ET-1 Expression Research Articles

Perk heterozygosity ameliorates chronic hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in male rats

ABSTRACT Background Pulmonary hypertension (PH) is a rare and deadly disease characterized by remodeling of the pulmonary vasculature and increased pulmonary artery pressure. hypobaric pulmonary hypertension (HPH) is clinically classified as group 4 of pulmonary hypertension and has a poor prognosis . Previous reports showed that HPH was associated with increased endoplasmic reticulum (ER) stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) is an ER-associated stress protein. However, to date, its physiological effects on HPH and RVF development remains unknown. This study aimed to assess PERK’s role in HPH and RV function using in vivo experimental model. Methods Perk-knockout male Sprague–Dawley rats were generated and were housed in either a hypobaric chamber or in a normoxic environment. After stimulation for 4 weeks, the hemodynamic parameters of the rats were measured. The heart and lungs were harvested for pathological observation. Blood was collected for the detection of inflammatory indexes. The right ventricle tissue was collected to assess phosphorylated-AKT, ROCK1, ET1, and MMP2 protein expression. Results: we firstly generated Perk+/− rats, Under normal conditions, Perk +/− rats showed no changes in mPAP(mean pulmonary artery pressure), RVHI(Right ventricular hypertrophy index), cardiomyocyte size and interstitial fibrosis, and pulmonary vascular remodeling. However, in response to chronic hypoxia, Perk +/− rats exhibited decreased in mPAP, RVHI, ventricular fibrosis, and lung remodeling compared to wild-type rats. Perk +/− rats also showed lower expression of phosphor-AKT, ROCK1, ET1, and MMP2 protein in response to chronic hypoxia. Conclusions These findings suggest that Perk heterozygosity protects against HPH and Perk may be a suitable target for treating HPH.

Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Fine‐tuning of G protein‐coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one‐step labeling method of multiple membrane‐embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETAR and ETBR), angiotensin II receptor type 1, and apelin. Wild type‐like G protein activation after N‐terminal modification was demonstrated for all receptor species. Using FRET‐competent dyes, a constitutive proximity between hetero‐receptors was limited to ETAR/ETBR. Further, we demonstrate, that ETAR expression regulates the signaling of co‐expressed ETBR. Our orthogonal peptide‐templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Effects of different enantiomers of amlodipine on lipid profiles and vasomotor factors in atherosclerotic rabbits.

This research aimed to describe the functions of vascular endothelial cells (VECs) in protecting target organs and the anti-atherosclerotic effects of different enantiomers of amlodipine on a rabbit model of atherosclerosis. Thirty male New Zealand white rabbits were randomly allocated to four groups (nA = 9, nB = 7, nC = 7, and nD = 7 rabbits): rabbits in group-A (control group) were fed a high-fat diet, group-B rabbits were fed a high-fat diet plus 2.5 mg/kg/day S-amlodipine, group-C rabbits were fed a high-fat diet plus 2.5 mg/kg/day R-amlodipine, and group-D rabbits were fed a high-fat diet plus 5 mg/kg/day racemic amlodipine. Different enantiomers of amlodipine did not influence lipid profiles and serum level of eNOS in the rabbit atherosclerosis model but decreased ET-1 expression to some extent. The serum NO and iNOS levels in the drug intervention groups were significantly reduced. No significant differences in the rabbits’ body weights were observed. At the 4th and 8th weeks, the serum lipid profiles significantly increased in high cholesterol diet groups. The serum ET-1 level was significantly increased in each group of rabbits at the 8th week. Both S-amlodipine and R-amlodipine may protect the endothelium by reducing the serum ET-1 level, downregulating iNOS expression.

High Glucose Induced Changes in Human VEC Phenotype in a 3D Hydrogel Derived From Cell-Free Native Aortic Root

Background: Valvular endothelial cells (VEC) have key roles in maintaining valvular integrity and homeostasis, and dysfunctional VEC are the initiators and major contributors to aortic valve disease in diabetes. Previous studies have shown that HG stimulated an inflammatory phenotype in VEC. Inflammation was shown to induce endothelial to mesenchymal transition (EndMT), a process extensively involved in many pathologies, including calcification of the aortic valve. However, the effect of HG on EndMT in VEC is not known. In addition, there is evidence that endothelin (ET) is a proinflammatory agent in early diabetes and was detected in aortic stenosis, but it is not known whether HG induces ET and endothelin receptors and whether endothelin modulates HG-dependent inflammation in VEC. This study aims to evaluate HG effects on EndMT, on endothelin and endothelin receptors induction in VEC and their role in HG induced VEC inflammation.Methods and Results: We developed a new 3D model of the aortic valve consisting of a hydrogel derived from a decellularized extracellular cell matrix obtained from porcine aortic root and human valvular cells. VEC were cultured on the hydrogel surface and VIC within the hydrogel, and the resulted 3D construct was exposed to high glucose (HG) conditions. VEC from the 3D construct exposed to HG exhibited: attenuated intercellular junctions and an abundance of intermediate filaments (ultrastructural analysis), decreased expression of endothelial markers CD31 and VE–cadherin and increased expression of the mesenchymal markers α-SMA and vimentin (qPCR and immunocytochemistry), increased expression of inflammatory molecules ET-1 and its receptors ET-A and ET-B, ICAM-1, VCAM-1 (qPCR and Immunocytochemistry) and augmented adhesiveness. Blockade of ET-1 receptors, ET-A and ET-B reduced secretion of inflammatory biomarkers IL-1β and MCP-1 (ELISA assay).Conclusions: This study demonstrates that HG induces EndMT in VEC and indicates endothelin as a possible target to reduce HG-induced inflammation in VEC.

The natural compound puerarin alleviates inflammation and apoptosis in experimental cell and rat preeclampsia models

Puerarin (Pue), an isoflavone derived from Radix puerariae, exerts anti-apoptosis and anti-inflammatory effects. However, the protective effect of Pue on PE is still unknown. The present study aimed to investigate whether Pue alleviates symptoms of PE and suppresses inflammation and apoptosis in vitro and in vivo. A cell model of PE was established by exposing HTR8/SVneo cells to LPS and an RNA-SEQ study was performed in LPS-stimulated HTR8/SVneo cells. We also established a rat model of PE by injecting pregnant rats with LPS and the basic preeclamptic symptoms were evaluated. Additionally, the placental histology, placental inflammation cytokines, and apoptosis markers were also measured. Pue protected HTR8/SVneo cells from LPS-evoked cytotoxicity, decreased the levels of sFlt-1, ET-1, and tPA in HTR8/SVneo cells. RNA-SEQ results revealed the significant changes in the expression levels of hub genes (TNF, IL-6, Jun, and NFKBIA) related to multiple inflammatory pathways, including the TNF signaling pathway, IL-17 signaling pathway, inflammatory disease, and NF-κB signaling pathway. After administration of Pue, we observed that LPS-evoked PE symptoms (hypertension, proteinuria, and fetal growth restriction), were reversed. Besides, Pue improved placental pathology change and reducing placental sFlt-1, ET-1, and tPA mRNA expression. Abnormal placental inflammatory cytokines (TNF, IL-6, IFN-γ, and IL-4) and apoptosis markers (Bcl-2, Bax, caspase-3, caspase-8, and caspase-9) expressions in the LPS-treated group were reversed after Pue treatment. Our findings revealed that Pue plays beneficial roles in PE models, and therefore possesses the therapeutic potential for prevention and treatment of PE.

Blood Pressure-Lowering Effect of Wine Lees Phenolic Compounds Is Mediated by Endothelial-Derived Factors: Role of Sirtuin 1.

The antihypertensive effect of wine lees powder (WLPW) from a Cabernet grape variety was related to its high content in flavanols and anthocyanins compounds. This study investigates the involvement of endothelial-derived factors and SIRT1 in its bioactivity. Spontaneously hypertensive rats (SHR) were orally administered water or WLPW (125 mg/kg bw). Posteriorly, both groups were intraperitoneally administered saline, Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthesis inhibitor, indomethacin, a prostacyclin synthesis inhibitor, or sirtinol, an inhibitor of sirtuins. Blood pressure (BP) was recorded before and 6 h after WLPW administration. In an additional experiment, SHR were administered water or WLPW and endothelial expressions of eNos, Sirt1, Nox4, and Et1 were determined. The BP-lowering properties of WLPW were abolished by L-NAME and partially reduced by indomethacin, demonstrating that WLPW antihypertensive effect was mediated by changes in NO availability, although prostacyclin also contributed to this activity. Moreover, BP-lowering effect was reduced by sirtinol, indicating that WLPW decreased BP in a SIRT1-dependent manner. Furthermore, WLPW upregulated eNos and Sirt1 and downregulated Nox4 and Et1 endothelial gene expression. These results evidence the vasoprotective effect of WLPW and show that its antihypertensive effect in SHR is endothelium dependent and mediated by SIRT1.

Downregulation of HIF-2α Enhances Apoptosis and Limits Invasion in Human Placental JEG-3 Trophoblast Cells.

Pre-eclampsia, one of the major disorders of pregnancy, is characterized by inadequate trophoblast invasion and defective trophoblast-mediated remodeling of placental vasculature. Hypoxia-inducible transcription factor (HIF)-2α plays a critical role in regulating cellular function of trophoblasts; however, its role in placental development and in the pathogenesis of pre-eclampsia remains elusive. CCK-8 assay was used to detect cell viability. Invasion assay was performed to determine the effect of HIF-2α on trophoblast function. Flow cytometry was used for detecting apoptosis and cell cycle. The mRNA and protein expressions of HIF-2α, VEGF, iNOS, and ET-1 were determined by quantitative real-time PCR and western blot techniques. The roles of HIF-2α in JEG-3 trophoblast cells were examined using siRNA technology. The presence of HIF-2α siRNA reduced the levels of cell viability after 48 h incubation, and the cell viability further reduced at 72 h. Besides, HIF-2α siRNA enhanced trophoblast apoptosis, as determined by flow cytometric measurement. Increased G1-phase and decreased S-phase cell population were induced by HIF-2α siRNA based on the determination of cell cycle distribution using propidium iodide staining. Furthermore, the invasive ability of JEG-3 trophoblasts was significantly reduced by HIF-2α siRNA. In addition, knockdown of the HIF-2α gene significantly decreased VEGF, iNOS, and ET-1 levels in JEG-3 human trophoblasts. Our findings provide preliminary evidence of the functions of HIF-2α in trophoblast biology and suggest that the downregulation of HIF-2α enhances cell apoptosis and limits trophoblast invasion.

Empagliflozin Inhibits IL-1β-Mediated Inflammatory Response in Human Proximal Tubular Cells.

SGLT2 inhibitor-related nephroprotection is—at least partially—mediated by anti-inflammatory drug effects, as previously demonstrated in diabetic animal and human studies, as well as hyperglycemic cell culture models. We recently presented first evidence for anti-inflammatory potential of empagliflozin (Empa) under normoglycemic conditions in human proximal tubular cells (HPTC) by demonstrating Empa-mediated inhibition of IL-1β-induced MCP-1/CCL2 and ET-1 expression on the mRNA and protein level. We now add corroborating evidence on a genome-wide level by demonstrating that Empa attenuates the expression of several inflammatory response genes in IL-1β-induced (10 ng/mL) normoglycemic HPTCs. Using microarray-hybridization analysis, 19 inflammatory response genes out of >30.000 human genes presented a consistent expression pattern, that is, inhibition of IL-1β (10 ng/mL)-stimulated gene expression by Empa (500 nM), in both HK-2 and RPTEC/TERT1 cells. Pathway enrichment analysis demonstrated statistically significant clustering of annotated pathways (enrichment score 3.64). Our transcriptomic approach reveals novel genes such as CXCL8/IL8, LOX, NOV, PTX3, and SGK1 that might be causally involved in glycemia-independent nephroprotection by SGLT2i.

Sex‐Specific Role of Endothelin‐A Receptors in the Cardiac Susceptibility to Ischemia/Reperfusion Insult in Adult Offspring Exposed to Prenatal Hypoxia

Introduction Fetal hypoxia, a common consequence of complicated pregnancies, leads to the development of cardiac dysfunction in adult offspring by impairing cardiac tolerance to an ischemia/reperfusion (I/R) insult. Extensive evidence demonstrates that cardiac I/R upregulates the endothelin-1 (ET-1) system leading to elevated levels of ET-1 (a peptide with potent physiological and pathophysiological effects on the cardiovascular system). Active ET-1 exerts its effects via the endothelin-A receptors (ETAR; of which enhanced activation has been associated with impaired post-I/R cardiac function) and endothelin-B receptors (ETBR; which are essential for post-I/R cardiac recovery). However, whether activation of ETAR contributes to cardiac dysfunction in adult offspring exposed to prenatal hypoxia is unknown. We hypothesize that, in adult offspring exposed to prenatal hypoxia, cardiac ETAR expression is increased, and its inhibition improves post-I/R cardiac recovery. Methods Pregnant Sprague-Dawley rats (n=4-6 dams/group) were exposed to normoxia (21% O2; normoxia) or hypoxia (11% O2; p-hypoxia) from gestational day 15-21 (term=22 days). Cardiac function was assessed ex vivo in 4-month old male and female offspring (n=1-2 offspring/dam) by subjecting isolated hearts to I/R (25 min. of ischemia with 40 min. of reperfusion). The contribution of ETAR to cardiac dysfunction was assessed by pre-infusion with an ETAR antagonist (ABT-627) before I/R (ABT-627 was recirculated during reperfusion). ETAR and ETBR levels were assessed in non-ischemic left ventricle tissues by Western blotting. Data (mean±SEM) were compared by two-way ANOVA (Sidak's post hoc test), or by unpaired t-test. Results : ABT-627 induced a dramatic reduction in cardiac recovery after I/R in prenatally hypoxic males compared to their respective controls (control: 60.5±16.3% vs ABT-627: 4.4±2.6%, p=0.009), without effect in normoxic males. In contrast, ABT-627 improved I/R recovery in prenatal hypoxia-exposed females (control: 53.5±10.6% vs ABT-627: 87±5.6%, p=0.05) with no effect in normoxic females. In males, exposure to prenatal hypoxia did not alter cardiac ETAR levels, but decreased ETBR levels compared to the normoxic group (normoxia: 100±3.2% vs p-hypoxia: 70.6±4%, p=0.0003), without changes in females. Conclusion Prenatal hypoxia alters the cardiac ET-1 system in a sex-specific manner. Inhibition of ETAR in prenatally hypoxic males impaired cardiac recovery post-I/R, potentially due to lower ETBR expression; while cardiac recovery was improved by ETAR inhibition in females. Activation of ETAR may thus be essential for cardiac tolerance to I/R in prenatally hypoxic males, while it may contribute to the development of cardiac dysfunction in females. Our data suggests that developmental influences and sex differences need to be considered during the implementation of therapeutic strategies to prevent the long-term effects of pregnancy complications on offspring cardiac health.

Therapeutic Effect of Endothelin-Converting Enzyme Inhibitor on Chronic Kidney Disease through the Inhibition of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome.

Chronic inflammation and oxidative stress significantly contribute to the development and progression of chronic kidney disease (CKD). The NOD-like receptor family pyrin containing domain-3 (NLRP3) inflammasome plays a key role in the inflammatory response. The renal endothelin (ET) system is activated in all cases of CKD. Furthermore, ET-1 promotes renal cellular injury, inflammation, fibrosis and proteinuria. Endothelin-converting enzymes (ECEs) facilitate the final processing step of ET synthesis. However, the roles of ECEs in CKD are not clear. In this study, we investigated the effects of ETs and ECEs on kidney cells. We found that ET-1 and ET-2 expression was significantly upregulated in the renal tissues of CKD patients. ET-1 and ET-2 showed no cytotoxicity on human kidney tubular epithelial cells. However, ET-1 and ET-2 caused endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation in tubular epithelial cells. The ECE inhibitor phosphoramidon induced autophagy. Furthermore, phosphoramidon inhibited ER stress and the NLRP3 inflammasome in tubular epithelial cells. In an adenine diet-induced CKD mouse model, phosphoramidon attenuated the progression of CKD by regulating autophagy, the NLRP3 inflammasome and ER stress. In summary, these findings showed a new strategy to delay CKD progression by inhibiting ECEs through autophagy activation and restraining ER stress and the NLRP3 inflammasome.

Tea polyphenols and Levofloxacin alleviate the lung injury of hepatopulmonary syndrome in common bile duct ligation rats through Endotoxin -TNF signaling

Background & AimsHepatopulmonary syndrome (HPS) is characterized by pulmonary vasodilation and arterial blood oxygen desaturation in patients with chronic liver disease. Generally, common bile duct ligation (CBDL) rats are a suitable experimental model for studying hepatopulmonary syndrome. Our previous study demonstrated that endotoxin surges markedly, followed by bacterial translocation and the loss of liver immune function in all the stages of CBDL, thereby contributing to the pathogenesis of HPS. However, the mechanisms behind the increase of the endotoxin and how to alleviate it have not yet been elucidated. Pulmonary injury induced by increased bilirubin, endotoxin, and inflammatory mediators occurs in the early and later stages of CBDL. This study assessed the effects of Tea polyphenols (TP) and Levofloxacin on endotoxin reduction and suppression of lung injury in HPS rats in the long and short term, respectively. MethodsMorphological change of pulmonary injury, HPS relative index, endotoxin concentration, and the activation extent of Malondialdehyde (MDA) and Myeloperoxidase (MPO) were evaluated in CBDL rats with or without TP and Levofloxacin for three weeks or six weeks. The inflammation factors of serum, lung tissue, and BALF were then compared at the same condition for the two time periods. This was followed by adoption of the network pharmacology approach, which was mainly composed of active component gathering, target prediction, HPS gene collection, network analysis, and gene enrichment analysis. Finally, the mRNA and protein levels of the inflammatory factors were studied and relative signaling expression was assessed using RT-PCR and Western blot analysis. ResultsThe obtained results indicated that the pulmonary injury manifestation was perceived and endotoxin, MDA, and MPO activation were markedly increased in the early and later stages of CBDL. TP and Levofloxacin treatment alleviated endotoxin infection and inflammation factor expression three weeks and six weeks after CBDL. In addition, Levofloxacin displayed a short time anti-bacterial effect, while TP exerted a long period function. TP and Levofloxacin also reduced TNF-α, TGF-β, IL-1β, PDGF-BB, NO, ICAM-1, and ET-1 expression on the mRNA or protein expression. With regard to the pharmacological mechanism, the network analysis indicated that 12 targets might be the therapeutic targets of TP and Levofloxacin on HPS, namely ET-1, NOs3, VEGFa, CCl2, TNF, Ptgs2, Hmox1, Alb, Ace, Cav1, and Mmp9. The gene enrichment analysis implied that TP and Levofloxacin probably benefited patients with HPS by modulating pathways associated with the AGE-RAGE signaling pathway, the TNF signaling pathway, the HIF-1 signaling pathway, the VEGF signaling pathway, and the IL-17 signaling pathway, Rheumatoid arthritis, Fluid shear stress, and atherosclerosis. Finally, the TNF-α level was mainly diminished on the protein level following CBDL. ConclusionsTP and Levofloxacin could alleviate pulmonary injury for short and long period, respectively, while at the same time preventing endotoxin and the development of HPS in CBDL rats. These effects are possibly associated with the regulation of the Endotoxin -TNF-α pathways.

Reactive Oxygen Species Are Essential for Vasoconstriction upon Cold Exposure.

Purpose We explored the role of ROS in cold-induced vasoconstriction and corresponding mechanism. Methods Three experiments were performed. First, we measured blood flow in human hands before and after cold exposure. Second, 24 mice were randomly divided into 3 groups: 8 mice received saline injection, 8 received subcutaneous Tempol injection, and 8 received intrathecal Tempol injection. After 30 min, we determined blood flow in the skin before and after cold exposure. Finally, we used Tempol, CCG-1423, and Go 6983 to pretreat HAVSMCs and HUVECs for 24 h. Then, cells in the corresponding groups were exposed to cold (6 h, 4°C). After cold exposure, the cytoskeleton was stained. Intracellular Ca2+ and ROS levels were measured by flow cytometry and fluorescence microscopy. We measured protein expression via Western blotting. Results In the first experiment, after cold exposure, maximum skin blood flow decreased to 118.4 ± 50.97 flux units. Then, Tempol or normal saline pretreatment did not change skin blood flow. Unlike intrathecal Tempol injection, subcutaneous Tempol injection increased skin blood flow after cold exposure. Finally, cold exposure for 6 h shrank the cells, making them narrower, and increased intracellular Ca2+ and ROS levels in HUVECs and HAVSMCs. Tempol reduced cell shrinkage and decreased intracellular Ca2+ levels. In addition, Tempol decreased intracellular ROS levels. Cold exposure increased RhoA, Rock1, p-MLC-2, ET-1, iNOS, and p-PKC expression and decreased eNOS expression. Tempol or CCG-1423 pretreatment decreased RhoA, Rock1, and p-MLC-2 levels in HAVSMCs. Furthermore, Tempol or Go 6983 pretreatment decreased ET-1, iNOS, and p-PKC expression and increased eNOS expression in HUVECs. Conclusion ROS mediate the vasoconstrictor response within the cold-induced vascular response, and ROS in blood vessel tissues rather than nerve fibers are involved in vasoconstriction via the ROS/RhoA/ROCK1 and ROS/PKC/ET-1 pathways in VSMCs and endothelial cells.

AGEs/RAGE blockade downregulates Endothenin-1 (ET-1), mitigating Human Umbilical Vein Endothelial Cells (HUVEC) injury in deep vein thrombosis (DVT)

ABSTRACT This study is aimed at identifying the roles of AGE/RAGE and ET-1 in deep vein thrombosis (DVT). Advanced glycation end products (AGEs) in glycated human serum albumin (M-HSA) were detected by ELISA. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis, followed by ELISA for the detection of inflammatory cytokine level and oxidative stress level in HUVECs. Immunofluorescence was performed to detect ET-1 and eNOS expression. The expression of specific proteins was assayed by western blot. As a result, decreased HUVEC viability was observed after stimulation with M-HSA, whereas RAGE inhibitor improved it. Cell apoptosis showed the opposite trend. Additionally, M-HSA-induced inflammatory cytokine release and oxidative stress of HUVECs were both alleviated by RAGE inhibitor. RAGE inhibitor also increased the levels of NO and eNOS while decreasing the level of ET-1 in M-HSA-stimulated HUVECs. Furthermore, decreased protein expression of Bax, cleaved-caspase3, RAGE, p65, ET-1 and iNOS was observed after treatment with RAGE inhibitor, in addition to increased protein expression of Bcl-2 and eNOS. In conclusion, blocking AGE/RAGE pathway downregulates ET-1, thereby mitigating HUVEC damage in DVT.

Evaluation of Propofol in Inhibiting Proliferation of Cardiac Fibroblasts in Angiotensin II-Induced Mouse

The present study was conducted to investigate the molecular mechanism of propofol in inhibiting the proliferation of mouse cardiac fibroblasts (CFs) induced by angiotensin II (Ag II). The ventricles of SPF mice from Kunming were cultured for the second to third generation of CFs under aseptic condition. On the basis of the different adding conditions, the mice were divided into five groups: (1) control group: no drug were added; (2) Ag II group: 100 nmol/L Ag II were added; (3) 10 μmol/L propofol + 100 nmol/L Ag II group; (4) 30 μmol/L propofol + 100 nmol/L Ag II group; (5) 50 μmol/L propofol + 100 nmol/L Ag II group. The effects of propofol on the proliferation of CFs induced by Ag II, the expression of CFs ET-1, the activity of NADPH oxidase and the formation of ROS were analyzed. In addition, our study also explored the potential role of Akt-eNOS-nitric oxide pathway regarding the inhibition of proliferation of Ag II induced CFs by propofol. We found that the proliferation of CFs, the secretion of ET-1, the activity of NADPH oxidase and the level of intracellular ROS and fibronectin expression were significantly increased after CFs exposure to Ag II for 24 h. The abovementioned indexes decreased significantly in CFs after treated with propofol for 24 h (10, 30, or 50 μmol/L) with significant statistical difference (P < 0.05). Akt and eNOS siRNA transfection significantly decreased the levels of Akt and eNOS protein, respectively. Blocking pathway of Akt-eNOS-nitric oxide decreased the inhibitory effect of propofol on Ag II-induced cell proliferation of CFs. Propofol exerts effect in inhibiting ET-1 and fibronectin expression and the formation of ROS induced by Ag II. Moreover, Akt-eNOS-nitric oxide signaling pathway may be involved in the effect of propofol on the proliferation of CFs induced by Ag II.

MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 μg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 μg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3'UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

Intermedin Alleviates Renal Ischemia-Reperfusion Injury and Enhances Neovascularization in Wistar Rats.

BackgroundIschemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and increases the risk of subsequently developing chronic kidney disease. Angiogenesis has been shown to play an important role in reducing renal injury after ischemia reperfusion. In this study, we investigated whether IMD could reduce renal IRI by promoting angiogenesis.MethodsThe kidneys of Wistar rats were subjected to 45 min of warm ischemia followed by 24 h of reperfusion. IMD was overexpressed in vivo using the vector pcDNA3.1-IMD transfected by an ultrasound-mediated system. The renal injury after ischemia reperfusion was assessed by detection of the serum creatinine concentration and histologic examinations of renal tissues stained by PAS and H&E. Real-time PCR and Western blotting were used to determine the mRNA and protein levels, respectively. Histological examinations were used to assess the expression of CD31, MMP2, MMP9, ET-1, VEGF and VEGFR2 in tissues.ResultsRenal function and renal histological damage were significantly ameliorated in IMD-transfected rats after ischemia reperfusion. Compared to the IRI, IMD significantly promoted angiogenesis. IMD also upregulated the protein and mRNA expression levels of VEGF and VEGFR2 and downregulated the expression level of MMP2, MMP9 and ET-1.ConclusionIMD could protect the kidney after renal ischemia-reperfusion injury by promoting angiogenesis and reducing the destruction of the perivascular matrix.

SIRT1-mediated deacetylation of NF-κB inhibits the MLCK/MLC2 pathway and the expression of ET-1, thus alleviating the development of coronary artery spasm.

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that causes total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR, and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation of VSMCs and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, 5-ethynyl-2'-deoxyuridine (EdU) assay, RT-qPCR, and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and upregulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway, and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.NEW & NOTEWORTHY Rat coronary artery spasm models exhibit reduced expression of SIRT1. Overexpression of SIRT1 inhibits contraction and proliferation of VSMCs. SIRT1 inhibits NF-κB through deacetylation to modulate VSMC contraction and proliferation. NF-κB activates the MLCK/MLC2 pathway. NF-κB upregulates ET-1 to modulate VSMC contraction and proliferation.

Abstract P001: Diurnal Dysfunction Of The Renal Endothelin System Is Associated With Obesity-induced Hypertension In Rats

Loss of endothelin (ET) control of Na + reabsorption results in salt-sensitive hypertension. Diet-induced obesity such as via high fat (HF) diet can produce salt-sensitive hypertension. The goal of this study was to determine if obesity produced by HF diet impairs the renal ET system. Male SD rats were fed either HF (45 Kcal% fat) or normal fat (10 Kcal% fat, NF) diet at 6 weeks of age and for 8 weeks duration. Telemetry transmitters were implanted for systolic blood pressure (SBP) and activity monitoring. During the last 2 weeks of the diet, HF and NF rats were sub-divided into 2 groups to receive an additional high salt (4% NaCl, HS) diet or continue on normal salt (0.3% NaCl, NS) diet. Urine was collected and food and water intakes monitored in 12-hr increments the final 2 days. All groups maintained a diurnal BP pattern. However, rats on HF had significantly higher SBP compared to NF (146 ± 1 vs 138 ± 2 mmHg during the active period, p=0.003, n= 12/group). 12-hr urine volume or Na + excretion values were similar between groups. However, urinary ET-1, a measure of intrarenal ET-1 production, was significantly lower in the HF compared to NF controls (0.92 ± 0.15 vs 1.52 ± 0.09 pg/12hrs, p= 0.0007, n= 12/group). Relative mRNA expression of ET-1 in the renal cortex collected at ZT12 was significantly lower in the HF vs. NF (0.8 ± 0.1 vs 1.3 ± 0.2, p= 0.008, n= 6/group). A similar reduction of ET-1 mRNA was observed in the OM of HF rats. However, there was no change in ET-1 mRNA in the IM of HF rats compared to NF (0.8 ± 0.1 vs 1.0 ± 0.2, p= 0.28, n= 8/group). HS diet led to a further increase in SBP in HF vs the HF/NS group (152 ± 2 vs 147 ± 1 mmHg, p=0.04, n= 8/group) while addition of HS to the NF group showed no significant increase in SBP (145 ± 2 vs 140 ± 3 mmHg, p=0.37 n= 6/group). The NF/HS group had a significant increase in active-time ET-1 excretion compared to NF/NS (4.0 ± 0.4 vs 1.5 ± 0.2 pg/12hrs, p&lt;0.0001, n= 6/group). ET-1 excretion was elevated in the HF/HS vs HF/NS rats (2.6 ± 0.3 vs 1.1 ± 0.1 pg/12hrs, p= 0.02, n= 8/group), but was significantly lower than NF/HS rats (4.0 ± 0.4 pg/12hrs p=0.009). These findings demonstrate that HF diets impair the renal ET-1 system and are consistent with our hypothesis that loss of renal ET-1 contributes to salt-sensitive hypertension in diet-induced obesity.

Local renal complement activation mediates immune kidney injury by inducing endothelin-1 signalling and inflammation in trichloroethylene-sensitised mice

Trichloroethylene (TCE) is a widely used industrial solvent that causes trichloroethylene hypersensitivity syndrome (THS) with multi-system damage, including kidney injury. Clinical studies have shown that the complement system is important for TCE-induced kidney injury. Our previous study found excessive deposition of complement C3, mainly on the glomerulus, indicating that local renal complement is activated after TCE sensitisation. However, whether local renal complement activation mediates TCE-induced immune kidney injury and the underlying mechanisms remain unknown. Therefore, we established a TCE percutaneous sensitisation BALB/c mouse model to explore the mechanisms by pretreating with or without the complement activation antagonist, cathepsin L inhibitor (CatLi). As expected, more C3 and C3a were detected mainly on glomerulus of TCE positive sensitisation (TCE+) mice. Renal dysfunction and pathological damage were also clearly observed in TCE+ mice. Moreover, the mRNA and protein expression of ET-1 increased significantly with local renal complement activation after TCE sensitisation, leading to cytokines release and inflammation. In addition, activation of p38MAPK and NF-κBp65 pathways were detected in kidneys of TCE+ mice, and CatLi pretreatment decreased these changes through complement activation antagonisation. Our research uncovered a novel role of local renal complement activation during immune kidney injury after TCE sensitisation through induction of ET-1 signalling and inflammation.

Endocan, ET-1, and ETAR Expression Profiles in Unicystic Ameloblastoma, Multicystic Ameloblastoma, and Ameloblastic Carcinoma

Background: Odontogenic lesions range from simple cysts to benign tumors and carcinomas. Unicystic ameloblastoma is a monocystic lesion and a less aggressive tumor compared to multicystic ameloblastoma. Ameloblastic carcinoma is the malignant variant of the multicystic ameloblastoma and may arise de novo or from malignant transformation of a long-standing multicystic ameloblastoma. Methods:We collected 54 tissue samples obtained from patients from 2000-2017 that were stored in the archives section of the Pathology Department of Taleghani Educational Hospital, Tehran, Iran. The specimens were processed for immunohistochemistry analysis. Immunostaining of the markers was assessed via quantitative methods. Statistical analysis was performed using one-way ANOVA and the chisquare test. Results: One-way ANOVA analysis and the chi-square test did not reveal any statistically significant differences between the expression levels of endocan, ET-1, and ETAR and lesion type. A positive correlation existed between ET-1 and ETAR expression levels in unicystic ameloblastoma and multicystic ameloblastoma (Pearson’s r = 0.506, P<0.002), and between ET-1 and ETAR expression levels in unicystic ameloblastoma and ameloblastic carcinoma (Pearson’s r = 0.376, P<0.024). Conclusion: This study revealed a positive correlation between the histological degree of lesion and endocan, ET-1, and ETAR expression levels. Hence, it might suggest that multicystic ameloblastoma develops from unicystic ameloblastoma. Over time, multicystic ameloblastoma may undergo a malignant transformation to ameloblastic carcinoma. Possibly, a simple cystic neoplasm can progress to a cystic lesion with invasion to the connective tissue wall and a gradual development into a true neoplasm (multicystic ameloblastoma), with potential for malignant transformation (ameloblastic carcinoma). Endocan, ET-1, and ETAR can be used as prognostic biomarkers for different variants of multicystic ameloblastoma and possible new targets for cancer therapy.