Gender differences in genotypic distribution of endothelin-1 gene and endothelin receptor A gene in pulmonary hypertension associated with rheumatic mitral valve disease.

INTRODUCTION: The female gender is a risk factor for idiopathic pulmonary arterial hypertension. However, it is unknown whether females with rheumatic mitral valve disease are more predisposed to develop pulmonary hypertension compared to males. AIM: We aimed to investigate whether there was a difference in genotypic distribution of endothelin-1 (ET-1) and endothelin receptor A (ETA) genes between female and male patients of pulmonary hypertension associated with rheumatic mitral valve disease (PH-MVD). METHODS: We compared prevalence of ET-1 gene (Lys198Asn) and ETA gene (His323His) polymorphisms according to gender in 123 PH-MVD subjects and 123 healthy controls. RESULTS: The presence of mutant Asn/Asn and either mutant Asn/Asn or heterozygous Lys/Asn genotypes of Lys198Asn polymorphism when compared to Lys/Lys in females showed significant association with higher risk (odds ratio [OR] 4.5; p =0.007 and OR 2.39; p =0.02, respectively). The presence of heterozygous C/T and either mutant T/T or heterozygous C/T genotypes of His323His polymorphism when compared to wild C/C genotype in females showed a significant association with higher risk (OR 1.96; p =0.047 and OR 2.26; p =0.01, respectively). No significant difference was seen in genotypic frequencies in males between PH-MVD subjects and controls. Logistic regression analysis showed that mutant genotype Asn/Asn (p =0.007) and heterozygous genotype Lys/Asn of Lys198Asn polymorphism (p =0.018) were independent predictors of development of PH in females. CONCLUSIONS: ET-1 and ETA gene polymorphisms were more prevalent in females than males in PH-MVD signifying that females with rheumatic heart disease may be more susceptible to develop PH.

ETAR silencing ameliorated neurovascular injury after SAH in rats through ERK/KLF4-mediated phenotypic transformation of smooth muscle cells.

Subarachnoid haemorrhage (SAH) is a devastating cerebrovascular disease which has a high morbidity and mortality. The phenotypic transformation of smooth muscle cells (SMCs) lead to neurovascular injury after SAH. However, the underlying mechanism remains unclear. In the present study, we aimed to investigate the potential role of ET-1/ETAR on the phenotypic transformation of SMCs after SAH. The models of SAH were established in vivo and vitro. We observed ET-1 secretion by endothelial cells was increased, and the phenotypic transformation of SMCs was aggravated after SAH. Knocking down ETAR inhibited the phenotypic transformation of SMCs, decreased the migration ability of SMCs in vitro. Moreover, Knocking down ETAR ameliorated cerebral ischaemia and alleviated dysfunction of neurological function in vivo. In addition, Exogenous ET-1 increased the migration ability of SMCs and aggravated the phenotypic transformation of SMCs in vitro, which were partly reversed by the antagonist of Erk1/2 - SCH772984. Taken together, our results demonstrated that endothelial ET-1 aggravated the phenotypic transformation of SMCs after SAH. Knocking down ETAR inhibited the phenotypic transformation of SMCs through ERK/KLF4 thus ameliorating neurovascular injury after SAH. We also revealed that ET-1/ETAR is a potential therapeutic target after SAH.

Endothelin receptors promote schistosomiasis-induced hepatic fibrosis via splenic B cells.

Endothelin receptors (ETRs) are activated by vasoactive peptide endothelins and involved in the pathogenesis of hepatic fibrosis. However, less is known about the role of ETRs in Schistosoma (S.) japonicum-induced hepatic fibrosis. Here, we show that the expression of ETRs is markedly enhanced in the liver and spleen tissues of patients with schistosome-induced fibrosis, as well as in murine models. Additional analyses have indicated that the expression levels of ETRs in schistosomiasis patients are highly correlated with the portal vein and spleen thickness diameter, both of which represent the severity of fibrosis. Splenomegaly is a characteristic symptom of schistosome infection, and splenic abnormality may promote the progression of hepatic fibrosis. We further demonstrate that elevated levels of ETRs are predominantly expressed on splenic B cells in spleen tissues during infection. Importantly, using a well-studied model of human schistosomiasis, we demonstrate that endothelin receptor antagonists can partially reverse schistosome-induced hepatic fibrosis by suppressing the activation of splenic B cells characterized by interleukin-10 (IL-10) secretion and regulatory T (Treg) cell-inducing capacity. Our study provides insights into the mechanisms by which ETRs regulate schistosomiasis hepatic fibrosis and highlights the potential of endothelin receptor antagonist as a therapeutic intervention for fibrotic diseases.

Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Covalent Inhibitor-Based One-Step Method for Endothelin Receptor A Immobilization: from Ligand Recognition to Lead Identification.

Protein immobilization is particularly significant in proteomics, interactomics, and in vitro drug screening. It is an essential primary step for numerous biological techniques that rely on immobilized proteins with controlled orientation, high conformational stability, and high activity (CHH). These have challenged the current immobilization strategy and demanded increasing efforts for an efficient method to meet the CHH immobilization in a single step. Herein, we proposed a covalent inhibitor-based, one-step method for G protein-coupled receptor (GPCR) immobilization inspired by the covalent reaction between an epidermal growth factor receptor (EGFR)-tag and its inhibitor ibrutinib. We immobilized endothelin receptor A (ETA) containing a fusion EGFR tag onto an ibrutinib-coated macroporous silica gel. The immobilized ETA proved to have demonstrable ligand-binding activity and specificity, thus resulting in a chromatographic technology allowing receptor-ligand interaction analysis and lead identification. Such immobilization method is attractable, owing to the properties of mild reacting conditions, fast rate, high yield, and good stability of the conjugated protein. It will be applicable to biochips, biosensors, and biocatalysts.

Endothelin-1 stimulates preadipocyte growth via the PKC, STAT3, AMPK, c-JUN, ERK, sphingosine kinase, and sphingomyelinase pathways.

Endothelin (ET)-1 regulates adipogenesis and the endocrine activity of fat cells. However, relatively little is known about the ET-1 signaling pathway in preadipocyte growth. We used 3T3-L1 preadipocytes to investigate the signaling pathways involved in ET-1 modulation of preadipocyte proliferation. As indicated by an increased number of cells and greater incorporation of bromodeoxyuridine (BrdU), the stimulation of preadipocyte growth by ET-1 depends on concentration and timing. The concentration of ET-1 that increased preadipocyte number by 51-67% was ~100 nM for ~24-48 h of treatment. ET-1 signaling time dependently stimulated phosphorylation of ERK, c-JUN, STAT3, AMPK, and PKCα/ßII proteins but not AKT, JNK, or p38 MAPK. Treatment with an ETAR antagonist, such as BQ610, but not ETBR antagonist BQ788, blocked the ET-1-induced increase in cell proliferation and phosphorylated levels of ERK, c-JUN, STAT3, AMPK, and PKCα/ßII proteins. In addition, pretreatment with specific inhibitors of ERK1/2 (U0126), JNK (SP600125), JAK2/STAT3 (AG490), AMPK (compound C), or PKC (Ro318220) prevented the ET-1-induced increase in cell proliferation and reduced the ET-1-stimulated phosphorylation of ERK1/2, c-JUN, STAT3, AMPK, and PKCα/ß. Moreover, the SphK antagonist suppressed ET-1-induced cell proliferation and ERK, c-JUN, STAT3, AMPK, and PKC phosphorylation, and the SMase2 antagonist suppressed ET-1-induced cell proliferation. However, neither the p38 MAPK antagonist nor the CerS inhibitor altered the effect of ET-1. The results indicate that ETAR, JAK2/STAT3, ERK1/2, JNK/c-JUN, AMPK, PKC, SphK, and SMase2, but not ETBR, p38 MAPK, or CerS, are necessary for the ET-1 stimulation of preadipocyte proliferation.

TRIM62 knockout protects against cerebral ischemic injury in mice by suppressing NLRP3-regulated neuroinflammation.

Cerebral stroke is a leading global cause for mortality and disability. However, its pathogenesis is still unclear. Most tripartite motif (TRIM) family proteins, including TRIM62, have E3 ubiquitin ligase activities, and have multiple functions in regulating cellular processes. Nevertheless, the effects of TRIM62 on cerebral stroke still remain vague. Here, we reported that TRIM62 expression was markedly up-regulated in oxygen and glucose deprivation (OGD)-treated microglial cells. After cerebral ischemia, significantly elevated expression of TRIM62 was detected in peri-infarct area of wild type (WT) mice. The TRIM62 knockout (KO) mice exhibited alleviated apoptosis and neuroinflammation in the ischemic brain, eventually attenuating the stroke outcomes. Both in vitro and in vivo studies showed that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome was dramatically activated in cerebral ischemia/reperfusion (I/R) conditions, while being ameliorated in TRIM62-KO mice, contributing to the suppression of neuroinflammatory response. Importantly, the in vitro experiments showed that OGD could induce the K63-ubiquitination of TRIM62 and the interaction between TRIM62 and NLRP3. In addition, adenovirus-regulated TRIM62 over-expression promoted the NLRP3 and nuclear factor κB (NF-κB) signaling, along with elevated interleukin-1ß (IL-1ß) and IL-18 transcriptional activities. Together, our results demonstrated that TRIM62 suppression was strongly protective in ischemic stroke through inhibiting NLRP3-regulated neuroinflammation.

A 14-bp insertion in endothelin receptor B-like (EDNRB2) is associated with white plumage in Chinese geese.

BACKGROUND: Gang goose is a native species with gray plumage in Sichuan, China. As a result of overhunting, the number of gray Gang geese has decreased dramatically. To keep the species from extinction, conservation work for Gang geese was undertaken. In the process of pure breeding of gray Gang geese, approximately 2% of the offspring of each generation were white. This study aims to explain the genetic mechanism of this phenomenon and provide reliable molecular markers for goose-related plumage color breeding. RESULTS: We used the method of pooled whole genome sequencing and Fst (fixation statistics) to identify the differentiation degree of alleles between gray Gang geese and white Gang geese from their offspring. In this way, EDNRB2, a key gene that affects the migration of melanoblasts, was identified. Then, the transcriptome was sequenced for the two geese plumage color populations, and the DEGs (differentially expressed genes) were analyzed. The results indicated that EDNRB2, as a possible candidate gene, had a significantly differential mRNA expression. In addition, a 14-bp insertion (NW_013185915.1: g. 750,748-750,735 insertion. CACAGGTGAGCTCT) in exon 3 of EDNRB2 was analyzed and found to have a significant association between gray geese and Chinese white breeds (P = 0.00), while this mutation was not found in European geese. Meanwhile, the insertion was homozygous in all the white geese we detected and heterozygous in gray geese, indicating that this mutation is recessive. Furthermore, this 14-bp insertion leads to a frameshift mutation in the EDNRB2 coding region and nonsense-mediated mRNA decay. CONCLUSION: Our study strongly suggests that the 14-bp insertion in exon 3 of the EDNRB2 gene is associated with the white plumage phenotype in Chinese geese. This study is the first to investigate the relationship between EDNRB2 and white plumage in geese.

Endothelin-1 induces a strong pressor effect in ball pythons (Python regius).

Endothelin-1 (ET-1) is a very potent vasoactive peptide released from endothelial cells, and ET-1 plays an important role in the maintenance and regulation of blood pressure in mammals. ET-1 signaling is mediated by two receptors: ETA and ETB. In mammals, ETA receptors are located on vascular smooth muscle where they mediate vasoconstriction. ETB receptors located on the endothelium mediate vasodilatation through the release of nitric oxide, whereas stimulation of ETB receptors placed on vascular smooth muscle leads to vasoconstriction. Less is known about ET-1 signaling in reptiles. In anaesthetized alligators, ET-1 elicits a biphasic blood pressure with a long-lasting initial decrease followed by a smaller increase in systemic blood pressure. In anaesthetized freshwater turtles, ET-1 causes a dose-dependent systemic vasodilatation mediated through ETB receptors. In the present study, we investigated the cardiovascular effects of ET-1 on the systemic and pulmonary vasculature of pythons. The presence of ETA and ETB receptors in the vasculature of pythons was verified by means of immunoblotting. Myography on isolated vessels revealed a dose-dependent vasoconstrictory response to ET-1 in both mesenteric and pulmonary arteries. Pressure measurements in recovered specimens revealed an ET-1-induced rise in systemic blood pressure supporting our in vitro findings. In conclusion, our study shows that ET-1 induces a strong pressor effect in the systemic circulation.

Genetic Characteristic and RNA-Seq Analysis in Transparent Mutant of Carp-Goldfish Nucleocytoplasmic Hybrid.

In teleost, pigment in the skin and scales played important roles in various biological processes. Iridophores, one of the main pigment cells in teleost, could produce silver pigments to reflect light. However, the specific mechanism of the formation of silver pigments is still unclear. In our previous study, some transparent mutant individuals were found in the carp-goldfish nucleocytoplasmic hybrid (CyCa hybrid) population. In the present study, using transparent mutants (TM) and wild type (WT) of the CyCa hybrid as a model, firstly, microscopic observations showed that the silver pigments and melanin were both lost in the scales of transparent mutants compared to that in wild types. Secondly, genetic study demonstrated that the transparent trait in the CyCa hybrid was recessively inherent, and controlled by an allele in line with Mendelism. Thirdly, RNA-Seq analysis showed that differential expression genes (DEGs) between wild type and transparent mutants were mainly enriched in the metabolism of guanine, such as hydrolase, guanyl nucleotide binding, guanyl ribonucleotide binding, and GTPase activity. Among the DEGs, purine nucleoside phosphorylase 4a (pnp4a) and endothelin receptor B (ednrb) were more highly expressed in the wild type compared to the transparent mutant (p < 0.05). Finally, miRNA-Seq analysis showed that miRNA-146a and miR-153b were both more highly expressed in the transparent mutant compared to that in wild type (p < 0.05). Interaction analysis between miRNAs and mRNAs indicated that miRNA-146a was associated with six DEGs (MGAT5B, MFAP4, GP2, htt, Sema6b, Obscn) that might be involved in silver pigmentation.

Endothelin neurotransmitter signalling controls zebrafish social behaviour.

The formation of social groups is an adaptive behaviour that can provide protection from predators, improve foraging and facilitate social learning. However, the costs of proximity can include competition for resources, aggression and kleptoparasitism meaning that the decision whether to interact represents a trade-off. Here we show that zebrafish harbouring a mutation in endothelin receptor aa (ednraa) form less cohesive shoals than wild-types. ednraa-/- mutants exhibit heightened aggression and decreased whole-body cortisol levels suggesting that they are dominant. These behavioural changes correlate with a reduction of parvocellular arginine vasopressin (AVP)-positive neurons in the preoptic area, an increase in the size of magnocellular AVP neurons and a higher concentration of 5-HT and dopamine in the brain. Manipulation of AVP or 5-HT signalling can rescue the shoaling phenotype of ednraa-/- providing an insight into how the brain controls social interactions.

The endothelin 1 and endothelin receptor A gene polymorphisms increase the risk of developing papillary thyroid cancer.

The endothelin (EDN) axis (EDN1 and EDN1 receptor A, EDNRA) is involved in cellular growth, differentiation, invasiveness, and tumor progression in several cancers. We wanted to examine the possible impact of single nucleotide polymorphisms (SNPs) of EDN1 and EDNRA genes on papillary thyroid cancer (PTC) development and general characteristics of PTC. Study population consist of 113 PTC patients and 185 controls. EDN1 (G5665T, T-1370G) and EDNRA (C TT70G, G-231A) SNPs were investigated by real-time PCR. The GG genotype of EDNRA + 70 SNP was associated with threefold increased PTC risk (p = 0.01), and the combined CG + GG genotype was 2.48 fold higher among PTC patients compared to controls. The variant EDNRA - 231 allele was overrepresented in PTC patients according to controls (p = 0.05). The combined GT + TT genotype of EDN1 5665 SNP was related with late (age after 40 years) PTC onset (p = 0.04), and was more prominent among male patients with PTC according to females (p = 0.03). No significant associations between PTC and - 1370 SNP were found. There were no relationships between laboratory parameters and investigated polymorphisms. The EDNRA + 70 SNP was associated with PTC development. The EDN1 5665 SNP was linked with increased risk for late PTC onset and was more prominent among male patients with PTC.

News from the endothelin-3/EDNRB signaling pathway: Role during enteric nervous system development and involvement in neural crest-associated disorders.

The endothelin system is a vertebrate-specific innovation with important roles in regulating the cardiovascular system and renal and pulmonary processes, as well as the development of the vertebrate-specific neural crest cell population and its derivatives. This system is comprised of three structurally similar 21-amino acid peptides that bind and activate two G-protein coupled receptors. In 1994, knockouts of the Edn3 and Ednrb genes revealed their crucial function during development of the enteric nervous system and melanocytes, two neural-crest derivatives. Since then, human and mouse genetics, combined with cellular and developmental studies, have helped to unravel the role of this signaling pathway during development and adulthood. In this review, we will summarize the known functions of the EDN3/EDNRB pathway during neural crest development, with a specific focus on recent scientific advances, and the enteric nervous system in normal and pathological conditions.

New drugs and emerging therapeutic targets in the endothelin signaling pathway and prospects for personalized precision medicine.

During the last thirty years since the discovery of endothelin-1, the therapeutic strategy that has evolved in the clinic, mainly in the treatment of pulmonary arterial hypertension, is to block the action of the peptide either at the ET(A) subtype or both receptors using orally active small molecule antagonists. Recently, there has been a rapid expansion in research targeting ET receptors using chemical entities other than small molecules, particularly monoclonal antibody antagonists and selective peptide agonists and antagonists. While usually sacrificing oral bio-availability, these compounds have other therapeutic advantages with the potential to considerably expand drug targets in the endothelin pathway and extend treatment to other pathophysiological conditions. Where the small molecule approach has been retained, a novel strategy to combine two vasoconstrictor targets, the angiotensin AT(1) receptor as well as the ET(A) receptor in the dual antagonist sparsentan has been developed. A second emerging strategy is to combine drugs that have two different targets, the ET(A) antagonist ambrisentan with the phosphodiesterase inhibitor tadalafil, to improve the treatment of pulmonary arterial hypertension. The solving of the crystal structure of the ET(B) receptor has the potential to identify allosteric binding sites for novel ligands. A further key advance is the experimental validation of a single nucleotide polymorphism that has genome wide significance in five vascular diseases and that significantly increases the amount of big endothelin-1 precursor in the plasma. This observation provides a rationale for testing this single nucleotide polymorphism to stratify patients for allocation to treatment with endothelin agents and highlights the potential to use personalized precision medicine in the endothelin field.

Up-regulation of contractile endothelin receptors by airborne fine particulate matter in rat mesenteric arteries via activation of MAPK pathway.

Fine particle matters (PM2.5) is a well-known risk factor for cardiovascular diseases. However, the underlying molecular mechanisms are largely unknown. Vascular hyper-reactivity plays an important roles in the pathogenesis of cardiovascular diseases. The present study was designed to investigate a hypothesis that PM2.5 up-regulated endothelin receptors in mesenteric artery and the potential underlying mechanisms. Rat mesenteric arteries were cultured with PM2.5. The artery contractile responses were recorded by a sensitive myograph. ETB and ETA receptor expressions of mRNA and protein were assessed by quantitative real-time PCR, Western blotting, and immunohistochemistry, respectively. Results showed that ETB receptor agonist, sarafotoxin 6c induced a negligible contraction in fresh artery segments, while ETA receptor agonist, ET-1 induced an obvious contraction. After organ culture, the contraction curve mediated by ETB and ETA receptors were shifted toward the left. PM2.5 1.0 µg/ml cultured for 16 h further enhanced ETB and ETA receptor-mediated contractile responses with a markedly increased maximal contraction. The organ culture enhanced ETB and ETA receptor mRNA and protein levels from fresh arteries, which were further increased by PM2.5. The U0126 (MEK/ERK1/2 inhibitor) and SB203580 (p38 inhibitor) significantly attenuated both organ cultured-induced and PM2.5-induced up-regulation of ETB receptor. U0126 also suppressed organ culture-increased and PM2.5-increased expressions of ETA receptor. SB203580 only suppressed PM2.5-induced enhanced expressions of ETA receptor In conclusion, airborne PM2.5 up-regulates ETB and ETA receptors of mesenteric artery via p38 MAPK and MEK/ERK1/2 MAPK pathways.

Is endothelin gene polymorphism associated with postoperative atrial fibrillation in patients undergoing coronary artery bypass grafting?

BACKGROUND: The mechanism of development of atrial fibrillation (AF) in patients undergoing coronary artery bypass grafting (CABG) has not been clearly defined, and the involvement of multiple factors such as advanced age, withdrawal of ß-blockers, inadequate atrial protection, and electrolyte imbalance, particularly hypomagnesemia has been documented by several authors. Despite all the available pharmacologic prophylaxis, incidence of AF still remains high in this group of patients. This unexplained cause could be genetic inheritance of endothelin-1 (ET-1) gene which is thought to have a pro-arrhythmogenic effect. AIM: This study aims to investigate the relationship between plasma ET-1 concentrations, ET-1 gene polymorphisms in loci -1370 T/G, -134 (3A/4A) Ins/del, Lys198Asn (G/T), and occurrence of AF in patients undergoing CABG. METHODOLOGY: Ninety-eight nonrelated, nondiabetic patients over a period of 4 years undergoing routine CABG were selected for the present study. All patients were genotyped for three single nucleotide polymorphisms (SNPs) in loci -1370 T/G, -134 (3A/4A) Ins/del, and Lys198Asn (G/T) in the ET-1 gene by gene sequencing. The plasma ET-1 concentrations were measured using an ET immunoassay. RESULTS: Plasma ET-1 concentrations were higher in AF+ group (P = 0.001) as compared to AF- group. The allele frequencies between AF+ and AF- group were significantly different only with respect to the Lys198Asn (G/T) SNP of the ET-1 gene. CONCLUSION: The study described the possible correlation of polymorphism of ET gene in CABG population from India. The ET-1 gene might play a disease-modifying role in atrial fibrillation.

Cross-sex transplantation alters gene expression and enhances inflammatory response in the transplanted kidneys.

Kidney transplantation (KTX) is a life-saving procedure for patients with end-stage renal disease. Expression levels of many genes in the kidney vary between males and females, which may play an essential role in the sex differences in graft function. However, whether these differences are affected after cross-sex-KTX is unknown. In the present study, we assessed postoperative changes in genotype, function, and inflammatory responses of the grafts in same-sex- and cross-sex-KTX. Single kidney transplants were performed between same and different sex C57BL/6 mice paired into four combination groups: female donor/female recipient (F/F); male donor/male recipient (M/M); female donor/male recipient (F/M); and male donor/female recipient (M/F). The remnant native kidney was removed 4 days posttransplant. Expression levels of genes related to the contractility of the afferent arteriole and tubular sodium reabsorption were assessed. Same-sex-KTX did not significantly alter the magnitude or sex difference pattern of gene expression in male or female grafts. Cross-sex-KTX showed an attenuated sex difference in gene expressions. The measurements of endothelin 1, endothelin ETA receptor, Na+-K--2Cl cotransporter 2 (NKCC2), and epithelial Na+ channels (ENaC) subunits exhibited decreases in M/F compared with M/M and increases in F/M compared with F/F. There were no significant differences in hemodynamics or sodium excretion in response to acute volume expansion for any sex combinations. Cross-sex-KTX stimulated more robust inflammatory responses than same-sex-KTX. IL-6 and KC mRNA levels elevated 5- to 20-fold in cross-sex-KTX compared with same-sex-KTX. In conclusion, cross-sex-KTX alters gene expression levels and induces inflammatory responses, which might play an important role in long-term graft function.

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox-sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin-secreting Rin-m5f ß-cells and its role in autocrine and paracrine MP-mediated cell crosstalk under conditions of oxidative stress. MP from aPC-treated primary endothelial (EC) or ß-cells were applied to H2 O2 -treated Rin-m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26-stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR-1 expression in EC and to a lesser extent in ß-cells. MP from aPC-treated EC (eMaPC ) exhibited high EPCR and annexin A1 content, protected ß-cells, restored insulin secretion and were captured by 80% of ß cells in a phosphatidylserine and ANXA1-dependent mechanism. eMP activated EPCR/PAR-1 and ANXA1/FPR2-dependent pathways and up-regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H2 O2 -treated rat islets with increased viability (62% versus 48% H2 O2 ), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.

Hippocampal Endothelin-1 decreases excitability of pyramidal neurons and produces anxiolytic effects.

Anxiety disorders contribute to the pathophysiology of psychiatric diseases, including major depression, substance abuse, and schizophrenia. The hippocampus is important for anxiety modulation. However, the mechanisms that control the neuronal activity of the hippocampus in anxiety are still not clear. We found that Endothelin-1 (ET1) mRNA in the hippocampus was down-regulated in high-anxiety mice. Neutralizing endogenous ET1 in the hippocampal CA1 enhanced anxiety-like behaviors. We next revealed that most expression of ET1 and its receptors in the CA1 takes place in pyramidal neurons, and the ET1 signaling pathway directly regulated the excitability of CA1 pyramidal neurons and glutamatergic synaptic neurotransmission. Finally, we proved that neutralizing endogenous CA1 ET1 produces anxiogenic effects on low-anxiety mice, whereas infusing exogenous ET1 into the CA1 alleviates the anxiety susceptibility of high-anxiety mice. Together, these results indicate that ET1 signaling is critical in maintaining the excitability of glutamatergic neurons in the hippocampus and, thus, in modulating anxiety-like behaviors. Because ET1 is a risk factor for ischemic stroke, our findings might also help to explain the potential mechanism of emotional abnormality in stroke.

Airborne fine particulate matter alters the expression of endothelin receptors in rat coronary arteries.

Exposure to airborne fine particulate matter (PM2.5) is associated with cardiovascular diseases. However, a comprehensive understanding of the underlying mechanisms by which PM2.5 induces or aggravates these diseases is still insufficiently clear. The present study investigated whether PM2.5 alters the expression of the endothelin subtype B (ETB) and endothelin subtype A (ETA) receptors in the coronary artery and examined the underlying mechanisms. Rat coronary artery segments were cultured with PM2.5 in the presence or absence of MEK/ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was measured by myography. ETB and ETA receptor expression was evaluated using RT-PCR, western blot and immunohistochemistry. Compared with fresh arteries, the cultured coronary arteries showed a significantly enhanced contraction mediated by the ETB receptor and an unaltered contraction mediated by the ETA receptor. Culture with PM2.5 significantly enhanced the contraction and the mRNA and protein expression levels of the ETB and ETA receptors in the coronary arteries, suggesting that PM2.5 induces an upregulation of ETA and ETB receptors. In addition, the PM2.5-induced increases in ETB- and ETA-mediated vasoconstriction and receptor expressions could be notably decreased by MEK1/2 inhibitor, U0126 and Raf inhibitor, SB386023, suggesting that the upregulation of ETB and ETA receptors is related with MEK/ERK1/2 pathway. In conclusion, PM2.5 induces the ETB and ETA receptor upregulation in rat coronary arteries, and the MEK/ERK1/2 pathway may be involved in this process.