Lycium barbarum glycopeptide promotes neuroprotection in ET-1 mediated retinal ganglion cell degeneration.

BACKGROUND: Vascular dysregulation is one of the major risk factors of glaucoma, and endothelin-1 (ET-1) may have a role in the pathogenesis of vascular-related glaucoma. Fruit extract from Lycium Barbarum (LB) exhibits anti-ageing and multitarget mechanisms in protecting retinal ganglion cells (RGC) in various animal models. To investigate the therapeutic efficacy of LB glycoproteins (LbGP) in ET-1 induced RGC degeneration, LbGP was applied under pre- and posttreatment conditions to an ET-1 mouse model. Retina structural and functional outcomes were characterised using clinical-based techniques. METHODS: Adult C57BL/6 mice were randomly allocated into four experimental groups, namely vehicle control (n = 9), LbGP-Pretreatment (n = 8), LbGP-Posttreatment (day 1) (n = 8) and LbGP-Posttreatment (day 5) (n = 7). Oral administration of LbGP 1 mg/Kg or PBS for vehicle control was given once daily. Pre- and posttreatment (day 1 or 5) were commenced at 1 week before and 1 or 5 days after intravitreal injections, respectively, and were continued until postinjection day 28. Effects of treatment on retinal structure and functions were evaluated using optical coherence tomography (OCT), doppler OCT and electroretinogram measurements at baseline, post-injection days 10 and 28. RGC survival was evaluated by using RBPMS immunostaining on retinal wholemounts. RESULTS: ET-1 injection in vehicle control induced transient reductions in arterial flow and retinal functions, leading to significant RNFL thinning and RGC loss at day 28. Although ET-1 induced a transient loss in blood flow or retinal functions in all LbGP groups, LbGP treatments facilitated better restoration of retinal flow and retinal functions as compared with the vehicle control. Also, all three LbGP treatment groups (i.e. pre- and posttreatments from days 1 or 5) significantly preserved thRNFL thickness and RGC densities. No significant difference in protective effects was observed among the three LbGP treatment groups. CONCLUSION: LbGP demonstrated neuroprotective effects in a mouse model of ET-1 induced RGC degeneration, with treatment applied either as a pretreatment, immediate or delayed posttreatment. LbGP treatment promoted a better restoration of retinal blood flow, and protected the RNFL, RGC density and retinal functions. This study showed the translational potential of LB as complementary treatment for glaucoma management.

CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1.

Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

Activation of ETAR and ETBR in myocardial tissue characterizes heart failure induced by experimental autoimmune myocarditis.

BACKGROUND: Endothelial dysfunction is characterized by an imbalance between endothelium-derived vasodilatory and vasoconstrictive effects and may play an important role in the development of heart failure. An increasing number of studies have shown that endothelial-derived NO-mediated vasodilation is attenuated in heart failure patients. However, the role of endothelin-1 (ET-1) in heart failure remains controversial due to its different receptors including ET-1 receptor type A (ETAR) and ET-1 receptor type B (ETBR). The aim of this study was to determine whether ET-1 and its receptors are activated and to explore the role of ETAR and ETBR in heart failure induced by myocarditis. METHODS: We constructed an animal model of experimental autoimmune myocarditis (EAM) with porcine cardiac myosin. Twenty rats were randomized to the control group (3 weeks, n = 5), the extended control group (8 weeks, n = 5), the EAM group (3 weeks, n = 5), the extended EAM group (8 weeks, n = 5). HE staining was used to detect myocardial inflammatory infiltration and the myocarditis score, Masson's trichrome staining was used to assess myocardial fibrosis, echocardiography was used to evaluate cardiac function, ELISA was used to detect serum NT-proBNP and ET-1 concentrations, and immunohistochemistry and western blotting were used to detect ETAR and ETBR expression in myocardial tissue of EAM-induced heart failure. Subsequently, a model of myocardial inflammatory injury in vitro was constructed to explore the role of ETAR and ETBR in EAM-induced heart failure. RESULTS: EAM rats tended to reach peak inflammation after 3 weeks of immunization and developed stable chronic heart failure at 8 weeks after immunization. LVEDd and LVEDs were significantly increased in the EAM group compared to the control group at 3 weeks and 8 weeks after immunization while EF and FS were significantly reduced. Serum NT-proBNP concentrations in EAM (both 3 weeks and 8 weeks) were elevated. Therefore, EAM can induce acute and chronic heart failure due to myocardial inflammatory injury. Serum ET-1 concentration and myocardial ETAR and ETBR protein were significantly increased in EAM-induced heart failure in vivo. Consistent with the results of the experiments in vivo, ETAR and ETBR protein expression levels were significantly increased in the myocardial inflammatory injury model in vitro. Moreover, ETAR gene silencing inhibited inflammatory cytokine TNF-α and IL-1ß levels, while ETBR gene silencing improved TNF-α and IL-1ß levels. CONCLUSIONS: ET-1, ETAR, and ETBR were activated in both EAM-induced acute heart failure and chronic heart failure. ETAR may positively regulate EAM-induced heart failure by promoting myocardial inflammatory injury, whereas ETBR negatively regulates EAM-induced heart failure by alleviating myocardial inflammatory injury.

Hemodynamics During Development and Postnatal Life.

A well-developed heart is essential for embryonic survival. There are constant interactions between cardiac tissue motion and blood flow, which determine the heart shape itself. Hemodynamic forces are a powerful stimulus for cardiac growth and differentiation. Therefore, it is particularly interesting to investigate how the blood flows through the heart and how hemodynamics is linked to a particular species and its development, including human. The appropriate patterns and magnitude of hemodynamic stresses are necessary for the proper formation of cardiac structures, and hemodynamic perturbations have been found to cause malformations via identifiable mechanobiological molecular pathways. There are significant differences in cardiac hemodynamics among vertebrate species, which go hand in hand with the presence of specific anatomical structures. However, strong similarities during development suggest a common pattern for cardiac hemodynamics in human adults. In the human fetal heart, hemodynamic abnormalities during gestation are known to progress to congenital heart malformations by birth. In this chapter, we discuss the current state of the knowledge of the prenatal cardiac hemodynamics, as discovered through small and large animal models, as well as from clinical investigations, with parallels gathered from the poikilotherm vertebrates that emulate some hemodynamically significant human congenital heart diseases.

Endothelial Dysfunction in Obesity and Therapeutic Targets.

Parallel to the increasing prevalence of obesity in the world, the mortality from cardiovascular disease has also increased. Low-grade chronic inflammation in obesity disrupts vascular homeostasis, and the dysregulation of adipocyte-derived endocrine and paracrine effects contributes to endothelial dysfunction. Besides the adipose tissue inflammation, decreased nitric oxide (NO)-bioavailability, insulin resistance (IR), and oxidized low-density lipoproteins (oxLDLs) are the main factors contributing to endothelial dysfunction in obesity and the development of cardiorenal metabolic syndrome. While normal healthy perivascular adipose tissue (PVAT) ensures the dilation of blood vessels, obesity-associated PVAT leads to a change in the profile of the released adipo-cytokines, resulting in a decreased vasorelaxing effect. Higher stiffness parameter ß, increased oxidative stress, upregulation of pro-inflammatory cytokines, and nicotinamide adenine dinucleotide phosphate (NADP) oxidase in PVAT turn the macrophages into pro-atherogenic phenotypes by oxLDL-induced adipocyte-derived exosome-macrophage crosstalk and contribute to the endothelial dysfunction. In clinical practice, carotid ultrasound, higher leptin levels correlate with irisin over-secretion by human visceral and subcutaneous adipose tissues, and remnant cholesterol (RC) levels predict atherosclerotic disease in obesity. As a novel therapeutic strategy for cardiovascular protection, liraglutide improves vascular dysfunction by modulating a cyclic adenosine monophosphate (cAMP)-independent protein kinase A (PKA)-AMP-activated protein kinase (AMPK) pathway in PVAT in obese individuals. Because the renin-angiotensin-aldosterone system (RAAS) activity, hyperinsulinemia, and the resultant IR play key roles in the progression of cardiovascular disease in obesity, RAAS-targeted therapies contribute to improving endothelial dysfunction. By contrast, arginase reciprocally inhibits NO formation and promotes oxidative stress. Thus, targeting arginase activity as a key mediator in endothelial dysfunction has therapeutic potential in obesity-related vascular comorbidities. Obesity-related endothelial dysfunction plays a pivotal role in the progression of type 2 diabetes (T2D). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (thiazolidinedione), is a popular drug for treating diabetes; however, it leads to increased cardiovascular risk. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin (EMPA) significantly improves endothelial dysfunction and mortality occurring through redox-dependent mechanisms. Although endothelial dysfunction and oxidative stress are alleviated by either metformin or EMPA, currently used drugs to treat obesity-related diabetes neither possess the same anti-inflammatory potential nor simultaneously target endothelial cell dysfunction and obesity equally. While therapeutic interventions with glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide or bariatric surgery reverse regenerative cell exhaustion, support vascular repair mechanisms, and improve cardiometabolic risk in individuals with T2D and obesity, the GLP-1 analog exendin-4 attenuates endothelial endoplasmic reticulum stress.

Effects of Bosentan on Hypoxia, Inflammation and Oxidative Stress in Experimental Blunt Thoracic Trauma Model.

Background and Objectives: In this study, we aimed to investigate the effects of bosentan, an endothelin receptor antagonist, on endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), nuclear factor-kappa B (NF-κB), and tumor necrosis factor (TNF)-α as inflammation markers, pro-oxidant antioxidant balance (PAB), and total antioxidant capacity (TAC) levels as oxidative stress parameters in lung tissues of rats in an experimental model of pulmonary contusion (PC) induced by blunt thoracic trauma. Materials and Methods: Thirty-seven male Sprague-Dawley rats were divided into five groups. C: The control group (n = 6) consisted of unprocessed and untreated rats. PC3 (n = 8) underwent 3 days of PC. PC-B3 (n = 8) received 100 mg/kg bosentan and was given orally once a day for 3 days. The PC7 group (n = 7) underwent 7 days of PC, and PC-B7 (n = 8) received 100 mg/kg bosentan and was given orally once a day for 7 days. Results: ET-1, NF-κB, TNF-α, HIF-1α, and PAB levels were higher, while TAC activity was lower in all groups compared with the control (p < 0.05). There was no significant difference in ET-1 and TNF-α levels between the PC-B3 and PC-B7 groups and the control group (p < 0.05), while NF-κB, HIF-1α, and PAB levels were still higher in both the PC-B3 and PC-B7 groups than in the control group. Bosentan decreased ET-1, NF-κB, TNF-α, HIF-1α, and PAB and increased TAC levels in comparison to the nontreated groups (p < 0.05). Conclusions: Bosentan decreased the severity of oxidative stress in the lungs and reduced the inflammatory reaction in rats with PC induced by blunt thoracic trauma. This suggests that bosentan may have protective effects on lung injury mechanisms by reducing hypoxia, inflammation, and oxidative stress. If supported by similar studies, bosentan can be used in both pulmonary and emergency clinics to reduce ischemic complications, inflammation, and oxidative stress in some diseases that may be accompanied by ischemia.

NLRP3 activation contributes to endothelin-1-induced erectile dysfunction.

In the present study, we hypothesized that endothelin (ET) receptors (ETA and ETB ) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3-/- and caspase-/- mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ETA receptor antagonist reversed the effect. The ETB receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1ß levels in a concentration-dependent manner (100 nM-10 µM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ETA - and ETB -mediated activation of NLRP3 in mouse CC via Ca2+ -dependent ROS generation.

Research progress on the role of ET-1 in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the common complications of diabetes mellitus, which usually progresses to end-stage renal disease and causes great damage to the health of patients. Endothelin-1 (ET-1), a molecule closely associated with the progression of DKD, has increased expression in response to high glucose stimulation and is involved in hemodynamic changes, inflammation, glomerular and tubular dysfunction in the kidney, causing an increase in proteinuria and a decrease in glomerular filtration function, ultimately leading to glomerulosclerosis and renal failure. This paper aims to review the molecular level changes, regulatory mechanisms, and mechanisms of action of ET-1 under DKD, clinical trials of ET-1 receptor antagonists in recent years and current problems, to provide basic information and new research directions and ideas for the treatment of DKD and ET-1-related research.

Metformin and simvastatin synergistically suppress endothelin 1-induced hypoxia and angiogenesis in multiple cancer types.

Multiple cancers have been reported to be associated with angiogenesis and are sensitive to anti-angiogenic therapies. Vascular normalization, by restoring proper tumor perfusion and oxygenation, could limit tumor cell invasiveness and improve the effectiveness of anticancer treatments. However, the underlying anticancer mechanisms of antiangiogenic drugs are still unknown. Metformin (MET) and simvastatin (SVA), two metabolic-related drugs, have been shown to play important roles in modulating the hypoxic tumor microenvironment and angiogenesis. Whether the combination of MET and SVA could exert a more effective antitumor effect than individual treatments has not been examined. The antitumor effect of the synergism of SVA and MET was detected in mouse models, breast cancer patient-derived organoids, and multiple tumor cell lines compared with untreated, SVA, or MET alone. RNA sequencing revealed that the combination of MET and SVA (but not MET or SVA alone) inhibited the expression of endothelin 1 (ET-1), an important regulator of angiogenesis and the hypoxia-related pathway. We demonstrate that the MET and SVA combination showed synergistic effects on inhibiting tumor cell proliferation, promoting apoptosis, alleviating hypoxia, decreasing angiogenesis, and increasing vessel normalization compared with the use of a single agent alone. The MET and SVA combination suppressed ET-1-induced hypoxia-inducible factor 1α expression by increasing prolyl hydroxylase 2 (PHD2) expression. Furthermore, the MET and SVA combination showed a more potent anticancer effect compared with bosentan. Together, our findings suggest the potential application of the MET and SVA combination in antitumor therapy.

GARP on hepatic stellate cells is essential for the development of liver fibrosis.

BACKGROUND & AIMS: Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis. METHODS: The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-ß. Moreover, cell contraction of HSCs in the context of TGF-ß activation was tested in a GARP-dependent fashion. RESULTS: Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-ß and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage. CONCLUSIONS: GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-ß. Considering that systemic blockade of TGF-ß has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-ß activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease.

Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex.

BACKGROUND: Reduction of histone deacetylase (HDAC) 2 expression and activity may contribute to amplified inflammation in patients with severe asthma. Connective tissue growth factor (CTGF) is a key mediator of airway fibrosis in severe asthma. However, the role of the HDAC2/Sin3A/methyl-CpG-binding protein (MeCP) 2 corepressor complex in the regulation of CTGF expression in lung fibroblasts remains unclear. METHODS: The role of the HDAC2/Sin3A/MeCP2 corepressor complex in endothelin (ET)-1-stimulated CTGF production in human lung fibroblasts (WI-38) was investigated. We also evaluated the expression of HDAC2, Sin3A and MeCP2 in the lung of ovalbumin-induced airway fibrosis model. RESULTS: HDAC2 suppressed ET-1-induced CTGF expression in WI-38 cells. ET-1 treatment reduced HDAC2 activity and increased H3 acetylation in a time-dependent manner. Furthermore, overexpression of HDAC2 inhibited ET-1-induced H3 acetylation. Inhibition of c-Jun N-terminal kinase, extracellular signal-regulated kinase, or p38 attenuated ET-1-induced H3 acetylation by suppressing HDAC2 phosphorylation and reducing HDAC2 activity. Overexpression of both Sin3A and MeCP2 attenuated ET-1-induced CTGF expression and H3 acetylation. ET-1 induced the disruption of the HDAC2/Sin3A/MeCP2 corepressor complex and then prompted the dissociation of HDAC2, Sin3A, and MeCP2 from the CTGF promoter region. Overexpression of HDAC2, Sin3A, or MeCP2 attenuated ET-1-stimulated AP-1-luciferase activity. Moreover, Sin3A- or MeCP2-suppressed ET-1-induced H3 acetylation and AP-1-luciferase activity were reversed by transfection of HDAC2 siRNA. In an ovalbumin-induced airway fibrosis model, the protein levels of HDAC2 and Sin3A were lower than in the control group; however, no significant difference in MeCP2 expression was observed. The ratio of phospho-HDAC2/HDAC2 and H3 acetylation in the lung tissue were higher in this model than in the control group. Overall, without stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex inhibits CTGF expression by regulating H3 deacetylation in the CTGF promoter region in human lung fibroblasts. With ET-1 stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex is disrupted and dissociated from the CTGF promoter region; this is followed by AP-1 activation and the eventual initiation of CTGF production. CONCLUSIONS: The HDAC2/Sin3A/MeCP2 corepressor complex is an endogenous inhibitor of CTGF in lung fibroblasts. Additionally, HDAC2 and Sin3A may be of greater importance than MeCP2 in the pathogenesis of airway fibrosis.

Endothelial cell-derived MMP19 promotes pulmonary fibrosis by inducing E(nd)MT and monocyte infiltration.

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in remodeling the extracellular matrix and in the pathogenesis of idiopathic pulmonary fibrosis (IPF). MMP19, which is an MMP, was significantly upregulated in hyperplastic alveolar epithelial cells in IPF lung tissues and promoted epithelial-mesenchymal transition (EMT). Recent studies have demonstrated that endothelial-to-mesenchymal transition (E(nd)MT) contributes to pulmonary fibrosis. However, the role of MMP19 in pulmonary vascular injury and repair and E(nd)MT remains unclear. METHODS: To determine the role of MMP19 in E(nd)MT and pulmonary fibrosis. MMP19 expressions were determined in the lung endothelial cells of IPF patients and bleomycin (BLM)-induced mice. The roles of MMP19 in E(nd)MT and endothelial barrier permeability were studied in the MMP19 cDNA-transfected primary human pulmonary microvascular endothelial cells (HPMECs) and MMP19 adenoassociated virus (MMP19-AAV)-infected mice. The regulatory mechanism of MMP19 in pulmonary fibrosis was elucidated by blocking its interacting proteins SDF1 and ET1 with AMD3100 and Bosentan, respectively. RESULTS: In this study, we found that MMP19 expression was significantly increased in the lung endothelial cells of IPF patients and BLM-induced mice compared to the control groups. MMP19 promoted E(nd)MT and the migration and permeability of HPMECs in vitro, stimulated monocyte infiltration into the alveolus, and aggravated BLM-induced pulmonary fibrosis in vivo. SDF1 and Endothelin-1 (ET1) were physically associated with MMP19 in HPMECs and colocalized with MMP19 in endothelial cells in IPF patient lung tissues. AMD3100 and bosentan alleviated the fibrosis induced by MMP19 in the BLM mouse model. CONCLUSION: MMP19 promoted E(nd)MT by interacting with ET1 and stimulated monocyte infiltration into lung tissues via the SDF1/CXCR4 axis, thus aggravating BLM-induced pulmonary fibrosis. Vascular integrity regulated by MMP19 could be a promising therapeutic target for suppressing pulmonary fibrosis. Video abstract.

The circadian clock protein PER1 is important in maintaining endothelin axis regulation in Dahl salt-sensitive rats.

Endothelin-1 (ET-1) is a peptide hormone that acts on its receptors to regulate sodium handling in the kidney's collecting duct. Dysregulation of the endothelin axis is associated with various diseases, including salt-sensitive hypertension and chronic kidney disease. Previously, our lab has shown that the circadian clock gene PER1 regulates ET-1 levels in mice. However, the regulation of ET-1 by PER1 has never been investigated in rats. Therefore, we used a novel model where knockout of Per1 was performed in Dahl salt-sensitive rat background (SS Per1 -/-) to test a hypothesis that PER1 regulates the ET-1 axis in this model. Here, we show increased renal ET-1 peptide levels and altered endothelin axis gene expression in several tissues, including the kidney, adrenal glands, and liver in SS Per1 -/- compared with control SS rats. Edn1 antisense lncRNA Edn1-AS, which has previously been suggested to be regulated by PER1, was also altered in SS Per1 -/- rats compared with control SS rats. These data further support the hypothesis that PER1 is a negative regulator of Edn1 and is important in the regulation of the endothelin axis in a tissue-specific manner.

Comparison of ET-1 and eNOS expressions in yak testes at different developmental stages.

Yak has strong adaptability to plateau hypoxia environment. However, the endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) are important regulators in blood oxygen transportation. Yak testes: newborn (3 days), young (1 years), adult (4 years) and old (9 years) were collected for microscopic analyses using haematoxylin and eosin staining (H&E), immunohistochemistry and immunofluorescence, as well as Western blot to compare the expression of ET-1 and eNOS. Furthermore, the levels of ET-1 mRNA and eNOS mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The results showed that ET-1 mRNA and eNOS mRNA in old yaks were higher than other developmental stages (p < .01). And the levels of ET-1 and eNOS protein increased with age. Immunohistochemistry and immunofluorescence showed that ET-1 and eNOS were mainly localized in gonocytes and spermatogenic membrane of newborn yaks. These two factors were expressed in both Leydig cells of young yaks and endothelial cells of adult yaks. In old yaks, ET-1 was mainly expressed in Sertoli cells, while eNOS was obviously positive in capillaries and Leydig cells. Therefore, the positive results of ET-1 and eNOS in gonocyte and spermatogenic basement were closely related to the development of testes. The expression of Leydig and Sertoli cells indicated that they played an important role in testes function. The expression in endothelial cells or interstitial capillaries, suggesting that they are involved in the regulation of microcirculation in yak testes. This study could provide clues for further revealing the regulation of yak testicular blood vessels in alpine cold and hypoxic environments.

E-Selectin, ICAM-1, and ET-1 Biomarkers Address the Concern of the Challenging Diagnosis of Interstitial Lung Disease in Patients with Autoimmune Diseases.

Interstitial lung disease (ILD) constitutes the most critical comorbidity in autoimmune diseases (ADs) and its early diagnosis remains a challenge for clinicians. Accordingly, we evaluated whether E-selectin, ICAM-1, and ET-1, key molecules in endothelial damage, could be useful biomarkers for the detection of AD-ILD+. We recruited patients with rheumatoid arthritis (RA)-ILD+ (n = 21) and systemic sclerosis (SSc)-ILD+ (n = 21). We included comparison groups of patients: RA-ILD- (n = 25), SSc-ILD- (n = 20), and idiopathic pulmonary fibrosis (IPF) (n = 21). Serum levels of these proteins were determined by ELISA. E-selectin, ICAM-1, and ET-1 serum levels were increased in RA-ILD+ and IPF patients in comparison to RA-ILD- patients. Additionally, SSc-ILD+ and IPF patients exhibited higher ICAM-1 levels than those with SSc-ILD-. The ability of E-selectin, ICAM-1, and ET-1 to discriminate RA-ILD+ from RA-ILD- patients, and ICAM-1 to distinguish SSc-ILD+ from SSc-ILD- patients was confirmed using ROC curve analysis. Furthermore, elevated levels of ET-1 and E-selectin correlated with lung function decline in RA-ILD+ and SSc-ILD+ patients, respectively. In conclusion, our findings support the relevant role of E-selectin, ICAM-1, and ET-1 in RA-ILD+ patients as well as of ICAM-1 in SSc-ILD+ patients, constituting potential screening blood biomarkers of ILD in AD. Moreover, this study suggests ET-1 and E-selectin as possible indicators of worsening lung function in RA-ILD+ and SSc-ILD+ patients, respectively.

Axial spondyloarthritis and inflammatory bowel disease: association between disease activity and endothelial dysfunction markers.

OBJECTIVE: We aimed to assess patients with axial spondyloarthritis (axSpA) and inflammatory bowel disease (IBD) for disease activity and serum markers of endothelial dysfunction. METHODS: We studied 161 patients (123 males, 38 females) with axSpA: 153 with ankylosing spondylitis and 8 with non-radiographic axSpA, and 30 healthy controls (HC). We collected: age; sex; disease duration; extra-articular symptoms (IBD and acute anterior uveitis), comorbidities; human leukocyte antigen B27 status; and treatment. We measured serum interleukin (IL)-6, interleukin-18, IL-23, vascular endothelial growth factor (VEGF) epidermal growth factor (EGF), asymmetric dimethylarginine (ADMA), endothelin-1 (ET-1), and fetuin-A levels. RESULTS: IBD was diagnosed in 19 (11.8%) patients with axSpA. Compared to patients with axSpA without IBD, those with IBD had higher serum C-reactive protein (CRP) level (p = 0.05), erythrocyte sedimentation rate (ESR) (p = 0.005), and serum ET-1 levels (p = 0.01). In patients with axSpA and IBD, ET-1 levels correlated positively with CRP level (p = 0.006) and ESR (p = 0.02), and ADMA levels with visual analog scale scores (p = 0.01). Patients with axSpA and IBD had higher serum levels of IL-6 (p = 0.01), IL-18 (p = 0.005), and ADMA (p = 0.01) and lower serum levels of fetuin-A (p = 0.01) than did controls. CONCLUSIONS: Patients with axSpA and IBD had higher levels of disease activity, as assessed by ESR and CRP and ET-1 levels, than did patients with axSpA without IBD. Compared to HC, patients with axSpA and IBD had increased IL-18, ADMA levels and decreased fetuin-A level.

Preparation, Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle.

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC50 values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 µM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.

Serum Collected from Preeclamptic Pregnancies Drives Vasoconstriction of Human Omental Arteries-A Novel Ex Vivo Model of Preeclampsia for Therapeutic Development.

New-onset maternal hypertension is a hallmark of preeclampsia, driven by widespread endothelial dysfunction and systemic vasoconstriction. Here, we set out to create a new ex vivo model using preeclamptic serum to cause injury to the endothelium, mimicking vascular dysfunction in preeclampsia and offering the potential to evaluate candidate therapeutic interventions. Human omental arteries were collected at caesarean section from normotensive pregnant patients at term (n = 9). Serum was collected from pregnancies complicated by preterm preeclampsia (birth < 34 weeks' gestation, n = 16), term preeclampsia (birth > 37 weeks' gestation, n = 5), and healthy gestation-matched controls (preterm n = 16, term n = 12). Using wire myography, we performed ex vivo whole vessel assessment where human omental arteries were treated with increasing doses of each serum treatment (2−20%) and vasoreactivity was assessed. All pregnant serum treatments successfully drove vasoconstriction; no significant difference was observed in the degree of vasoconstriction when exposed to preeclamptic or control serum. We further demonstrated the ability of esomeprazole (a candidate therapeutic for preeclampsia; 0.1−100 µM) to drive vasorelaxation of pre-constricted vessels (only with serum from preeclamptic patients). In summary, we describe a novel human physiological model of preeclamptic vascular constriction. We demonstrate its exciting potential to screen drugs for their therapeutic potential as treatment for vasoconstriction induced by preeclampsia.

Endothelin receptor type A is involved in the development of oxaliplatin-induced mechanical allodynia and cold allodynia acting through spinal and peripheral mechanisms in rats.

Oxaliplatin, a platinum-based chemotherapeutic agent, frequently causes severe neuropathic pain typically encompassing cold allodynia and long-lasting mechanical allodynia. Endothelin has been shown to modulate nociceptive transmission in a variety of pain disorders. However, the action of endothelin varies greatly depending on many variables, including pain causes, receptor types (endothelin type A (ETA) and B (ETB) receptors) and organs (periphery and spinal cord). Therefore, in this study, we investigated the role of endothelin in a Sprague-Dawley rat model of oxaliplatin-induced neuropathic pain. Intraperitoneal administration of bosentan, a dual ETA/ETB receptor antagonist, effectively blocked the development or prevented the onset of both cold allodynia and mechanical allodynia. The preventive effects were exclusively mediated by ETA receptor antagonism. Intrathecal administration of an ETA receptor antagonist prevented development of long-lasting mechanical allodynia but not cold allodynia. In marked contrast, an intraplantar ETA receptor antagonist had a suppressive effect on cold allodynia but only had a partial and transient effect on mechanical allodynia. In conclusion, ETA receptor antagonism effectively prevented long-lasting mechanical allodynia through spinal and peripheral actions, while cold allodynia was prevented through peripheral actions.

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.