Endothelin-1 Secretion Research Articles

Topiramate Reduces Aortic Cross-Clamping-Induced Lung Injury in Male Rats.

Topiramate (TPM) decreases cytokine release and generation of reactive oxygen species (ROS). Cytokine and endothelin-1 (ET-1) secretion and ROS formation play an important role in ischemia-reperfusion (I/R) injury. We aimed to evaluate whether TPM prevents damage occurring in lung tissue during I/R. A total of 27 Wistar albino rats were divided into three groups of nine. To the I/R group, two hours of ischemia via infrarenal abdominal aorta cross-ligation and then two hours of reperfusion process were applied. TPM (100 mg/kg/day) orally for seven days was administered in the TPM treatment group. After the last dose of TPM treatment, respectively, two hours of ischemia and two hours of reperfusion were applied in this group. Tumor necrosis factor-alpha (TNF-α) (p < 0.05), malondialdehyde (MDA) (p < 0.05), myeloperoxidase (MPO) (p < 0.05) and ET-1 (p < 0.05) levels of TPM treatment group's lung tissue were significantly lower than for the I/R group. Caspase-3 and histopathological damage were rather lower than that of the I/R group. During I/R, lung damage occurs due to excessive TNF-α and ET-1 release and ROS generation. TPM could well reduce development of lung damage by decreasing cytokine and ET-1 release and levels of ROS produced.

Intradialytic hypertension is associated with low intradialytic arterial oxygen saturation.

ABSTRACTBackgroundThe pathophysiology of a paradoxical systolic blood pressure (SBP) rise during hemodialysis (HD) is not yet fully understood. Recent research indicated that 10% of chronic HD patients suffer from prolonged intradialytic hypoxemia. Since hypoxemia induces a sympathetic response we entertained the hypothesis that peridialytic SBP change is associated with arterial oxygen saturation (SaO2).MethodsWe retrospectively analyzed intradialytic SaO2 and peridialytic SBP change in chronic HD patients with arteriovenous vascular access. Patients were followed for 6 months. We defined persistent intradialytic hypertension (piHTN) as average peridialytic SBP increase ≥10 mmHg over 6 months. Linear mixed effects (LME) models were used to explore associations between peridialytic SBP change and intradialytic SaO2 in univariate and adjusted analyses.ResultsWe assessed 982 patients (29 872 HD treatments; 59% males; 53% whites). Pre-dialysis SBP was 146.7 ± 26.5 mmHg and decreased on average by 10.1 ± 24.5 mmHg. Fifty-three (5.7%) patients had piHTN. piHTN patients had lower intradialytic SaO2, body weight and interdialytic weight gain. LME models revealed that with every percentage point lower mean SaO2, the peridialytic SBP change increased by 0.46 mmHg (P < 0.001). This finding was corroborated in multivariate analyses.ConclusionWe observed an inverse relationship between intradialytic SaO2 and the blood pressure response to HD. These findings support the notion that hypoxemia activates mechanisms that partially blunt the intradialytic blood pressure decline, possibly by sympathetic activation and endothelin-1 secretion. To further explore that hypothesis, specifically designed prospective studies are required.

Andrographolide inhibits hypoxia-induced hypoxia-inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP-5 pathways in EA.hy926 cells.

Andrographolide is a potent anti-inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET-1) is an endothelium-derived vasoconstrictor with pro-inflammatory properties secreted in response to hypoxia. Mitogen-activated protein kinase phosphatase 5 (MKP-5) is a dual-specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia-induced HIF-1α expression and ET-1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP-5 plays in andrographolide's inhibition of hypoxia-induced expression of HIF-1α and ET-1. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl2 . Andrographolide enhanced HO-1 and MKP-5 expression and cellular cGMP content in addition to inhibiting hypoxia-induced ROS generation. Concomitantly, the HO-1 byproduct CO and the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) increased MKP-5 expression, and pretreatment with CO and 8-Br-cGMP inhibited hypoxia-induced HIF-1α and ET-1 expression. Transfection of HO-1 siRNA or pretreatment with the HO-1 inhibitor ZnPP-9 or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide-induced MKP-5 expression. Moreover, silencing MKP-5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia-induced p38 MAPK activation and HIF-1α expression. The inhibition of hypoxia-induced HIF-1α and ET-1 expression by andrographolide is likely associated with HO-1/CO/cGMP/MKP-5 pathways, which is involved in inhibiting hypoxia-induced p38 MAPK activation.

Keratinocyte Sonic Hedgehog Upregulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

Antihypertensive effect of piperitenone oxide on spontaneously hypertensive rat by regulating calcium balance and reducing endothelin-1 secretion

The purpose of this study was to investigate the antihypertensive mecha-nisms of piperitenone oxide in spontaneously hypertensive rat (SHR). Compared to control SHR, piperitenone oxide reduced the systolic blood pressure and serum endothelium-1 (ET-1). The nitric oxide (NO) production and ET-1 secretion were significantly inhibited in the noradrenaline-exposed bovine aortic endothelial cells. Piperitenone oxide showed endothelium-independent but concentration-dependent relaxation of the artery rings. The responsible mechanisms appear to be related to the modulation of ET-1 and NO levels. The vasorelaxant effect of piperitenone oxide is independent of endothelium-derived mediators, which is associated with interfering intracellular calcium homeostasis by blocking Ca2+ influx, as well as intracellular Ca2+ release.&#x0D; Video Clip of Methodology:&#x0D; 2 min 58 sec: Full Screen Alternate

Intertraffic of Endothelin 1 and Thrombospondin 1 between lungs and myocard via pulmonary circulation can alter cardiac loads, tissue integrity and atrial blood coagulability

Similar expression patterns of mRNA profiles for Endothelin 1 (ET-1) and Thrombospondin 1 (TSP1) from GeneAtlas U133A, gcrma, suggest that these two mediators are dominantly synthesized in the myocard and lungs. This paper proposes that intertraffic of these two mediators between myocard and lungs via pulmonary and coronary vessels optimizes cardiopulmonary functions and maintains their tissue integrity. A controlled delivery of endocrine mediators to the left and to the right heart is done by the coronary sinus (CS) that drains venous blood to the right atrium, and Thebesian veins (TVs) that open in all four heart cavities. Myocard and pulmonary capillaries are connected as a bidirectional portal system. Mediators from lungs can directly influence myocardial cells after entering the coronary circulation, while mediators in the myocardial venous blood that drain into the right heart will initially affect lungs. Strain induced myocardial ET-1 secretion into the right heart and pulmonary circulation increases the right heart afterload by constricting pulmonary vasculature. The same action reduces the left heart preload. Pulmonary ET-1 secretion can protect lungs from overperfusion by increasing the left heart afterload through constriction of peripheral arterioles. Chronic tissue overexposure to ET-1 leads to pulmonary and myocardial fibrosis. TSP1 availability is important in tissues under mechanical stress, since TSP1 is an adhesive glycoprotein involved in cell-to-cell and cell-to-matrix interactions. Pulmonary and myocardial TSP1 secretion that enter the fibrillating left atrium can mimic actions of the platelet-derived TSP1 in promoting the thrombus formation.

Role of Autophagy in HIV-1 Matrix Protein p17-Driven Lymphangiogenesis.

AIDS-related lymphomas (ARLs) are expected to increase in the future since combined antiretroviral therapy (cART) enhances the life expectancy of HIV-1-infected (HIV+) patients but does not affect the occurrence of ARLs to the same extent as that of other tumors. Lymphangiogenesis is essential in supporting growth and metastatic spreading of ARLs. HIV-1 does not infect the neoplastic B cells, but HIV-1 proteins have been hypothesized to play a key role in sustaining a prolymphangiogenic microenvironment in lymphoid organs. The HIV-1 matrix protein p17 is detected in blood and accumulates in the germinal centers of lymph nodes of HIV+ patients under successful cART. The viral protein displays potent lymphangiogenic activity in vitro and in vivo This is, at least in part, mediated by the secretion of the lymphangiogenic factor endothelin-1, suggesting that activation of a secretory pathway sustains the lymphangiogenic activity of p17. Here, we show that the p17 lymphangiogenic activity occurs on human lymph node-derived lymphatic endothelial cells (LN-LECs) under stress conditions only and relies entirely on activation of an autophagy-based pathway. In fact, induction of autophagy by p17 promotes lymphangiogenesis, whereas pharmacological and genetic inhibition of autophagy inhibits p17-triggered lymphangiogenesis. Similarly, the vasculogenic activity of p17 was totally inhibited in autophagy-incompetent mice. Our findings reveal a previously unrecognized role of autophagy in lymphangiogenesis and open the way to identify novel treatment strategies aimed at inhibiting aberrant tumor-driven lymphangiogenesis in HIV+ patients.IMPORTANCE AIDS-related lymphomas (ARLs) are the most common malignancies in HIV-1-infected (HIV+) patients after the introduction of combined antiretroviral therapy (cART). Lymphangiogenesis is of critical importance in sustaining growth and metastasis of ARLs. Indeed, enhanced lymphangiogenesis occurs in the lymph nodes of HIV+ patients under successful cART. The HIV-1 matrix protein p17 is detected in blood and accumulates in the lymph node germinal centers even in the absence of virus replication. Several findings suggest a key role for p17 as a microenvironmental factor capable of promoting lymphangiogenesis. Here, we show that p17 promotes lymphangiogenesis of human lymph node-derived lymphatic endothelial cells (LN-LECs). The lymphangiogenic activity of p17 is sustained by an autophagy-based pathway that enables LN-LECs to release prolymphangiogenic factors into the extracellular microenvironment. Our findings indicate that specific targeting of autophagy may provide an important new tool for treating ARLs.

Endothelial cell-derived endothelin-1 is involved in abnormal scar formation by dermal fibroblasts through RhoA/Rho-kinase pathway.

Hypertrophic scars and keloids are characterized by excessive dermal deposition of extracellular matrix due to fibroblast-to-myofibroblast differentiation. Endothelin-1 (ET-1) is primarily produced by vascular endothelial cells and plays multiple roles in the wound-healing response and organ fibrogenesis. In this study, we investigated the pathophysiological significance of ET-1 and involvement of RhoA, a member of the Rho GTPases, in hypertrophic scar/keloid formation. We found that ET-1 expression on dermal microvascular endothelial cells (ECs) in hypertrophic scars and keloids was higher than that in normal skin and mature scars. We also confirmed that ET-1 induced myofibroblast differentiation and collagen synthesis in cultured human dermal fibroblasts through the RhoA/Rho-kinase pathway. Finally, since hypertrophic scar/keloid formation was most prominent in areas exposed to mechanical stretch, we examined how mechanical stretch affected ET-1 secretion in human dermal microvascular ECs, and found that mechanical stretch increased ET-1 gene expression and secretion from ECs. Taken together, these results suggest that dermal microvascular ECs release ET-1 in response to mechanical stretch, and thereby contribute to the formation of hypertrophic scars and keloids through the RhoA/Rho-kinase pathway.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α-induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

AMP-activated protein kinase mediates the effects of lipoprotein-associated phospholipase A2 on endothelial dysfunction in atherosclerosis.

The present study aimed to investigate the effects of lipoprotein-associated phospholipase A2 (Lp-PLA2) on endothelial dysfunction in an in vitro cell model of atherosclerosis, and to determine whether AMP-activated protein kinase (AMPK) mediates the effects of Lp-PLA2 on endothelial dysfunction. A total of 392 patients with coronary artery disease (CAD), including various sub-conditions, were recruited, and the plasma concentrations of Lp-PLA2 were evaluated. In addition, an in vitro model of atherosclerosis was established by exposing human umbilical vein endothelial cells (HUVECs) to oxidized low-density lipoprotein (oxLDL). SB-435495 was used to inhibit Lp-PLA2, and compound C was used to suppress AMPK expression. Lp-PLA2, AMPKα and phosphorylated-AMPKα (T172) expression in HUVECs were evaluated using western blot analysis. The concentrations of nitric oxide (NO), endothelin 1 (ET-1), intercellular adhesion molecule 1 (ICAM-1) and platelet/endothelial cell adhesion molecule 1 (PECAM-1) in cell culture supernatant were determined using commercially available ELISA kits. MTT assays were employed to indicate changes in cell viability. The current study found the plasma Lp-PLA2 levels were elevated in the CAD patients with stable angina pectoris, unstable angina pectoris, acute coronary syndromes and acute myocardial infarction, compared with a healthy control population. In addition, the in vitro results showed that Lp-PLA2 expression levels were elevated in oxLDL-exposed HUVECs. Lp-PLA2 suppression could increase cell viability, induce the production of NO and decrease the secretion of ET-1, in addition to suppressing the expression of cell adhesion molecules, including ICAM-1 and PECAM-1 in oxLDL-exposed HUVECs. The expression of AMPKα and phosphorylated-AMPKα (T172) was regulated by Lp-PLA2, and AMPK suppression was able to reverse the effects of Lp-PLA2 with regard to cell viability, endothelial vasorelaxation capacity and the secretion of adhesion molecules in oxLDL-exposed HUVECs. In conclusion, the present study provides initial evidence that Lp-PLA2 is able to cause endothelial dysfunction in an in vitro model of atherosclerosis, and the effects of Lp-PLA2 on endothelial dysfunction was at least partially a result of the downregulation of AMPKα, thus contributing to the progression of atherosclerosis.

BETA-BLOCKERS AND RENOPROTECTION: THE POTENTIAL OF CARVEDILOL

The review presented the importance of carvedilol using in terms of renoprotection in renal dysfunction at the pre-dialysis stage of the disease in order to reduce the risk of progression of chronic kidney diseases (CKD) and the development of cardiovascular complications. Immune and non-immune mechanisms (proteinuria, dyslipidemia, anemia, arterial hypertension) of renal dysfunction progression in patients with CKD of inflammatory and non-inflammatory origin are described. Moreover, with the slowing down of the glomerular filtration rate in CKD, the role of non-immune factors in the development of cardiovascular complications becomes very important. In contrast to non-selective and some β 1-selective beta-blockers, the use of beta-adenoblocker with vasodilating activity, in particular carvedilol, makes it possible to prevent the onset of the terminal stage of CKD. Carvedilol, being a lipophilic beta-adrenoblocker of the third generation with alpha-blocking properties, influences the possible mechanisms of renoprotection: antihypertensive (including in combined antihypertensive therapy), anti-inflammatory, antiproliferative, anti-apoptotic, antioxidant, antiplatelet and others. Carvedilol due to the vasodilating effect softens the stress of the parietal shear, exerting a retarding action on the progression of CKD. Carvedilol with a pronounced vasodilating effect and a long half-life significantly reduces central arterial pressure that is also an important renoprotective mechanism in the treatment of patients with renal dysfunction. Carvedilol has an important renoprotective mechanism in CKD – inhibition of the secretion of the potent vasoconstrictor endothelin. In the metabolic syndrome, in which there is a significant risk of developing renal dysfunction, carvedilol levels the imbalance of adipokine secretion, insulin resistance, sodium and water retention, and the activation of renin-angiotensin-aldosterone and sympathoadrenal systems. Carvedilol at the early stages of CKD development shows predominantly antihypertensive action due to inhibition of the renin-angiotensin-aldosterone system activity directly in the kidneys. At the late stage of the disease, the drug is able to retain residual kidney function. That is, carvedilol can be used at all stages of CKD development, regardless of the etiology of kidney damage.

The Endothelin Receptor Etb Plays a Crucial Role for Recruitment of Neutrophils to the Vascular Wall in Sickle Cell Disease

Introduction: Although Sickle Cell Disease (SCD) is due to abnormal hemoglobin, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), play a key role in the pathophysiology of the disease, particularly in the vaso-occlusive events. Adhesion of PMNs to activated endothelium is critical in SCD and might contribute to vaso-occlusion; thus targeting PMN interactions with the endothelium may represent a good opportunity for new therapeutics. We looked for candidate mediators that may be involved in PMN activation and recruitment in tissues. We focused on endothelin-1 (ET-1) since (i) high levels of cytokines have been reported in SCD patients; (ii) we previously demonstrated that ET-1 receptors blockade in the SAD mouse model of SCD leads to reversal or prevention of vaso-occlusive events, nitrative stress, kidney and lung damage, and even death. While demonstrating that ET receptor (ETR) antagonism inhibited a tonic ET-1-dependent vasoconstriction during experimental vaso-occlusive crisis, we observed unexpected inhibition of PMN recruitment in lungs and kidneys. We postulated that activation of ETRs might stimulate a pathogenic proinflammatory role for PMNs in SCD.Methods: In the present study, we combinedintravital videomicroscopy of the cremaster muscle microcirculation in SAD mice and quantitative microfluidic fluorescence microscopy of human blood to investigate the involvement of the ETRs in the interaction between neutrophils and endothelial cells.Results: Experiments performed both on SAD mice and SCD patients indicate that blocking ETRs hinders PMN recruitment to endothelial cells in inflammatory conditions (Fig.1). In SAD mice, we showed that the ETRs are involved at several steps of PMN microvascular recruitment. Rolling adhesion involves the ETB receptor only; firm adhesion and post-adhesive dynamic events with transmigration involve both the ETA and the ETB receptors. Inhibition experiments performed with the highly selective BQ788 antagonist (specific for ETB receptor) provide potent anti-inflammatory action in SAD mice. In SCD patients and healthy volunteers, we evidenced that human PMNs display functional ETB receptors that trigger a downstream calcium signaling leading to enhanced adhesion to endothelial cells. Furthermore, we investigated the expression of preproET-1 mRNA and the secretion of ET-1 in human PMNs from SCD patients and healthy controls and we highlighted the ability of PMNs to produce and secrete ET-1. We also found an unexpected stimulatory role for the ETB receptor in PMN adhesion to endothelial cells in laminar flow conditions. Last, we showed that this abnormal adhesion involves ETB receptors on both endothelial cells and PMNs.Conclusion: SAD mice and SCD patients both provide consistent evidence for a powerful anti-adhesion role of ETB blockade. We emphasize that our work is the first to unravel the impact of the ET receptors on the different phases of PMN-endothelial interaction, further complementing early evidence that endothelins are chemoattractants for neutrophils in vitro. Our findings involving a potent contribution of the ETB receptor to vascular inflammation are novel as well as the fact that this pathogenic phenomenon is found in sickle but not in normal mice. Overall, the aforementioned findings indicated that human neutrophils display functional ETB receptors with calcium signaling capability, and we confirmed that human PMNs synthesize ET-1 that may be involved in autocrine and paracrine pathophysiological actions. Thus, the ET-1- ETB axis should be considered a cytokine-like potent proinflammatory pathway in SCD. If endothelin receptors antagonists prove safe and effective for preventing or treating acute vasoocclusive events in the clinical setting, they should include anti- ETB potency and may provide major benefits for lung and renal integrity, quality of life, and survival of SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 12560: Effect of Somah Solution on Hypoxia/Reoxygenation Induced Coronary Artery Endothelial Injury: A Comparison to Current Ex-Vivo Heart Perfusates

Introduction: Ex-Vivo Heart Perfusion (EVHP) has been proposed to expand cardiac donation by enabling resuscitation and preservation of hearts donated after circulatory death. Hypoxia/reoxygenation endothelial injury affects organ viability and patient’s outcomes. STEEN Solution is accepted as the primary component for perfusion of other solid organs. However, there is lack of evidence to determine the optimal perfusate composition to maintain cardiac viability during longer periods of perfusion. Somah solution has been proposed as a novel cardioprotective solution optimized to meet cardiac metabolism and confer enhanced protective effects against reperfusion injury. Our aim was to compare the effects of STEEN and Somah on endothelial reoxygenation injury in vitro. Methods: Human coronary artery endothelial cells were exposed to 16 hours of hypoxia in culture medium followed by 16 hours of reoxygenation treated with Celsior, STEEN or Somah to simulate the EVHP setting. Endothelial activation markers’ gene expression was assessed through qRT-PCR; supernatant was collected to quantify endothelin-1 (ET-1) with ELISA; and quantitative neutrophil adhesion assay was performed to evaluate endothelial function. Results: Treatment with Somah demonstrated decreased expression of cell surface adhesion molecules E-Selectin (Me 0.133±0.12, So -0.073±0.34, ST 1.624±0.46, Ce 0.999±0.45 fold-change from hypoxia; p=0.002) and VCAM-1 (Me -0.004±0.20, So -0.363±0.43, ST 0.428±0.39, Ce 0.597±0.13 fold-change from hypoxia; p=0.023), and vasoactive molecule MCP-1 (Me 0.225±0.72, So -0.510±0.70, ST 1.500±0.49, Ce 0.829±0.28 fold-change from hypoxia; p=0.015). Celsior and Somah reduced cell secretion of ET-1 (Me 395 pg/ml, So 20 pg/ml, ST 448 pg/ml, Ce 16 pg/ml; n=4, p&lt;0.05). Reoxygenation injury increased neutrophil adhesion to endothelial cells; there was a non-statistically significant trend towards protection against this injury with Somah. Conclusions: Somah exerts protective effect against reoxygenation injury when compared to STEEN in vitro. It may have utility in enhancing cardiac organ preservation during EVHP. Future studies in a pre-clinical setting should be conducted to address this question.

BMPRII influences the response of pulmonary microvascular endothelial cells to inflammatory mediators.

Mutations in the bone morphogenetic protein receptor (BMPR2) gene have been observed in 70% of patients with heritable pulmonary arterial hypertension (HPAH) and in 11-40% with idiopathic PAH (IPAH). However, carriers of a BMPR2 mutation have only 20% risk of developing PAH. Since inflammatory mediators are increased and predict survival in PAH, they could act as a second hit inducing the development of pulmonary hypertension in BMPR2 mutation carriers. Our specific aim was to determine whether inflammatory mediators could contribute to pulmonary vascular cell dysfunction in PAH patients with and without a BMPR2 mutation. Pulmonary microvascular endothelial cells (PMEC) and arterial smooth muscle cells (PASMC) were isolated from lung parenchyma of transplanted PAH patients, carriers of a BMPR2 mutation or not, and from lobectomy patients or lung donors. The effects of CRP and TNFα on mitogenic activity, adhesiveness capacity, and expression of adhesion molecules were investigated in PMECs and PASMCs. PMECs from BMPR2 mutation carriers induced an increase in PASMC mitogenic activity; moreover, endothelin-1 secretion by PMECs from carriers was higher than by PMECs from non-carriers. Recruitment of monocytes by PMECs isolated from carriers was higher compared to PMECs from non-carriers and from controls, with an elevated ICAM-1 expression. CRP increased adhesion of monocytes to PMECs in carriers and non-carriers, and TNFα only in carriers. PMEC from BMPR2 mutation carriers have enhanced adhesiveness for monocytes in response to inflammatory mediators, suggesting that BMPR2 mutation could generate susceptibility to an inflammatory insult in PAH.

Inhibitory effect of fentanyl citrate on the release of endothlin-1 induced by bradykinin in melanoma cells.

Our previous study showed that the μ-opioid receptor agonist fentanyl citrate inhibits endothelin-1-and bradykinin-mediated pain responses in mice orthotopically inoculated with melanoma cells. We also demonstrated that bradykinin induces endothelin-1 secretion in melanoma cells. However, the analgesic mechanisms of fentanyl citrate remain unclear. Thus, the present study was conducted to determine whether fentanyl citrate affects bradykinin-induced endothelin-1 secretion in B16-BL6 melanoma cells. The amount of endothelin-1 in the culture medium was measured using an enzyme immunoassay. The expression of endothelin-1, kinin B2 receptors, and μ-opioid receptors in B16-BL/6 melanoma cells was determined using immunocytochemistry. Fentanyl citrate inhibited bradykinin-induced endothelin-1 secretion. The inhibitory effect of fentanyl citrate on the secretion of endothelin-1 was attenuated by the μ-opioid receptor antagonist naloxone methiodide. The immunoreactivities of endothelin-1, kinin B2 receptors, and μ-opioid receptors in B16-BL6 melanoma cells were observed. These results suggest that fentanyl citrate regulates bradykinin-induced endothelin-1 secretion through μ-opioid receptors in melanoma cells.

Role of vascular remodeling markers in the development of osteoporosis in idiopathic pulmonary arterial hypertension

To define the role of circulating biomarkers for the metabolism of collagen and intercellular substance and vascular remodeling in the development of osteoporosis (OP) in idiopathic pulmonary arterial hypertension (IPAH). Functional hemodynamic parameters, bone mineral density (BMD) in the lumbar spine and femoral neck and the serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/TIMP-1 complex, C-terminal telopeptide of collagen type 1 (CITP), and endothelin-1 (ET-1) were determined in 27 high-risk IPAH patients and 30 healthy volunteers. OP in IPAH was detected in 50% of the examinees. The serum levels of CITP, MMP-9, TIMP-1, and ET-1 proved to be higher in the high-risk IPAH patients than in the healthy volunteers. There was a direct correlation between BMD and six-minute walk test and an inverse correlation with total pulmonary vascular resistance (TPVR). Serum TMIP-1 levels correlated with cardiac index and TPVR; ET-1 concentrations were directly related to pulmonary artery systolic pressure, cardiac index, and TPVR. Inverse relationships were found between BMD and circulating CITP, MMP-9, TMIP-1, MMP-9/TMIP-1, and ET-1. At the same time, there was only a tendency towards a positive correlation between serum CITP and ET-1 concentrations. The results of the investigation confirm that endothelin system dysregulation plays a leading role in the development of persistent hemodynamic disorders in high-risk IPAH and suggest that it is involved in the development of osteopenic syndrome. Enhanced ET-1 secretion initiates bone loss possibly via activation of connective tissue matrix destruction.

Nuclear factor-κB is involved in oxyhemoglobin-induced endothelin-1 expression in cerebrovascular muscle cells of the rabbit basilar artery

The present research was designed to investigate whether endothelin-1 (ET-1) secretion can be induced by oxyhemoglobin and whether nuclear factor κB (NF-κB) is involved in the regulation of ET-1 transcription in cerebrovascular muscle cells. Cerebrovascular muscle cells isolated from a rabbit basilar artery were stimulated by oxyhemoglobin (OxyHb) and ET-1 production was increased significantly in the supernatant. Inhibition of NF-κB with pyrrolidine dithiocarbamate and small interfering RNA decreased the expression of ET-1. Nuclear translocation of NF-κB and the degradation of IkB-α was observed with the stimulation of OxyHb. The supernatant obtained from cerebrovascular muscle cells stimulated by OxyHb produced contractions in arterial rings and was blocked by the ET-1 receptor antagonist (BQ-123). The time course of the OxyHb-induced contractions of the basilar artery rings correlated with the time course of the OxyHb-induced ET-1 secretion. The contraction of the basilar artery rings induced by OxyHb was attenuated when the artery rings were preincubated with pyrrolidine dithiocarbamate and SN50 (20 and 10 µM, respectively). These results indicate that cerebrovascular muscle cells may be an important source of ET-1 production after subarachnoid hemorrhage. NF-κB was involved in the expression of ET-1 and the inhibition of the NF-κB pathway may be beneficial for the treatment of cerebral vasospasm.

Andrographolide inhibits hypoxia-induced HIF-1α-driven endothelin 1 secretion by activating Nrf2/HO-1 and promoting the expression of prolyl hydroxylases 2/3 in human endothelial cells.

Andrographolide, the main bioactive component of the medicinal plant Andrographis paniculata, has been shown to possess potent anti-inflammatory activity. Endothelin 1 (ET-1), a potent vasoconstrictor peptide produced by vascular endothelial cells, displays proinflammatory property. Hypoxia-inducible factor 1α (HIF-1α), the regulatory member of the transcription factor heterodimer HIF-1α/β, is one of the most important molecules that responds to hypoxia. Changes in cellular HIF-1α protein level are the result of altered gene transcription and protein stability, with the latter being dependent on prolyl hydroxylases (PHDs). In this study, inhibition of pro-inflammatory ET-1 expression and changes of HIF-1α gene transcription and protein stability under hypoxia by andrographolide in EA.hy926 endothelial-like cells were investigated. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl2. We found that hypoxia stimulated the production of reactive oxygen species (ROS), the expression of HIF-1α mRNA and protein, and the expression and secretion of ET-1. These effects, however, were attenuated by co-exposure to andrographolide, bilirubin, and RuCO. Silencing Nrf2 and heme oxygenase 1 (HO-1) reversed the inhibitory effects of andrographolide on hypxoia-induced HIF-1α mRNA and protein expression. Moreover, andrographolide increased the expression of prolyl hydroxylases (PHD) 2/3, which hydroxylate HIF-1α and promotes HIF-1α proteasome degradation, with an increase in HIF-1α hydroxylation was noted under hypoxia. Inhibition of p38 MAPK abrogated the hypoxia-induced increases in HIF-1α mRNA and protein expression as well as ET-1 mRNA expression and secretion. Taken together, these results suggest that andrographolide suppresses hypoxia-induced pro-inflammatory ET-1 expression by activating Nrf2/HO-1, inhibiting p38 MAPK signaling, and promoting PHD2/3 expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 918-930, 2017.

Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch

TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of “forefront” signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.

CTRP9 Ameliorates Pulmonary Arterial Hypertension Through Attenuating Inflammation and Improving Endothelial Cell Survival and Function.

Endothelial dysfunction and inflammation are believed to be 2 primary instigators of pulmonary arterial hypertension (PH). C1q/TNF-related protein 9 (CTRP9) plays important roles in anti-inflammation and improvement of epithelial function. However, the role of CTRP9 in the progression of PH remains still unclear. In this study, the role and mechanism of CTRP9 in the PH progression were explored. First, serum CTRP9 contents and CTRP9 mRNA expression in the pulmonary artery epithelial cells from patients with PH were detected. Our data on enzyme-linked immunosorbent assay and real-time quantitative Polymerase Chain Reaction showed that CTRP9 mRNA and protein content were markedly downregulated in the patients with PH. Then the pcDNA-CTRP9 expression vector or CTRP9 siRNA was transfected into the primary pulmonary artery epithelial cells from the patients with PH in vitro. CTRP9 overexpression significantly improved endothelial NOS protein expression and reduced the secretion of endothelin-1 (ET-1) and matrix metalloproteinase-2 (MMP-2), whereas knockdown of CTRP9 sharply reduced eNOS protein expression and promoted the secretion of ET-1 and MMP-2 in the cultured human epithelial cells. Moreover, the levels of phosphatidylinositol 3-kinase (PI3K) and pAkt were reduced in the epithelial cells and CTRP9 overexpression activated the PI3K/Akt pathway. CTRP9 could inhibit cell apoptosis and eNOS expression reduction in the cells pretreated with the PI3K/Akt inhibitor LY294002 and resist LY294002-induced ET-1 and MMP-2 secretion. Finally, to verify the role of CTRP9 in the progression of PH in vivo, the pcDNA-CTRP9 expression vector or CTRP9 siRNA was intravenously injected into rats with PH. Pulmonary arterial pressures of the rats were notably reduced by the pcDNA-CTRP9 injection and elevated by the CTRP9 siRNA injection. In conclusion, CTRP9 ameliorated PH by attenuating inflammation and improving endothelial cell survival and function.