribute to a number of NOS/NO-related phenomena (e.g. nitrite bioactivity, selective protein S-(N-)nitrosation, endothelium and erythrocyte-dependent vasodilation, some neural and immune NOS functions) and predicts new NO-related discoveries, diagnostics and therapeutics.

OBJECTIVE To compare demographic data, prenatal and postnatal characteristics, laboratory data, and outcomes in a cohort of premature infants with spontaneous ileal perforation (SIP), surgical necrotizing enterocolitis (sNEC) and matched controls. METHODS A retrospective case-control study of infants with intestinal perforation with a birth weight (BW) less than 2,000 grams and gestational age (GA) less than 34 weeks and infants without perforation matched for BW (±150 grams) and GA (±1week). RESULTS 130 premature infants were included, 30 infants with SIP, 35 infants with sNEC and 65 control infants. The median age of onset was 5 days postnatal age in SIP versus 25 days in sNEC (p

Objective To describe the pharmacokinetics and pharmacodynamics of milrinone in infants with persistent pulmonary hypertension of the newborn (PPHN) and to explore the impact of age on milrinone disposition. Design Randomized, open label pilot study. Setting Multicenter; level 3 and level 4 neonatal intensive care units. Patients Six infants ≥34 weeks' gestational age and Intervention Intravenous milrinone lactate in one of two dosing regimens: (1) low dose, 20 mcg/kg bolus followed by 0.2 mcg/kg/minute, and (2) standard dose, 50 mcg/kg bolus followed by 0.5 mcg/kg/minute. Measurements and Main Results The final structural model was a two-compartment disposition model with interindividual variability estimated on clearance (CL). The estimated value of CL is 7.65 mL/minute/3.4 kg (3.05 mL/minute/kg). The addition of age improved the precision of the CL estimate, and CL increased with chronological age in days. The oxygenation index was highly variable within each participant and improved with time. There were no observed safety concerns in either dosing group. Conclusion The CL of milrinone in newborns with PPHN is reduced and increases with age. In this pilot study, we did not see significant pharmacodynamic or safety effects associated with drug exposure.

Most outcomes of COVID-19 are associated with dysfunction of the vascular system, particularly in the lung. Inhalation of nitric oxide (NO) gas is currently being investigated as a treatment for patients with moderate to severe COVID-19. In addition to the expected vasodilation effect, it has been also suggested that NO potentially prevents infection by SARS-CoV-2. Since NO is an unstable radical molecule that is easily oxidized by multiple mechanisms in the human body, it is practically difficult to control its concentration at lesions that need NO. Inorganic nitrate and/or nitrite are known as precursors of NO that can be produced through chemical as well enzymatic reduction. It appears that this NO synthase (NOS)-independent mechanism has been overlooked in the current developing of clinical treatments. Here, I suggest the missing link between nitrate and COVID-19 in terms of hypoxic NO generation.

IntroductionPostoperative acute kidney injury (AKI) is a common complication in cardiac surgery. Levels of intravascular haemolysis are strongly associated with postoperative AKI and with prolonged (>90 min) use of cardiopulmonary bypass (CPB). Ferrous plasma haemoglobin released into the circulation acts as a scavenger of nitric oxide (NO) produced by endothelial cells. Consequently, the vascular bioavailability of NO is reduced, leading to vasoconstriction and impaired renal function. In patients with cardiovascular risk factors, the endothelium is dysfunctional and cannot replenish the NO deficit. A previous clinical study in young cardiac surgical patients with rheumatic fever, without evidence of endothelial dysfunction, showed that supplementation of NO gas decreases AKI by converting ferrous plasma haemoglobin to ferric methaemoglobin, thus preserving vascular NO. In this current trial, we hypothesised that 24 hours administration of NO gas will reduce AKI following CPB in patients with endothelial dysfunction.MethodsThis is a single-centre, randomised (1:1) controlled, parallel-arm superiority trial that includes patients with endothelial dysfunction, stable kidney function and who are undergoing cardiac surgery procedures with an expected CPB duration >90 min. After randomisation, 80 parts per million (ppm) NO (intervention group) or 80 ppm nitrogen (N2, control group) are added to the gas mixture. Test gases (N2or NO) are delivered during CPB and for 24 hours after surgery. The primary study outcome is the occurrence of AKI among study groups. Key secondary outcomes include AKI severity, occurrence of renal replacement therapy, major adverse kidney events at 6 weeks after surgery and mortality. We are recruiting 250 patients, allowing detection of a 35% AKI relative risk reduction, assuming a two-sided error of 0.05.Ethics and disseminationThe Partners Human Research Committee approved this trial. Recruitment began in February 2017. Dissemination plans include presentations at scientific conferences, scientific publications and advertising flyers and posters at Massachusetts General Hospital.Trial registration numberNCT02836899.

Depending on their levels, source of generation, and subcellular locations, reactive oxygen species (ROS) are known to have paradoxical effects on coronary vascular endothelium. At low concentrations, ROS contribute to physiological signaling pathways that regulate vascular endothelial cell (EC) growth and survival. At higher concentrations, or with prolonged exposure, ROS can exacerbate endothelial cell injury and trigger apoptosis. In this chapter, oxidant-dependent and oxidant-independent angiogenic and vasomotor signaling pathways will be discussed in-depth, including the structures of oxidant-producing enzymes, their agonists, and their related signaling pathways in EC. Vascular endothelial growth factor (VEGF), a major growth factor involved in the maintenance of EC health, vasomotor tone, and angiogenesis, will also be discussed. VEGF utilizes both reactive oxygen species (ROS)-dependent and ROS-independent arms of EC signaling.

Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx. RESUMO Objetivo: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes. Métodos: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato. Resultados: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM). Conclusão: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).

Acute pulmonary hypertension (PH) complicates the course of several cardiovascular, pulmonary and other systemic diseases in children. An acute rise of RV afterload, either as exacerbating chronic PH of different aetiologies (eg, idiopathic pulmonary arterial hypertension (PAH), chronic lung or congenital heart disease), or pulmonary hypertensive crisis after corrective surgery for congenital heart disease, may lead to severe circulatory compromise. Only few clinical studies provide evidence on how to best treat children with acute severe PH and decompensated RV function, that is, acute RV failure. The specific treatment in the intensive care unit should be based on the underlying pathophysiology and not only be focused on so-called 'specific' or 'tailored' drug therapy to lower RV afterload. In addition therapeutic efforts should aim to optimise RV preload, and to achieve adequate myocardial perfusion, and cardiac output. Early recognition of patients at high risk and timely initiation of appropriate therapeutic measures may prevent the development of severe cardiac dysfunction and low cardiac output. In patients not responding adequately to pharmacotherapy, (1) novel surgical and interventional techniques, temporary mechanical circulatory support with extracorporeal membrane oxygenation, (2) pumpless lung assist devices (3) and/or lung or heart-lung transplantation should be timely considered. The invasive therapeutic measures can be applied in a bridge-to-recovery or bridge-to-lung transplant strategy. This consensus statement focuses on the management of acute severe PH in the paediatric intensive care unit and provides an according treatment algorithm for clinical practice.

Persistent pulmonary hypertension of the newborn (PPHN) is the most common neonatal form and mostly reversible after a few days with improvement of the underlying pulmonary condition. When pulmonary hypertension (PH) persists despite adequate treatment, the severity of parenchymal lung disease should be assessed by chest CT. Pulmonary vein stenosis may need to be ruled out by cardiac catheterisation and lung biopsy, and genetic workup is necessary when alveolar capillary dysplasia is suspected. In PPHN, optimisation of the cardiopulmonary situation including surfactant therapy should aim for preductal SpO2between 91% and 95% and severe cases without post-tricuspid-unrestrictive shunt may receive prostaglandin E1 to maintain ductal patency in right heart failure. Inhaled nitric oxide is indicated in mechanically ventilated infants to reduce the need for extracorporal membrane oxygenation (ECMO), and sildenafil can be considered when this therapy is not available. ECMO may be indicated according to the ELSO guidelines. In older preterm infant, where PH is mainly associated with bronchopulmonary dysplasia (BPD) or in term infants with developmental lung anomalies such as congenital diaphragmatic hernia or cardiac anomalies, left ventricular diastolic dysfunction/left atrial hypertension or pulmonary vein stenosis, can add to the complexity of the disease. Here, oral or intravenous sildenafil should be considered for PH treatment in BPD, the latter for critically ill patients. Furthermore, prostanoids, mineralcorticoid receptor antagonists, and diuretics can be beneficial. Infants with proven or suspected PH should receive close follow-up, including preductal/postductal SpO2measurements, echocardiography and laboratory work-up including NT-proBNP, guided by clinical improvement or lack thereof.

There is growing preclinical as well as clinical evidence supporting remote ischemic preconditioning (RIPC), in which short cycles of non-fatal ischemia followed by reperfusion to an organ or tissue distant from the heart elicits cardioprotection. It is the most practical, non-invasive, cost-free, and clinically compatible, secure procedure for reducing ischemia-reperfusion induced injury. The use of a conventional blood pressure cuff on the upper or lower limb in eliciting cardioprotection has expedited its clinical applicability. Endothelium has been documented to respond very quickly to blood flow and hypoxia by releasing different humoral factors such as endothelium derived releasing factor, endothelium derived contracting factor, endothelium derived hyperpolarizing factor. In recent years, there have been studies suggesting the key role of endothelial derived factors in RIPC induced cardioprotection. The signaling cascade involves nitric oxide, gap junctions, epoxyeicosatrienoic (EETs) acids, Ca-activated K + channels, angiotensin II, thromboxane A 2, superoxide anions and prostacyclin. The present review describes the role of these endothelial derived factors in RIPC induced cardioprotection with possible mechanisms.

Purpose/Aim of the study: the retinal relaxing factor (RRF) is an unidentified paracrine factor, which is continuously released from retinal tissue and causes smooth muscle cell relaxation. This study tried to identify the cellular source of the RRF. Furthermore, the possible RRF release by voltage-dependent sodium channel activation and the calcium-dependency of the RRF release were investigated. MATERIALS AND METHODS mouse femoral arteries were mounted in myograph baths for in vitro isometric tension measurements. The vasorelaxing effect of chicken retinas, which contain no vascular cells, and of solutions incubated with MIO-M1 or primary Muller cell cultures were evaluated. The RRF release of other retinal cells was investigated by using cell type inhibitors. Concentration-response curves of veratridine, a voltage-dependent sodium channel activator, were constructed in the presence or absence of mouse retinal tissue to evaluate the RRF release. The calcium-dependency of the RRF release was investigated by evaluating the vasorelaxing effect of RRF-containing solutions made out of chicken retinas in the absence or presence of calcium. RESULTS Chicken retinas induced vasorelaxation, whereas solutions incubated with Muller cell cultures did not. Moreover, the gliotoxin DL-α-aminoadipic acid, the microglia inhibitor minocycline, and the tetrodotoxin-resistant voltage-dependent sodium channel 1.8 inhibitor A-803467 could not reduce the RRF-induced relaxation. Concentration-response curves of veratridine were not enlarged in the presence of retinal tissue, and RRF-containing solutions made in the absence of calcium induced a substantial, but reduced vasorelaxation. CONCLUSIONS the RRF is not released from vascular cells and probably neither from glial cells. The retinal cell type that does release the RRF remains unclear. Veratridine does not stimulate the RRF release in mice, and the RRF release in chickens is calcium-dependent as well as calcium-independent.