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Despite the achievements in the detection of calcium deposits in the walls of blood vessels, there is practically no data on the relationship of calcification of the coronary arteries with clinical and laboratory indicators of calcification in the blood, and the mechanisms of this process have not been fully established. The aim of the work was to establish the relationship between the severity of vascular calcification and clinical and laboratory markers of vascular calcification to improve the effectiveness of the diagnosis of diseases of the cardiovascular system and optimize therapy. The data obtained during the study indicate a high prevalence of vascular calcification in patients with atherosclerosis. Estimates of the calcium index and traditional risk factors are not always sufficient to predict cardiovascular complications. Thus, the identification of specific laboratory markers of calcification and predisposition to calcinosis is very relevant at the present time. Studies have shown that atherosclerosis with vascular calcification is combined with the development of chronic systemic inflammation and inflammation of the vascular wall. At the same time, there are elevated levels of C-reactive protein, endothelin, homocysteine, lipid metabolism indicators, and reduced levels of fetuin-A in the blood, which allows us to recommend these laboratory indicators to prevent cardiovascular complications.

Purpose: To investigate the efficacy of the combined use of sildenafil and magnesium sulfate in the treatment of persistent pulmonary hypertension of the newborn (PPHN), and its influence on hemodynamics.Methods: A total of 174 children with persistent pulmonary hypertension who were treated in Ganzhou People’s Hospital, Ganzhou, China were selected and randomly assigned to joint group (JG) and control group (CG), with 87 patients in each group. The CG group received magnesium sulfate, while the JG group received sildenafil plus magnesium sulphate. The respiratory parameters of the children were analyzed using blood gas analyzer, while their hemodynamic indices were evaluated using color Doppler echocardiography. The levels of cytokines and inflammatory factors were determined by enzyme-linked immunosorbent assay (ELISA).Results: Time taken for symptom disappearance, oxygen therapy, and hospitalization period were shorter in JG than in CG (p < 0.05). Post-treatment, the respiratory parameters (PaO2, PaCO2, and SaO2) in both groups s improved, with lower levels of PaO2 and PaCO2, and a higher level of SaO2 in JG (p < 0.05). Following treatment, the levels of systemic vascular resistance (SVR), posterior pulmonary vascular resistance (PVR) and pulmonary artery pressure (PA) in JG were significantly reduced, relative to CG (p < 0.05). Similarly, the expression of endothelin -1 (ET-1), brain natriureticpeptide (BNP), and angiotensin 1 (ANG-1) improved, with lower levels of ET-1 and BNP, and a higher level of ANG-1 in JG (p < 0.05). There was post-treatment reduction as well in IL-6 and TNF-α, with lower levels in JG (p < 0.05). Patients in JG showed higher total treatment effectiveness and a lowerincidence of adverse reactions than those in CG (p < 0.05).Conclusion: The combined use of sildenafil and magnesium sulfate enhances the management of PPHN, ameliorates respiratory parameters, hemodynamics, and levels of cytokines and inflammatory factors. These findings provide evidence-based medical references for a new treatment strategy for PPHN.

Objective: to study the effect of testosterone (T) levels on laboratory and instrumental markers of endothelial dysfunction (ED). Materials and methods: the study included 276 male patients with type 2 diabetes mellitus (DM). General clinical studies were carried out, analysis of parameters of carbohydrate metabolism, the content of hormones (total T, SHBG, free T, estradiol, LH, FSH, prolactin, TSH, DHEA) were performed. Endothelial secretory function was assessed using markers such as: nitric oxide (NO), endothelial NO synthase type 3, endothelin, ICAM-1, VCAM-1, p- and e-selectins, cadherin, PAI-1, VEGF-1. Additionally, the content of biologically active substances affecting endothelial function was studied: homocysteine B, C-reactive protein (CRP), osteoprotegerin, leptin, resistin, adiponectin. The vasomotor function of the endothelium was assessed by ultrasound examination of the endothelium-dependent vasodilation (EDVD) of the brachial artery (BA) during the reactive hyperemia test; in addition, the thickness of the intima-media complex (TIM) of the carotid arteries was measured. Correlation analysis was performed using Spearman’s method. Results: the level of total T is interrelated with the instrumental parameters of the endothelial function: the TIM of the carotid arteries (r = -0.26; p = 0.009), the time of maximum BA vasodilation development (r = -0.41; p Conclusion: T deficiency is associated with a deterioration in the vasomotor function of the endothelium: a decrease in EDVD along with an increase in the time of maximum BA vasodilation development and impaired endothelial secretory function: an increase in the concentrations of VCAM-1, ICAM-1, cadherin, VEGF and, on the contrary, a decrease in NO levels. A decrease in T levels is accompanied by an increase in the content of CRP, resistin, leptin and a decrease in adiponectin, which aggravates the dysfunction of the endothelium.

Актуальність. Ожиріння і цукровий діабет (ЦД) розглядаються як найважливіші фактори ризику перебігу артеріальної гіпертензії (АГ), що призводять до різних ускладнень. Однією з причин погіршення перебігу АГ є дисфункція ендотелію. Застосування різних схем комбінованої терапії призводить і до поліпшення стану ендотелію. Метою роботи було вивчення динаміки функції ендотелію при лікуванні хворих на АГ у поєднанні з ожирінням і ЦД. Матеріали та методи. Обстежено 54 хворих з визначенням антропометричних даних, функції ендотелію сонної артерії і аорти, ендотеліну, сумарної кількості нітратів/нітритів. Результати. В обстежуваних групах (як з нефіксованою базисною терапією, так і з додаванням трьохкомпонентної фіксованої комбінації) відбувалося зниження як систолічного (на 7,36 і 6,36 % відповідно, р < 0,05), так і діастолічного (на 9,2 і 8,29 % відповідно, р 0,1), товщина комплексу інтима­медіа (ТКІМ) — на 5,48 % (р > 0,1), рівень ендотеліну­1 — на 31,27 % (р > 0,1), тоді як рівень Total NO підвищився на 8,35 % (р > 0,1), а ендотелійзалежної вазодилатації (ЕЗВД) — на 10,5 % (р > 0,1). При комбінованій фіксованій комбінації гіпотензивних препаратів відбувалися більш виражені зміни з боку артеріальної стінки: ІЖА знизився на 10,96 % (р < 0,05), ТКІМ — на 8 %, ендотелін­1 — на 31,52 % (р < 0, 05), у той час як Total NO збільшився на 8,7 % (р < 0,05), а ЕЗДВ — на 11,8 % (р < 0,05). Висновки. У хворих на АГ з ожирінням і цукровим діабетом відбувається збільшення жорсткості артерій, товщини комплексу інтима­медіа, підвищення рівня ендотелінемії і зниження ЕЗВД і рівня загальних нітратів. Фіксована базисна гіпотензивна терапія стимулює ЕЗВД і істотно покращує механізми авторегуляції і пружно­еластичні властивості судин м’язового типу, статистично вірогідно краще знижує систолічний артеріальний тиск.

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Introduction The podocyte is thought to be the mainly affected cell type in focal segmental glomerulosclerosis (FSGS). However, recent studies have also indicated a role for glomerular endothelial cells and podocyte−endothelial crosstalk in FSGS development. An experimental model for podocyte injury showed that increased endothelin-1 (ET-1) signaling between podocytes and endothelial cells induces endothelial oxidative stress and subsequent podocyte loss. In the current study, we investigated endothelial endothelin receptor A (ETAR) expression in patients with FSGS and its association with podocyte injury and glomerular oxidative stress. Methods We selected 39 biopsy samples of patients with FSGS and 8 healthy control subjects, and stained them for ETAR, nephrin and 8-oxo-guanine, a DNA lesion caused by oxidative damage. Glomeruli with ETAR-positive endothelium and with nephrin loss were scored, and the 8-oxo-guanine−positive glomerular area was measured. Results The mean percentage of glomeruli with ETAR-positive endothelial cells in patients with FSGS was higher compared to that in healthy control subjects (52% vs. 7%; P < 0.001). The presence of glomerular ETAR-positive endothelium was strongly associated with nephrin loss both on the biopsy level (rho = 0.47; P < 0.01), as on the level of individual glomeruli (odds ratio = 2.0; P < 0.001). Moreover, glomeruli with ETAR-positive endothelium showed more 8-oxo-guanine−positive staining (1.9% vs. 2.4%; P = 0.037). Finally, 8-oxo-guanine positivity in glomeruli was associated with increased levels of proteinuria. Conclusion Taking together our findings, we show that ETAR is increased in glomerular endothelial cells of patients with FSGS and associated with podocyte damage and glomerular oxidative stress. These findings support the hypothesis that ET-1 signaling in glomerular endothelial cells contributes to disease development in patients with FSGS., Graphical abstract

Purpose of review To summarize new clinical findings of endothelin receptor antagonists (ERA) in various etiologies of kidney disease targeted in clinical trials. Recent findings Endothelin-1 is a multifunctional peptide with potential relevance to glomerular and tubulointerstitial kidney diseases. The phase 3 SONAR trial demonstrated a significant reduction in clinically relevant kidney outcomes for patients with diabetic kidney disease (DKD) after long-term treatment with the ERA, atrasentan, in addition to blockade of the renin-angiotensin-aldosterone system. Promising preclinical disease models and small clinical trials in non-DKD resulted in the initiation of phase 3 trials investigating the effects of long-term treatment with ERA in patients with immunoglobulin A (IgA) nephropathy and focal segmental glomeruloscelerosis (FSGS). The mechanisms by which ERA protects the kidneys have been extensively studied with evidence for the protection of tubule cells, podocytes, mesangial cells, the endothelial glycocalyx, and a reduction in glomerular perfusion pressure. The occurrence of fluid retention during ERA treatment, particularly in susceptible populations, necessitates strategies to support safe and effective treatment. Summary Treatment with ERA induces long-term kidney protection in DKD. Phase 3 trials are underway to investigate ERA effects in patients with IgA nephropathy and FSGS.

Purpose. To assess the effectiveness of curative complex consisting of photodynamic therapy (PDT) + intravenous laser blood irradiation (ILBI) + local tumor irradiation (TR) at hemostatic parameters at the initial stages of squamous cell skin cancer (SCSC).Material and methods. The present work analyzes SCSC course in 185 patients who had outpatient and inpatient treatment in medical institutions of cities Engels and Saratov in 2015–2019. 129 (69.7%) males and 56 females (31.2%). Patients were divided into three groups. Group I – 74 (40%) patients who had combined treatment: surgery with wide tumor excision under intravenous anesthesia + PDT + ILBI + TR; Group II – 111 (60%) patients who had only surgical treatment. Group III was created for controlling and comparing the obtained laboratory parameters; it included 17 patients without established pathology.Results. It was found out that hemostatic disorders in SCSC are characterized by increased activity of AT III and vWF and by simultaneous decrease of tPA-PAI-1 complex as well as by increased activity of VIIIa factor and resistance of Va factor – indices which indicate dysfunction of endothelial vascular wall. Combination of PDT + ILBI + TR prior to surgical treatment in patients with squamous cell skin cancer at the initial stages of the disease allow to normalize homeostatic indices and reduce the number of complications.Conclusion. 1. Squamous cell skin cancer at the initial stages changes the hemostatic system. 2. Effects of SCSC mechanism may be characterized as follows: damage of the endothelium vascular wall which is confirmed by endothelin increase; it results in vasoconstriction and increased blood procoagulant activity and, ultimately, in changes of hemostatic parameters. 3. Combination of PDT + ILBI + TR applied before surgery in patients with squamous cell skin cancer at the initial stages of the disease normalizes homeostatic indices and reduces the number of complications.

Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator.

We reported that high salt (HS) intake stimulates renal collecting duct (CD) endothelin (ET) type B receptor (ETBR)/nitric oxide (NO) synthase 1β (NOS1β)-dependent NO production inhibiting the epithelial sodium channel (ENaC) promoting natriuresis. However, the mechanism underlying the HS-induced increase of NO production is unclear. Histone deacetylase 1 (HDAC1) responds to increased fluid flow, as can occur in the CD during HS intake. The renal inner medulla (IM), in particular the IMCD, has the highest NOS1 activity within the kidney. Hence, we hypothesized that HS intake provokes HDAC1 activation of NO production in the IM. HS intake for 1 wk significantly increased HDAC1 abundance in the IM. Ex vivo treatment of dissociated IM from HS-fed mice with a selective HDAC1 inhibitor (MS-275) decreased NO production with no change in ET-1 peptide or mRNA levels. We further investigated the role of the ET-1/ETBR/NOS1β signaling pathway with chronic ETBR blockade (A-192621). Although NO was decreased and ET-1 levels were elevated in the dissociated IM from HS-fed mice treated with A-192621, ex vivo MS-275 did not further change NO or ET-1 levels suggesting that HDAC1-mediated NO production is regulated at the level or downstream of ETBR activation. In split-open CDs from HS-fed mice, patch clamp analysis revealed significantly higher ENaC activity after MS-275 pretreatment, which was abrogated by an exogenous NO donor. Moreover, flow-induced increases in mIMCD-3 cell NO production were blunted by HDAC1 or calcium inhibition. Taken together, these findings indicate that HS intake induces HDAC1-dependent activation of the ETBR/NO pathway contributing to the natriuretic response.

Sex dependent differences in coronary artery vasoregulation may be due to variations in responses to endogenous vasoactive compounds including endothelin (ET-1) and nitric oxide (NO).Septal coronary arteries (200 μm) from healthy, sexually mature male, female and ovariectomized (i.e. surgical menopause) Sprague-Dawley rats were used. Myogenic tone, measured by pressure myography, was initially determined for all vessel segments studied before and after exposure to the nonselective ETMyogenic tone was 26 ± 7% in male, 20 ± 7% in female (p = 0.04 versus male) and 24 ± 3% in ovariectomized (p = NS versus male/female) vessels. Antagonism of ET-1 receptors produced a greater reduction in myogenic tone in male, compared to female rats over a similar range of intraluminal pressure (20-80 mmHg). Robust constrictor responses to cumulative concentrations of ET-1 were observed in all vessels; however, male rats exhibited greater sensitivity to vasoconstrictor effects of ET-1. After exposure to L-NAME vessel responses to ET-1 were normalized in male and female (not studied in ovariectomized) groups.These findings confirm marked sex differences for myogenic tone and vessel constrictor responses to ET-1 in coronary resistance vessels. Results also suggest greater sensitivity to vasoconstrictor effects of ET-1 in male coronary resistance vessels.

ZusammenfassungIn der Therapie der pulmonalarteriellen Hypertonie (PAH) kommen zahlreiche Medikamentenklassen zum Einsatz, u. a. Endothelinrezeptorantagonisten (ERA) und Phosphodiesterase-5-(PDE-5-)Inhibitoren. In einem humanen Ex-vivo-Modell sollte überprüft werden, ob durch die Kombination zweier Substanzklassen ein höherer Effekt erzielt werden kann oder – bei gleichem Effekt – eine niedrigere Dosierung der Einzelsubstanzen ausreicht. Wir etablierten ein Organbadmodell, welches uns die In-vitro-Untersuchung der dosisabhängigen Effekte von ERA und PDE-5-Inhibitoren auf die durch Norepinephrin und Endothelin-1 induzierte Kontraktilität humaner Pulmonalgefäße sowie den Vergleich von Mono- und Dualtherapie ermöglichte. Auch wenn die Übertragung der Ex-vivo-Daten auf die Situation im Patienten mit Vorsicht erfolgen muss, so hat sich das Organbad dennoch als hilfreiches Instrument zur Evaluation der dosisabhängigen Effekte von ERA, PDE-5-Inhibitoren und deren Kombination erwiesen. Die Wirksamkeit der Kombinationstherapie und das Potenzial zur Dosisreduktion waren in diesem Modell abhängig von den verwendeten Konzentrationen und vom Einfluss der Vorerkrankungen auf die Blutgefäßfunktion. Diese Arbeit beschreibt die bisherigen und wichtigsten Ergebnisse unserer experimentellen Untersuchungen und gibt einen Ausblick auf zukünftige Projekte.

Background/aim Vimentin3 (Vim3) was recently described as a tumour marker for the direct discrimination between benign and malignant kidney tumours. Here, we examined its expression in prostate cancer (PCa) cell lines and the regulation of its expression by endothelin receptors. Materials and methods Prostate cancer cell lines (PC3, DU145, LNCap) were incubated with endothelin 1 (ET-1), BQ123 [endothelin A receptor (ETAR) antagonist], BQ788 [endothelin B receptor (ETBR) antagonist], BQ123+ET-1, BQ788+ET-1 for 24 h and a scratch assay was performed. Cell extracts were analysed by western blotting and qRT-PCR. Results ET-1 induced Vim3 overexpression. Blocking the ETBR in the different prostate cancer cell lines yielded a higher migration rate, whereby Vim3 expression was significantly increased. Conclusion Vim3 concentration increases in cell lines without a functional ETBR and may be used as a marker for PCas where ETBR is frequently methylated.

Aim To study the association between concentrations of endothelial dysfunction (ED) markers and arterial hypertension (AH) in people who were exposed to long-term action of “low-dose” ionizing radiation.Material and methods The study subjects were men of middle age (45–55 years) who were workers of the Siberian Integrated Chemical Plant with the length of service on the shop floor of at least 5 years. The subjects were divided into the main group (n=96) consisting of workers with grade 1-2 AH and the control group (n=48) consisting of arbitrarily healthy workers. Both groups contained workers who had been exposed to long-term occupational low-intensity irradiation (γ-radiation) and those not exposed to this irradiation. The study evaluated risk factors for cardiovascular diseases, presence of concomitant diseases, blood biochemistry (concentrations of glucose, high-sensitivity C-reactive protein (hsCRP), total cholesterol, low-density lipoproteins, high-density lipoproteins, triglycerides, creatinine, and ED markers, including endothelin, angiotensin II, von Willebrand factor, C-type natriuretic peptide, tissue plasminogen activator, tumor necrosis factor α (TNF-α), and homocysteine, major clinical data, total dose of external irradiation, and the content of 239Pu in the body.Results AH was associated primarily with excessive body weight and severity of atherogenic dyslipidemia and homocysteinemia. Higher plasma concentrations of TNF-α and a tendency to increasing hsCRP in the AH group, as distinct from the control group of arbitrarily healthy men, indicated a proinflammatory shift. The ED markers were related with clinical data of AH patients and associated with the lipid profile and increased blood concentrations of inflammatory mediators. The radiation exposure did not change the ED marker array in AH patients, which did not allow recommendation of the studied plasma indexes for detection of vascular endothelial injury in workers with AH of the Siberian Integrated Chemical Plant.Conclusion The study results evidenced the absence of adverse effects of long-term occupational exposure to low-intensity radiation on the vascular endothelium as evaluated by ED markers. In men aged 45–55 years, AH was associated primarily with excessive body weight, homocysteinemia, and atherogenic dyslipidemia.

The G protein-coupled estrogen receptor 1 (GPER1) mediates rapid estrogenic signaling. We recently reported that activation of GPER1 in the renal medulla evokes endothelin-1-dependent natriuresis in female, but not male, rats. However, the involvement of the ET receptors, ETA and ETB, underlying GPER1 natriuretic action remain unclear. In this study, we used genetic and pharmacologic methods to identify the contributions of ETA and ETB in mediating this female-specific natriuretic effect of renal medullary GPER1. Infusion of the GPER1-selective agonist G1 (5 pmol/kg per minute) into the renal medulla for 40 minutes increased Na+ excretion and urine flow in anesthetized female ETB-deficient (ETB def) rats and littermate controls but did not affect blood pressure or urinary K+ excretion in either group. Pretreatment with the selective ETA inhibitor ABT-627 (5 mg/kg, intravenous) abolished G1-induced natriuresis in ETB def rats. To further isolate the effects of inhibiting either receptor alone, we conducted the same experiments in anesthetized female Sprague-Dawley (SD) rats pretreated or not with ABT-627 and/or the selective ETB inhibitor A-192621 (10 mg/kg, intravenous). Neither antagonism of ETA nor antagonism of ETB receptor alone affected the G1-induced increase in Na+ excretion and urine flow in SD rats. However, simultaneous antagonism of both receptors completely abolished these effects. These data suggest that ETA and ETB receptors can mediate the natriuretic and diuretic response to renal medullary GPER1 activation in female rats. SIGNIFICANCE STATEMENT: Activation of G protein-coupled estrogen receptor 1 (GPER1) in the renal medulla of female rats evokes natriuresis via endothelin receptors A and/or B, suggesting that GPER1 and endothelin signaling pathways help efficient sodium excretion in females. Thus, GPER1 activation could be potentially useful to mitigate salt sensitivity in females.

Hypertension and kidney involvement are common in patients with autoimmune disease. Sodium intake is linked to hypertension in both human and animal studies. Evidence suggests that dietary salt may be an important environmental factor that promotes autoimmune activity. Therefore, we hypothesized that a long-term high-salt diet would accelerate the progression of autoimmunity, hypertension, and albuminuria during systemic lupus erythematosus (SLE), an autoimmune disease that predominantly affects young women and has a high prevalence of hypertension and renal disease. To test this hypothesis, an established experimental model of SLE (female NZBWF1 mice) that develops hypertension and renal disease was used. SLE mice were fed a high-salt (4% NaCl) or normal (0.4% NaCl) diet for 24 wk beginning at 10 wk of age and ending at 34 wk of age, a time by which female NZBWF1 mice typically have hypertension and exhibit signs of renal disease. Plasma anti-dsDNA autoantibodies were measured as an indicator of active SLE disease, and urinary albumin was monitored longitudinally as a marker of renal disease. Arterial pressure was measured in conscious, freely moving mice at 34 wk of age. Urinary endothelin-1 (ET-1) excretion, renal endothelin A and B receptor protein expression, and renal mRNA expression of NOS1, NOS2, NOX2, MCP-1, TNF-α, serum- and glucocorticoid-regulated kinase 1, and interleukin-2 (IL-2) were assessed to determine the impact on gene products commonly altered by a high-salt diet. SLE mice fed a high-salt diet had increased circulating autoantibodies, but the high-salt diet did not significantly affect albuminuria or arterial pressure. Urinary ET-1 excretion was increased, whereas renal endothelin A receptor and IL-2 expression were decreased in response to a high-salt diet. These data suggest that a chronic high-salt diet may not accelerate cardiovascular and renal consequences commonly associated with SLE.

Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor (ETA) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor (ETB) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment.

Coronary artery spasm (CAS) causes serious ischaemic cardiac disease. The hyper-reactivity of vascular smooth muscle cells, which ascribe the up-regulation of endothelin receptor B type, is a key abnormality responsible for the pathological constriction of arteries in CAS. In this study, the nanocarriers were prepared and optimised for the delivery of U0126, which is an inhibitor of the mitogen-activated protein kinase, to treat pathological constriction of arteries. First, cationic amphipathic starch and hyaluronic acid were used in the encapsulation of U0126 to form U0126 nanoparticles (NPs). The shape of the U0126 NPs was spherical with a typical core–shell structure. The size was 94.1 ± 10.2 nm and the zeta potential was −20.3 ± 5.1 mV. Maximal encapsulation efficiencies and drug loading of U0126 in the NPs were 96.2 ± 3.1 and 8.2 ± 2.5%, respectively. U0126 NPs showed very great stability and could perform an effective targeted delivery. An arterial tissue culture model was used in the investigation of anti-constriction. In comparison with treatment with the free drug, the U0126 NPs significantly decreased the effective dosage, enhanced the effect of anti-constriction, and down-regulated the endothelin type B receptor in the culture model. These results suggest that U0126 NPs have a significant potential for the treatment of CAS.

The endothelin system has an important role in bone modelling during orthodontic tooth movement (OTM); however, little is known about the involvement of endothelin B receptors (ETB) in this process. The aim of this study was to evaluate the role of ETB in bone modelling during OTM using ETB knockout rats (ETB-KO). Thirty-two male rats were divided into 4 groups (n = 8 per group): the ETB-KO appliance group, ETB-KO control group, wild type (ETB-WT) appliance group, and ETB-WT control group. The appliance consisted of a super-elastic closed-coil spring placed between the first and second left maxillary molar and the incisors. Tooth movement was measured on days 0 and 35, and maxillary alveolar bone volume, osteoblast, and osteoclast volume were determined histomorphometrically on day 35 of OTM. Next, we determined the serum endothelin 1 (ET-1) level and gene expression levels of the osteoclast activity marker cathepsin K and osteoblast activity markers osteocalcin and dentin matrix acidic phosphoprotein 1 (DMP1) on day 35. The ETB-KO appliance group showed significantly lower osteoblast activity, diminished alveolar bone volume and less OTM than the ETB-WT appliance group. Our results showed that ETB is involved in bone modelling in the late stage of OTM.

BACKGROUND: The impact of donor-specific anti-HLA antibodies (DSA) on antibody-mediated rejection (AMR) and kidney allograft failure is well established. However, the relevance of non-HLA antibodies remains unclear. METHODS: We investigated 13 pretransplant non-HLA antibodies and their association with histology of AMR (AMRh) and kidney allograft failure. We included single kidney recipients (n = 203) with AMRh, according to the Banff 2017 classification and matched AMRh-free controls (n = 219). Non-HLA antibodies were assessed using multiplex Luminex assay. RESULTS: Of the selected non-HLA antibodies (against agrin, adipocyte plasma membrane-associated protein, Rho GDP-dissociation inhibitor 2 [ARHGDIB], Rho guanine nucleotide exchange factor 6, angiotensin-II type 1 receptor, endothelin type A receptor, lamin B1, BPI fold-containing family B member 1, peroxisomal trans-2-enoyl-coenzyme A reductase, phospholipase A2 receptor, protein kinase C zeta type, tubulin beta-4B class IVb, vimentin), only antibodies against ARHGDIB (adjusted median fluorescence intensity [aMFI] ≥ 1000), a minor histocompatibility antigen, associated with graft failure, in univariate and multivariate models (hazard ratio = 2.7; 95% confidence interval [CI],1.3-5.4; P = 0.007). There was a 19.5-fold (95% CI, 6.0-63.9; P < 0.0001) increased risk of graft failure in patients positive for both DSA and anti-ARHGDIB antibodies (aMFI ≥ 1000) versus patients negative for both DSA and anti-ARHGDIB antibodies, compared with a 4.4-fold (95% CI, 2.4-8.2; P < 0.0001) increased risk in patients with only DSA, and a 4.1-fold (95% CI, 1.4-11.7; P = 0.009) increased risk in patients with only anti-ARHGDIB antibodies above 2000 aMFI. AMRh associated with increased intrarenal expression of the ARHGDIB gene. In the absence of AMRh and DSA, anti-ARHGDIB antibodies were not clearly associated with graft failure. CONCLUSIONS: The presence of pretransplant anti-ARHGDIB antibodies has an additive effect in patients with DSA on the risk of graft failure via AMRh. Other investigated non-HLA antibodies, including antibodies against angiotensin-II type 1 receptor, did not contribute to risk stratification and could not explain the histology of AMR in the absence of DSA. ispartof: TRANSPLANTATION vol:104 issue:7 pages:1462-1471 ispartof: location:United States status: published

Background The five-year survival rate of lung adenocarcinoma patients (LUAD) is very low,and the methods of predicting survival are a great obstacle for LUAD therapies. Endothelin receptor type B (EDNRB) gene is associated with tumorigenesis. In this study, we aimed to evaluate the predictive value of EDNRB on LUAD. Methods Survival analyses was performed to assess the correlation between EDNRB expression and survival of LUAD patients from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) datasets. Gene set enrichment analysis (GSEA) was conducted to illustrate possible biological functions of EDNRB. Laboratory methods were used to verify the function of EDNRB in LUAD. Results The TCGA results showed that a low expression of EDNRB was found in LUAD patients which led to poor outcome and worse survival, compared with the high expression in GSEA results which suggested that expression of EDNRB might be associated with regulation of the ERK pathway. Laboratory results suggested that EDNRB could inhibit the proliferation and migration of LUAD H1299 cells. Conclusions EDNRB is a potential prognostic marker for LUAD patients and might exert its functions by regulating the ERK pathway in LUAD.

Supplemental Digital Content is available in the text., GPR81 (G-protein-coupled receptor 81) is highly expressed in adipocytes, and activation by the endogenous ligand lactate inhibits lipolysis. GPR81 is also expressed in the heart, liver, and kidney, but roles in nonadipose tissues are poorly defined. GPR81 agonists, developed to improve blood lipid profile, might also provide insights into GPR81 physiology. Here, we assessed the blood pressure and renal hemodynamic responses to the GPR81 agonist, AZ′5538. In male wild-type mice, intravenous AZ′5538 infusion caused a rapid and sustained increase in systolic and diastolic blood pressure. Renal artery blood flow, intrarenal tissue perfusion, and glomerular filtration rate were all significantly reduced. AZ′5538 had no effect on blood pressure or renal hemodynamics in Gpr81−/− mice. Gpr81 mRNA was expressed in renal artery vascular smooth muscle, in the afferent arteriole, in glomerular and medullary perivascular cells, and in pericyte-like cells isolated from kidney. Intravenous AZ′5538 increased plasma ET-1 (endothelin 1), and pretreatment with BQ123 (endothelin-A receptor antagonist) prevented the pressor effects of GPR81 activation, whereas BQ788 (endothelin-B receptor antagonist) did not. Renal ischemia-reperfusion injury, which increases renal extracellular lactate, increased the renal expression of genes encoding ET-1, KIM-1 (Kidney Injury Molecule 1), collagen type 1-α1, TNF-α (tumor necrosis factor-α), and F4/80 in wild-type mice but not in Gpr81−/− mice. In summary, activation of GPR81 in vascular smooth muscle and perivascular cells regulates renal hemodynamics, mediated by release of the potent vasoconstrictor ET-1. This suggests that lactate may be a paracrine regulator of renal blood flow, particularly relevant when extracellular lactate is high as occurs during ischemic renal disease.

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. The current study explored the effect of mmLDL on the endothelin type A (ETA) receptor in mouse mesenteric arteries in vivo, as well as the role of autophagy in this process. mmLDL was injected via the caudal vein, and the Class III PI3K autophagic pathway inhibitor 3-methyladenine (3-MA) was injected intraperitoneally. The animals were divided into physiological saline (NS), mmLDL, and mmLDL + 3-MA groups. The dose-effect curve of endothelin-1- (ET-1-) induced mesenteric artery contraction was measured using myography, while ETA receptor mRNA expression was detected using real-time polymerase chain reactions, and the protein levels of the ETA receptor, class III PI3K, Beclin-1, LC3 II/I, p62, NF-κB, and p-NF-κB were observed using Western blot analysis. mmLDL significantly strengthened ET-1-induced contraction (the Emax value increased from 184.87 ± 7.46% in the NS group to 319.91 ± 20.31% in the mmLDL group (P<0.001), and the pEC50 value increased from 8.05 ± 0.05 to 9.11 ± 0.09 (P<0.01). In addition to upregulating the protein levels of Class III PI3K, Beclin-1, and LC3 II/I and downregulating that of p62, mmLDL significantly increased the mRNA expression and protein level of the ETA receptor and increased the protein level of p-NF-κB. However, these effects were significantly inhibited by 3-MA. mmLDL activates autophagy via the Class III PI3K/Beclin-1 pathway and upregulates the ETA receptor via the downstream NF-κB pathway. Understanding the effect of mmLDL on the ETA receptor and the underlying mechanisms may provide a new idea for the prevention and treatment of cardiovascular diseases.

Donation after circulatory death (DCD) can expand the donor pool effectively. A gap remains in outcome between DCD livers and living donor livers, warranting improved DCD liver quality and urgent resolution. Bone marrow mesenchymal stem cells (BMMSCs) can regulate immunity, participate in the anti-inflammatory response, and secrete cytokines. We investigated the effect of BMMSCs combined with normothermic machine perfusion (NMP) on DCD liver quality, and the role of microcirculation therein. Rat thoracic aortas were clipped to obtain DCD livers, and a rat NMP system was established. The DCD livers were grouped by preservation method: normal, static cold storage (SCS), NMP (P), and BMMSCs plus NMP (BP); storage time was up to 8 h. Liver function in outflow perfusate was detected by biochemical methods; liver tissue histopathology was observed by hematoxylin–eosin staining; hepatocyte ultrastructure was observed by transmission electron microscopy; hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling; liver microcirculation–related indicators were detected by immunofluorescence, immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay. Compared with SCS, P and BP significantly improved liver function and liver histological damage, reduced hepatocyte apoptosis, and repaired hepatocyte mitochondrial damage after 6 h in vitro. BP also significantly inhibited intrahepatic macrophage activation and intercellular adhesion, improved endothelial damage, and significantly improved endothelin 1–nitric oxide balance and microcirculation perfusion. In conclusion, BP can improve DCD liver microcirculation and quality. The mechanism may be the improvement of improve hepatic sinusoidal endothelial injury and microcirculation perfusion by inhibiting macrophage activation and intercellular adhesion. Electronic supplementary material The online version of this article (10.1007/s00441-020-03202-z) contains supplementary material, which is available to authorized users.

Angiopoietin-1, an angiogenic factor, stabilizes brain microvessels through Tie-2 receptor tyrosine kinase. In traumatic brain injury, blood-brain barrier (BBB) disruption is an aggravating factor that induces brain edema and neuroinflammation. We previously showed that BQ788, an endothelin ETB receptor antagonist, promoted recovery of BBB function after lateral fluid percussion injury (FPI) in mice. To clarify the mechanisms underlying BBB recovery mediated by BQ788, we examined the involvements of the angiopoietin-1/Tie-2 signal. When angiopoietin-1 production and Tie-2 phosphorylation were assayed by quantitative reverse transcription polymerase chain reaction and western blotting, increased angiopoietin-1 production and Tie-2 phosphorylation were observed in 7-10 days after FPI in the mouse cerebrum, whereas no significant effects were obtained at 5 days. When BQ788 (15 nmol/day, i.c.v.) were administered in 2-5 days after FPI, increased angiopoietin-1 production and Tie-2 phosphorylation were observed. Immunohistochemical observations showed that brain microvessels and astrocytes contained angiopoietin-1 after FPI, and brain microvessels also contained phosphorylated Tie-2. Treatment with endothelin-1 (100 nM) decreased angiopoietin-1 production in cultured astrocytes and the effect was inhibited by BQ788 (1 μM). Five days after FPI, increased extravasation of Evans blue dye accompanied by reduction in claudin-5, occludin, and zonula occludens-1 proteins were observed in mouse cerebrum while these effects of FPI were reduced by BQ788 and exogenous angiopoietin-1 (1 μg/day, i.c.v.). The effects of BQ788 were inhibited by co-administration of a Tie-2 kinase inhibitor (40 nmol/day, i.c.v.). These results suggest that BQ788 administration after traumatic brain injury promotes recovery of BBB function through activation of the angiopoietin-1/Tie-2 signal.

To study effect of nanomedicine on lower extremity deep vein thrombosis (LEDVT) and its correlation with susceptibility genes (SGs), preliminary nanoparticle LSA was constructed using lauric acid and stearic acid, and CTAB-HAuCL4-TEOS (CHT) was obtained using cetyltrimethyl ammonium bromide (CTAB), chloroauric acid (HAuCL4), and tetraethoxysilane (TEOS) to prepare UK-LSA-CHT by combining with urokinase (UK). 20 rabbits were grouped into UK-LSA-CHT, LSA, UK, and D0 group. Human umbilical vein endothelial cells (HUVEC) were selected for cytotoxicity test. The plasma endothelin (ET-1), interleukin 6 (IL-6), and nuclear factor κβ (NF-κβ) of the rabbits were detected with double antibody sandwich method. Expression of platelet activating factor (PAF) was detected by immumohistochemical staining. Genotype distribution was detected with the second-generation sequencing technology (SGST). The results showed that expressions of ET-1, IL-6, and NF-κβ) in UK-LSA-CHT group were lower than those in the LSA group and the UK group after 12 hours (P < 0.05). Frequency of allele G and T, and GT and TT genotype ratio of SG NOS3 rs1799983 in UK -LSA-CHT group were less than those in LSA and UK group (P < 0.05). UK-LSA-CHT group had observably lower value in PAF positive expression than LSA and UK groups (P < 0.05). In short, UK-LSA-CHT had good biocompatibility and safety and good therapeutic effect on LEDVT. N0S3 rs1799983 alleles G and T may be the key risk factors for the occurrence and development of LEDVT.

Endothelin-1 (ET-1) is involved in the regulation of steroidogenesis. Additionally, patients with castration-resistant prostate cancer (PCa) have a higher ET-1 plasma concentration than those with localized PCa and healthy individuals. The aim of the present study was to evaluate the effect of ET-1 on steroidogenesis enzymes, androgen receptor (AR) and testosterone (T) production in PCa cells. The expression levels of endothelin receptors in prostate tissue from patients with localized PCa by immunohistochemistry, and those in LNCaP and PC3 cells were determined reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Furthermore, the expression levels of ET-1 were determined in LNCaP and PC3 cells by RT-qPCR and western blotting. The ET-1 receptor activation was evaluated by intracellular calcium measurement, the expression levels of AR and enzymes participating in steroidogenesis [cytochrome P450 family 11 subfamily A member 1 (CyP11A1), cytochrome P450 family 17 subfamily A member 1, aldo-keto reductase family member C2 and 3β-hydroxysteroid dehydrogenase/isomerase 2 (3β HSD2)] were determined by western blotting and T concentration was determined by ELISA using PC3 cells. The present results revealed higher expression levels of endothelin A receptor (ETAR) in tissues obtained from samples of patients with PCa with a low Gleason Score. No changes were identified for endothelin B receptor (ETBR). PC3 cells expressed higher levels of ET-1 and ETAR, while LNCaP cells exhibited higher expression levels of ETBR. Blocking of ETAR and endothelin B receptor decreased the expression levels of CyP11A1 and 3β HSD2 enzymes and AR in PC3 cells, as well as T secretion. These findings suggested that ET-1 has a potential role in modulating the intratumoral steroidogenesis pathway and might have relevance as a possible therapeutic target.

Introduction: Previous studies in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX) have shown that besides pharmacological blockade of the renin-angiotensin system (RAS) also increasing kidney tissue epoxyeicosatrienoic acids (EET) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for degradation of EETs, and endothelin type A (ETA) receptor blockade retards chronic kidney disease (CKD) progression. This prompted us to evaluate if this progression will be alleviated by the addition of sEH inhibitor and ETA receptor antagonist to the standard complex blockade of RAS (angiotensin-converting enzyme inhibitor plus angiotensin II type 1 receptor blocker) in rats with established CKD. Methods: The treatment regimens were initiated 6 weeks after 5/6 NX in TGR, and the follow-up period was 60 weeks. Results: The addition of sEH inhibition to RAS blockade improved survival rate, further reduced albuminuria and renal glomerular and kidney tubulointerstitial injury, and attenuated the decline in creatinine clearance – all this as compared with 5/6 NX TGR treated with RAS blockade alone. Addition of ETA receptor antagonist to the combined RAS and sEH blockade not only offered no additional renoprotection but, surprisingly, also abolished the beneficial effects of adding sEH inhibitor to the RAS blockade. Conclusion: These data indicate that pharmacological strategies that combine the blockade of RAS and sEH could be a novel tool to combat the progression of CKD. Any attempts to further extend this therapeutic regimen should be made with extreme caution.

Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-β receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis.

Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. Hypoxia is known as one of the major risk factors for the development of NEC. Endothelin, known to regulate vasoconstriction, has two receptors (A and B). However, the role of endothelin receptor B (EDNRB) in neonatal intestinal injury remains unclear. We aimed to investigate whether EDNRB is involved in NEC pathophysiology. Following ethical approval (#44032), EDNRB hetero knockout mice pups (EDNRB±) and their wild-type (WT) littermates were studied. NEC was induced from postnatal day 5–9 (P5–P9) by hypoxia, gavage feeding of formula and administration of lipopolysaccharide. On P9, the ileum was harvested. NEC induction in WT mice was associated with mucosal injury. However, EDNRB± NEC mice had reduced mucosal injury. Similarly, EDNRB± mice had significantly lower expression of IL-6 mRNA compared to WT NEC mice. Pimonidazole immunostaining was also significantly lower in EDNRB± compared to WT NEC, suggesting reduced tissue hypoxia. Partial knockout of EDNRB results in reduced NEC severity and reduced tissue hypoxia. Intestinal perfusion and hypoxia are important elements of NEC pathogenesis. These findings are relevant to the understanding of NEC pathophysiology and to the development of novel preventive strategies for NEC.

Background Congenital cardiovascular malformations are the most common birth defects affecting children. Several of these defects occur in structures developing from neural crest cells. One of the key signaling pathways regulating cardiac neural crest cell (CNCC) development involves the endothelin-A receptor (Ednra). However, the exact function of Ednra signaling in CNCC is unknown. Results The fate mapping of CNCC in Ednra embryos indicated that the migration of these cells is aberrant in the cardiac outflow tract (OFT), but not in the pharyngeal arches. This premature arrest of CNCC migration occurs independently of CNCC proliferation and apoptosis changes and major gene expression changes. Analysis of the Rho family of small GTPases in the mutant embryos revealed that Cdc42 failed to localize normally in the CNCC migrating in the OFT. The inhibition of Cdc42 activity in cultured embryos recapitulated the migratory phenotype observed in Ednra mice. Further analyses revealed that Cdc42 is part of the signaling pathway activated by endothelin specifically in OFT CNCC to control their migration. Conclusions These results indicated that the activation of Cdc42 by endothelin signaling is important for CNCC migration in the OFT but this pathway is not involved in mandibular or pharyngeal arch artery patterning.

The effects of Konjac mannan oligosaccharides (KMOS) on diphenoxylate-induced constipation in mice were investigated in the present study. After administration with KMOS at 1800 mg/kg·bw, the weight and number of black feces within 24 h, and the gastrointestinal transit ratio were distinctly increased by 96.8%, 156.5% and 45.3%, respectively. Furthermore, KMOS regulated the levels of enteric nervous system parameters, including motilin, somatostatin, substance P, acetyl cholinesterase, endothelin and vasoactive intestinal peptide. Additionally, KMOS could recover the characteristics of small intestine villi and inhibit the reduction of mucosal thickness. KMOS administration produced more short chain fatty acids and lactic acid compared with the control group. The availability of 5-hydroxytryptamine and the expression of serotonin transporter were ameliorated in the KMOS treatment groups. Collectively, these findings suggested that KMOS could be considered as a functional food material for regulating intestinal function and alleviating constipation.

Endothelin receptor A (ETA) is a G protein–coupled receptor and a major therapeutic target for pulmonary arterial hypertension (PAH). We took a novel approach and developed an antagonistic monoclonal antibody, getagozumab, specifically against ETA. Getagozumab displayed a Kd value of 8.7 nM and an IC50 value of 37.9 nM in the cell-based assays. Getagozumab could significantly lower pulmonary arterial pressure in both hypoxia-induced and monocrotaline (MCT)-induced PAH monkey models and further attenuate the pulmonary arterial and right ventricular hypertrophy in MCT-induced PAH monkeys. The preclinical studies demonstrated that getagozumab is safe, long lasting, and efficacious. Getagozumab may provide a new and effective treatment for PAH patients.

Hirschsprung disease (HSCR) is a common cause of intestinal obstruction in the newborn. Hirschsprung-associated enterocolitis (HAEC) is a significant and life-threatening complication of HSCR, affecting up to 60% of patients. Animal models of endothelin receptor B (EdnrB) mutation reliably model human HSCR and HAEC. We previously demonstrated intestinal dysbiosis and a gut-specific deficiency of B-lymphocyte–produced secretory IgA (sIgA), the primary effector molecule of mucosal immunity, in mice with homozygous neural crest cell–conditional deletion of EdnrB (EdnrB(NCC−/−)). To determine mechanisms for sIgA deficiency, we examined intrinsic and extrinsic aspects of B-lymphocyte development and function. Expression of the endothelin axis components [endothelin-1 (ET-1), endothelin-3 (ET-3), endothelin receptor A (EdnrA), EdnrB] were determined over a developmental time course. B-lymphocyte survival and Ig production were assayed in vitro. Polymeric Ig receptor (pIgR)–mediated IgA transport into the intestinal lumen was interrogated. We found endothelin axis component (EdnrA, EdnrB, ET-1, ET-3) expression in developing extramedullary hematopoietic organs and that some splenic B lymphocytes express EdnrB. Splenic B lymphocytes from EdnrB(NCC−/−) mice showed no intrinsic defect in survival vs. wild-type (WT) B lymphocytes. In vitro stimulation of splenic B lymphocytes demonstrated decreased IgA, IgG, and IgM production in EdnrB(NCC−/−) vs. WT mice. Additionally, small intestinal pIgR was decreased ∼50% in EdnrB(NCC−/−) mice. These results suggest an intrinsic B-lymphocyte defect in antibody production as well as an extrinsic defect in IgA transport in the EdnrB(NCC−/−) model of HAEC. Our results are consistent with human HAEC observations of decreased luminal sIgA and mouse models of other inflammatory bowel diseases, in which decreased pIgR is seen in concert with a dysregulated microbiota. Finally, our results suggest targeting the dysbiotic microbiome and pIgR-mediated sIgA transport as potential therapeutic approaches in prevention and treatment of HAEC.—Medrano, G., Cailleux, F., Guan, P., Kuruvilla, K., Barlow-Anacker, A. J., Gosain, A. B-lymphocyte–intrinsic and –extrinsic defects in secretory immunoglobulinA production in the neural crest–conditional deletion of endothelin receptor B model of Hirschsprung-associated enterocolitis.

Background: Hypoxia plays an important role in the vascular tone of pulmonary circulation via the vasculature and parenchymal tissue. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, plays a role in inflammation in mononuclear cells. Nitric oxide synthase (NOS), which generates nitric oxide (NO)/cyclic 3', 5'-monophosphate (cGMP), is coexpressed with ET-1 in many cell types. The aim of this study was to assess whether hypoxia induces the production of ET-1 and associated expression of NOS, NO/cGMP and chemokines in rat alveolar macrophages (AMs). Methods: NR8383 cells were cultured under hypoxic (1% oxygen) conditions for 0, 2, 4, 8 and 12 hours. Levels of ET-1, inducible NOS (iNOS), phosphorylated iNOS (p-iNOS), nitrite/nitrate (NOx), cGMP and monocyte chemoattractant protein-1 (MCP-1) were measured. Results: ET-1, p-iNOS, NOx, and cGMP increased significantly in AMs after 4 hours of hypoxia (p < 0.05). ET-1 and MCP-1 mRNA increased after 8 hours (p < 0.05). The protein expression of ET-1, MCP-1, and p-iNOS increased in a time-dependent manner, while iNOS expression decreased with time. Conclusions: The changes in ET-1, p-iNOS, and the NO/cGMP pathway in AMs may help elucidate the mechanisms in the hypoxic lung. Understanding changes in the endothelin axis in hypoxic AMs is a crucial first step to unravel its role in pulmonary circulation.

Introduction It is well established that upregulation of endothelin type B (ETB) receptors in vascular smooth muscle cells plays a role in pathophysiological artery remodeling as response to ischemia and atherosclerosis. This study aimed to investigate the ETB receptors function and localization under normal physiological remodeling. Specifically, in the guinea pig uterine arteries during pregnancy. Methods Uterine artery contractility was assessed with sarafotoxin 6c and endothelin-1 in wire-myography in uterine arteries from non-pregnant and pregnant guinea pigs at gestational day 37 ± 5. Localization of ETB receptors, proliferation marker Ki-67, and SMC differentiation marker SM22α in uterine arteries were investigated with immunohistochemistry. Results Uterine arteries from pregnant guinea pigs showed significantly increased ETB receptor-mediated vasoconstriction compared to uterine arteries from non-pregnant and to coronary arteries from pregnant guinea pigs (p Discussion Our results suggest that ETB receptors are expressed in dedifferentiated proliferating SMCs of uterine arteries in pregnant guinea pigs. This study provides novel insight into the function and expression of ETB receptors in uterine vascular remodeling during pregnancy.

The present study was designed to investigate the effect of late phase of whole body hypoxic preconditioning on endothelial-dependent vasorelaxation and cardioprotection from ischemia-reperfusion injury in spontaneously hypertensive rats (SHR). Hypoxic preconditioning was performed by subjecting rats to four episodes of alternate exposure to low O2 (8%) and normal air O2 of 10 min each. After 24 h, the mesenteric arteries and hearts were isolated to determine the vascular function and cardioprotection from ischemia-reperfusion (I/R) injury on the Langendorff apparatus. There was a significant impairment in acetylcholine-induced relaxation in norepinephrine precontracted arteries (endothelium-dependent function) and increase in I/R-induced myocardial injury in SHR in comparison to Wistar Kyoto rats (WKY). However, hypoxic preconditioning significantly restored endothelium-dependent relaxation in SHR and attenuated I/R injury in both SHR and WKY. Hypoxic preconditioning also led to an increase in the levels of endothelin-1 (not endothelin-2 or −3), vascular endothelial growth factor-A (VEGF-A) and HIF-1α levels. Pretreatment with bevacizumab (anti-VEGF-A) and bosentan (endothelin receptor blocker) significantly attenuated hypoxic preconditioning-induced restoration of endothelium-dependent relaxation and cardioprotection from I/R injury. These interventions also attenuated the levels of VEGF-A and HIF-1α without modulating the endothelin-1 levels. It may be concluded that an increase in the endothelin-1 levels with a subsequent increase in HIF-1α and VEGF expression may possibly contribute in improving endothelium-dependent vasorelaxation and protecting hearts from I/R injury in SHR during late phase of whole body hypoxic preconditioning.

Objectives Preeclampsia (PE), hypertension with proteinuria during pregnancy, is associated with an altered CD4+ T cell population and chronic inflammation compared to normal pregnant (NP) women. Previous studies from the Zenclussen lab show that adoptive transfer of Th1-like splenocytes increases MAP, fetal rejection and inflammatory cytokines when transferred to NP mice, but the mechanisms causing this increased MAP and abnormalities were not further investigated. We believe CD4+ T cells play an important role to increase Endothelin (ET-1) and ROS as mediators of the pathophysiology associated with PE. Therefore, the objective of this study was to determine if CD4+ T cells isolated from placentas of PE patients cause PE symptoms during pregnancy compared to those collected from NP women. Methods CD4+ T cells were isolated from placentas of PE and NP women using magnetic anti-CD4 antibodies, cultured in TexMACS medium (Miltenyi Biotec) with IL-2 at 37 °C in 5% CO 2 , and injected intraperitoneally into pregnant, nude-athymic rats on day 12 of gestation. On day 18, carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. Results MAP was significantly increased from 125 ± 3 mmHg in rats injected with NP T cells to 140 ± 9 mmHg in rats injected with PE T cells. Circulating IL-6 and TNF- α were increased in rats exposed to PE vs NP CD4+ T cells (TNF- α -PE = 142.4 pg/mL, NP = 79.4 pg/mL; IL-6 -PE = 311.6 pg/mL, NP = 277.8 pg/mL). Cortical ET-1 mRNA expression was increased 4.5 fold in rats exposed to PE CD4+ T cells vs those exposed to NP CD4+ T cells ( p = 0.06). Conclusions These data suggest an important role for placental CD4+ T cells from PE patients to cause inflammation, renal abnormalities and thus hypertension during pregnancy. Disclosures A.C. Harmon: None. D.C. Cornelius: None. K. Wallace: None. J. Moseley: None. B. LaMarca: None.

Background The pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1. Methods Male and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10−12 to 10−8 M) and sarafotoxin 6c (S6c, ETB agonist, 10−12 to 10−8 M) using the blood-perfused juxtamedullary nephron preparation. Results Control afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 μm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10−8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10−8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10−8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10−12 to 10−9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation. Conclusion These data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.

Background: Ca2+ plays an important role in the regulation of vasoconstriction. Ca2+ signaling is regulated by a number of Ca2+-handling proteins. However, whether differences in Ca2+ handling affect the regulation of vasoconstriction in different arteries remains elusive. Objective: To determine whether differences in Ca2+ handling affect the response to vasoconstrictors in different arteries. Methods: Arterial ring contraction was measured using a Multi Myograph System. Vascular smooth muscle cells (VSMCs) were digested with type 2 collagenase in DMEM, then intracellular calcium concentration was measured with the Ca2+ probe fluo-4/AM in the isolated cells. Calcium-related proteins were assayed by Western blotting. Results: Phenylephrine did not induce ­coronary arterial contraction. There were differences in ­5-hydroxytryptamine, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2a, and endothelin 1-induced vasoconstriction in different solutions between coronary and renal arteries. Vasoconstrictions in the presence of Bay K8644 were stronger in coronary than in renal arteries. Store-operated calcium (SOC) channels could mediate Ca2+ influx in VSMCs of both groups. SOC channels did not participate in the contraction of coronary arteries. In addition, there were significant differences in the expressions of receptors and ion channels between the two groups. Conclusions: Ca2+ handling contributed to the different responses to vasoconstrictors between coronary and renal arteries.

Objective To observe the lipid-lowering effect of Typha angustifolia extract (TAE) in Sprague Dawley rats. Methods The rats were randomly divided into six groups, including the hypercholesterolemic diet (HYD) group. Ad libitum food was given to the animals for 60 d to establish dyslipidemia models. The rats were received an ig administration of 1 mL medicine daily. After 120 d the animals were sacrificed by cervical dislocation; Blood was collected to measure total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), nitric oxide (NO), and endothelin (ET) plasma concentration; Livers were collected to measure ApoE mRNA and protein expression; Morphologic changes of aorta ventralis tissue were also observed. Results Compared with HYD group, TAE had the ability of reducing TC, TG, LDL-C, NO, and ET (P Conclusion These results suggested that TAE was capable of effectively decreasing the circulating lipids levels and enhancing the protective effects of artery.

Objective Mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Long-term exposure to human ET-1 (hET-1) in mice inducibly overexpressing hET-1 in the endothelium (ieET-1) caused sustained BP elevation. ET-1 has been shown to stimulate the release of aldosterone. Whether aldosterone plays a role in hET-1 overexpression-induced BP elevation and vessel injury is unknown. Method Nine- to 12-week-old male ieET-1 mice and control mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in the endothelial cells (ieCre) were treated with tamoxifen for 5 days and studied 3 months later. Results Endothelial hET-1 overexpression increased plasma aldosterone levels, which was reversed by 2-week treatment with atrasentan, an endothelin type A receptor blocker. Aldosterone synthase and cryptochrome 2 adrenal cortex mRNA expression was decreased in ieET-1 mice. Two-week treatment with eplerenone, a mineralocorticoid receptor antagonist, reduced systolic BP by 10 mmHg in ieET-1 mice during rest time. Saline challenge-induced sodium excretion and renal cortex thiazide-sensitive sodium-chloride cotransporter mRNA expression were decreased in ieET-1 mice. The sensitivity of mesenteric arteries to contraction by norepinephrine was increased in ieET-1 mice, and was abrogated by eplerenone treatment, whereas sensitivity of endothelium-independent relaxation responses to sodium nitroprusside was enhanced. Resistance artery remodeling was reduced in eplerenone-treated ieET-1 vs. ieET-1 and ieCre mice. Conclusion These results demonstrate that aldosterone contributes to BP elevation and vascular norepinephrine sensitivity and remodeling caused by hET-1 overexpression in endothelium in mice.

Objectives This study was designed to test our hypothesis that angiotensin II (Ang II) upregulates endothelin (ET) receptors in vascular smooth muscle cells (VSMCs). Methods Rat superior mesenteric artery (SMA) without endothelium was cultured in serum-free medium for 24 h in the presence of Ang II with or without metformin or nicotinamide. In vivo, rats were implanted subcutaneously with a mini-osmotic pump infusing AngII (500 ng/kg/min) for 4 weeks. The level of protein expression was determined using Western blotting. The contractile response to ET receptor agonists was studied using sensitive myography. Caudal artery blood pressure (BP) was measured using non-invasive tail-cuff plethysmography. Key findings The results showed that Ang II significantly increased ET receptors and decreased phosphorylated-adenosine monophosphate-activated protein kinase α (p-AMPKα) in SMA. Furthermore, metformin significantly inhibited Ang II-upregulated ET receptors and upregulated Ang II-decreased sirtuin 1 (Sirt1). However, this effect was reversed by nicotinamide. Moreover, the in-vivo results showed that metformin not only inhibited Ang II-induced upregulation of ET receptors but also recovered Ang II-decreased p-AMPKα and Sirt1. In addition, metformin significantly inhibited Ang II-elevated BP. However, the effect was reversed by nicotinamide, except for p-AMPKα. Conclusions Ang II upregulated ET receptors in VSMCs to elevate BP by inhibiting AMPK, thereby inhibiting Sirt1.

Background: Aim of this study was to evaluate morphologic features of healthy saphenous vein and internal thoracic artery used in coronary artery bypass surgery and compare results. Materials and methods: Ten specimens of saphenous veins and ten of internal thoracic arteries used for coronary artery bypass graft were obtained from 20 patients. Histological routine and immunohistochemical staining was performed with: endothelin (ET), tissue inhibitor of metalloproteinase 2 (TIMP2), metallomembranoproteinase 2 (MMP2), transforming growth factor beta (TGF β), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), protein gene product 9.5 (PGP9.5), vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM). A semiquantitative evaluation method was used. Results: There was found: moderate positive endothelin-containing cells in both blood vessel types; moderate positive MMP2 cells and moderate to numerous positive TIMP2 cells in veins. In arteries – occasionally marked positive MMP2 cells and negative TIMP2; moderate to numerous positive VEGF endothelial cells on small blood vessels in vein wall and occasional in artery wall; numerous TGFβ structures in veins and abundance of VCAM, ICAM positive cells, few in arteries; few HGF positive structures in veins, negative in arteries; In veins few PGP9,5 positive nerve fibres, in arteries - moderate. Moderate TUNEL reaction positive apoptotic cells in veins and few to moderate in arteries. Conclusions: V.saphena magna grafts are characterized by more intensive modelation plasticity. Number of VEGF, VCAM and ICAM found in v.saphena magna proves the possible tendency of graft failure on basis of local blood supply intensification. Appearance of endothelin positive cells indicate the similar homeostasis condition in endotheliocytes in both – vein and artery grafts.

The endothelium is the single-cell monolayer that lines the entire vasculature. The endothelium has a barrier function to separate blood from organs and tissues but also has an increasingly appreciated role in anti-coagulation, vascular senescence, endocrine secretion, suppression of inflammation and beyond. In modern times, endothelial cells have been identified as the source of major endocrine and vaso-regulatory factors principally the dissolved lipophilic vosodilating gas, nitric oxide and the potent vascular constricting G protein receptor agonists, the peptide endothelin. The role of the endothelium can be conveniently conceptualized. Continued investigations of the mechanism of endothelial dysfunction will lead to novel therapies for cardiovascular disease. In this review, we discuss the impact of endothelial dysfunction on cardiovascular disease and assess the clinical relevance of endothelial dysfunction.

In brain disorders, reactive astrocytes, which are characterized by hypertrophy of the cell body and proliferative properties, are commonly observed. As reactive astrocytes are involved in the pathogenesis of several brain disorders, the control of astrocytic function has been proposed as a therapeutic strategy, and target molecules to effectively control astrocytic functions have been investigated. The production of brain endothelin-1 (ET-1), which increases in brain disorders, is involved in the pathophysiological response of the nervous system. Endothelin B (ETB) receptors are highly expressed in reactive astrocytes and are upregulated by brain injury. Activation of astrocyte ETB receptors promotes the induction of reactive astrocytes. In addition, the production of various astrocyte-derived factors, including neurotrophic factors and vascular permeability regulators, is regulated by ETB receptors. In animal models of Alzheimer’s disease, brain ischemia, neuropathic pain, and traumatic brain injury, ETB-receptor-mediated regulation of astrocytic activation has been reported to improve brain disorders. Therefore, the astrocytic ETB receptor is expected to be a promising drug target to improve several brain disorders. This article reviews the roles of ETB receptors in astrocytic activation and discusses its possible applications in the treatment of brain disorders.

Cardiac hypertrophy is a key pathophysiological process in the heart in response to stress cues. Although taking place in cardiomyocytes, the hypertrophic response is influenced by other cell types, both within the heart and derived from circulation. In the present study we investigated the myeloid-specific role of megakaryocytic leukemia 1 (MKL1) in cardiac hypertrophy. Following transverse aortic constriction (TAC), myeloid MKL1 conditional knockout (MFCKO) mice exhibit an attenuated phenotype of cardiac hypertrophy compared to the WT mice. In accordance, the MFCKO mice were protected from excessive cardiac inflammation and fibrosis as opposed to the WT mice. Conditioned media collected from macrophages enhanced the pro-hypertrophic response in cardiomyocytes exposed to endothelin in an MKL1-dependent manner. Of interest, expression levels of macrophage derived miR-155, known to promote cardiac hypertrophy, were down-regulated in the MFCKO mice compared to the WT mice. MKL1 depletion or inhibition repressed miR-155 expression in macrophages. Mechanistically, MKL1 interacted with NF-κB to activate miR-155 transcription in macrophages. In conclusion, our data suggest that MKL1 may contribute to pathological hypertrophy via regulating macrophage-derived miR-155 transcription.

The endothelial glycoprotein thrombomodulin regulates coagulation, vascular inflammation and apoptosis. In the kidney, thrombomodulin protects the glomerular filtration barrier by eliciting crosstalk between the glomerular endothelium and podocytes. Several glomerular pathologies are characterized by a loss of glomerular thrombomodulin. In women with pre-eclampsia, serum levels of soluble thrombomodulin are increased, possibly reflecting a loss from the glomerular endothelium. We set out to investigate whether thrombomodulin expression is decreased in the kidneys of women with pre-eclampsia and rats exposed to an angiogenesis inhibitor. Thrombomodulin expression was examined using immunohistochemistry and qPCR in renal autopsy tissues collected from 11 pre-eclamptic women, 22 pregnant controls and 11 hypertensive non-pregnant women. Further, kidneys from rats treated with increasing doses of sunitinib or sunitinib in combination with endothelin receptor antagonists were studied. Glomerular thrombomodulin protein levels were increased in the kidneys of women with pre-eclampsia. In parallel, in rats exposed to sunitinib, glomerular thrombomodulin was upregulated in a dose-dependent manner, and the upregulation of glomerular thrombomodulin preceded the onset of histopathological changes. Selective ETAR blockade, but not dual ETA/BR blockade, normalised the sunitinib-induced increase in thrombomodulin expression and albuminuria. We propose that glomerular thrombomodulin expression increases at an early stage of renal damage induced by antiangiogenic conditions. The upregulation of this nephroprotective protein in glomerular endothelial cells might serve as a mechanism to protect the glomerular filtration barrier in pre-eclampsia.

Pregnancy-associated malaria is often associated with adverse pregnancy outcomes. Placental circulatory impairments are an intriguing and unsolved component of malaria pathophysiology. Here, we uncovered a Toll-like receptor 4 (TLR4)-TRIF-endothelin axis that controls trophoblast motility and is linked to fetal protection during Plasmodium infection. In a cohort of 401 pregnancies from northern Brazil, we found that infection during pregnancy reduced expression of endothelin receptor B in syncytiotrophoblasts, while endothelin expression was only affected during acute infection. We further show that quantitative expression of placental endothelin and endothelin receptor B proteins are differentially controlled by maternal and fetal TLR4 alleles. Using murine malaria models, we identified placental autonomous responses to malaria infection mediated by fetally encoded TLR4 that not only controlled placental endothelin gene expression but also correlated with fetal viability protection. In vitro assays showed that control of endothelin expression in fetal syncytiotrophoblasts exposed to Plasmodium-infected erythrocytes was dependent on TLR4 via the TRIF pathway but not MyD88 signaling. Time-lapse microscopy in syncytiotrophoblast primary cultures and cell invasion assays demonstrated that ablation of TLR4 or endothelin receptor blockade abrogates trophoblast collective motility and cell migration responses to infected erythrocytes. These results cohesively substantiate the hypothesis that fetal innate immune sensing, namely, the TRL4-TRIF pathway, exerts a fetal protective role during malaria infection by mediating syncytiotrophoblast vasoregulatory responses that counteract placental insufficiency.

Retinitis pigmentosa (RP) and macular dystrophy (MD) are prevalent retinal degenerative diseases associated with gradual photoreceptor death. These diseases are often caused by genetic mutations that result in degeneration of the retina postnatally after it has fully developed. The Prominin-1 gene (Prom1) is a causative gene for RP and MD, and Prom1- knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and stenosis of retinal blood vessels. The mechanisms underlying progression of such degeneration have remained unknown, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. High-throughput analysis revealed that expression of the endothelin-2 gene (Edn2) was markedly up-regulated in the Prom1-deficient retina during this period. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice that had been reared in the dark. Finally, treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis, and retinal vessel stenosis in Prom1-KO mice. Our findings suggest that inhibitors of endothelin signalling may delay the progression of RP and MD and therefore warrant further study as potential therapeutic agents for these diseases.