Endothelin-1 Secretion Research Articles

Carnosine: Potential aid for diabetes and cardiovascular disease.

In 2015 more than 250 scientific papers published in international journals analyzed the potential beneficial effects of oral administration of carnosine in a wide range of diseases. There is strong evidence that demonstrates the ability of carnosine to improve cellular metabolism and muscle performance. Carnosine is a powerful antioxidant, acts as a buffer system of pH slowing muscular acidosis, and increases the release of nitric oxide enhancing endothelial function (1). The interest around this dipetide (β-alanyl-l-histidine) present in large amounts in skeletal muscle is derived from the demonstration that in humans the oral administration of carnosine increases exercise tolerance and functional capacity in both healthy subjects and recently in patients with heart failure and depression (2, 3). Animal studies have suggested that oral supplementation of carnosine can prevent diabetes and non-cardiovascular complications associated with this disease such as diabetes-induced nephropathy and ocular diseases (4, 5). There is a strong relationship between cardiovascular disease and diabetes (5). The identification of novel treatments for diabetes and the control of risk factors for obesity, metabolic syndrome, and sedentary lifestyle are still the objectives of the research. The article by de Courten et al. (6) in this issue examines for the first time the effect of oral administration of carnosine in overweight subjects randomized to receive 2 mg per day of carnosine or placebo for 3 months. The results show carnosine's ability to hamper insulin resistance and associated fasting hyperinsulinemia. In addition, carnosine supplementation normalized 2-h glucose and decreased 2-h insulin levels in individuals with impaired glucose tolerance. Although this study was methodologically well conducted, there are some important limitations: 1) the number of patients is limited and the cardiovascular risk profile was rather low because there were no patients with hypertension and lipid profile was normal; 2) the authors used indirect methods to calculate the plasma levels of insulin and its release; 3) it is unclear whether any changes in the diet were able to alter the impact of carnosine in the final results. Despite these observations, this preliminary data confirm the results of many studies that attribute carnosine's ability to prevent the formation of advanced lipoxidation end-products (ALEs) and advanced glycoxidation end-products (AGEs) (1). These molecules are associated with cell aging and are directly involved in the processes that induce hyperglycemia and subsequently diabetes; they have also been associated with the early onset of microvascular complications typical of diabetes. The mechanisms by which AGEs alter cell activity by stimulating atherosclerosis and diabetes include: glycation of intracellular proteins, activation through specific receptor (RAGE) that increases the secretion of endothelin-1, vascular endothelial growth factor (VEGF), and numerous cytokines. Hipkiss et al. have widely documented the ability of carnosine to inhibit the formation of AGEs and ALEs (7). Carnosine has also showed the ability to regulate the autonomic nervous system by preserving or increasing β-cells within the pancreas. This mechanism is responsible for the increase in pancreatic insulin secretion and the subsequent reduction of release of glucagon. The study by de Courten et al. (6) suggests the use of carnosine proved free of side effects as a potential treatment for the reduction of hyperglycemia and hyperinsulinemia in individuals at risk of developing diabetes. The authors are to be commended for their study which adds another piece to the puzzle of the complex relationships among carnosine and metabolism. These data in sedentary individuals with overweight and obesity still require replication in other large studies before carnosine supplementation can become part of the standard medical approach for the metabolic syndrome.

Low-Level Blast Exposure Increases Transient Receptor Potential Vanilloid 1 (TRPV1) Expression in the Rat Cornea

ABSTRACTBackground: Blast-related ocular injuries sustained by military personnel have led to rigorous efforts to elucidate the effects of blast exposure on neurosensory function. Recent studies have provided some insight into cognitive and visual deficits sustained following blast exposure; however, limited data are available on the effects of blast on pain and inflammatory processes. Investigation of these secondary effects of blast exposure is necessary to fully comprehend the complex pathophysiology of blast-related injuries. The overall purpose of this study is to determine the effects of single and repeated blast exposure on pain and inflammatory mediators in ocular tissues.Methods: A compressed air shock tube was used to deliver a single or repeated blast (68.0 ± 2.7 kPa) to anesthetized rats daily for 5 days. Immunohistochemistry was performed on ocular tissues to determine the expression of the transient receptor potential vanilloid 1 (TRPV1) channel, calcitonin gene-related peptide (CGRP), substance P (SP), and endothelin-1 (ET-1) following single and repeated blast exposure. Neutrophil infiltration and myeloperoxidase (MPO) expression were also assessed in blast tissues via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis, respectively.Results: TRPV1 expression was increased in rat corneas exposed to both single and repeated blast. Increased secretion of CGRP, SP, and ET-1 was also detected in rat corneas as compared to control. Moreover, repeated blast exposure resulted in neutrophil infiltration in the cornea and stromal layer as compared to control animals.Conclusion: Single and repeated blast exposure resulted in increased expression of TRPV1, CGRP, SP, and ET-1 as well as neutrophil infiltration. Collectively, these findings provide novel insight into the activation of pain and inflammation signaling mediators following blast exposure.

Therapeutic potential of endothelin inhibitors in canine hemangiosarcoma

AimsHemangiosarcoma (HSA) that originates from vascular endothelial cells is the most common splenic malignant neoplasm in dogs, as it accounts for approximately 20% of all canine soft tissue sarcomas. In this study, inhibitory effects of endothelin receptor antagonists on the growth of HSA cells were examined using cell lines established from canine HSA. Main methodsThe preproendothelin-1 (PPET-1), endothelin type A receptor (ETA) and endothelin type B receptor (ETB) mRNA expression levels in HSA cell lines (n=5) were analyzed quantitatively by real-time RT-PCR. These levels were compared with those in HSA tissues (n=11) and those in normal splenic tissues (n=6). ETA and ETB protein expression was examined by western blot. The production and secretion of endothelin-1 (ET-1) and big ET-1 by cell lines were analyzed by measuring the levels in the culture medium by ELISA. The inhibitory effects of endothelin receptor antagonists (ambrisentan, BQ788 and bosentan) on cell growth were evaluated by WST-8 assay. Key findingsThe PPET1 and ETA mRNA expression levels were elevated in HSA tissues and HSA cell lines compared with normal tissues. In cell lines, the production of ET-1 and big ET-1 peptide as well as the expression of ETA protein were detected, but the levels of ETB were not measured. Ambrisentan and bosentan inhibited growth activity in cell lines. Ambrisentan was more effective than bosentan. SignificanceThese findings demonstrate the importance of the ETA axis in canine HSA as well as the potential of ETA inhibitors in the treatment of canine HSA.

TIMAP-protein phosphatase 1-complex controls endothelin-1 production via ECE-1 dephosphorylation

Endothelin induced signaling pathways can affect blood pressure and vascular tone, but the influence of endothelins on tumor cells is also significant. We have detected elevated endothelin-1 secretion from TIMAP (TGF-β inhibited membrane associated protein) depleted vascular endothelial cells. The autocrine signaling activated by the elevated endothelin-1 level through the ETB receptors evoked an angiogenic-like phenotype, the cells assumed an elongated morphology, and enhanced tube formation and wound healing abilities. The depleted protein, TIMAP, is a highly specific and abundant protein in the endothelial cells, and it is a regulatory/targeting subunit for the catalytic subunit of protein phosphatase 1 (PP1c). Protein-protein interaction between the TIMAP-PP1c complex and the endothelin converting enzyme-1 (ECE-1) was detected, the latter of which is a transmembrane protein that produces the biologically active 21-amino acid form of endothelin-1 from proendothelin. The results indicate that silencing of TIMAP induces a reduction in TIMAP-PP1c activity connected to ECE-1. This leads to an increase in the amount of ECE-1 protein in the plasma membrane and a consequent increase in endothelin-1 secretion. Similarly, activation of PKC, the kinase responsible for ECE-1 phosphorylation increased ECE-1 protein level in the membrane fraction of the endothelial cells. The elevated ECE-1 level was mitigated in time in normal cells, but was clearly preserved in TIMAP-depleted cells. Overall, our results indicate that PKC-phosphorylated ECE-1 is a TIMAP-PP1c substrate and this phosphatase complex has an important role in endothelin-1 production of EC through the regulation of ECE-1 activity.

Endothelin regulates intermittent hypoxia-induced lipolytic remodelling of adipose tissue and phosphorylation of hormone-sensitive lipase.

Obstructive sleep apnoea syndrome is characterized by repetitive episodes of upper airway collapse during sleep resulting in chronic intermittent hypoxia (IH). Obstructive sleep apnoea syndrome, through IH, promotes cardiovascular and metabolic disorders. Endothelin-1 (ET-1) secretion is upregulated by IH, and is able to modulate adipocyte metabolism. Therefore, the present study aimed to characterize the role of ET-1 in the metabolic consequences of IH on adipose tissue in vivo and in vitro. Wistar rats were submitted to 14 days of IH-cycles (30 s of 21% FiO2 and 30 s of 5% FiO2 ; 8 h day(-1) ) or normoxia (air-air cycles) and were treated or not with bosentan, a dual type A and B endothelin receptor (ETA-R and ETB-R) antagonist. Bosentan treatment decreased plasma free fatty acid and triglyceride levels, and inhibited IH-induced lipolysis in adipose tissue. Moreover, IH induced a 2-fold increase in ET-1 transcription and ETA-R expression in adipose tissue that was reversed by bosentan. In 3T3-L1 adipocytes, ET-1 upregulated its own and its ETA-R transcription and this effect was abolished by bosentan. Moreover, ET-1 induced glycerol release and inhibited insulin-induced glucose uptake. Bosentan and BQ123 inhibited these effects. Bosentan also reversed the ET-1-induced phosphorylation of hormone-sensitive lipase (HSL) on Ser(660) . Finally, ET-1-induced lipolysis and HSL phosphorylation were also observed under hypoxia. Altogether, these data suggest that ET-1 is involved in IH-induced lipolysis in Wistar rats, and that upregulation of ET-1 production and ETA-R expression by ET-1 itself under IH could amplify its effects. Moreover, ET-1-induced lipolysis could be mediated through ETA-R and activation of HSL by Ser(660) phosphorylation.

ETA and ETB receptors contribute to neuropeptide Y-induced secretion of endothelin-1 in right but not left human ventricular endocardial endothelial cells

Our recent work showed that neuropeptide Y-induced secretion of endothelin-1 (ET-1) in left and right human ventricular endocardial endothelial cells (hLEECs or hREECs respectively) via the activation of neuropeptide Y2 or Y5 receptors depending on the cell type. The aim of this study was to verify whether hLEECs or hREECs secretion of ET-1 induced by NPY is due, in part, to the activation of ETA and/or ETB receptors by the secreted ET-1. Using the technique of indirect immunofluorescence coupled to real 3-D confocal microscopy, as well as ELISA, our results show that in hREECs, the NPY-induced release of ET-1 seems to be due, in part, to the activation of both ETA and ETB receptors. On the other hand, in hLEECs, ETA and ETB receptors do not contribute to the ET-1 released by NPY. Therefore, our results suggest that the NPY-induced release of ET-1 in EECRs is due to NPY receptor activation and the subsequent activation of the ETA and ETB receptors by the released ET-1. However, the release of ET-1 by NPY in hLEECs is mainly due to NPY receptor activation. Furthermore, this secretory process of ET-1 is different between the right and left ventricular cells and highlights the important tuning roles that right and left ventricular EECs possess as well as their contribution to the physiological and pathophysiological states of the underlying heart muscle.

Two angiotensin-converting enzyme-inhibitory peptides from almond protein and the protective action on vascular endothelial function.

This study aimed to discover and prepare novel angiotensin converting enzyme (ACE) inhibitory peptides from almond protein and further evaluate the effect on endothelial function of human umbilical vascular endothelial cells (HUVECs). Almond protein was hydrolyzed using a two-stage alcalase-protamex hydrolysis process, and the hydrolysates were subjected to a series of separations, ultrafiltration, gel filtration chromatography, and reversed-phased preparative chromatography, to obtain the active peptides. Seven ACE inhibitory fractions with the molecular weight below 1.5 kDa were isolated and prepared, and two purified ACE inhibitory peptides with the IC50 values of 67.52 ± 0.05 and 43.18 ± 0.07 μg mL(-1), were identified as Met-His-Thr-Asp-Asp and Gln-His-Thr-Asp-Asp, respectively. Then the effect of two ACE inhibitory peptides on the endothelial function of HUVECs was evaluated. Results showed that the two potent ACE inhibitory peptides significantly regulated the release of nitric oxide and endothelin in HUVECs. These results suggest that almond peptides have potential as an antihypertensive nutraceuticals or a functional food ingredient.

Hypertension in an Animal Model of HELLP Syndrome is Associated With Activation of Endothelin 1

Women with hypertensive forms of pregnancy such as hemolysis-elevated liver enzymes-low platelet syndrome have increased circulating endothelin 1; however, the relationship between hypertension and endothelin 1 has not been studied. Using an animal model, we sought to determine whether there was an increased activation/dysfunction of endothelin 1, the effect of endothelin 1 receptor-A blockade on hypertension and other manifestations of hemolysis, elevated liver enzymes, and low platelets syndrome. On gestational day 12, timed-pregnant rats were infused with soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEndoglin; 4.7 and 7 µg/kg) via mini-osmotic pumps for 8 days. A subset of rats were treated with receptor-A antagonist (ABT-627, 5mg/kg) for 8 days. Rats with hemolysis-elevated liver enzymes-low platelet syndrome had significantly increased hypertension (P = .0001), circulating endothelin 1 (P = .03), and a significant 3.3- and 7.2-fold increase in preproendothelin messenger RNA (mRNA) expression in the placenta and liver (P = .01 and .04). Urinary protein:creatinine ratio was significantly increased in these animals (P = .0007), and circulating factors from these rats stimulated a significant increase in endothelial cell secretion of endothelin 1 (P = .001) in an in vitro assay. Blockade of the endothelin 1 receptor A significantly decreased hypertension (P = .001), circulating endothelin 1, and interleukin 17 (P = .004 and .003), placental preproendothelin mRNA expression (P = .016), and urinary protein:creatinine ratio (P = .007) in rats with hemolysis-elevated liver enzymes-low platelet syndrome. Blockade of the endothelin 1 receptor A significantly decreased hemolysis (P = .009), liver enzymes (P = .011), and significantly increased platelet levels (P = .03) and decreased circulating CD4+ and CD8+ T lymphocytes (P = .0004 and .0001) in rats infused with sFlt-1 and sEndoglin. These data support the hypothesis that endothelin 1 activation has a critical role in pathophysiology of as hemolysis-elevated liver enzymes-low platelet syndrome.

CAMP induction by ouabain promotes endothelin-1 secretion via MAPK/ERK signaling in beating rabbit atria.

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na+-K+-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 µmol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 µmol/L), an inhibitor for reverse mode of Na+-Ca2+ exchangers (NCX), but did not by L-type Ca2+ channel blocker nifedipine (1.0 µmol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 µmol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 µmol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 µmol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 µmol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

Effect of Sancaijiangtang on plasma nitric oxide and endothelin-1 levels in patients with type 2 diabetes mellitus and vascular dementia: a single-blind randomized controlled trial

ObjectiveTo observe the effect of Sancaijiangtang powders on plasma nitric oxide and endothelin-1 levels. We sought to identify the common pathological link and mechanism of action for Traditional Chinese medicine in type 2 diabetes mellitus and vascular dementia, and to explicate the material basis for treating the different diseases with the same method in Traditional Chinese Medicine. MethodsIn total, 168 patients with type 2 diabetes mellitus and vascular dementia were enrolled in the study, and randomly divided into two groups by simple randomization. Patients in the treatment group received oral Sancaijiangtang powders with pioglitazone hydrochloride three times daily, while patients in the control group received pioglitazone hydrochloride alone. The treatment course was for 12 weeks. Mini-mental state examinations (Chinese version) and Montreal Cognitive Assessments (Beijing version) were performed, and fasting plasma glucose, fasting insulin, hemoglobin A1c, homeostasis model assessment of insulin resistance, plasma nitric oxide and endothelin-1 levels were measured before and after the treatment. ResultsThe post-treatment levels for all measurements in both groups were better than pre-treatment levels (P < 0.05). The post-treatment levels for all measurements in the treatment group were better than the levels measured in the control group (P < 0.05). ConclusionType 2 diabetes mellitus and vascular dementia have common pathological mechanisms for insulin resistance and endothelium dysfunction. Sancaijiangtang powders could improve the release of nitric oxide and inhibit the secretion of endothelin-1. Therefore, the material basis exists for treating the different diseases with the same method in Traditional Chinese Medicine.

Protective Effects of Infliximab on Lung Injury Induced by Methotrexate

IntroductionMethotrexate (MTX) is used to treat cancers, several forms of arthritis and other rheumatic conditions, although MTX may cause pulmonary toxicity related to the production of free oxygen radicals, various cytokines. Infliximab (IB) with its potent effect on tumor necrosis factor-alpha (TNF-α) inhibition also inhibits the release of endothelin-1 (ET-1). We aimed to investigate whether IB reduces pulmonary damage induced by an overdose of MTX. MethodThe rats were divided into 3 groups of 8 animals. The control group was given only saline. One dose of 20mg/kg MTX intraperitoneal was administered in the MTX group. IB 7mg/kg was given to the MTX+IB (MI) group. Three days after IB was administered, 20mg/kg MTX was given. Five days after MTX was administered, all rats were sacrificed. ResultsThe TNF-α, ET-1, malondialdehyde (MDA), myeloperoxidase (MPO) and caspase-3 levels in MTX group were significantly higher than in control groups of TNF-α (P=.001), ET-1 (P=.001), MDA (P=.001), MPO (P=.001) and caspase-3 levels (P=.001) and MI groups of TNF-α (P=.009), ET-1 (P=.001), MDA (P=.047), MPO (P=.007) and caspase-3 levels (P=.003). The MI group had less histopathological damage in lung tissue than the MTX group. ConclusionOverdose of MTX leads to cytokine release and the formation of reactive oxygen species in addition to increased ET-1 secretion release that causes lung damage. IB, as a potent proinflammatory agent, TNF-α blocker, can decrease ET-1 release and oxidative stress, it may show significant protective effects in lung tissue against damage caused by MTX overdose.

Efectos protectores de infliximab sobre el daño pulmonar inducido por metotrexato

IntroductionMethotrexate (MTX) is used to treat cancers, several forms of arthritis and other rheumatic conditions, although MTX may cause pulmonary toxicity related to the production of free oxygen radicals, various cytokines. Infliximab (IB) with its potent effect on tumor necrosis factor-alpha (TNF-α) inhibition also inhibits the release of endothelin-1 (ET-1). We aimed to investigate whether IB reduces pulmonary damage induced by an overdose of MTX. MethodThe rats were divided into 3 groups of 8 animals. The control group was given only saline. One dose of 20mg/kg MTX intraperitoneal was administered in the MTX group. IB 7mg/kg was given to the MTX+IB (MI) group. Three days after IB was administered, 20mg/kg MTX was given. Five days after MTX was administered, all rats were sacrificed. ResultsThe TNF-α, ET-1, malondialdehyde (MDA), myeloperoxidase (MPO) and caspase-3 levels in MTX group were significantly higher than in control groups of TNF-α (P=.001), ET-1 (P=.001), MDA (P=.001), MPO (P=.001) and caspase-3 levels (P=.001) and MI groups of TNF-α (P=.009), ET-1 (P=.001), MDA (P=.047), MPO (P=.007) and caspase-3 levels (P=.003). The MI group had less histopathological damage in lung tissue than the MTX group. ConclusionOverdose of MTX leads to cytokine release and the formation of reactive oxygen species in addition to increased ET-1 secretion release that causes lung damage. IB, as a potent proinflammatory agent, TNF-α blocker, can decrease ET-1 release and oxidative stress, it may show significant protective effects in lung tissue against damage caused by MTX overdose.

Lycopene inhibits cyclic strain‐induced endothelin‐1 expression through the suppression of reactive oxygen species generation and induction of heme oxygenase‐1 in human umbilical vein endothelial cells

Lycopene is the most potent active antioxidant among the major carotenoids, and its use has been associated with a reduced risk for cardiovascular disease (CVD). Endothelin-1 (ET-1) is a powerful vasopressor synthesized by endothelial cells and plays a crucial role in the pathophysiology of CVD. However, the direct effects of lycopene on vascular endothelial cells have not been fully described. This study investigated the effects of lycopene on cyclic strain-induced ET-1 gene expression in human umbilical vein endothelial cells (HUVECs) and identified the signal transduction pathways that are involved in this process. Cultured HUVECs were exposed to cyclic strain (20% in length, 1Hz) in the presence or absence of lycopene. Lycopene inhibited strain-induced ET-1 expression through the suppression of reactive oxygen species (ROS) generation through attenuation of p22(phox) mRNA expression and NAD(P)H oxidase activity. Furthermore, lycopene inhibited strain-induced ET-1 secretion by reducing ROS-mediated extrace-llular signal-regulated kinase (ERK) phosphorylation. Conversely, lycopene treatment enhanced heme oxygenase-1 (HO-1) gene expression through the activation of phosphoinositide 3-kinase (PI3K)/Akt pathway, followed by induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation; in addition, HO-1 silencing partially inhibited the repressive effects of lycopene on strain-induced ET-1 expression. In summary, our study showed, for the first time, that lycopene inhibits cyclic strain-induced ET-1 gene expression through the suppression of ROS generation and induction of HO-1 in HUVECs. Therefore, this study provides new valuable insight into the molecular pathways that may contribute to the proposed beneficial effects of lycopene on the cardiovascular system.

Resveratrol inhibits inflammation and ameliorates insulin resistant endothelial dysfunction via regulation of AMP‐activated protein kinase and sirtuin 1 activities

Resveratrol is a phytoalexin with beneficial effects on human health. The aim of the present study was to investigate the effects of resveratrol on endothelial dysfunction involved in insulin signaling and inflammation. Endothelial cells were stimulated with palmitate (PA) to induce insulin resistance characterized by a loss of insulin-mediated nitric oxide (NO) production. Diabetes was induced in rats by fructose feeding. The effects of resveratrol and the mechanisms involved were investigated using an aortic relaxation assay and Western blot analysis. In endothelial cells, 0.1-10 μmol/L resveratrol suppressed IκB kinase β (IKKβ)/nuclear factor-κB phosphorylation, as well as tumor necrosis factor-α and interleukin-6 production, and restored the insulin receptor substrate-1 (Irs-1)/Akt/endothelial NO synthase signaling pathway. Furthermore, resveratrol effectively inhibited the mitogenic actions of insulin by decreasing the secretion of endothelin-1 and plasminogen activator inhibitor-1. It also positively regulated AMP-activated kinase (AMPK) and sirtuin 1 (SIRT1) activation, which contributed to the inhibition of inflammation implicated in endothelial insulin resistance. Stimulation with PA and long term-fructose feeding impaired insulin-mediated vessel dilation in rat aorta, whereas pretreatment of aortic rings with resveratrol (0.1-10 μmol/L) or treatment of rats with 5 or 20 mg/kg resveratrol counteracted these changes. The results indicate that resveratrol inhibits inflammation and facilitates insulin phosphatidylinositol 3-kinase signaling by beneficial modulation of IRS-1 function partly via regulation of AMPK and SIRT1 activity in the endothelium.

Agonistic Autoantibodies to the Angiotensin II Type 1 Receptor Enhance ANGII Binding on Vascular Endothelial Cells

Preeclamptic women produce agonistic autoantibodies to the Angiotensin II type 1 receptor (AT1-AA) and exhibit increased sensitivity to angiotensin II (ANG II). Together, AT1-AA and ANGII increased blood pressure, endothelin-1 (ET-1), oxidative stress in pregnant rats. However, cellular mechanisms for such responses remain unknown. Our objective in this study was to examine the effect of AT1-AA on ANGII binding to the AT1 receptor on human vascular umbilical endothelial cells (HUVEC) and the subsequent release of ET-1. Methods: HUVECs were incubated for 1 hour with FITC labelled ANGII (10nM, 50nM, 100nM and 200nM) with or without AT1-AA (1:100), losartan [LOS (10µM)], or a specific inhibitor of AT1-AAs [7AA(5µg/ml)]. ANGII binding was determined by FITC labeled ANGII florescence at 490nm excitation and 522nm emission wavelengths and ET-1 was measured by ELISA. Results: At 1 hour incubation, bound ANG II to AT1 R was approximately 1.0+/-0.11 and 5.2±1.3 pmols/mg at 100 and 200 nM concentrations, which drastically increased with the AT1-AA to 20+/-5 and 65+/-3, and was decreased with AT1-AA inhibitory peptide (40 +/-3 at the 200nM ANGII) and losartan. Interestingly, AT1-AA induced a 2-fold increase in ET-1 above that with ANGII alone at physiological doses of 10nM and 50nM, which sharply declined at higher doses of ANGII (100, 200 & 500nM). Conclusion AT1-AA greatly increases the binding affinity of ANGII for the AT1 R on HUVECs and secretion of ET-1 at physiological ANGII concentrations supporting a role for the AT1-AA to enhance ANGII effects during preeclampsia.

Возможности коррекции дисфункции эндотелия на фоне комбинированной антигипертензивной терапии у больных артериальной гипертонией с сахарным диабетом 2-го типа

To evaluate the impact of a 12-week combined antihypertensive therapy with enalapril + indapamide on endothelial dysfunction in patients with arterial hypertension (AH) and diabetes mellitus (DM) type 2.30 patients with AH stage II-III and DM type 2 age from 40 to 65 years were included into research. The combined antihypertensive therapy with enalapril 28.6 ± 1.9 + indapamide 2.5 ± 0 mg/day was assigned for 12 weeks. We studied endothelial function by determining the concentration of NO metabolites and endothelin-1 in serum and urine, the results of occlusion test.After 12-week therapy, 100% of the patients achieved BP goals. There was no impairment of carbohydrate metabolism. Endothelial function was improved in hypertensive patients with T2DM: there were increases in both serum and urinary NO production (by 381 and 48.2%, respectively) and decreases in urinary endothelin-1 secretion (by 56.3%). The number of patients with normal microcirculation increased from 13.3 to 53.3% (p < 0.001) by reducing the number of patients with abnormal (hyperemic and stagnant-stasic) types of microcirculation.Twelve-week treatment with the combined antihypertensive medication. The combined antihypertensive therapy with enalapril + indapamide is highly effective and safe for recovering endothelial function in hypertensive patients with T2DM.

Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling

BackgroundParacrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. ObjectiveTo resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. Methods and ResultsRT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes. ConclusionsThese results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A).

Substance P Stimulates Endothelin 1 Secretion via Endothelin-Converting Enzyme 1 and Promotes Melanogenesis in Human Melanocytes

Substance P (SP) is a well-known neuropeptide implicated in the wound-healing process. The wound occasionally causes a pigmented scar. In the present study, we examined whether increased levels of SP affected melanogenesis. When human melanocytes were treated with SP, the melanin content increased and the pigmentation process accelerated in a dose-dependent manner. In addition to melanogenesis-related genes, the expression of neurokinin 1 receptor, endothelin 1 (EDN1), and EDN receptor type B (EDNRB) also increased at both the messenger RNA and protein levels. Interestingly, secreted EDN1 was observed in the melanocyte culture medium, and this phenomenon was significantly enhanced by SP treatment. Through knockdown experiments using small interfering RNAs (siRNAs), we confirmed that endothelin-converting enzyme 1 (ECE1), EDN1, and EDNRB were involved in SP-induced pigmentation and found that EDN1 secretion was affected by ECE1 and EDN1 siRNAs, but not by EDNRB siRNA. These findings indicate that ECE1 is essential for EDN1 secretion in melanocytes and that EDNRB functions downstream of secreted EDN1 to increase the cAMP levels and activate the melanogenesis-related phosphorylation cascade. This study provides in vitro evidence for a melanogenic function of SP in the skin and suggests that the SP-related signal is a potent target for regulating stress- or wound-induced pigmentation.

High glucose induces the release of endothelin-1 through the inhibition of hydrogen sulfide production in HUVECs.

Hydrogen sulfide (H(2)S) has recently been identified as an endogenous gaseous signaling molecule. In the vascular system, the formation of H(2)S is catalyzed by cystathionine γ‑lyase (CSE). Previous studies have demonstrated the protective effects of H(2)S on ischemic injury in various types of tissue. However,, little is known about the role of H(2)S in diabetes-associated vascular diseases. Thus, the aim of the present study was to examine the possible role of H(2)S in high glucose-induced vascular dysfunction, and to explore the underlying mechanisms. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical veins. The levels of H(2)S following treatment with various levels of glucose were determined and the secretion of endothelin-1 (ET-1) was measured by ELISA. The mRNA and protein expression of CSE in the HUVECs was determined by real-time RT-PCR and western blot analysis, respectively. Treatment with high glucose (25 mmol/l) for 48 h significantly increased the secretion of ET-1 by HUVECs, with the concomitant suppression of H(2)S production and CSE protein expression. The increase in exogenous H(2)S levels through the administration of sodium hydrosulfide (NaHS) attenuated the high glucose-induced downregulation of CSE protein expression, and significantly inhibited the secretion of ET-1. These results suggest that the downregulation of CSE protein expression and the subsequent decrease in H(2)S production play a role in high glucose-induced vascular dysfunction possibly by increasing the secretion of ET-1 by endothelial cells.

ASSA14-14-06 Expressions of endothelin-1 in primary cultured atrial myocytes after rapid electrical-field stimulation

<h3>Background</h3> To study the expressions of endothelin-1 at early stages of atrial fibrillation in a rapid paced primary cultured atrial myocyte model. <h3>Methods</h3> Primary rat atrial myocytes were cultured and a rapid paced cell model was established. The polymerase chain reaction (RT-PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) were applied to detect the messenger ribonucleic acid (mRNA) expression and secretion of endothelin-1. <h3>Results</h3> mRNA expression of endothelin-1 increased after 6 h of rapid pacing and continued to elevate in a time dependent manner. Similarly, changes of endothelin-1 secretion were paralleled. <h3>Conclusion</h3> Expressions of endothelin-1 increased in early phase of rapid paced atrial myocytes. It implicated the endothelin-1 was implicated in pathological remodelling of atrial fibrillation.