ET-1 mRNA Research Articles

Endothelin receptor-antagonists suppress lipopolysaccharide-induced cytokine release from alveolar macrophages of non-smokers, smokers and COPD subjects

Smoking-induced COPD is characterized by chronic airway inflammation, which becomes enhanced by bacterial infections resulting in accelerated disease progression called exacerbation. Alveolar macrophages (AM) release endothelin-1 (ET-1), IL-6, CCL-2 and MMP-9, all of which are linked to COPD pathogenesis and exacerbation. ET-1 signals via ETA- and ETB-receptors (ETAR, ETBR). This is blocked by endothelin receptor antagonists (ERAs), like bosentan, which targets both receptors, ETAR-selective ambrisentan and ETBR-specific BQ788. Therefore, ERAs could have anti-inflammatory potential, which might be useful in COPD and other inflammatory lung diseases. We hypothesized that ERAs suppress cytokine release from AM of smokers and COPD subjects induced by lipopolysaccharide (LPS), the most important immunogen of gram-negative bacteria. AM were isolated from the broncho-alveolar lavage (BAL) of n=29 subjects (11 non-smokers, 10 current smokers without COPD, 8 smokers with COPD), cultivated and stimulated with LPS in the presence or absence of ERAs. Cytokines were measured by ELISA. Endothelin receptor expression was investigated by RT-PCR and western blot. AM expressed ETAR and ETBR mRNA, but only ETBR protein was detected. LPS and ET-1 both induced IL-6, CCL-2 and MMP-9. LPS-induced IL-6 release was increased in COPD versus non-smokers and smokers. Bosentan, ambrisentan and BQ788 all partially reduced all cytokines without differences between cohorts. Specific ETBR inhibition was most effective. LPS induced ET-1, which was exclusively blocked by BQ788. In conclusion, LPS induces ET-1 release in AM, which in turn leads to CCL-2, IL-6 and MMP-9 expression rendering AM sensitive for ERAs. ERAs could have anti-inflammatory potential in smoking-induced COPD.

Cardiovascular protective effect of polysaccharide from Ophiopogon japonicus in diabetic rats

Ophiopogon japonicus (Thunb.) Ker-Gawl is a well known traditional Chinese medicine used to treat cardiovascular and chronic inflammatory diseases for thousands of years. The present study was set up to investigate the protective effects of O. japonicus polysaccharide (OJP1) on cardiovascular injuries in diabetic rats. Results showed that OJP1 significantly reduced the MDA concentration and increased the activities of GPx, CAT and SOD in heart of diabetic rats. The levels of AGE, hs-CRP, sICAM-1, NO and ET-1 in diabetic rats were significantly reversed by OJP1 treatment. In addition, the level of ET-1 mRNA was decreased significantly, whereas eNOS mRNA level was increased after administration of OJP1. Meanwhile, the histopathological analysis showed that OJP1 alleviates the heart injury in diabetic rats. Together, these results suggest that OJP1 maintains the antioxidant enzyme levels and improves cardiovascular performance in diabetic rats.

Loss expression of micro ribonucleic acid (miRNA)-200c induces adverse post-surgical prognosis of advanced stage non-small cell lung carcinoma and its potential relationship with ETAR messenger RNA.

BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. As micro ribonucleic acid (miRNA)-200 and ETAR may play an essential role in the process of epithelial to mesenchymal transition (EMT) simultaneously, the purpose of this study was to detect the expression of miRNA-200c and ETAR messenger (m)RNA and assess their prognostic significance in early stage NSCLC.MethodsOur study included 78 advanced stage (IIB, IIIA, IIIB) NSCLC patients. All patients were smokers. Using quantitative reverse transcriptase polymerase chain reaction analysis, we detected the expression of miRNA-200c and ETAR mRNA and assessed their correlation by χ2 test. Time to progression was used as the recurrent index and was assessed by univariate and multivariate analysis in the Cox hazard model.ResultsBoth miRNA-200c and ETAR mRNA expression are associated with N stage and tumor node metastasis (TNM) stage in a series of advanced NSCLC patients. Among N stage and TNM stage patients, significant differences were found in IIB (P = 0.0126), IIIB (P = 0.0107) and N0 (P = 0.0023) and in N1 + N2 groups (P = 0.0133). Using both univariate and multivariate survival analyses, we found that miRNA-200c (hazard ratio [HR] = 0.352, 95% confidence interval [CI]: 0.187–0.662) and ETAR mRNA (HR = 2.500 95% CI: 1.345–4.647) were independent prognostic factors, independent of TNM stage (HR = 2.414, 95% CI: 1.600–3.642) and differentiation (HR = 1.530, 95% CI: 1.050–2230).Conclusions miRNA-200c induces an expedient surgical survival, whereas ETAR mRNA has the reverse prognosis in advanced stage NSCLC patients. A potential relationship exists in that miRNA-200c targets ETAR mRNA during EMT.

The lung endothelin system: a potent therapeutic target with bosentan for the amelioration of lung alterations in a rat model of diabetes mellitus

The aim of this study is to show the effect of a new mechanism on endothelin (ET) receptors in the physiopathology of diabetes-related pulmonary injury. We tested the hypothesis that dual ET-1 receptor antagonism via bosentan can reverse diabetes-induced lung injury. The rats (24 male) were separated into four groups: group 1 (HEALTHY): Control group; group 2 (DM): Streptozotocin 60 mg/kg (i.p.); group 3 (DM + BOS-1): Diabetes + bosentan 50 mg/kg per-os; group 4 (DM + BOS-2): Diabetes + bosentan 100 mg/kg per-os. The bosentan treatment was initiated immediately after the onset of STZ-induced diabetes and continued for 6 weeks. In the treatment group, SOD activity was significantly increased, although GSH and MDA levels and TNF-α and TGF-β gene expression were decreased. Bosentan 50 mg/kg and bosentan 100 mg/kg showed a significantly down-regulatory effect on ET-1, ET-A, and ET-B mRNA expression. In conclusion, increased endothelin levels in the lung associated with diabetes may be one cause of endothelial dysfunction, cytokine increase, and oxidant/antioxidant imbalance in the pathogenesis of complications that may develop during diabetes. With its multiple effects, bosentan therapy may be an effective option against complications that may develop in association with diabetes.

Increased Glomerular ET‐1 in Female Sickle Cell Mice is Abolished by Chronic Hydroxyurea Treatment

Sickle cell disease (SCD) is associated with glomerular nephropathy leading to the chronic kidney disease. Endothelin (ET‐1), a powerful vasoconstrictor and pro‐inflammatory mediator, is increased in SCD patients and recent evidence suggests it can contribute to the development of glomerulopathy and decline in renal function in SCD. Currently, hydroxyurea is the only therapy demonstrating benefits for SCD patients due to its ability to increase fetal hemoglobin. Therefore, the aim of the study was to determine if elevated glomerular ET‐1 mRNA expression in sickle cell mice is reduced by hydroxyurea treatment and if sex differences exist in this response. Experiments utilized homozygous knockout‐transgenic sickle mice (humanized) from our in‐house colony. Hydroxyurea was administrated via drinking water and the concentration adjusted daily according to intake (50mg/kg/day) for 7 days. Glomeruli were isolated for ET‐1 mRNA. Female sickle cell mice had a 3‐fold increase in glomerular ET‐1 mRNA at baseline compared to male (3.07±0.21 vs. 1.07±0.16 fold change, p<0.05). After treatment with hydroxyurea female sickle cell mice had a significant reduction in ET‐1 expression (from 3.07±0.21 to 1.21±0.12 fold change, p<0.05). Hydroxyurea did not significantly alter glomerular ET‐1 production in male sickle cell. These data support the hypothesis that chronic treatment with hydroxyurea can reduce glomerular ET‐1 production, and possible renal pathologies associated with SCD. Further, sex differences in glomerular ET‐1 expression may play an important role in the development of sickle cell nephropathy.

Sex Differences in Renal Inner Medullary ET‐1 Gene Expression Levels with Increasing Medullary Osmolality

We previously reported that exposing the renal medulla to hyperosmotic solutions enhances urinary ET‐1 excretion in male rats. We have also seen that female rats have a more robust renal ET‐1 natriuretic capacity compared to males. Therefore, we determined whether intramedullary infusion of hyperosmotic saline has a differential effect on ET‐1 gene expression in inner medullas from anesthetized male and female rats. Isosmotic saline (284 mosmol/kgH2O) was infused into the renal medullary interstitium (500 μl/h) during a 60 min equilibration period and 30 min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mosmol/kgH2O) for two further 30 min periods. At the end of each experiment, the renal inner medulla was isolated for ET‐1 mRNA assessment using the real‐time quantitative polymerase chain reaction (RT‐PCR) technique. Compared with isosmotic saline, hyperosmotic saline infused in the renal medulla caused significant increases in urine flow in both male and female rats (from 6.3±0.9 to 12.2±1.7 and from 8.2±1.3 to 15.0±2.8 μl/min, respectively). Sodium excretion was also significantly enhanced, in both males and females (from 0.6±0.1 to 8.3±1.9 and from 0.9±0.2 to 5.8±1.0 μl/min, respectively). Inner medullary ET‐1 expression in male rats was significantly increased after 60 min of medullary interstitial infusion of hyperosmotic saline (from 1.0± 0.1 to 1.4± 0.1 fold change, p<0.05). In contrast, ET‐1 expression did not change with medullary hyperosmotic infusion in females. These data indicate that an increase in renal inner medullary ET‐1 could play a more important role in the diuretic and natriuretic response to intramedullary hyperosmolality in male compared to female rats.

The effects of PBDE-209 exposure during pregnancy on placental ET-1 and eNOS expression and the birth weight of offspring

Decabrominated diphenyl ether (PBDE-209) is a persistent organic pollutant. Gestational exposure to PBDE-209 can accumulate in pregnant women and fetuses via the placenta and umbilical cord, affecting perinatal outcome. In this study, pregnant Sprague–Dawley (SD) rats were randomly divided into five groups and intragastrically administered peanut oil (vehicle) 1, 5 and 10mg/kg by body weight (b.w.) of PBDE-209, or nothing (control) from day 0 (G0) to day 21 (G21) gestation, respectively. Placental samples were collected on G21 by cesarean section. The mRNA and protein expressions of ET-1, eNOS and iNOS in the placenta were examined using qRT-PCR and Western blot, respectively. Total nitric oxide (NO) in the placenta was measured using a specific ELISA kit. Compared with the control and vehicle groups, the mRNA expression of ET-1 and iNOS in the placenta was gradually and significantly increased after exposure to increasing concentrations of PBDE-209 (P<0.05), while the mRNA level of eNOS in the placenta was gradually and significantly reduced after exposure to increasing concentrations of PBDE-209 (P<0.05). The expression trends of ET-1, eNOS and iNOS proteins were consistent with those of mRNA expression. Interestingly, the production of total NO was significantly increased after exposure to 5 and 10mg/kg b.w. PBDE-209 (P<0.05). Finally, the birth weight of the offspring rats was significantly reduced after maternal exposure to 5 and 10mg/kg b.w. PBDE-209 compared with the control and vehicle groups (P<0.05). These results suggest that PBDE-209 exposure during pregnancy upregulates ET-1 and iNOS expression, but decreases eNOS expression in the placenta, as well as reduces the birth weight of offspring.

Expression and clinical significance of the HIF-1a/ET-2 signaling pathway during the development and treatment of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a major health problem in reproductive-aged women worldwide, but the precise pathogenesis of PCOS remains unclear. Our previous study revealed that hypoxia-inducible factor (HIF)-1a mediated endothelin (ET)-2 signaling plays an important role in ovulation in rats. Therefore, the present study used a PCOS rat model to test the hypotheses that HIF-1a signaling is expressed and inhibited in ovaries during PCOS formation and that the HIF-1a/ET-2 signaling pathway is a target of dimethyldiguanide (DMBG) in the clinical treatment of PCOS. First, the development of a PCOS model and the effect of DMBG treatment were examined through ovarian histology and serum hormone levels, which were consistent with previous reports. Second, HIF-1a and ET-2 expression were detected by immunohistochemistry and western blot. The results showed decreased HIF-1a/ET-2 expression in the ovaries of PCOS rats, whereas DMBG treatment reversed the protein decreases and improved the PCOS symptoms. Third, to understand the molecular mechanism, HIF-1a/ET-2 mRNA expression was also examined. Interestingly, HIF-1a mRNA increased in the ovaries of PCOS rats, while ET-2 mRNA decreased, indicating that HIF-1a protein degradation may be involved in POCS development and treatment. Finally, HIF prolyl hydroxylase (PHD) activity was examined to further clarify the contribution of HIF-1a signaling to the development and treatment of PCOS. The results suggested that the inhibition of HIF-1a/ET-2 signaling may be caused by increased PHD activity in PCOS. DMBG-treated PCOS may further activate HIF-1a signaling at least partly through inhibiting PHD activity. Taken together, these results indicate that HIF-1a signaling is inhibited in a PCOS rat model through increasing PHD activity. DMBG treatment improved PCOS by rescuing this pathway, suggesting that HIF-1a signaling plays an important role in the development and treatment of PCOS. This HIF-1a-mediated ET-2 signaling pathway may be an important mechanism regulating PCOS formation and treatment in mammalian ovaries in vivo and should be a new clinical target for PCOS prevention and treatment in the future.

Flow regulation of endothelin-1 production in the inner medullary collecting duct.

Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na(+) and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm(2) for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca(2+), chelation of intracellular Ca(2+), or inhibition of Ca(2+) signaling (calmodulin, Ca(2+)/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca(2+) entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca(2+) was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na(+) channels or the mitochondrial Na(+)/Ca(2+) exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca(2+)-dependent signaling pathways that stimulate ET-1 synthesis.

Adaptations of the endothelin system after exercise training in a porcine model of ischemic heart disease.

To the test the hypothesis that exercise training would increase endothelin-mediated vasoconstriction in collateral-dependent arteries via enhanced contribution of ET(A). An ameroid constrictor was surgically placed around the proximal LCX artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomized into sedentary or exercise-training (treadmill; 5 days/week; 14 weeks) groups. Subsequently, arteries (~150 μm diameter) were isolated from collateral-dependent and nonoccluded myocardial regions and studied. Following exercise training, ET-1-mediated contraction was significantly enhanced in collateral-dependent arteries. Exercise training induced a disproportionate increase in the ET(A) contribution to the ET-1 contractile response in collateral-dependent arteries, with negligible contributions by ET(B). In collateral-dependent arteries of sedentary pigs, inhibition of ET(A) or ET(B) did not significantly alter ET-1 contractile responses in collateral-dependent arteries, suggesting compensation by the functionally active receptor. These adaptations occurred without significant changes in ET(A), ET(B), or ECE mRNA levels but with significant exercise-training-induced elevations in endothelin levels in both nonoccluded and collateral-dependent myocardial regions. Taken together, these data reveal differential adaptive responses in collateral-dependent arteries based upon physical activity level. ET(A) and ET(B) appear to compensate for one another to maintain contraction in sedentary pigs, whereas exercise-training favors enhanced contribution of ET(A).

Molecular adaptations in vasoactive systems during acute stroke in salt-induced hypertension

Investigations regarding hypertension and dietary sodium, both factors that influence stroke risk, have previously been limited to using genetically disparate treatment and control groups, namely the stroke-prone, spontaneously hypertensive rat and Wistar-Kyoto rat. In this investigation, we have characterized and compared cerebral vasoactive system adaptations following stroke in genetically identical, salt-induced hypertensive, and normotensive control mice. Briefly, ANP(+/-) (C57BJ/6 × SV129 background) mice were fed chow containing either 0.8% NaCl (NS) or 8.0% NaCl (HS) for 7 weeks. Transient cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Infarct volumes were measured 24-h post-reperfusion and the mRNA expression of five major vasoactive systems was characterized using qPCR. Along with previous publications, our data validate a salt-induced hypertensive state in ANP(+/-) mice fed HS chow as they displayed left ventricular hypertrophy, increased systolic blood pressure, and increased urinary sodium excretion. Following MCAO, mice fed HS exhibited larger infarct volumes than their dietary counterparts. In addition, significant up-regulation in Et-1 and Nos3 mRNA expression in response to salt and stroke suggests implications with increased cerebral damage in this group. In conclusion, our data demonstrate increased cerebral susceptibility to stroke in salt-induced hypertensive mice. More importantly, however, we have characterized a novel method of investigating hypertension and stroke with the use of genetically identical treatment and control groups. This is the first investigation in which genetic confounding variables have been eliminated.

ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus.

This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O₂) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment. These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

Abstract 294: CD4 + T cells Isolated from Preeclamptic Women Produce a Preeclampsia-Like Phenotype in Pregnant Nude-Athymic Rats

Preeclampsia (PE) is the development of hypertension, with proteinuria, during the second half of pregnancy and is associated with an altered immune function, resulting in a chronic state of inflammation, which is believed to play an important role to increase vasoconstrictors such as Endothelin (ET-1) and autoantibodies as well as much of the pathophysiology associated with PE. It has been previously shown that women with PE have an altered CD4 + T cell population compared to normal pregnant women. In addition, Ana Zenclussen’s lab has shown that adoptive transfer of activated T helper 1 like splenocytes increased blood pressure, fetal rejection and cytokines secreted from uterine cells when transferred to normal pregnant mice. However, mechanisms causing the increased blood pressure and renal abnormalities were not further investigated. The purpose of this study was to determine if CD4 + T cells isolated from placentas of PE patients have altered consequences during pregnancy compared to those collected from normal pregnant (NP) women that may further define their role in causing the pathophysiology of PE. 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 first timed 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. MAP was significantly increased from 125±3 mmHg in rats injected with NP CD4 + T cells to 140±9 mmHg in rats injected with PE CD4 + T cells. Circulating inflammatory cytokines IL-6 and TNF-α were measured and found to be 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). Renal cortical ET-1 mRNA expression was found to be increased 4.5 fold in rats exposed to PE CD4+ T cells compared to those exposed to NP CD4+ T cells (p=0.06). These data suggest an important role for placental CD4 + T cells stimulated in women with PE to cause hypertension and inflammation and renal abnormalities during the course of this disease.

Anti-inflammatory and pro-angiogenic effects of placental mesenchymal stromal cells conditioned media on preeclamptic placental tissue.

s / Placenta 35 (2014) A1eA112 A87 increasing doses (0-100mM) on the following outputs 1) heme-oxygenase1 expression (HO-1, key antioxidant enzyme) 2) production of sFlt1 and sEng and 3) assays of endothelial dysfunction 4) endothelial tube formation. Endothelial dysfunction was modeled by adding TNFa or preeclamptic (PE) patient serum (to induce dysfunction) and subsequently adding PPIs. Results: All PPIs induced a significant dose dependent increase in HO-1 mRNA and protein (average 4-5 fold increase). Furthermore, PPIs induced nuclear translocation of Nrf2 (transcription factor). PPIs caused a significant dose dependent reduction in mRNA expression of sFlt1 variants (e15a and i13), and reduced secretion of both sFlt1 and sEng (dose dependently) in all three cell types. PPIs rescued both TNFa and PE serum induced endothelial dysfunction (evidenced by increased VCAM1 and endothelin-1 expression, but this was significantly attenuated by adding PPIs (decreased VCAM and ET-1 mRNA and protein levels). Finally, PPIs reversed TNFainduced disruption of in vitro endothelial cell tube formation. Summary and conclusions: In primary human tissues, PPIs potently 1) induced HO-1, 2) reduced sFlt1 and sEng secretion and 3) quenched endothelial dysfunction 4) rescued endothelial tube formation. PPIs may represent a highly novel candidate therapeutic for severe preeclampsia. Given PPIs are safe in pregnancy, they could be fast-tracked to clinical trials. P2.81-N. ANTI-INFLAMMATORY AND PRO-ANGIOGENIC EFFECTS OF PLACENTAL MESENCHYMAL STROMAL CELLS CONDITIONED MEDIA ON PREE CLAMPTIC PLACENTAL TISSUE. Anna Maria Nuzzo, Domenica Giuffrida, Ettore Piccoli, Cristian Zenerino, Rossella Barrile, Tullia Todros, Alessandro Rolfo Dept. of Surgical Sciences, University of Turin, Turin, Italy Objective: Placenta-derived Mesenchymal Stromal Cells (PDMSCs) are renowned for their unique anti-inflammatory and pro-angiogenic trophic activities. Indeed, we hypothesized that PDMSCs-derived molecules could counteract the production of pro-inflammatory cytokines and antiangiogenic factors typical of preeclamptic (PE) placentae. To verify our hypothesis, herein we investigated the expression of Macrophage-migration Inhibitory Factor (MIF), Tumor Necrosis Factor a (TNF-a) and Interleukin 6 (IL-6) cytokines, and anti-angiogenic soluble Fms-like tyrosine kinase-1 (sFlt1) in PE villous explants treated by PDMSCs conditioned media (CM). Methods: PDMSCs were isolated from control (n1⁄430) and PE (n1⁄430) placentae. At passage 5, control and PE-PDMSC were plated (1x105 cells/ ml) in DMEM without FBS. Conditioned media (CM) were collected after 48h. PE (n1⁄448) villous explants were treated for 72h using control PDMSCs CM. MIF, TNF-a, IL-6 and sFlt1 mRNA expression were evaluated by Real Time PCR. Results:Normal PDMSCs CM treatment significantly decreasedMIF, TNF-a, IL-6 and sFlt-1 mRNA levels (p<0.05, 3 Fold decrease) in PE villous explants relative to untreated controls. Conclusion: Our results suggest that PDMSCs CM could be used to neutralize the exacerbated inflammation and impaired angiogenesis typical of PE placentae. These data open to the development of innovative PDMSCs-based therapeutic tools for the treatment of preeclampsia. P2.82. A COMBINATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE 3’UNTRANSLATED REGION OF HLA-G IS ASSOCIATED WITH PREECLAMPSIA Kevin Quach , Stephanie Grover , Shlomit Kenigsberg , Clifford Librach a,b,c CReATe Fertility Centre, Toronto, Ontario, Canada; Women’s College Hospital, Toronto, Ontario, Canada; University of Toronto, Toronto,

Endothelin receptor inhibition with bosentan delays onset of liver injury in streptozotocin-induced diabetic condition.

This study was designed to investigate the protective effects of bosentan an orally active non-peptide mixed ETA/ETB receptor antagonist, on liver injury in streptozotocin-induced diabetic rats. 24 Albino-Wistar rats were randomly divided into 4 groups: healthy (Group 1), diabetic (Group 2) (60 mg/kg of streptozotocin i.p.), diabetic treated with bosentan 50 mg/kg (Group 3) and diabetic treated with bosentan 100 mg/kg (Group 4). The treatment of bosentan was initiated after streptozocin injection and continued for 60 days. Liver from diabetic rats showed significant increase in malondialdehyde (MDA) level and significant decrease in glutathione (GSH), and superoxide dismutase (SOD) activity. Endothelin (ET-1), tumor necrosis factor (TNF-α) and transforming growth factor beta (TGF-β) gene expression significantly increased in the diabetic groups in the rat liver tissue. Bosentan treatment showed a significant up-regulatory effect on ET-1, TNF-α and TGF-β mRNA expression. Results from histopathological evaluation of the liver were in accordance with our biochemical and molecular results. These data provide clear evidence that bosentan treatment is associated with promising hepatoprotective effect against diabetes-induced liver damage via reduction of cell inflammation and oxidative damage. These data suggest that ET receptors may be an important actor in diabetes-related liver damage, and blockage of these receptors may become a target for preventing diabetic complications in the future.

Vascular endothelin (ET‐1) mediates skin Na+ storage in response to chronic increases in salt intake (860.13)

We have recently implicated extrarenal vascular ET‐1 in the body’s integrated response to a chronic high salt intake by mediating storage of Na+ within the skin. We hypothesize that upregulation of vascular ET‐1 in response to a high salt diet leads to the recruitment of macrophages and lymphocytes to the interstitium of the skin, which promote lymph hyperplasia, all of which provide a buffer for extracellular Na+ homeostasis. In the present study, we determined if vascular endothelial cell ET‐1 knockout mice (VEET KO) have reduced vascular cell adhesion molecule expression and increased skin Na+ storage in response to a high salt diet. After 2 weeks of a high NaCl (4%) diet, VEET KO mice have higher total skin Na/water ratio than floxed controls (0.112±0.007 vs. 0.096±0.003 mmol/ml respectively). Floxed control mice have elevated ET‐1 mRNA in the aorta compared to animals on low salt (1.38±0.21 vs. 1.05±0.14 fold change respectively, n=6), a response that was completely blunted in VEET KO mice (1.07±0.12 vs. 1.05±0.16 fold change, n=5). There was no change in vascular cell adhesion molecule‐1 between groups. These data support our hypothesis that vascular endothelial derived ET‐1 plays a critical role in mediating skin storage of Na+ in response to a high salt intake.Grant Funding Source: Supported by NHLBI and AHA

Valproic acid attenuates blood pressure, vascular remodeling and modulates ET-1 expression in L-NAME induced hypertensive rats

The present study was considered to assess the antihypertensive and antioxidant effect of valproic acid, against Nω- nitro-L arginine methyl ester hydrochloride (L-NAME) induced hypertension in male Wistar rats. Hypertension was prompted in adult male albino rats of the Wistar strain, weighing 180–220g, by oral administration of the L-NAME (40mg/kg body weight/day) in drinking water for 4 weeks. The L- NAME hypertensive rats revealed significant (P<0.05) rise in the systolic and diastolic blood pressure, heart rate, water intake and heart weight L-NAME hypertensive rats also revealed significant (P<0.05) increase in the levels of thiobarbituric acid reactive substances, lipid hydroperoxides in plasma and tissues (heart and aorta), and significant (P<0.05) drop in the body weight, nitrite and nitrate levels in plasma and aorta. Activities of enzymic antioxidants such as superoxide dismutase, catalase and glutathione peroxidase in erythrocyte and tissues and the levels of non-enzymic antioxidant such as reduced glutathione in plasma and tissues, ET-1 mRNA expression in aorta was significantly (P<0.05) increased in L-NAME rats. Valproic acid (VPA) supplementation (100mg/kg) daily for four weeks brought back all the above parameters to near normal level. The above outcomes were confirmed by the histopathological examination. No significant (P<0.05) effect was observed in control rats treated with valproic acid (100mg/kg). These results suggest that valproic acid performed as an antihypertensive and antioxidant agent against L-NAME induced hypertension.

Smooth muscle specific disruption of the endothelin-A receptor in mice reduces arterial pressure, and vascular reactivity and affects vascular development

AimsThe role of vascular smooth muscle endothelin A receptors (ETA) in development and normal physiology remains incompletely understood. To address this, mice were generated with smooth muscle-specific knockout (KO) of ETA. Main methodsMice were homozygous for loxP-flanked exons 6–8 of the EDNRA gene (floxed) or were also hemizygous for a transgene expressing Cre recombinase under control of the smooth muscle-specific SM22 promoter (KO mice). Key findingsGenotyping at 17days postnatal yielded a 10:1 ratio of floxed:KO mice. Smooth muscle actin staining of embryos at day E10.5 revealed increased tortuosity in dorsal aortae while E12.5 embryos had mandibular, vascular and thymic abnormalities. Mice surviving to weaning developed and bred normally. ETA KO mice aged 2–3months manifested EDNRA gene recombination in all organs tested. Aortas from KO mice had a >90% reduction in ETA mRNA content, but no differences in ET-1 or ETB mRNA levels. Addition of 0.01–100nM ET-1 to isolated femoral arteries from floxed, but not KO, mice dose-dependently decreased vessel diameter (up to 80% reduction in the presence of ETB blockade). Intravenous infusion of ET-1 into floxed, but not KO, mice increased mean arterial pressure (MAP) (by ~10mmHg). Telemetric analysis revealed decreased MAP in KO mice (reduced by ~7–10mmHg) when fed a high salt diet. SignificanceSmooth muscle ETA is important for normal vascular, mandibular and thymic development and is involved in the maintenance of arterial pressure under physiological conditions.

Intermittent hypoxia-induced increases in reactive oxygen species activate NFATc3 increasing endothelin-1 vasoconstrictor reactivity

Sleep apnea (SA), defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension, peripheral vascular disease, stroke, and sudden cardiac death. We have shown that intermittent hypoxia with CO2 supplementation (IH), a model for SA, increases blood pressure and circulating ET-1 levels, upregulates lung pre-pro ET-1 mRNA, increases vasoconstrictor reactivity to ET-1 in rat small mesenteric arteries (MA) and increases vascular reactive oxygen species (ROS). NFAT activity is increased in the aorta (AO) and MA of mice exposed to IH in an ET-1-dependent manner, and the genetic ablation of the isoform NFATc3 prevents IH-induced hypertension. We hypothesized that IH causes an increase in arterial ROS generation, which activates NFATc3 to increase vasoconstrictor reactivity to ET-1. In support of our hypothesis, we found that IH increases ROS in AO and MA. In vivo administration of the SOD mimetic tempol during IH exposure prevents IH-induced increases in NFAT activity in mouse MA and AO. We found that IH causes an NFATc3-dependent increase in vasoconstrictor reactivity to ET-1, accompanied by an increase in vessel wall [Ca2+]. Our results indicate that IH exposure causes an increase in arterial ROS to activate NFATc3, which then increases vasoconstrictor reactivity and Ca2+ response to ET-1. These studies highlight a novel regulatory pathway, and demonstrate the potential clinical relevance of NFAT inhibition to prevent hypertension in SA patients.

Protective effects of simultaneous splenectomy on small-for-size liver graft injury in rat liver transplantation

Splenectomy is an effective technique in living donor liver transplantation (LDLT) with small-for-size (SFS) liver grafts for overcoming SFS liver graft injury. However, the protective mechanism of splenectomy is still unclear. The aim of this study was to investigate how splenectomy could attenuate SFS graft injury through the measurement of biochemical factors, particularly the expression of endothelin (ET)-1, which is a key molecule of microcirculatory disorders by mediating sinusoidal vasoconstriction. We performed rat orthotopic liver transplantation using SFS liver grafts with or without splenectomy. We investigated intragraft expression of ET-1 mRNA and hepatic protein levels of ET-1. In addition, portal pressure, hepatic injury and morphological changes, and survival rate were evaluated. In result, intragraft ET-1 mRNA expression after SFS liver transplantation was significantly downregulated by splenectomy, and hepatic expression of ET-1 in SFS grafts was rarely observed. Splenectomy inhibited the increase in portal pressure, ameliorated SFS liver graft injury and improved the graft survival rate after SFS liver transplantation. In conclusion, splenectomy improved the SFS liver injury and decreased the expression of ET-1 by attenuating portal hypertension on SFS liver transplantation. Downregulation of intragraft ET-1 expression plays important roles in the protective mechanism of splenectomy in SFS liver transplantation.