Methylation analysis of DCC gene in saliva samples is an efficient method for non-invasive detection of superficial hypopharyngeal cancer.

BACKGROUND: Advances in upper gastrointestinal endoscopic technology have enabled early detection and treatment of hypopharyngeal cancer. However, in-depth pharyngeal observations require sedation and are invasive. It is important to establish a minimally invasive and simple evaluation method to identify high-risk patients. METHODS: Eighty-seven patients with superficial hypopharyngeal cancer and 51 healthy controls were recruited. We assessed the methylation status of DCC, PTGDR1, EDNRB, and ECAD, in tissue and saliva samples and verified the diagnostic accuracy by methylation analyses of their promoter regions using quantitative methylation-specific PCR. RESULTS: Significant differences between cancer and their surrounding non-cancerous tissues were observed in the methylation values of DCC (p = 0.003), EDNRB (p = 0.001), and ECAD (p = 0.043). Using receiver operating characteristic analyses of the methylation values in saliva samples, DCC showed the highest area under the curve values for the detection of superficial hypopharyngeal cancer (0.917, 95% confidence interval = 0.864-0.970), compared with those for EDNRB (0.680) and ECAD (0.639). When the cutoff for the methylation values of DCC was set at ≥0.163, the sensitivity to detect hypopharyngeal cancer was 82.8% and the specificity was 90.2%. CONCLUSIONS: DCC methylation in saliva samples could be a non-invasive and efficient tool for early detection of hypopharyngeal cancer in high-risk patients.

High incidence of EDNRB gene mutation in seven southern Chinese familial cases with Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HSCR) is the leading cause of neonatal functional intestinal obstruction, which has been identified in many familial cases. HSCR, a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and seven chromosomal loci, with RET and EDNRB as its major genes. We present a genetic investigation of familial HSCR to clarify the genotype-phenotype relationship. METHODS: We performed whole exome sequencing (WES) on Illumina HiSeq X Ten platform to investigate genetic backgrounds of core family members, and identified the possibly harmful mutation genes. Mutation carriers and pedigree relatives were validated by Sanger sequencing for evaluating the gene penetrance. RESULTS: Four familial cases showed potential disease-relative variants in EDNRB and RET gene, accounting for all detection rate of 57.1%. Three familial cases exhibited strong pathogenic variants as frameshift or missense mutations in EDNRB gene. A novel c.367delinsTT mutation of EDNRB was identified in one family member. The other two EDNRB mutations, c.553G>A in family 2 and c.877delinsTT in family 5, have been reported in previous literatures. The penetrance of EDNRB variants was 33-50% according mutation carries. In family 6, the RET c.1858T>C (C620R) point mutation has previously been reported to cause HSCR, with 28.5% penetrance. CONCLUSION: We identified a novel EDNRB (deleted C and inserted TT) mutation in this study using WES. Heterozygote variations in EDNRB gene were significantly enriched in three families and RET mutations were identified in one family. EDNRB variants showed an overall higher incidence and penetrance than RET in southern Chinese families cases.

Genome-wide detection of selection signatures in Jianli pigs reveals novel cis-regulatory haplotype in EDNRB associated with two-end black coat color.

BACKGROUND: Jianli pig, a renowned indigenous breed in China, has the characteristics of a two-end black (TEB) coat color, excellent meat quality, strong adaptability and increased prolificacy. However, there is limited information available regarding the genetic diversity, population structure and genomic regions under selection of Jianli pig. On the other hand, the genetic mechanism of TEB coat color has remained largely unknown. RESULTS: In this study, the whole genome resequencing of 30 Jianli pigs within a context of 153 individuals representing 13 diverse breeds was performed. The population structure analysis revealed that Jianli pigs have close genetic relationships with the Tongcheng pig breed, their geographical neighbors. Three methods (observed heterozygosity, expected heterozygosity, and runs of homozygosity) implied a relatively high level of genetic diversity and, a low inbreeding coefficient in Jianli compared with other pigs. We used Fst and XP-EHH to detect the selection signatures in Jianli pigs compared with Asian wild boar. A total of 451 candidate genes influencing meat quality (CREBBP, ADCY9, EEPD1 and HDAC9), reproduction (ESR1 and FANCA), and coat color (EDNRB, MITF and MC1R), were detected by gene annotation analysis. Finally, to fine-map the genomic region for the two-end black (TEB) coat color phenotype in Jianli pigs, we performed three signature selection methods between the TEB coat color and no-TEB coat color pig breeds. The current study, further confirmed that the EDNRB gene is a candidate gene for TEB color phenotype found in Chinese pigs, including Jinhua pigs, and the haplotype harboring 25 SNPs in the EDNRB gene may promote the formation of TEB coat color. Further ATAC-seq and luciferase reporter assays of these regions suggest that the 25-SNPs region was a strong candidate causative mutation that regulates the TEB coat color phenotype by altering enhancer function. CONCLUSION: Our results advanced the understanding of the genetic mechanism behind artificial selection, and provided further resources for the protection and breeding improvement of Jianli pigs.

Multiple gene-drug prediction tool reveals Rosiglitazone based treatment pathway for non-segmental vitiligo.

Vitiligo is a skin disease characterized by selective loss of melanocytes, which seriously affects the appearance and causes great psychological stress to patients. In this study, we performed a comprehensive analysis of two vitiligo microarray datasets from the GEO database using bioinformatics tools to identify 297 up-regulated mRNAs and 186 down-regulated mRNAs, revealing important roles for pathways related to melanin synthesis, tyrosine metabolism, and inflammatory factors, such as "PPAR signaling pathway", "tyrosine metabolism", "nonalcoholic fatty liver disease (NAFLD) pathway", "melanogenesis", and "IL-17 signaling pathway". Combining the Search Tool for Interacting Chemicals (STITCH) database 5.0 and the drug-gene interaction database 3.0 (DGIdb), we identified that the PPAR-γ agonist rosiglitazone may promote melanin synthesis via EDNRB. Next, we investigated the mechanism of rosiglitazone and PPAR-γ pathway in promoting melanin production. Consistent with the results of bioinformatics analysis, the expression levels of PPAR-γ, EDNRB, and TYR were significantly reduced in human non-segmental vitiligo skin along with the reduction of MITF, a key gene for epidermal melanogenesis. Meanwhile, rosiglitazone increased melanin synthesis capacity in melanocytes and zebrafish by activating PPAR-γ and upregulating TYR, TYRP-1, and TYRP-2. Conversely, treatment of melanocytes with the PPAR-γ antagonist GW resulted in inhibition of melanin synthesis and expression of melanin-related factors. At the same time, simultaneous treatment of rosiglitazone with GW reversed the inhibitory effect of GW on melanin synthesis. In this study, we identified that rosiglitazone, an important insulin sensitizer, promotes melanin synthesis in melanocytes by increasing PPAR-γ activity and upregulating the expression levels of EDNRB and TYR. These findings may provide new ideas for exploring the pathogenesis and potential therapeutic targets of non-segmental vitiligo.

Elevated renal afferent nerve activity in a rat model of endothelin B receptor deficiency.

Renal nerves have been an attractive target for interventions aimed at lowering blood pressure; however, the specific roles of renal afferent (sensory) versus efferent sympathetic nerves in mediating hypertension are poorly characterized. A number of studies have suggested that a sympathoexcitatory signal conveyed by renal afferents elicits increases in blood pressure, whereas other studies identified sympathoinhibitory afferent pathways. These sympathoinhibitory pathways have been identified as protective against salt-sensitive increases in blood pressure through endothelin B (ETB) receptor activation. We hypothesized that ETB-deficient (ETB-def) rats, which are devoid of functional ETB receptors except in adrenergic tissues, lack appropriate sympathoinhibition and have lower renal afferent nerve activity following a high-salt diet compared with transgenic controls. We found that isolated renal pelvises from high salt-fed ETB-def animals lack a response to a physiological stimulus, prostaglandin E2, compared with transgenic controls but respond equally to a noxious stimulus, capsaicin. Surprisingly, we observed elevated renal afferent nerve activity in intact ETB-def rats compared with transgenic controls under both normal- and high-salt diets. ETB-def rats have been previously shown to have heightened global sympathetic tone, and we also observed higher total renal sympathetic nerve activity in ETB-def rats compared with transgenic controls under both normal- and high-salt diets. These data indicate that ETB receptors are integral mediators of the sympathoinhibitory renal afferent reflex (renorenal reflex), and, in a genetic rat model of ETB deficiency, the preponderance of sympathoexcitatory renal afferent nerve activity prevails and may contribute to hypertension.NEW & NOTEWORTHY Here, we found that endothelin B receptors are an important contributor to renal afferent nerve responsiveness to a high-salt diet. Rats lacking endothelin B receptors have increased afferent nerve activity that is not responsive to a high-salt diet.

Role of Brain Endothelin Receptor Type B (ETB) in the Regulation of Tyrosine Hydroxylase in the Olfactory Bulb of DOCA-Salt Hypertensive Rats.

BACKGROUND: We previously reported that endothelins (ETs) regulate tyrosine hydroxylase (TH) activity and expression in the olfactory bulb (OB) of normotensive and hypertensive animals. Applying an ET receptor type A (ETA) antagonist to the brain suggested that endogenous ETs bind to ET receptor type B (ETB) to elicit effects. OBJECTIVE: The aim of the present work was to evaluate the role of central ETB stimulation on the regulation of blood pressure (BP) and the catecholaminergic system in the OB of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. METHODS: DOCA-salt hypertensive rats were infused for 7 days with cerebrospinal fluid or IRL-1620 (ETB receptor agonist) through a cannula placed in the lateral brain ventricle. Systolic BP (SBP) and heart rate were recorded by plethysmography. The expression of TH and its phosphorylated forms in the OB were determined by immunoblotting, TH activity by a radioenzymatic assay, and TH mRNA by quantitative real-time polymerase chain reaction. RESULTS: Chronic administration of IRL-1620 decreased SBP in hypertensive rats but not in normotensive animals. Furthermore, the blockade of ETB receptors also decreased TH-mRNA in DOCA-salt rats, but it did not modify TH activity or protein expression. CONCLUSION: These findings suggest that brain ETs through the activation of ETB receptors contribute to SBP regulation in DOCA-salt hypertension. However, the catecholaminergic system in the OB does not appear to be conclusively involved although mRNA TH was reduced. Present and previous findings suggest that in this salt-sensitive animal model of hypertension, the OB contributes to chronic BP elevation.

MiRNA-19b-3p downregulates the endothelin B receptor in gastric cancer cells to prevent angiogenesis and proliferation.

MiR19b-3p acts as a tumor suppressor gene in various cancers, but its function in gastric cancer is unknown. This study investigated the role of miR19b-3p in angiogenesis and the proliferation of human gastric cancer cells targeting ETBR expression. Cell proliferation in SGC-7901 cells, cell transfection, luciferase reporter assay, detection of endothelin B receptor mRNAs by RT-qPCR, and a Western blot assay were carried out. RT-qPCR expression analysis showed a prominent (p<0.01) downregulation of miR19b-3p in SGC-7901 cells, which was inversely correlated with a substantial increase (p<0.01) in the endothelin B receptor (ETBR). However, overexpression of miR19b-3p in SGC-7901 cells with its mimic (p<0.01) resulted in the loss of cell viability in the MTT assay. This effect was reversed (p<0.01) by the inhibitor. Western blot analysis revealed that ETBR was significantly (p<0.01) decreased by miR19b-3p overexpression compared with that of the negative control or its inhibitor. Based on bioinformatics tools and luciferase reporter assays, we found that miR19b-3p interacts with the 3'untranslated region (3'UTR) of ETBR. Restoring miR19b-3p overexpression with its mimic led to downregulation of ETBR in gastric cancer cells (SGC-7901), which significantly (p<0.01) decreased the expression of vascular endothelial growth factor A (VEGF-A). These findings were considerably reversed by miR19b-3p inhibitors (p<0.01). The results indicated that miR19b-3p exerts its molecular action by targeting ETBR at the post-transcriptional level by regulating angiogenesis and proliferation by overexpressing miR19b-3p as a potential treatment target for gastric cancer.

The evolution of white-tailed jackrabbit camouflage in response to past and future seasonal climates.

The genetic basis of adaptive traits has rarely been used to predict future vulnerability of populations to climate change. We show that light versus dark seasonal pelage in white-tailed jackrabbits (Lepus townsendii) tracks snow cover and is primarily determined by genetic variation at endothelin receptor type B (EDNRB), corin serine peptidase (CORIN), and agouti signaling protein (ASIP). Winter color variation was associated with deeply divergent alleles at these genes, reflecting selection on both ancestral and introgressed variation. Forecasted reductions in snow cover are likely to induce widespread camouflage mismatch. However, simulated populations with variation for darker winter pelage are predicted to adapt rapidly, providing a trait-based genetic framework to facilitate evolutionary rescue. These discoveries demonstrate how the genetic basis of climate change adaptation can inform conservation.

Identification of nine novel variants across PAX3, SOX10, EDNRB, and MITF genes in Waardenburg syndrome with next-generation sequencing.

BACKGROUND: Waardenburg syndrome (WS) is a hereditary, genetically heterogeneous disorder characterized by variable presentations of sensorineural hearing impairment and pigmentation anomalies. This study aimed to investigate the clinical features of WS in detail and determine the genetic causes of patients with clinically suspected WS. METHODS: A total of 24 patients from 21 Han-Taiwanese families were enrolled and underwent comprehensive physical and audiological examinations. We applied targeted next-generation sequencing (NGS) to investigate the potential causative variants in these patients and further validated the candidate variants through Sanger sequencing. RESULTS: We identified 19 causative variants of WS in our cohort. Of these variants, nine were novel and discovered in PAX3, SOX10, EDNRB, and MITF genes, including missense, nonsense, deletion, and splice site variants. Several patients presented with skeletal deformities, hypotonia, megacolon, and neurological disorders that were rarely seen in WS. CONCLUSION: This study revealed highly phenotypic variability in Taiwanese WS patients and demonstrated that targeted NGS allowed us to clarify the genetic diagnosis and extend the genetic variant spectrum of WS.

Sex Differences in Diurnal Sodium Handling During Diet-Induced Obesity in Rats.

BACKGROUND: Emerging evidence over the past several years suggests that diurnal control of sodium excretion is sex dependent and involves the renal endothelin system. Given recent awareness of disruptions of circadian function in obesity, we determined whether diet-induced obesity impairs renal handling of an acute salt load at different times of day and whether this varies by sex and is associated with renal endothelin dysfunction. METHODS: Male and female Sprague-Dawley rats were placed on a high-fat diet for 8 weeks before assessing renal sodium handling and blood pressure. RESULTS: Male, but not female, rats on high fat had a significantly reduced natriuretic response to acute NaCl injection at the beginning of their active period that was associated with lower endothelin 1 (ET-1) excretion, lower ET-1 mRNA expression in the cortex and outer medulla as well as lower ETB receptor expression in the outer medulla of the high-fat rats. Obese males also had significantly higher blood pressure (telemetry) that was exacerbated by adding high salt to the diet during the last 2 weeks. While female rats developed hypertension with a high-fat diet, they were not salt sensitive and ET-1 excretion was unchanged. CONCLUSIONS: These data identify diet-induced obesity as a sex-specific disruptive factor for maintaining proper sodium handling. Although high-fat diets induce hypertension in both sexes, these data reveal that males are at greater risk of salt-dependent hypertension and further suggest that females have more redundant systems that can be productive against salt-sensitive hypertension in at least some circumstances.

Mutations in rhodopsin, endothelin B receptor, and CC chemokine receptor 5 in large animals: Modeling human diseases.

G protein-coupled receptors (GPCRs) are the largest family of cell membrane receptors involved in modulating almost all physiological processes by transducing extracellular signals into the cytoplasm. Dysfunctions of GPCR-regulated signaling result in diverse human diseases, making GPCRs the most popular drug targets for human medicine. Large animals share higher similarities (in physiology and metabolism) with humans than rodents. Similar to findings in human genetics, diverse diseases caused by mutations in GPCR genes have also been discovered in large animals. Rhodopsin, endothelin B receptor, and CC chemokine receptor type 5 have been shown to be involved in human retinitis pigmentosa, Hirschsprung disease, and HIV infection/AIDS, respectively, and several mutations of these GPCRs have also been identified from large animals. The large animals with naturally occurring mutations of these GPCRs provide an opportunity to gain a better understanding of the pathogenesis of human diseases, and can be used for preclinical trials of therapies for human diseases. In this review, we aim to summarize the naturally occurring mutations of these three GPCRs in large animals and humans.

Genetic heterogeneity in GJB2, COL4A3, ATP6V1B1 and EDNRB variants detected among hearing impaired families in Morocco.

BACKGROUND: Deafness is the most prevalent human sensorineural defect. It may occur as a result of an external auditory canal involvement, or a deficiency in the sound conduction mechanism, or an impairment of the cochlea, the cochlear nerve or central auditory perception. The genetic causes are the most common, as approximately 70% of hearing disorders are of hereditary origin, divided into two groups, syndromic (associated with other symptoms) and no syndromic (isolated deafness). METHODS: A whole exome sequencing was performed to identify the genetic cause of hearing loss in six Moroccan families and Sanger sequencing was used to validate mutations in these genes. THE RESULTS: The results of four out of the six families revealed four genetic variants in the genes GJB2, COL4A3, ATP6V1B1 and EDNRB responsible for non-syndromic and syndromic hearing loss. Multiple Bioinformatics programs and molecular modelling predicted the pathogenic effect of these mutations. CONCLUSIONS: We identified in Moroccan deaf patients four homozygous mutations. These results show the importance of whole exome sequencing to identify pathogenic mutations in heterogeneous disorders with multiple genes responsible.

Inhibitors of the MAPK/ NF-κB pathway attenuate the upregulation of the ETB receptor mediated by high glucose in vascular smooth muscle cells.

BACKGROUND: Increased vascular smooth muscle cell (VSMC) endothelin type B (ETB) receptor expression is involved in cardiovascular diseases. High glucose (HG) in diabetes is closely related to cardiovascular complications. Although diabetes upregulates VSMC endothelin subtype B (ETB) receptors, its mechanism is still unclear. Our aim is to investigate the mechanism of HG-induced ETB receptors in VSMCs. METHODS: Rat superior mesenteric arteries (SMAs) without endothelium were cultured in medium without serum for 24 h. HG with or without mitogen-activated protein kinase (MAPK) signaling pathway inhibitors and downstream nuclear factor-kappaB (NF-κB) inhibitors was coincubated with SMAs. A sensitive myograph detected the contractile responses to sarafotoxin 6c. Western blotting and immunofluorescence staining were used to determine protein expression. RESULTS: HG promoted the expression of VSMC ETB receptors in rat SMAs and enhanced the ETB receptor-induced contractile response. The results showed that HG increased vascular smooth muscle cell (VSMC) ETB receptor expression and ETB receptor-induced contractile responses in rat SMAs. Both extracellular signal-related kinase 1 and 2 (ERK1/2) inhibitors (U0126) and P38 inhibitors (SB203580) significantly inhibited HG-increased VSMC ETB receptors. However, a C-jun terminal kinase (p-JNK) inhibitor (SP600125) did not affect HG- upregulated VSMC ETB receptors. Further study showed that NF-κB using an IκB kinase inhibitor (wedelolactone) also significantly inhibited HG-increased VSMC ETB receptors. CONCLUSION: In conclusion, HG upregulated the VSMC ETB receptor by activating the ERK1/2- or P38- NF-κB signaling pathway.

A large Canadian cohort provides insights into the genetic architecture of human hair colour.

Hair colour is a polygenic phenotype that results from differences in the amount and ratio of melanins located in the hair bulb. Genome-wide association studies (GWAS) have identified many loci involved in the pigmentation pathway affecting hair colour. However, most of the associated loci overlap non-protein coding regions and many of the molecular mechanisms underlying pigmentation variation are still not understood. Here, we conduct GWAS meta-analyses of hair colour in a Canadian cohort of 12,741 individuals of European ancestry. By performing fine-mapping analyses we identify candidate causal variants in pigmentation loci associated with blonde, red and brown hair colour. Additionally, we observe colocalization of several GWAS hits with expression and methylation quantitative trait loci (QTLs) of cultured melanocytes. Finally, transcriptome-wide association studies (TWAS) further nominate the expression of EDNRB and CDK10 as significantly associated with hair colour. Our results provide insights on the mechanisms regulating pigmentation biology in humans.

Structural heart defects associated with ETB mutation, a cause of Hirschsprung disease.

BACKGROUND: HSCR, a colonic neurocristopathy affecting 1/5000 births, is suggested to associate with cardiac septal defects and conotruncal malformations. However, we question subtle cardiac changes maybe more commonly present due to multi-regulations by HSCR candidate genes, in this instance, ETB. To investigate, we compared the cardiac morphology and quantitative measurements of sl/sl rat to those of the control group. METHODS: Eleven neonatal rats were generated from heterozygote (ETB+/-) crossbreeding. Age and bodyweight were recorded at time of sacrifice. Diffusion-staining protocols with 1.5% iodine solution was completed prior to micro-CT scanning. All rats were scanned using an in vivo micro-CT scanner, Caliper Quantum FX, followed by two quality-control scans using a custom-built ex vivo micro-CT system. All scans were reviewed for gross cardiac dysmorphology. Micro-CT data were segmented semi-automatically post-NLM filtering for: whole-heart, LV, RV, LA, RA, and aortic arch. Measurements were taken with Drishti. Following image analysis, PCR genotyping of rats was performed: five sl/sl rats, three wildtype, and three heterozygotes. Statistical comparisons on organ volume, growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control group. RESULTS: Cardiac morphology and constituents were preserved. However, significant volumetric reductions were recorded in sl/sl rats with respect to the control: whole heart (38.70%, p value = 0.02); LV (41.22%, p value = 0.01), RV (46.15%, p value = 0.02), LA (44.93%, p value = 0.06), and RA (39.49%, p value = 0.02). Consistent trend was observed in growth rate (~ 20%) and organ-volume/bodyweight ratios (~ 25%). On the contrary, measurements on aortic arch demonstrated no significant difference among the two groups. CONCLUSION: Despite the presence of normal morphology, significant cardiac growth retardation was detected in sl/sl rat, supporting the likely association of cardiac anomalies with HSCR, at least in ETB-/- subtype. Structural reduction was likely due to a combination of failure to thrive from enteric dysfunction, alterations to CaNCC colonization, and importantly coronary hypoperfusion from elevated ET-1/ETA-mediated hypervasoconstriction. Little correlation was detected between aortic arch development and sl/sl rat, supporting minor ETB role in large vessels. Although further clinical study is warranted, HSCR patients may likely require cardiac assessment in view of potential congenital cardiac defects.

Prenatal hypoxia induced ETBR activation and abnormal ROS signalling in pulmonary artery cells of rat offspring.

Pulmonary arterial hypertension is a progressive disorder characterized by remodeling and increased small pulmonary arteries resistance. Endothelin-1 (ET-1) was related to PAH and ET-1 receptors were up-regulated selectively in the lung when exposed to toxic factor hypoxia. However, the role of ET-1 signaling in the pathogenesis of prenatal hypoxia-induced pulmonary abnormalities remains to be elucidated. Pregnant rats were divided into prenatal hypoxia (10.5 % O2 from gestational day 4-21) and control group. Their three-month-old offspring male rats were tested for vascular functions and molecular analysis, DNA methylation was assessed for cellular hypoxia. Functional testing showed that ET-1-mediated vasoconstriction was enhanced, and the expressions of endothelin A receptor/B receptor (ETAR/ETBR), inositol 1,4,5-trisphosphate receptor, type 1, and the sensitivity of calcium channels were increased in the small pulmonary arteries following prenatal hypoxia. q-PCR and DHE staining showed that the expressions of NADPH oxidase 1/4 (Nox1/4) were up-regulated, along with the increased production of superoxide anion. Furthermore, superoxide anion promoted ET-1-mediated pulmonary artery contraction. In the pulmonary artery smooth muscle cell experiments, q-PCR, Western Blot, CCK8 and DHE staining showed that the expressions of ETBR, Nox1/4, and superoxide anion were increased by hypoxia, along with promoted cell proliferation. 2,2,6,6-Tetramethyl-1-piperidinyloxy reversed hypoxia-induced cell proliferation. ETBR antagonist BQ788 inhibited hypoxia-increased expressions of Nox1/4, superoxide anion production, and proliferation of cells. Moreover, methylation analysis indicated that hypoxia decreased the methylation levels of the ETBR promoter in the pulmonary artery smooth muscle cells. The results indicated that prenatal toxic factor hypoxia resulted in abnormal ETBR activation, which enhanced ET-1-mediated vasoconstriction of pulmonary arteries and pulmonary artery smooth muscle cell proliferation through ETBR/Nox1/4-derived ROS pathway.

Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease.

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Brain size reductions associated with endothelin B receptor mutation, a cause of Hirschsprung's disease.

BACKGROUND: ETB has been reported to regulate neurogenesis and vasoregulation in foetal development. Its dysfunction was known to cause HSCR, an aganglionic colonic disorder with syndromic forms reported to associate with both small heads and developmental delay. We therefore asked, "is CNS maldevelopment a more general feature of ETB mutation?" To investigate, we reviewed the micro-CT scans of an ETB-/- model animal, sl/sl rat, and quantitatively evaluated the structural changes of its brain constituents. METHODS: Eleven neonatal rats generated from ETB+/- cross breeding were sacrificed. Micro-CT scans were completed following 1.5% iodine-staining protocols. All scans were reviewed for morphological changes. Selected organs were segmented semi-automatically post-NLM filtering: TBr, T-CC, T-CP, OB, Med, Cer, Pit, and S&I Col. Volumetric measurements were made using Drishti rendering software. Rat genotyping was completed following analysis. Statistical comparisons on organ volume, organ growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control groups based on autosomal recessive inheritance. One-way ANOVA was also performed to evaluate potential dose-dependent effect. RESULTS: sl/sl rat has 16.32% lower body weight with 3.53% lower growth rate than the control group. Gross intracranial morphology was preserved in sl/sl rats. However, significant volumetric reduction of 20.33% was detected in TBr; similar reductions were extended to the measurements of T-CC, T-CP, OB, Med, and Pit. Consistently, lower brain and selected constituent growth rates were detected in sl/sl rat, ranging from 6.21% to 11.51% reduction. Lower organ volume/bodyweight ratio was detected in sl/sl rats, reflecting disproportional neural changes with respect to body size. No consistent linear relationships exist between ETB copies and intracranial organ size or growth rates. CONCLUSION: Although ETB-/- mutant has a normal CNS morphology, significant size reductions in brain and constituents were detected. These structural changes likely arise from a combination of factors secondary to dysfunctional ET-1/ET-3/ETB signalling, including global growth impairment from HSCR-induced malnutrition and dysregulations in the neurogenesis, angiogenesis, and cerebral vascular control. These changes have important clinical implications, such as autonomic dysfunction or intellectual delay. Although further human study is warranted, our study suggested comprehensive managements are required for HSCR patients, at least in ETB-/- subtype.

Genome-wide association analysis reveals that EDNRB2 causes a dose-dependent loss of pigmentation in ducks.

BACKGROUND: Birds have various plumage color patterns, and spot is a common phenotype. Herein, we conducted genome-wide association studies (GWAS) in a population of 225 ducks with different sized black spots to reveal the genetic basis of this phenomenon. RESULTS: First, we quantified the black spot phenotype within the duck population. The results showed that the uncolored area of the body surface first appeared on the ventral side. With increasing duck age, the area of the black spots was highly conserved across the whole body surface. The GWAS results identified a 198 kb (Chr4: 10,149,651 bp to 10,348,068 bp) genetic region that was significantly associated with the black spot phenotype. The conditional GWAS and linkage disequilibrium (LD) analysis further narrowed the ultimate candidate region to 167 kb (Chr4: 10,180,939 bp to 10,348,068 bp). A key gene regulating melanoblast migration and differentiation, EDNRB2 (Endothelin B receptor-like), was found in the candidate region and having significant mRNA expression level changes in embryonic duck skin tissue with different spot sizes. The significant SNPs (single nucleotide polymorphisms) associated with the EDNRB2 gene were annotated, and two mutations (Chr4: 10,180,939 T > C and Chr4: 10,190,671 A > T) were found to result in the loss of binding sites for two trans-factors, XBP1 and cMYB. The phenotypic effect of these two mutations suggested that they can regulate the size of black spots in a dose-dependent manner, and Chr4: 10,180,939 T > C was the major allele locus. CONCLUSIONS: Our results revealed that EDNRB2 was the gene responsible for the variation in duck body surface spot size. Chr4: 10,180,939 T > C was the major allele that explained 49.5 % (dorsal side) and 32.9 % (ventral side) of the variation in duck body surface spot size, while 32.1 % (dorsal side) and 19.1 % (ventral side) of the variation could be explained by Chr4: 10,190,671 A > T. The trans-factor prediction also suggested that XBP1 and cMYB have the potential to interact with EDNRB2, providing new insights into the mechanism of action of these genes.

Sorafenib not only impairs endothelium-dependent relaxation but also promotes vasoconstriction through the upregulation of vasoconstrictive endothelin type B receptors.

As a VEGF-targeting agent, sorafenib has been used to treat a number of solid tumors but can easily lead to adverse vascular effects. To elucidate the underlying mechanism, rat mesenteric arteries were subjected to organ cultured in the presence of different concentrations of sorafenib (0, 3, 6 and 9 mg/L) with or without inhibitors (U0126, 10-5 M; SB203580, 10-5 M; SP200126, 10-5 M) of MAPK kinases, and then acetylcholine- or sodium nitroprusside-induced vasodilation and sarafotoxin 6c-induced vasoconstriction were monitored by a sensitive myograph. The NO synthetases, the nitrite levels, the endothelial marker CD31,the ETB and ETA receptors and the phosphorylation of MAPK kinases were studied. Next, rats were orally administrated by sorafenib for 4 weeks (7.5 and 15 mg/kg/day), and their blood pressure, plasma ET-1, the ETB and ETA receptors and the phosphorylation of MAPK kinases in the mesenteric arteries were investigated. The results showed that sorafenib impairs endothelium-dependent vasodilation due to decreased NO levels and the low expression of eNOS and iNOS. Weak staining for CD31 indicated that sorafenib induced endothelial damage. Moreover, sorafenib caused the upregulation of vasoconstrictive ETB receptors, the enhancement of ETB receptor-mediated vasoconstriction and the activation of JNK/MAPK. Blocking the JNK, ERK1/2 and p38/MAPK signaling pathways by using the inhibitors significantly abolished ETB receptor-mediated vasoconstriction. Furthermore, it was observed that the oral administration of sorafenib caused an increase in blood pressure and plasma ET-1, upregulation of the ETB receptor and the activation of JNK in the mesenteric arteries. In conclusion, sorafenib not only impairs endothelium-dependent vasodilatation but also enhances ETB receptor-mediated vasoconstriction, which may be the causal factors for hypertension and other adverse vascular effects in patients treated with sorafenib.