Upregulation Of Endothelin-1 Research Articles

Catecholamine treatment induces reversible heart injury and cardiomyocyte gene expression

BackgroundCatecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved.ResultsC57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment.ConclusionsThe results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment.

Effect of Endothelin-1 on Resting Blood Pressure and the Blood Pressure Response to Hypoxia in Healthy Young Men

Objectives: Upregulation of endothelin-1 (ET-1) and its receptors have been linked to increases in hypoxic sensitivity of the carotid chemoreceptors and the development of hypertension. As such, chemoreflex sensitization has been shown in pre-clinical models to be attenuated by ET-1 receptor blockade. Herein, we sought to assess a role for ET-1 in the maintenance of resting blood pressure (BP) in healthy young men and determine whether chemoreflex control of BP could be altered by ET-1 blockade. We hypothesized endothelin receptor antagonism with oral bosentan would lower resting BP in healthy young men as well as the acute BP response to hypoxia. Methods: Twenty-four healthy young men (31±5 yrs, 26±3 kg/m2) completed two study visits (control, bosentan) separated by a minimum of 1 week. The nonspecific endothelin receptor antagonist bosentan (62.5 mg) was taken by mouth twice daily for 3 days prior to the second study visit. On each visit, beat-by-beat BP was assessed under 3 inspiratory air conditions: 1) steady-state normoxia (FiO2 0.21), 2) chemoreflex excitation produced by acute, graded hypoxia (FiO2 0.05 – 0.21), 3) chemoreflex inhibition elicited by transient hyperoxia (FiO2 1.00). Results: Oral bosentan treatment resulted in an increase in plasma ET-1 (0.9±0.9 to 1.3±0.6 pg/mL, p=0.004), supporting receptor blockade. Resting diastolic and mean BP were reduced following 3 days of bosentan treatment compared to control (diastolic: 73±5 to 69±7 mmHg, p=0.007; mean: 93±7 to 88±7 mmHg, p=0.005) with no change in systolic BP (p=0.507). Notably, the BP response to both acute hypoxia (-0.48±0.38 to -0.25±0.31 mmHg/%, p=0.004) and hyperoxia (main effect of bosentan, p=0.025) were attenuated following bosentan. Conclusions: Acute oral bosentan treatment resulted in a reduction in resting BP in healthy young men. The effect of endothelin receptor inhibition with bosentan further attenuated the rise in BP during chemoreflex excitation via acute, graded hypoxia as well as the fall in BP during chemoreflex inhibition (transient hyperoxia). Together these data support a role for ET-1 in control of resting BP in healthy young men, possibly through a chemoreceptor-mediated mechanism. NIH HL130339 (JL), Mayo Clinic Center for Biomedical Discovery (JL, SB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of acupuncture on pulmonary function and airway smooth muscle spasm in asthma rats

To observe the effects of acupuncture at "Kongzui" (LU 6) and "Yuji" (LU 10) on the latent period of inducing asthma, pulmonary function and the expression of endothelin-1 (ET-1) and metallothionein-2 (MT-2) in asthma rats, and to explore the possible mechanism of acupuncture in alleviating airway smooth muscle spasm and improving the acute attack of asthma. A total of 40 male SD rats of SPF-grade were randomly divided into a normal group, a model group, a medication group and an acupuncture group, 10 rats in each group. Except for the normal group, ovalbumin sensitization method was used to establish the asthma model in the other 3 groups. Salbutamol nebulization was adopted in the medication group, while acupuncture was applied at bilateral "Kongzui" (LU 6) and "Yuji" (LU 10) in the acupuncture group. The intervention was given once a day for 14 days in the two groups. The latent period of inducing asthma and pulmonary function were observed, the levels of ET-1 and tumor necrosis factor (TNF)-α in serum and bronchoalveolar lavage fluid (BALF) were detected by ELISA method, the morphology of the airway was observed by Masson staining, the ultrastructure of the airway smooth muscle was observed by transmission electron microscopy, the mRNA and protein expression of ET-1 and MT-2 in lung tissue was detected by real-time PCR and Western blot methods. Compared with the normal group, in the model group, the latent period of inducing asthma was shortened (P<0.01); the airway resistance (RL) was increased while the dynamic compliance (Cdyn) was decreased (P<0.01, P<0.05); the levels of ET-1 and TNF-α in serum and BALF were increased (P<0.01); collagen fibers and collagen depositions were found around the bronchi, airway smooth muscle was thickened, the cell damage was severe and mitochondria were swollen; the mRNA and protein expression of ET-1 was increased while the mRNA and protein expression of MT-2 was decreased (P<0.01). Compared with the model group, in the acupuncture group, the latent period of inducing asthma was prolonged (P<0.05), the RL was decreased while the Cdyn was increased (P<0.01, P<0.05). Compared with the model group, in the medication group and the acupuncture group, the levels of ET-1 and TNF-α in serum and BALF were decreased (P<0.01, P<0.05); collagen fibers and collagen depositions around the bronchi were reduced, the thickened airway smooth muscle was lightened, the cell damage was improved; the mRNA and protein expression of ET-1 was decreased while the mRNA and protein expression of MT-2 was increased (P<0.01). Compared with the medication group, the mRNA expression of MT-2 was increased in the acupuncture group (P<0.05). Acupuncture at "Kongzui" (LU 6) and "Yuji" (LU 10) can improve the pulmonary function and alleviate the airway smooth muscle spasm in rats with asthma. Its mechanism may be related to the down-regulation of ET-1 expression and up-regulation of MT-2 expression.

Dual Blockade of TGF-β Receptor and Endothelin Receptor Synergistically Inhibits Angiotensin II-Induced Myofibroblast Differentiation: Role of AT1R/Gαq-Mediated TGF-β1 and ET-1 Signaling.

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-β1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-β1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-β1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT1R/Gαq axis in adult human cardiac fibroblasts (HCFs). Following AT1R stimulation, Gαq protein, not Gβγ subunit, was required for upregulation of TGF-β1 and ET-1. Moreover, dual inhibition of TGF-β and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT1R/Gαq cascade transduced signals to TGF-β1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ETAR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-β receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-β1 and ET-1 are major effectors of AT1R/Gαq cascade, and therefore, negative regulation of TGF-β and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.

Fibrocytes boost tumor-supportive phenotypic switches in the lung cancer niche via the endothelin system

Fibrocytes are bone marrow–derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies.

Ufl1 deficiency causes skin pigmentation by up-regulation of Endothelin-1.

Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.

Acid-Mediated Kidney Injury Across the Spectrum of Metabolic Acidosis.

Metabolic acidosis affects about 15% of patients with chronic kidney disease. As kidney function declines, the kidneys progressively fail to eliminate acid, primarily reflected by a decrease in ammonium and titratable acid excretion. Several studies have shown that the net acid load remains unchanged in patients with reduced kidney function; the ensuing acid accumulation can precede overt metabolic acidosis, and thus, indicators of urinary acid or potential base excretion, such as ammonium and citrate, may serve as early signals of impending metabolic acidosis. Acid retention, with or without overt metabolic acidosis, initiates compensatory responses that can promote tubulointerstitial fibrosis via intrarenal complement activation and upregulation of endothelin-1, angiotensin II, and aldosterone pathways. The net effect is a cycle between acid accumulation and kidney injury. Results from small- to medium-sized interventional trials suggest that interrupting this cycle through base administration can prevent further kidney injury. While these findings inform current clinical practice guidelines, large-scale clinical trials are still necessary to prove that base therapy can limit chronic kidney disease progression or associated adverse events.

Endothelin-1 response to whole-body vibration in obese and normal weight individuals.

Upregulation of endothelin‐1 (ET‐1) is the hallmark of various cardiovascular diseases (CVD). The purpose of the present study was to assess the ET‐1 response to an acute bout of whole‐body vibration (WBV) in humans and to determine the role of adiposity. Twenty‐two participants volunteered for the study; they were grouped into overweight/obese [(OW/OB): n = 11, Age: 33 ± 4 years, Body mass index (BMI): 35 ± 10 kg/m2] or normal weight [(NW): n = 11, Age: 28 ± 7 years, BMI: 21 ± 2 kg/m2]. Participants engaged in 10 cycles of WBV exercise (1 cycle = 1 min WBV followed by 30 s of rest). Blood samples were analyzed for ET‐1 pre‐WBV (PRE), immediately post (POST), 1 h (1H), 3 h (3H), and 24 h (24H) post‐WBV. There was a significant time main effect of WBV on circulating ET‐1 (F = 12.5, p < 0.001); however, the ET‐1 response was similar (F = 0.180, p = 0.677) between groups. Specifically, compared to PRE, a significant increase in ET‐1 was observed at 1H (p = 0.017) and 3H (p = 0.025). In addition, concentrations of ET‐1 were significantly lower at 24H compared to PRE (p = 0.019), 1H (p < 0.001), and 3H (p < 0.001). Maximal oxygen uptake during WBV was similar between the two groups. Acute WBV resulted in an initial rise in ET‐1, followed by a significantly lower ET‐1 at 24H in both groups. Findings support the utility of routine WBV exercise to elicit a decrease in ET‐1 and improve CVD risk, similar to what has been reported with traditional modes of exercise.

Abstract 8859: Hematopoietic Calr Frameshift Mutation Exacerbates Hypoxia-Induced Pulmonary Hypertension in Mice

Introduction: Calreticulin ( CALR ) frameshift is the second frequent driving mutation in myeloproliferative neoplasms (MPNs). MPNs are complicated with pulmonary hypertension (PH), but the mechanistic relevance of the CALR mutations on PH has not yet been clarified. Methods and Results: We generated a 10-bp deletion knock-in mice with murine Calr exon 9 mimicking the human CALR mutations. To investigate whether the hematopoietic CALR mutation contributes to PH, a bone marrow transplantation (BMT) was applied using the donor BM cells from Calr knock-in mice or control wild-type (WT) mice. Four weeks after BMT, the recipient mice transplanted with BM cells from Calr knock-in mice (Del-R) and WT mice (WT-R) were exposed to chronic hypoxia. Three weeks after starting chronic hypoxia exposure, the right ventricular systolic pressure and right ventricle/left ventricle-plus-septum weight ratio in the Del-R mice were significantly increased compared to the WT-R mice. Lung histology showed an acceleration of pulmonary arterial muscularization and medial wall thickness in Del-R mice after chronic hypoxia. Increases in macrophage infiltration was similarly observed in both WT-R and Del-R mice after chronic hypoxia. Endothelin-1 expression and STAT3 phosphorylation were significantly upregulated in the lung of Del-R mice compared to the WT-R mice in response to chronic hypoxia. Using Calr Del/CAG-EGFP double transgenic mice, we visualized the accumulations of donor-derived macrophages in pulmonary arterial regions. BM-derived macrophages carrying the Calr mutation showed increases in endothelin-1 expression, indicating that BM-derived macrophages with the Calr mutation played important roles in the pulmonary arterial remodeling. Conclusion: The frameshifted Calr mutations presented a novel mechanism of PH through BM-derived macrophages accompanied by upregulation of endothelin-1.

Testosterone attenuates hypoxia-induced hypertension by affecting NRF1-mediated transcriptional regulation of ET-1 and ACE.

Hypertension induced by hypoxia at high altitude is one of the typical symptoms of high-altitude reactions (HARs). Emerging evidence indicates that endothelial abnormalities, including increases in angiotensin-2 (Ang-2) and endothelin-1 (ET-1), are closely associated with hypertension. Thus, low blood oxygen-induced endothelial dysfunction through acceleration of Ang-2 and ET-1 synthesis may alleviate HARs. In this study, we investigated the effects of hypoxia on rat blood pressure (BP) and endothelial injury. We found that BP increased by 10 mmHg after treatment with 10% O2 (~5500 m above sea level) for 24 h. Consistently, serum Ang-2 and ET-1 levels were increased along with decreases in NO levels. In endothelial cells, angiotensin-1-converting enzyme (ACE) and ET-1 expression levels were upregulated. Interestingly, nuclear respiratory factor 1 (NRF1) levels were also upregulated, consistent with the changes in ACE and ET-1 levels. We further demonstrated that NRF1 transcriptionally activated ACE and ET-1 by directly binding to their promoter regions, suggesting that the endothelial cell dysfunction induced by hypoxia was due to NRF1-dependent upregulation of ACE and ET-1. Surprisingly, testosterone supplementation showed significant protective effects on BP, while castration induced even higher BPs in rats exposed to hypoxia. We further showed that physiological testosterone repressed NRF1 expression in vivo and in vitro and thereby reduced Ang-2 and ET-1 levels, which was dependent on hypoxia. In summary, we have identified that physiological testosterone protects against hypoxia-induced hypertension through inhibition of NRF1, which transcriptionally regulates ACE and ET-1 expression.

Role of mitochondrial dynamics in the response of endothelial cells to shear stress during early phase of atherosclerosis

Mitochondria are dynamic organelles altering their inner and outer membranes through fission and fusion mechanisms. In response to shear stress, the mitochondria in endothelial cells (ECs) continuously undergo fusion and fission mechanisms. We hypothesized that alteration in mitochondrial dynamics could alter ECs function leading to vascular damages. For that, we investigated the role of OPA1 and DRP1 deficiency in ECs and vascular homeostasis using mice lacking fusion protein OPA1 and fission protein DRP1(heterozygous Opa1 ± & Drp1 ± mice). We investigated mitochondrial dynamics-related proteins expression level in ECs submitted to a disturbed shear stress (evidenced by eNOS down-regulation and endothelin-1 up-regulation) and observed a reduction in fusion protein OPA1 mRNA expression and an increase in fission protein DRP1 mRNA expression level. The opposite was observed in ECs submitted to disturbed shear stress. After acute stimulation of ECs to Histamine, cells lacking OPA1 and DRP1 protein have shown a decreased intracellular Ca2+ release, compared to control cells. Besides, these cells showed a decreased capability of migration compared to control cells. As shear stress determines atherogenesis in large arteries, we investigated mitochondrial dynamics-related proteins in various areas in the mouse aorta. In the areas submitted to low shear stress (inner part of the aortic cross), OPA1 mRNA expression was reduced and DRP1 mRNA expression was increased compared to aortic areas subjected to high shear stress. These observations led to investigate atherosclerosis in Opa1± and Drp1 ± mice. These mice, when crossed with Ldlr−/− mice and fed high fat diet, develop more atherosclerosis in Opa1 ± and less in Drp1 ± mice compared to WT. Thus, disregulation in mitochondrial dynamics proteins altered endothelium dependent functions in atherosclerotic mice, and increasing plaque formation with reduced fusion.

Pathological Hyperinsulinemia and Hyperglycemia in the Impaired Glucose Tolerance Stage Mediate Endothelial Dysfunction Through miR-21, PTEN/AKT/eNOS, and MARK/ET-1 Pathways.

BackgroundImpaired glucose tolerance (IGT) is an important prediabetic stage characterized by elevated concentrations of glucose and insulin in the blood. The pathological hyperglycemia and hyperinsulinemia in IGT may regulate the expression of microRNA-21 (miR-21) and affect the downstream insulin signaling pathways, leading to endothelial cell dysfunction and early renal damage.MethodsThe individual and combined effects of insulin and glucose were investigated using human glomerular endothelial cells (HGECs). The expression levels of miR-21, and PTEN/AKT/eNOS and MAPK/ET-1 pathway proteins in the treated cells were measured. The levels of nitric oxide (NO) and endothelin-1 (ET-1) secreted by the cells were also measured. The role of miR-21 in mediating the regulatory effects of insulin and glucose was assessed by overexpression/inhibition of this miRNA using mimics/inhibitor.ResultsHigh (>16.7 mmol/L) concentration of glucose upregulated the expression of miR-21, leading to the activation and inhibition of the PTEN/AKT/eNOS and MAPK/ET-1 pathways, and upregulation of NO and downregulation of ET-1 secretion, respectively. High (>25 ng/mL) concentration of insulin downregulated the expression of miR-21, and lead to the activation of the MAPK/ET-1 and inhibition of the PTEN/AKT/eNOS pathway, thereby upregulating the expression of ET-1 and downregulating the secretion of NO. MiR-21 was observed to play a key role by directly controlling the activation of the insulin signaling pathways when the cells were cotreated with different concentrations of insulin and glucose. The expression of miR-21 was found to be dependent on the relative concentration of insulin and glucose. Under simulated conditions of the IGT stage (8.3 mmol/L glucose + 50 ng/mL insulin), the inhibitory effect of high insulin concentration on miR-21 expression in the cells attenuated the activation by high glucose concentration, resulting in the downregulation of miR-21, upregulation of ET-1 and downregulation of NO secretion.ConclusionTaken together, these results indicate that high insulin and glucose concentrations regulate the secretory function of glomerular endothelial cells in opposite ways by regulating the expression of miRNA-21. Pathological concentrations of insulin and glucose in the IGT stage may lead to a decrease in miR-21 expression, thereby disordering the secretion of vasoactive factors, resulting in renal tubule ischemia.

Effect of Shuangdan Mingmu capsule, a Chinese herbal formula, on oxidative stress-induced apoptosis of pericytes through PARP/GAPDH pathway

BackgroundDiabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway.MethodsNetwork pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC).ResultsOverproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor.ConclusionsSDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

HIF-1α Enhances Vascular Endothelial Cell Permeability Through Degradation and Translocation of Vascular Endothelial Cadherin and Claudin-5 in Rats With Burn Injury.

The mechanism underlying burn injury-induced enhanced vascular endothelial permeability and consequent body fluid extravasation is unclear. Here, the rat aortic endothelial cells (RAECs) were treated with the serum derived from rats with burn injury to elucidate the mechanism. Sprague-Dawley (SD) rats were grouped as follows (10 rats/group): control, 2, 4, 8, 12, and 24 hours postburn groups. The heart, liver, kidney, lung, jejunum, and ileum of rats injected with 2% Evans blue (EB) through the tail vein were excised to detect the EB level in each organ. The serum levels of hypoxia-inducible factor-1α (HIF-1α) and endothelin-1 (ET-1) were examined using enzyme-linked immunosorbent assay (ELISA). The effect of serum from 12-hour postburn group on the membrane permeability of RAEC monolayer, as well as on the mRNA and protein levels of ET-1, endothelin receptor A (ETA), ETB, and zonula occludens (ZO-1), was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The membrane permeability of GV230/HIF-1α-transfected or shRNA-HIF-1α-transfected RAECs, as well as the expression levels of HIF-1α, ET-1, ETA, ETB, vascular endothelial (VE)-cadherin, and claudin-5, was analyzed using qRT-PCR and western blotting, whereas the localization of VE-cadherin and claudin-5 was examined using immunofluorescence. The serum HIF-1α and ET-1 levels in the burn groups, which peaked at 12 hours postburn, were significantly upregulated (P < .01) when compared with those in the control group. Additionally, the serum HIF-1α levels were positively correlated with vascular permeability. Compared with the shRNA-negative control-transfected RAECs, the shRNA-II/HIF-1α-transfected RAECs exhibited downregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and upregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). Compared with the GV230-transfected RAECs, the GV230/HIF-1α-transfected RAECs exhibited upregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and downregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). The GV230/HIF-1α-transfected RAECs exhibited degradation and translocation of VE-cadherin and claudin-5. In addition to degradation of VE-cadherin and claudin-5, HIF-1α mediated enhanced endothelial cell permeability through upregulation of ET-1, ETA, and ETB, and downregulation of ZO-1 and VE-cadherin in rats with burn injury.

Modulation of Cytokine-Induced Astrocytic Endothelin-1 Production as a Possible New Approach to the Treatment of Multiple Sclerosis.

Background: In the human central nervous system (CN), resting astrocytes do not visually show endothelin-1 (ET-1)-like immunoreactivity. In patients with multiple sclerosis (MS), an inflammatory disorder of the CNS, high levels of ET-1 are found in reactive astrocytes in demyelinated plaques. ET-1 may contribute to the pathology of MS by interrupting the blood-brain-barrier, enhancing inflammatory responses, excitotoxicity and reducing cerebral blood flow. Methods: We used the human astrocytoma cell line 1321N1 to investigate the role of inflammatory cytokines involved in MS lesions (IL-1β, TNF-α, IFN-γ, LPS, IL-10, TGF-β) on astrocytic ET-1 upregulation. Prucalopride, rolipram, fenofibrate, fluoxetine, simvastatin, daglutril, and resveratrol were investigated as potential candidate drugs to suppress cytokine-induced astrocytic ET-1 production. Effects on ET-1 production were measured using both ELISA and RT-qPCR. Results and Conclusions: ET-1 secretion by astrocytoma cells was only stimulated by the pro-inflammatory cytokines IL-1β and TNF-α. Fluoxetine, simvastatin, and resveratrol significantly inhibited this IL-1β- and TNF-α-induced ET-1 production. Simvastatin and resveratrol significantly reduced ET-1 mRNA levels, indicating an effect at the level of transcription. Fluoxetine significantly reduced endothelin converting enzyme-1 mRNA levels, suggesting and effect at the level of protein-processing. The required concentrations of simvastatin (>0.1 µM) and resveratrol (>10 µM) cannot be achieved in humans using pharmacologically accepted doses. Fluoxetine exerted a significant inhibitory effect on ET-1 secretion at a concentration of 5 µM, which is pharmacologically achievable in human brain, but the effect was modest (<50% suppression) and probably not sufficient to obtain a clinically relevant ET-1 effect. Our in vitro model can be a useful screening tool in the development of new drugs to suppress astrocytic ET-1 production. The effect of simvastatin was for the most part mediated via the mevalonate pathway, suggesting that this might be an interesting target for further drug development.

The G Protein-Coupled Bile Acid Receptor TGR5 (Gpbar1) Modulates Endothelin-1 Signaling in Liver.

TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs), and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype (WT) littermates were fed a diet supplemented with 1% LCA for 84 h. Liver injury and gene expression changes induced by the LCA diet revealed an enrichment of pathways associated with inflammation, proliferation, and matrix remodeling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signaling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs, dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signaling and modulates portal pressure.

Altered mitochondrial dynamics induced endothelial cell dysfunction and increased atherosclerosis in the mouse

Mitochondria are dynamic organelles altering their inner and outer membranes through fission and fusion mechanisms. In a response to shear stress, the mitochondria in endothelial cells (ECs) continuously undergo fusion and fission mechanisms. We hypothesized that alteration in mitochondria dynamic could alter EC functions leading to vascular damage. We investigated the role of Opa1 deficiency in ECs and vascular homeostasis using mice lacking the fusion protein Opa1 (heterozygous Opa1 ± mice). Endothelium-mediated dilatation was lower in Opa1 ± than in Opa1 +/+ mice, without change in vascular contractility in isolated arteries. Flow-mediated ECs alignment was reduced in Opa1± compared to Opa1 +/+ mice. We then investigated mitochondrial dynamics-related proteins expression level in ECs submitted to a disturbed shear stress (evidenced by eNOS down-regulation and endothelin-1 up-regulation) and observed a reduction in fusion protein Opa1 mRNA expression and an increase in fission protein Drp1 mRNA expression level. As shear stress determines atherogenesis in large arteries, we investigated mitochondrial dynamics-related proteins in various areas in the mouse aorta. In the area submitted to low shear stress, Opa1 mRNA expression was reduced and Drp1 mRNA expression was increased compared to other parts of the aorta submitted to higher shear stress. These observations led to investigate atherosclerosis in Opa1± mice. These mice, when crossed with LDLr-/- mice and fed high-fat diet, develop more atherosclerosis than Opa1 +/+ mice. Thus, reduced mitochondrial fusion altered endothelium-dependent functions and exacerbated plaque formation in atherosclerotic mice. And, in order to investigate the mechanism by which the ECs respond to shear stress, we are studying the effect of mitochondrial dynamic proteins on endothelial cells alignment and functions.

Blocking C/EBP β protects vascular endothelial cells from injury induced by intermittent hypoxia.

Intermittent hypoxia (IH) can damage endothelial cells and lead to apoptosis in obstructive sleep apnea-hypopnea syndrome (OSAHS). Hypoxia induces apoptosis in endothelial cells via upregulation of endothelin-1 (ET-1) and hypoxia inducible factor-1 alpha (HIF-1α) plays a key role in the hypoxic stress response. We investigated an approach to diminish the negative effect of HIF-1α while maintaining its protective effect. Human umbilical vein endothelial cells (HUVECs) were subjected to sustained hypoxia (SH) or IH for 24h, and the responses of HIF-1α, CCAAT/enhancer binding protein beta (C/EBP β), and endothelin-1 (ET-1) were assessed by western blotting. A luciferase reporter system was employed to verify the potential binding site (transcription factor binding site, TFBS) for C/EBP β in the ET-1 promoter. The specificity of regulation of ET-1 by HIF-1α via C/EBP β was evaluated by a lentiviral system. The effects of silencing of C/EBP β on IH-induced apoptosis, vascular endothelial growth factor (VEGF) protein levels, proliferation, and in vitro tube formation were studied. We found that IH significantly increased HIF-1α, C/EBP β, and ET-1 in HUVECs. Knockdown of HIF-1α or C/EBP β inhibited the upregulation of ET-1 induced by IH. Blocking C/EBP β impaired IH-induced apoptosis but did not affect VEGF expression, proliferation, or in vitro tube formation. C/EBP β was shown to mediate increased ET-1 transcription by HIF-1α through the TFBS, 5'-GTTGCCTGTTG-3', in ET-1 promoter. Silencing of C/EBP β can suppress apoptosis but does not affect the protective role of HIF-1α in the hypoxic stress response.

Inhaled nitric oxide protects cerebral autoregulation through prevention of impairment of ATP and calcium sensitive K channel mediated cerebrovasodilation after traumatic brain injury

Hypotension and low cerebral perfusion pressure are associated with low cerebral blood flow, cerebral ischemia, and poor outcomes after traumatic brain injury (TBI). Cerebral autoregulation is impaired after TBI, contributing to poor outcome. In prior studies, ERK mitogen activated protein kinase (MAPK) and ET-1 had been observed to be upregulated and contribute to impairment of cerebral autoregulation and histopathology after fluid percussion brain injury (FPI). Activation of ATP and Calcium sensitive (Katp and Kca) channels produce cerebrovasodilation and contribute to autoregulation, both impaired after TBI. Upregulation of ERK MAPK and endothelin-1 (ET-1) produces K channel function impairment after CNS injury. Inhaled nitric oxide (iNO) has recently been observed to prevent impairment of cerebral autoregulation and hippocampal CA1 and CA3 neuronal cell necrosis after FPI via block of upregulation of ERK MAPK and ET-1. We presently investigated whether iNO prevented impairment of Katp and Kca-mediated cerebrovasodilation after FPI in pigs equipped with a closed cranial window. Results show that pial artery dilation in response to the Katp agonist cromakalim, the Kca agonist NS1619, PGE2 and the NO releaser sodium nitroprusside (SNP) were blocked by FPI, but such impairment was prevented by iNO administered at 2 h post injury. Protection lasted for at least 1 h after iNO administration was stopped. Using vasodilaton as an index of function, these data indicate that iNO prevents impairment of cerebral autoregulation and limits histopathology after TBI through protection of K channel function via blockade of ERK MAPK and ET-1.

TSC1 deletion in fibroblasts alleviates lipopolysaccharide-induced acute kidney injury.

Mechanistic target of rapamycin complex 1 (mTORC1) signaling is active in inflammation, but its involvement in septic acute kidney injury (AKI) has not been shown. mTORC1 activation (p-S6) in renal fibroblasts was increased in a mouse AKI model induced by 1.5 mg/kg lipopolysaccharide (LPS). Deletion of tuberous sclerosis complex 1 (TSC1), an mTORC1 negative regulator, in fibroblasts (Fibro-TSC1-/-) inhibited the elevation of serum creatinine and blood urea nitrogen in AKI compared with that in TSC1fl/fl control mice. Endothelin-1 (EDN1) and phospho-Jun-amino-terminal kinase (p-JNK) were up-regulated in Fibro-TSC1-/- renal fibroblasts after LPS challenge. Rapamycin, an mTORC1 inhibitor, and bosentan, an EDN1 antagonist, eliminated the difference in renal function between TSC1fl/fl and Fibro-TSC1-/- mice after LPS injection. Rapamycin restored LPS-induced up-regulation of EDN1, endothelin converting enzyme-1 (ECE1), and p-JNK in TSC1-knockdown mouse embryonic fibroblasts (MEFs). SP600125, a Jun-amino-terminal kinase (JNK) inhibitor, attenuated LPS-induced enhancement of EDN1 and ECE1 in TSC1-knockdown MEFs without a change in phospho-S6 ribosomal protein (p-S6) level. The results indicate that mTORC1-JNK-dependent up-regulation of ECE1 elevated EDN1 in TSC1-knockout renal fibroblasts and contributed to improvement of renal function in Fibro-TSC1-/- mice with LPS-induced AKI. Renal fibroblast mTORC1 plays an important role in septic AKI.