ET-1 mRNA Research Articles

Sulfatase 2 mediates, partially, the expression of endothelin-1 and the additive effect of Ang II-induced endothelin-1 expression by CXCL8 in vascular smooth muscle cells from spontaneously hypertensive rats

The extracellular sulfatases (Sulfs), sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), have an important role in cell signaling by modulating the 6-O-sulfation of heparan sulfate proteoglycans (HSPGs) on the cell surface. Gene expression and enzyme activity of Sulfs are elevated in hypertensive vascular smooth muscle cells (VSMCs) compared to those in normotensive VSMCs. CXC-chemokine ligand (CXCL) 8 has a pathogenic role in the development and progression of hypertension. In this study, we investigated the effect of Sulfs on the expression of CXCL8-induced endothelin (ET)-1, a hypertensive mediator, in VSMCs from spontaneously hypertensive rats (SHR). Expression of ET-1 and elevation of angiotensin (Ang) II-induced ET-1 expression by CXCL8 were reduced in Sulf2 small interfering RNA (siRNA)-transfected SHR VSMCs. But, downregulation of Sulf1 did not affect the expression of CXCL8-induced ET-1 and additive effect of CXCL8 on Ang II-induced ET-1 expression in SHR VSMCs. CXCL8-induced ET-1 expression and the additive effect of CXCL8 on Ang II-induced ET-1 expression were dependent on the Ang II type 1 receptor (AT1 R) pathway, not the Ang II type 2 receptor (AT2 R) pathway. In addition, downregulation of Sulf2 reduced the expression of CXCL8-induced AT1 R and abrogated the additive effect of CXCL8 on Ang II-induced AT1 R expression in SHR VSMCs. Sulf2 mediated, partially, the expression of ET-1 and the additive expression of Ang II-induced ET-1 mRNA by CXCL8 via the AT1 R pathway in SHR VSMCs. These findings suggest that Sulf2 is an up-regulatory factor in the additive action of CXCL8 via the AT1 R pathway on Ang II-induced ET-1 expression in VSMCs under hypertension environment.

Temporal changes of the endothelin system in human cytotrophoblasts during the first trimester of pregnancy.

The first trimester of pregnancy is characterized by continuous proliferation, invasion and differentiation of cytotrophoblasts. These processes are precisely controlled both, in space and time by molecules such as endothelin-1 (ET-1). ET-1 is expressed in human first trimester trophoblast and is known to stimulate cytotrophoblast proliferation through endothelin A and B receptor subtypes (ET(A) and ET(B)), and cytotrophoblast invasion through ET(B). However, temporal changes of the ET system during the first trimester of pregnancy have not been previously studied. This study tested the hypothesis that ET-1 release, ET(A) and ET(B) expression are increased towards the end of the first trimester of pregnancy (weeks 10-12 vs. weeks 6-9), resulting in increased cytotrophoblast proliferation and invasion. Tissue samples were obtained from 17 surgical pregnancy interruptions (week 6-9: n=9; week 10-12: n=8). After cytotrophoblast isolation, the invasive and proliferative phenotypes were immune-separated by an alpha(6)-integrin antibody. Both proliferative and invasive cytotrophoblasts were cultured separately on plastic or Matrigel for 24 h. ET-1 release into the culture medium of both cytotrophoblast subtypes was measured by radioimmunoassay. ET(A) and ET(B) mRNA expression was measured by RT-PCR, and the ET-1 effect on cytotrophoblast proliferation and invasion was determined using proliferation and invasion assays, respectively. ET-1 release increased from early to late first trimester of pregnancy in both proliferative (1.8-4.5 fold) and invasive cytotrophoblasts (9.3-28 fold), especially when cultured on Matrigel. This was paralleled by less ET(B) mRNA on invasive cytotrophoblasts independent of the time period in first trimester, whereas ET(A) expression was similar on proliferative an invasive cytotrophoblasts. Proliferation and invasion of cytotrophoblasts under control conditions decreased from early to late first trimester. ET-1 stimulated both processes at both periods with the most pronounced effect (7-fold) on invasion in late first trimester. The ET-1/ET-receptor system changes between weeks 6-9 and 10-12 in pregnancy. Our data suggest an autocrine and endocrine ET-1 effect, which is stronger in late than in early first trimester of pregnancy paralleled by different stimulatory effects on trophoblast invasion and proliferation. In general, this suggests time as an additional effector of the critical processes governing placental development in the first trimester of human pregnancy.

Nephron prorenin receptor deficiency alters renal medullary endothelin-1 and endothelin receptor expression.

The endothelin (ET) and prorenin/renin/prorenin receptor (PRR) systems have opposing physiological effects on collecting duct (CD) salt and water reabsorption. It is unknown if the CD ET and renin/PRR systems interact, hence we examined the effects of deleting CD renin or nephron PRR on CD ET system components. PRR knockout (KO) mice were polyuric and had markedly increased urinary ET-1 and inner medullary CD (IMCD) ET-1 mRNA. PRR KO mice had greatly increased IMCD ETA receptor mRNA and protein, while ETB mRNA and protein were decreased. Water loaded wild-type mice with similar polyuria as PRR KO mice had modestly increased urinary ET-1 excretion and inner medullary ET-1 mRNA, while inner medullary ETA and ETB mRNA or protein expression were unaffected. In contrast to PRR KO, CD prorenin/renin KO did not alter ET system components. Taken together, these results suggest that the nephron PRR is involved in regulating CD ET system expression, but this effect may be independent of CD-derived renin.

The glucagon-like peptide-1 receptor agonist liraglutide improves hypoxia-induced pulmonary hypertension in mice partly via normalization of reduced ET(B) receptor expression.

The glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide is an incretin hormone mimetic used in the treatment of diabetes. However, the effects of liraglutide on pulmonary hypertension (PH) and pulmonary endothelin (ET) system are unknown. Eight-week-old C57BL6/J mice were injected liraglutide or vehicle for 5 weeks. One week after injection, the mice were exposed to either room air (normoxia) or chronic hypoxia (10 % O(2)) for 4 weeks. The right ventricular systolic pressure (RVSP) was significantly higher in hypoxia + vehicle group than in normoxia + vehicle group. ET-1 mRNA expression in the lungs was comparable among all the groups. ET(B) mRNA and protein expression in the lungs was significantly lower in hypoxia + vehicle group than in normoxia + vehicle group. The above changes were normalized by liraglutide treatment. The expression of phospho-eNOS and phospho-AMPK proteins in the lungs was significantly higher in hypoxia + liraglutide group than in normoxia + vehicle group. We demonstrated for the first time that liraglutide effectively improved RVSP and RV hypertrophy in hypoxia-induced PH mice by activating eNOS through normalization of impaired ET(B) pathway and augmentation of AMPK pathway. Therefore, GLP-1R agonists can be promising therapeutic agents for PH.

Upregulation of Endothelin-1/Endothelin A Receptor Expression Correlates with Heparanase Expression in Ovarian Carcinoma.

Heparanase and endothelin-1/endothelin A receptor (ET-1/ETAR) expressions increase in cancer. This condition enhances tumor progression and correlates with poor survival. Limited data are documented regarding the role of heparanase and ET-1/ETAR in epithelial ovarian cancer (EOC). We sought to characterize the correlation between heparanase and ET-1/ETAR in EOC. Thirty patients with benign and malignant ovarian neoplasms were recruited in this study. Neoplasm subtypes were diagnosed by pathologists. RNA extraction was done in fresh frozen neoplasms while immunohistochemical (IHC) staining was done on ETAR, heparanase, and proliferation (Ki-67 antigen) in paraffin sections. Reverse transcriptase PCR was done to quantify the expression of preproET-1 (ppET-1), ETAR, and heparanase. ETAR and heparanase histoscores were done based on IHC staining. The Independent Samples t Test, ANOVA, and correlations were used for statistical analysis. Heparanase and ETAR histoscores, ppET-1 and ETAR mRNA levels, and Ki-67 were significantly higher in the group with EOC than in the benign or borderline group, regardless of the histopathological types. The heparanase histoscore correlated with the ETAR histoscore (r=0.484, P=0.007) and the ETAR mRNA level (r=0.551, P=0.003). The level of ppET-1 mRNA correlated with both ETAR mRNA level and ETAR histoscore (r=0.603, P=0.001 and r=0.455, P=028, respectively). The ovarian neoplasms with high ppET-1 mRNA levels also tended to have high heparanase mRNA levels; however, the correlation was weak (r=0.354, P=0.07). Ki-67 correlated with the heparanase and ETAR histoscores (r=0.381, P=0.038 and r=0.477, P=0.008, respectively). Heparanase and ETAR were upregulated in EOC, and the correlation between heparanase and ETAR expressions was also elucidated in the current study.

The effect of leech extracts on endothelial cell coagulation-related factors and endothelial dysfuction-related molecules

ABSTRACTObjective: This study was focused on screening leech extracts to identify those with little or no anti-coagulation effect or with significant anti-endothelial dysfunction activity.Methods: Different leech extracts were prepared by enzymolysis and microbial transformation and their cytotoxicity were measured by MTT assay. The effect of different leech extracts on mRNA expression of coagulation-related factors (PAI, vWF, tPA, PS, TFPI, TM) was quantified by RT-PCR. After identifying a leech extract with little anti-coagulatory effect, RT-PCR was then used to assess the effect of this extract on the mRNA expression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, IL-6).Results: 8 leech extracts were obtained, including 4 enzymatic extracts (LP, PHL, PTHL, CEHL) and 4 Lactobacillus metabolites (MRS, MRS-1, MRS-2, and MRS-3). Following optimization of conditions using MTT assays, we treated EA.hy926 cells with 0, 12.5, 25, 50 μg/mL of LP, PTHL, CEHL, MRS, MRS-1 or MRS-3 extract for 24 h. We found that PHL and MRS-1 had no significant effect on coagulation-related factors. Furthermore, treatment with 50 μg/mL PHL resulted in significant decreases in ET-1, iNOS, MCP-1, and IL-6 mRNA expression by 28.06%, 33.30%, 19.80%, and 52.34%, respectively.Conclusions: In the present study, we found that PHL, a pepsin hydrolysate of leech with little anti-coagulatory effect, could significantly suppress TNF-α induced mRNA overexpression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, and IL-6). These results provide a reliable experimental basis for identifying new anti-atherosclerosis therapeutics for long term use and with minimal bleeding side effects.

Co‐regulation of Renal Function Genes by the Circadian Clock Protein PER1 and Aldosterone

Hypertension is the leading cause of cardiovascular disease. A subset of hypertensive patients experience salt‐sensitive hypertension, which causes elevations in blood pressure (BP) after high salt intake. With large increases in dietary salt worldwide, the prevalence of hypertension has reached 30%. BP follows a circadian rhythm. The circadian clock is a highly conserved mechanism that allows organisms to adapt to the light/dark cycles of our environment. The molecular clock is comprised of core transcription proteins, such as the clock protein PER1. Further, the renin angiotensin aldosterone system (RAAS) is an important homeostatic mechanism to manage sodium and BP homeostasis. Previous results from our lab show that the circadian clock and RAAS both contribute to sodium and BP homeostasis. Based on these studies, we hypothesized that aldosterone and Per1 coordinately regulate genes that contribute to kidney function. We tested this hypothesis first by using a cell culture model from the renal cortical collecting duct. Cells were treated with aldosterone and/or siRNA to knockdown down PER1 expression. Using an unbiased microarray approach, we determined PER1 and aldosterone‐dependent changes in gene expression. Several hundred genes exhibited significant changes in expression in response to hormone treatment and PER1 knockdown. For the first time, we identified secreted frizzled related protein 1 (sFRP1), Claudin‐1, and Rhesus glycoprotein C (Rhcg) as target genes of aldosterone and PER1. The expression of sFRP1 demonstrated a 1.5‐fold increase in response to aldosterone (p<0.05, n=4), which was not affected in the absence of PER1, indicating that aldosterone and PER1 independently regulate sFRP1. In contrast, the 2.9‐fold (p<0.05, n=4) and 1.9‐fold (p<0.05, n=3) induction of Rhcg and Claudin‐1, respectively, by aldosterone was decreased significantly in the absence of PER1, suggesting that PER1 contributes to aldosterone‐mediated regulation of these genes. The ET‐1 gene is of particular interest due to its known role in the regulation of renal salt handling and BP. In the microarray study, knockdown of PER1 augmented the aldosterone response of ET‐1 mRNA by 3‐fold (p<0.05, n=4). Importantly, a similar effect was observed in the kidneys of Per1 knockout mice treated with a high salt diet plus an aldosterone analogue: loss of Per1 augmented the response of ET‐1 by 50% (p<0.01, n=6) compared to no change in WT mice. Interrogation of the database CircaDB revealed that Claudin‐1 and ET‐1 are both expressed with a significant circadian rhythm in the kidney (p<0.001). Together these data demonstrate that multiple genes critical to renal function are co‐regulated by the circadian clock PER1 and RAAS. These data may have important implications for understanding the underlying mechanisms of, and designing novel treatments for, salt‐sensitive hypertension in humans.Support or Funding InformationR01DK109570; R21AG052861; American Heart Association Grant‐in‐aid; Gatorade Trust Fund, Division of Nephrology, Department of Medicine, University of Florida; NF/SG VAMCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective role of β-patchoulene from Pogostemon cablin against indomethacin-induced gastric ulcer in rats: Involvement of anti-inflammation and angiogenesis

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) are most widely used as effective anti-inflammatory agents. However, their clinical application brings about inevasible gastrointestinal side effects. Pogostemon cablin is a traditional herbal medicine used for the treatment of gastrointestinal diseases in China. One of its representative components, the tricyclic triterpenoid β-patchoulone (β-PAE) has demonstrated great anti-inflammatory activity and gastroprotective effect against ethanol-induced gastric injury, but its protective effect against gastric ulcer induced by indomethacin is still unknown. PurposeTo assess the protective effect of β-PAE against ulcer produced by indomethacin and reveal the underlying pharmacological mechanism. Study designWe used an indomethacin-induced gastric ulcer model of rats in vivo. MethodsGastroprotective activity of β-PAE (10, 20, 40 mg/kg, i.g.) was estimated via indomethacin-induced gastric ulcer model in rats. Histopathological and histochemical assessment of ulcerated tissues were performed. Protein and mRNA expression were determined by Elisa, Western blotting and qRT-PCR. Resultsβ-PAE could inhibit ulcer formation. Histopathological and histochemical assessment macroscopically demonstrated that β-PAE alleviates indomethacin-induced gastric ulceration in dose-dependent manner. After administration of β-PAE, elevated tumor necrosis factor -α level was significantly decreased and the phosphorylation of JNK and IκB was markedly inhibited. β-PAE suppressed the levels of E-selectin, P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule and monocyte chemoattractant protein 1, as well as myeloperoxidase. Meanwhile, β-PAE increased cyclooxygenase enzyme activities (COX-1 and COX-2) to enhance the production of prostaglandin E2. Proangiogenic protein, vascular endothelial growth factor and its receptor fms-like tyrosine kinase-1 mRNA expression were promoted while anti-angiogenic protein, endostatin-1 and its receptor ETAR mRNA expression were decreased. Conclusionβ-PAE may provide gastroprotection in indomethacin-induced gastric ulcer in rats by reducing inflammatory response and improving angiogenesis.

Abstract P272: Preeclamptic Placental CD4+ T Cells Cause Preeclamptic Like Features in Normal Pregnant Nude Athymic Rats

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with a proinflammatory profile compared to normal pregnancy (NP). We hypothesize that CD4 + T cells play an important role to cause much of the pathophysiology associated with PE. To determine if CD4 + T cells isolated from PE patients cause PE symptoms during pregnancy compared to those collected from NP women, CD4 + T cells were isolated from placentas of both PE and NP women using magnetic separation with anti-CD4 antibodies, cultured in TexMACS medium with IL-2 at 37°C in 5% CO 2 , and injected intraperitoneally into pregnant, nude-athymic rats on day 12 of gestation. On day 18, carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. MAP was 125±3 mmHg in rats with NP T cells but increased to 140±9 mmHg in rats with PE T cells. Significant differences in circulating cytokines TNF-α, IL-6, IL-17 and sFlt-1 were found with 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; IL-17-PE= 7.054 pg/mL, NP=3.185 pg/mL; sFlt-1-PE=90.7 pg/mL, NP=58.2 pg/mL. However, there was no difference in Isoprostane levels or IL-2 between the two groups (Isoprostane-PE=3628 pg/mL, NP=3061 pg/mL; IL-2-PE=125 pg/mL, NP=125 pg/mL). ROS in tissues was measured using chemiluminescence and no difference was found in placenta while renal ROS showed an increased with PE CD4 + T cells vs NP CD4 + T cells (ROS-PE=5528 RLU/min/mg protein, NP=3372 RLU/min/mg protein). In addition, no difference in ET-1 mRNA was found in the placenta of nude rats receiving PE CD4+T cells, however, renal cortical ET-1 mRNA expression was increased 4.5 fold in rats with PE CD4+ T cells compared to those receiving to NP CD4+ T cells. These data indicate an important role for placental PE CD4 + T cells to cause inflammation and stimulate renal vasoactive pathways that may contribute to hypertension during pregnancy. This research is funded by R01HD067541-06

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ETA receptor (R) and ETBR on cancer cells. High levels of tumor ET-1 and ETAR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ETAR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ETAR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca2+ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.

High Dietary Fructose Intake on Cardiovascular Disease Related Parameters in Growing Rats.

The objective of this study was to determine the effects of a high-fructose diet on cardiovascular disease (CVD)-related parameters in growing rats. Three-week-old female Sprague Dawley rats were randomly assigned to four experimental groups; a regular diet group (RD: fed regular diet based on AIN-93G, n = 8), a high-fructose diet group (30Frc: fed regular diet with 30% fructose, n = 8), a high-fat diet group (45Fat: fed regular diet with 45 kcal% fat, n = 8) or a high fructose with high-fat diet group (30Frc + 45Fat, fed diet 30% fructose with 45 kcal% fat, n = 8). After an eight-week treatment period, the body weight, total-fat weight, serum glucose, insulin, lipid profiles and pro-inflammatory cytokines, abdominal aortic wall thickness, and expressions of eNOS and ET-1 mRNA were analyzed. The result showed that total-fat weight was higher in the 30Frc, 45Fat, and 30Frc + 45Fat groups compared to the RD group (p < 0.05). Serum triglyceride (TG) levels were highest in the 30Frc group than the other groups (p < 0.05). The abdominal aorta of 30Frc, 45Fat, and 30Frc + 45Fat groups had higher wall thickness than the RD group (p < 0.05). Abdominal aortic eNOS mRNA level was decreased in 30Frc, 45Fat, and 30Frc + 45Fat groups compared to the RD group (p < 0.05), and also 45Fat and 30Frc + 45Fat groups had decreased mRNA expression of eNOS compared to the 30Frc group (p < 0.05). ET-1 mRNA level was higher in 30Frc, 45Fat, and 30Frc + 45Fat groups than the RD group (p < 0.05). Both high fructose consumption and high fat consumption in growing rats had similar negative effects on CVD-related parameters.

Potential role for ET-2 acting through ETA receptors in experimental colitis in mice.

This study attempted to clarify the roles of endothelins and mechanisms associated with ETA/ETB receptors in mouse models of colitis. Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 1.5mg/animal) or dextran sulfate sodium (DSS, 3%). After colitis establishment, mice received Atrasentan (ETA receptor antagonist, 10mg/kg), A-192621 (ETB receptor antagonist, 20mg/kg) or Dexamethasone (1mg/kg) and several inflammatory parameters were assessed, as well as mRNA levels for ET-1, ET-2 and ET receptors. Atrasentan treatment ameliorates TNBS- and DSS-induced colitis. In the TNBS model was observed reduction in macroscopic and microscopic score, colon weight, neutrophil influx, IL-1β, MIP-2 and keratinocyte chemoattractant (KC) levels, inhibition of adhesion molecules expression and restoration of IL-10 levels. However, A192621 treatment did not modify any parameter. ET-1 and ET-2 mRNA was decreased 24h, but ET-2 mRNA was markedly increased at 48h after TNBS. ET-2 was able to potentiate LPS-induced KC production in vitro. ETA and ETB receptors mRNA were increased at 24, 48 and 72h after colitis induction. Atrasentan treatment was effective in reducing the severity of colitis in DSS- and TNBS-treated mice, suggesting that ETA receptors might be a potential target for inflammatory bowel diseases.

Role of portal venous pressure changes in the liver dysfunction caused by hepatic congestion after extended liver resection

Objective To explore the role of portal venous pressure changes in the liver dysfunction caused by hepatic congestion after extended liver resection. Methods The experimental study was adopted. According to the random number table, 90 Sprague-Dawley rats were divided into 3 groups, 30 in each group: 30 rats in the non-congestion group received 70% of liver resection (median lobe+ left lobe), 30 rats in the congestion group received 70% of liver resection (median lobe+ left lobe) with whole caudal lobe congestion by ligation of veins and 30 rats in the congestion+ splenectomy group received 70% of liver resection (median lobe+ left lobe) with whole caudal lobe congestion by ligation of veins and splenectomy. (1) Twenty rats in each group were used to make postoperative survival analysis.Ten rats in each group were used for related experiments. The portal venous pressures (PVPs) of 5 rats in each group were detected at postoperative 12 hours and 24 hours, and then blood and liver specimens were collected. (2) PVP changes were detected at postoperative 12 hours and 24 hours. (3) Clinical and biochemical test: level of total bilirubin (TBil) was tested at postoperative 12 hours and 24 hours. (4) Pathological examination: liver pathological damage was detected by HE staining. (5) The expression of CD68 macrophagocyte was detected by immunohistochemical staining. (6) The relative expressions of Cleaved Casepase-3 and hypoxia inducible factor-1α (HIF-1α) proteins at postoperative 24 hours were detected by Western blot. (7) The relative expressions of mRNA of vascular regulation related genes (ET-1/eNOS) and inflammatory factors (TNF-α and IL-6) were detected by real-time polymerase chain reaction (RT-PCR). (8) The hyaluronic acid (HA) was measured by enzyme-linked immuno-sorbent assay (ELISA). Measurement data with normal distribution were represented as ±s. Comparison among 3 groups was done using the ANOVA, and pairwise comparison was done by the LSD test. The postoperative 5-day survival curve was drawn by the Kaplan-Meier method, and the survival was compared using the Log-rank test. Results (1) Survival analysis: 5-day survival rate in the non-congestion group, congestion group and congestion+ splenectomy group were respectively 75%, 10% and 55%, with a statistically significant difference among the 3 groups (χ2=18.21, P<0.05). (2) Changes of PVPs and TBil: levels of PVP and TBil in the non-congestion group, congestion group and congestion+ splenectomy group were respectively (15.77±0.67)cmH2O, (18.33±0.28)cmH2O, (14.87±0.58)cmH2O, (1.48±0.10)μmol/L, (1.76±0.15)μmol/L, (1.62±0.11)μmol/L at postoperative 12 hours and (13.49±0.45)cmH2O, (16.96±0.82)cmH2O, (15.69±0.85)cmH2O, (1.47±0.11)μmol/L, (1.94±0.07)μmol/L, (1.67±0.11)μmol/L at postoperative 24 hours, showing statistically significant differences among 3 groups (F=56.53, 29.01, 6.81, 27.85, P<0.05). (3) Results of pathological examination: compared with non-congestion group, there were a lot of vacuolar cells with degeneration appearing in non-congestion liver tissues, severe liver cell swelling and hepatic sinus congestion in the congestion group at postoperative 24 hours. Compared with congestion group, vacuolar degeneration appearing in non-congestion liver tissues have some improvement in the congestion+ splenectomy group. (4) Immunohistochemical staining: compared with non-congestion group and congestion+ splenectomy group, the positive CD68 marked macrophages in the congestion group were increased at postoperative 24 hours. (5) Western blot assay: the relative expressions of Cleaved Casepase-3 and HIF-1α proteins in the non-congestion group, congestion group and congestion+ splenectomy group were 0.63±0.05, 1.17±0.18, 0.95±0.17 and 0.63±0.14, 1.48±0.08, 1.13±0.17, respectively, showing statistically significant differences among 3 groups (F=17.42, 50.58, P<0.05). (6) Results of RT-PCR: the relative expression of mRNA of ET-1/eNOS in the non-congestion group, congestion group and congestion+ splenectomy group was respectively 1.01±0.63, 2.09±0.27, 0.82±0.12 at postoperative 12 hours and 0.73±0.17, 2.16±0.94, 0.80±0.24 at postoperative 24 hours, showing statistically significant differences among 3 groups (F=62.91, 10.65, P<0.05). The relative expression of mRNA of TNF-α in the non-congestion group, congestion group and congestion+ splenectomy group was respectively 0.99±0.08, 127.80±13.15, 7.34±1.56 at postoperative 12 hours and 0.99±0.06, 116.62±13.32, 58.62±12.12 at postoperative 24 hours, showing statistically significant differences among 3 groups (F=436.77, 154.54, P<0.05). The relative expression of mRNA of IL-6 in the non-congestion group, congestion group and congestion+ splenectomy group was respectively 0.98±0.06, 1.87±0.34, 1.54±0.15 at postoperative 12 hours and 0.99±0.05, 2.02±0.27, 1.51±0.11 at postoperative 24 hours, with statistically significant differences among 3 groups (F=22.08, 46.71, P<0.05). (7) Results of ELISA: the level of HA in the non-congestion group, congestion group and congestion+ splenectomy group was respectively (149±9)ng/L, (200±19)ng/L, (174±9)ng/L at postoperative 12 hours and (136±16)ng/L, (202±13)ng/L, (91±11)ng/L at postoperative 24 hours, with statistically significant differences among 3 groups (F=19.23, 34.68, P<0.05). Conclusions On the basis of extended liver resection, a wide range of liver congestion through increasing PVP causes hepatic microcirculation disorders, hypoxia, inflammation, vacuoles degeneration cells, increased cells apoptosis, aggravated damage of liver function and increased mortality of rats. Splenectomy could reduce PVP and then improve the liver tissues damage caused by liver congestion, meanwhile, increase the survival rate of rats. Key words: Hepatic congestion; Hepatectomy; Portal venous pressure; Liver function

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model.

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.

Neuropathic pain induced by spinal cord injury: Role of endothelin ETA and ETB receptors

Spinal cord injury (SCI) is a devastating neurologic disorder that often inflicts neuropathic pain, which further impacts negatively on the patient’s quality of life. Endothelin peptides, which exert their effects via endothelin A (ETAR) and endothelin B (ETBR) receptors, can contribute to sensory changes associated with inflammatory and neuropathic pain, but their role in nociception following SCI is unknown. At different time points after subjecting male Wistar rats to surgery for compression-induced T10 level SCI, the spinal cord levels of ETAR and ETBR were assessed by Western blot and immunohistochemistry, and the corresponding mRNAs by real-time PCR, alongside recordings of behavioural responses to mechanical stimulation of the hind paws with von Frey hairs. SCI was associated with development of hind paw mechanical allodynia from day 14 onwards, and up-regulation of ETAR and ETBR mRNA in the spinal cord and dorsal root ganglia, and of ETAR protein in the spinal cord. SCI increased ETAR protein expression in spinal grey matter. Treatment on day 21 after surgery with the ETAR selective antagonist BQ-123 (40 and 90pmol, intrathecally) or the dual ETAR/ETBR antagonist bosentan (30 and 100mg/kg, orally) transiently reduced SCI-induced mechanical allodynia, but the ETBR antagonist BQ-788 was ineffective. Altogether, these data show that SCI upregulates ETAR expression in the spinal cord, which appears to contribute to the hind paw mechanical allodynia associated with this condition. Therapies directed towards blockade of spinal ETAR may hold potential to limit SCI-induced neuropathic pain.

Mixed Aqueous Extract of Salvia Miltiorrhiza Reduces Blood Pressure through Inhibition of Vascular Remodelling and Oxidative Stress in Spontaneously Hypertensive Rats

Background/Aims: Salvia miltiorrhiza (SM) contains four major aqueous active ingredients, which have been isolated, purified and identified as danshensu (DSS), salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and protocatechuic aldehyde (PAL), totally abbreviated as SABP. Although SM is often used to treat various cardiovascular diseases in traditional Chinese medicine, the efficacy and function of optimal compatibility ratio of SM's active ingredients (SABP) in the prevention and treatment of cardiovascular diseases remain uncertain. This study investigated antihypertensive effect and underlying mechanisms of SABP vs. SM lyophilized powder (SMLP) in spontaneously hypertensive rats (SHR) and to establish the ratio of the optimal compatibility of DSS, Sal-A, Sal-B and PAL in improving cardiovascular functions. Methods: The SHRs were treated with either SABP or SMLP and their systolic blood pressures (SBP) were monitored. The isolated thoracic aorta of SHRs was segregated for immunohistochemistry, Hematoxylin-Eosin stain and mRNA and protein expression of NOX4, TGF-β1, Col-I, ET-1, α-SMA and Smad7. Moreover, the adventitial fibroblasts (AFs) were isolated and cultured from SD rats' aorta and the reactive oxygen species (ROS) production was determined after SABP or SMLP treatment. Results: SABP, but not SMLP, significantly reduced SBP, which were accompanied by the inhibited morphological changes in the thoracic aorta and the reduced mRNA and protein expression of NOX4, TGF-β1, Col-I, ET-1 and α-SMA, but the increased Smad 7 expression in SHRs. Moreover, SABP also resulted in a decreased ROS production in AFs of SD rats. Conclusions: These results indicate that SABP, but not SMLP, treatment potently inhibits hypertension through improvements of vascular remodeling and oxidative stress. The present study provides new evidence that the efficacy and function from optimal compatibility ratio of SM active ingredients is much better than its lyophilized powder, which represents a strategy to develop SM's new beneficial effect in improving cardiovascular functions.

Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells

Objective: Investigation of the effect of endothelin receptor A (ETaR)-targeting small interfering RNA (siRNA) on rat vascular endothelial cellular hypoxia injury, as well as its underlying mechanism. Methods: An in vitro rat vascular smooth muscle cells - endothelial cells co-culture model was established and transfected with ETaR siRNA before hypoxia treatment. Cell culture supernatant, cellular protein and RNA were collected and examined at 0.5hrs, 1hrs, 2hrs, 4hrs, 8hrs, 16hrs, 24hrs and 48hrs of hypoxia with 1% oxygen. The time point at which the best silencing effect was achieved was chosen, eNOS inhibitor L-NAME was added, and post hypoxia cell culture supernatant, cellular protein and RNA was collected for further examination. Results: After hypoxic treatment, endothelial-1 (ET-1) and ETaR expression levels gradually increased as oxygen deprivation extended. ET-1 and ETaR expression levels were significantly lower in the ETaR siRNA group compared with the Hypoxia group (P<0.001). Such difference peaked at 4hrs of hypoxia. ELISA examination of cell culture supernatant revealed that the amount of ET-1 and TGF-βin the ETaR siRNA group were significantly lower compared to the Hypoxia group at all times, while the amount of NO and eNOS was higher. After 4 hrs of hypoxia, Smad2, Smad3, HIF-1, TNF-α, IFN-γ, IL-6, MCP-1, NF-κb, ET-1 and ANG II mRNA expression in endothelial cells and ETaR mRNA expression in A-10 cells of the ETaR siRNA group were lower than those of the Hypoxia siRNA group, while such results were much higher in the L-NAME group. Western Blot results showed lower expression of ETaR in the ETaR siRNA group compared with the hypoxia and negative siRNA groups, as well as significantly higher ETaR expression in the L-NAME group compared with the ETaR siRNA group. PI3K and p-AKT expression levels were mildly elevated after mild oxygen deprivation, and ETaR siRNA was able to enhance such elevation induced by hypoxia. In the L-NAME group, PI3K and p-AKT expression was much higher than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group. Conclusion: ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA.

Osmolar regulation of endothelin-1 production by the inner medullary collecting duct

AimsEndothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na+ and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. Main methodsET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. Key findingsFlow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca2+, however were not altered by blockade of downstream Ca2+-signaling pathways (calcineurin or NFATc), inhibition of cellular Ca2+ entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na+ channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. SignificanceBoth flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

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.