Identification of endothelin 2 as an inflammatory factor that promotes central nervous system remyelination.

The development of new regenerative therapies for multiple sclerosis is hindered by the lack of potential targets for enhancing remyelination. The study of naturally regenerative processes such as the innate immune response represents a powerful approach for target discovery to solve this problem. By 'mining' these processes using transcriptional profiling we can identify candidate factors that can then be tested individually in clinically-relevant models of demyelination and remyelination. Here, therefore, we have examined a previously described in vivo model of the innate immune response in which zymosan-induced macrophage activation in the retina promotes myelin sheath formation by oligodendrocytes generated from transplanted precursor cells. While this model is not itself clinically relevant, it does provide a logical starting point for this study as factors that promote myelination must be present. Microarray analysis of zymosan-treated retinae identified several cytokines (CXCL13, endothelin 2, CCL20 and CXCL2) to be significantly upregulated. When tested in a cerebellar slice culture model, CXCL13 and endothelin 2 promoted myelination and endothelin 2 also promoted remyelination. In studies to identify the receptor responsible for this regenerative effect of endothelin 2, analysis of both remyelination following experimental demyelination and of different stages of multiple sclerosis lesions in human post-mortem tissue revealed high levels of endothelin receptor type B in oligodendrocyte lineage cells. Confirming a role for this receptor in remyelination, small molecule agonists and antagonists of endothelin receptor type B administered in slice cultures promoted and inhibited remyelination, respectively. Antagonists of endothelin receptor type B also inhibited remyelination of experimentally-generated demyelination in vivo. Our work therefore identifies endothelin 2 and the endothelin receptor type B as a regenerative pathway and suggests that endothelin receptor type B agonists represent a promising therapeutic approach to promote myelin regeneration.

Epigenetic inactivation of endothelin-2 and endothelin-3 in colon cancer.

Endothelin-1 (ET-1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET-2 and ET-3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET-2 and ET-3 expression and determine the underlying mechanisms. Human primary colon cancers and carcinogen-induced rat colon tumors were subjected to real-time RT-PCR, immunoblotting and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation-specific PCR, bisulfite sequencing and pyrosequencing; and forced expression of ET-2 and ET-3 was conducted in human colon cancer cells followed by real-time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET-2 and ET-3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers and in a panel of human colon cancer cell lines. Forced expression of ET-2 and ET-3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET-2 and ET-3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re-expression of ET-2 and ET-3 as natural antagonists of ET-1 in colon cancer.

Production and binding of endothelin-2 (EDN2) in the rat ovary: endothelin receptor subtype A (EDNRA)-mediated contraction.

Endothelin-2 (EDN2)-mediated contraction has been proposed as a final mechanical signal facilitating ovulation. The objectives herein were to determine (1) whether ovarian endothelins were increased before ovulation; (2) whether a specific endothelin-converting enzyme (ECE) was mediating their production; (3) which receptor was facilitating ovarian contraction; and (4) whether receptor-specific antagonism affected ovulation. Follicular development was induced in immature rats with 10 IU pregnant mare serum gonadotrophin (PMSG) and the ovulatory cascade was initiated 48 h later with 10 IU human chorionic gonadotrophin (hCG). In Experiment 1, an immunoassay revealed that the ovarian concentration of endothelin peptide was increased 7-fold 12 h after hCG when compared with 48 h after PMSG (P < 0.05). In Experiment 2, real-time PCR indicated that mRNA for Ece1, but not Ece2, was increased in granulosa cells collected 12 h after hCG when compared with those collected before the ovulatory stimulus (P < 0.05). In Experiment 3, isometric tension analysis revealed that the contractile effect of EDN2 was mediated by endothelin receptor A (EDNRA), not B (EDNRB). In Experiment 4, no effect was observed on the rate of ovulation when rats were treated with an antagonist specific to EDNRA (BQ123) or EDNRB (BQ788), or when mice were treated with BQ123, BQ788 or BQ123 + BQ788. In conclusion, endothelin peptide is produced before ovulation and the contractile action of EDN2 within the ovary is facilitated via EDNRA. In addition, findings of this study indicate synergistic interactions among contractile factors affect ovulatory outcome, while the role of EDNRB alone in the process of ovulation requires further investigation.

Frequent loss of endothelin-3 (EDN3) expression due to epigenetic inactivation in human breast cancer.

INTRODUCTION: Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer. METHODS: EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome. RESULTS: Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate (P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA re-expression in vitro. Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression (P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation. CONCLUSIONS: EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland.

Characterization of CoCl2-induced reactive oxygen species (ROS): Inductions of neurite outgrowth and endothelin-2/vasoactive intestinal contractor in PC12 cells by CoCl2 are ROS dependent, but those by MnCl2 are not.

CoCl(2) and MnCl(2) are hypoxic mimetic agents. We previously found that expression of ET-2/VIC, one of hypoxia-related factors, and the induction of neurite outgrowth in PC12 cells through ROS induced by CoCl(2). MnCl(2) also are known to induce neurite outgrowth in PC12 cells. However, it is unclear whether the mechanism of the effect induced by these metals is same. In the present study, we evaluated biological effects induced by MnCl(2) and compared with those induced by CoCl(2). Furthermore, we analyzed sources of CoCl(2)-induced ROS generation. MnCl(2) up-regulated ET-2/VIC gene expression and ET-2/VIC peptide production as CoCl(2) did, but not affect ET-1 gene expression, in the neurite outgrowth of PC12 cells. NAC did not at all inhibit the effects induced by MnCl(2). Furthermore, addition of MnCl(2) to the culture medium did not generate ROS as CoCl(2) did. These results indicate that ET-2/VIC expression is a common pathway in neurite outgrowth induced by CoCl(2) and MnCl(2), but the effects induced by CoCl(2) are ROS dependent, whereas the effects induced by MnCl(2) are ROS independent. Taken together, the mechanism for the effects by CoCl(2) was different from that by MnCl(2). The ROS, were not decomposed by catalase or SOD, were rapidly generated by reaction of CoCl(2) mainly with components of HS rather than with FBS or DMEM. Some ROS generated by reaction of CoCl(2) with components of HS may participate in the observed neurite outgrowth of PC12 cells.

A role for endothelin-2 and its receptors in breast tumor cell invasion.

We have studied the role of endothelins (ET-1, ET-2 and ET-3) and ET receptors (ET-RA and ET-RB) in the invasive capacity of breast tumor cells, which express ET-1 and ET-2 as well as ET-RA and ET-RB. Of five human breast tumor cell lines tested, all expressed mRNAs for ET-1, ET-2, and ET-RB. ET-RA mRNA was expressed by four of five tumor cell lines. Breast tumor cells migrated toward ET-1 and ET-2 but not toward ET-3. Chemotaxis involved signaling via both receptors, and a pertussis toxin-sensitive p42/p44 mitogen-activated protein kinase (MAPK)-mediated pathway that could be inhibited by MAPK kinase (MEK)1/2 antagonists. Chemotaxis toward ETs did not involve p38 or stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) and was not inhibited by hypoxia. Incubation of tumor cells with ET-2 also increased chemotaxis toward the chemokines CXCL12 and CCL21. As well as inducing chemotaxis of tumor cells, ET-1 and ET-2 increased tumor cell invasion through Matrigel. Furthermore, stimulation of macrophage/tumor cell cocultures with ETs led to increased matrix metalloproteinase (MMP)-2 and -9 production by macrophages and a marked increase in invasion of tumor cells. Antagonism of either ET-RA or ET-RB decreased the invasion seen in ET-stimulated cocultures, as did a broad-spectrum MMP inhibitor. Immunohistochemical staining of human breast tumor sections showed increased ET and ET receptor protein expression by tumor cells in invasive ductal carcinoma compared with normal breast tissue or ductal carcinoma in situ. Furthermore, tumor cell ET and receptor expression was stronger at the invasive margin of invasive ductal carcinomas, in the lymphovascular space, and in lymph node metastases. ET expression often colocalized with ET-RB expression in all neoplastic tissue indicating a possible autocrine action of ETs. We suggest that expression of ETs and their receptors by human breast tumors, particularly in conjunction with a high macrophage infiltrate, may have a role in the progression of breast cancer and the invasion of tumor cells.

Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression.

ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor-alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.

Myocardial expression of endothelin-2 is altered reciprocally to that of endothelin-1 during ischemia of cardiomyocytes in vitro and during heart failure in vivo.

We and other groups have reported that endothelin (ET)-1 expression in the heart is altered in the setting of heart diseases. We have also reported that myocardial ET-1 is involved in the progression of heart failure, and that an ET receptor antagonist improves long-term survival in heart failure (Nature 384: 353-355, 1996). However, the role of myocardial ET-2 in disease states are not known. To characterize the role of ET-2, we used a) the failing hearts of rats with heart failure caused by myocardial infarction, and b) primary cultured cardiomyocytes subjected to hypoxia. In the failing heart in vivo, ET-1 mRNA increased by 390% compared with that in the non-failing heart, while ET-2 mRNA drastically decreased by 88%. Thus, gene expression of ET-1 and ET-2 was reciprocally altered in the failing heart in vivo. In in vitro studies, reciprocal alterations in ET-1 and ET-2 gene expression were also observed in isolated primary cultured cardiomyocytes, subjected to hypoxia. Specifically, acute hypoxic stress induced a significant increase (360% of the basal level) in ET-2 mRNA expression compared with that in normoxic cells, whereas it decreased ET-1 mRNA expression by 62% in primary cultured cardiomyocytes. Although these two crucial conditions, i.e., heart failure in vivo and acute hypoxic stress in vitro, are pathophysiologically distinct from each other, reciprocal alteration of ET-1 and ET-2 gene expression was observed in both cases. To further investigate the regulatory mechanism of the altered gene expression, luciferase analysis was performed using primary cultured cardiomyocytes. ET-2 promoter, which is the 5'-flanking region of preproET-2 gene (5'ET-2), showed a marked increase in luciferase activity during acute hypoxia. In contrast, the luciferase activity of 5'ET-1 (ET-1 promoter) did not change in response to hypoxic stress. The present study suggests that there are transcriptionally distinct regulatory mechanisms for ET-1 and ET-2 expression in cardiomyocytes, and therefore this study may provide a new aspect of cardiac ET system that not only ET-1 but also ET-2 can be participated in the pathophysiological conditions.

[Does captopril decrease endothelial production of endothelin?]. / Czy kaptopryl hamuje sródblonkowa synteze endoteliny?

Serum levels endothelin-1,2 aldosterone, renin activity were performed in 52 patients with chronic congestive heart failure during captopril therapy. The investigations were done before therapy, after 10-15 days therapy and after disappearance symptoms of heart failure. The levels endotelin-1,2,aldosterone and renin activity were determined using radioimmunologic methods. Fraction ejection and peripheral vascular resistance were determined by echocardiography technics. It was found that serum endothelin levels in patients with chronic congestive heart failure were increased proportionally to functional class of heart failure and decreased after disappearance symptoms of heart failure. An increase in plasma endothelin concentration has not correlated with ejection fraction, aldosterone concentration and renin activity. These studies have demonstrated that in patients with congestive heart failure on captopril therapy endothelin serum levels positively correlates with peripheral vascular resistance. No correlation was found between ejection fraction and activity renin-angiotensin-aldosterone system. These findings indicated that captopril therapy decreases endothelin production.

Expression of endothelin 2 and localized clear cell renal cell carcinoma.

Despite the rising incidence of clear cell renal cell carcinoma, the molecular events that support its development and progression remain unclear. Herein, we evaluate the association of endothelin 2 expression with both clear cell renal cell carcinoma development and progression-free survival. We conducted real-time polymerase chain reaction to determine endothelin 2 expression levels on 238 patients who underwent nephrectomy for localized clear cell renal cell carcinoma, 161 of whom also had adjacent normal kidney samples available for analysis. To evaluate associations with clear cell renal cell carcinoma development, linear mixed models were used to compare differential expression between tumor and a normal kidney as well as to explore interactions with clinicopathologic features. To evaluate associations with prognosis, Cox proportional hazards models were used to assess the association of progression-free survival and endothelin 2 expression in tumor tissue. Overall, endothelin 2 expression was higher in tumor samples versus patient-matched normal kidney samples, with an average fold change of 1.99 (95% confidence interval, 1.48-2.60; P < .0001). This overexpression in tumor versus normal kidney samples was more pronounced in low- compared with high-grade tumors (interaction, P = .0002), in early- compared with late-stage tumors (interaction, P = .001), and in tumors without compared with those with necrosis (interaction, P = .001). Moreover, an increasing endothelin 2 expression in tumors was associated with a longer progression-free survival (hazard ratio, 0.89; 95% confidence interval, 0.80-0.99; P = .03); however, after controlling for known clinicopathologic factors, this association was attenuated (hazard ratio, 0.99; 95% confidence interval, 0.89-1.09; P = .7). Up-regulation of endothelin 2 is a common and early event in localized clear cell renal cell carcinoma. Higher tumor expression of endothelin 2 is associated with a longer progression-free survival but not after adjustment for well-known pathologic indices. Thus, although endothelin 2 does not appear to be an independent prognostic marker, there is evidence of a putative role in clear cell renal cell carcinoma progression. If supportive mechanistic data can be produced, endothelin 2 could represent a potential target for chemopreventive or neoadjuvant therapeutics for clear cell renal cell carcinoma.

Structure of human endothelin-2 gene and demonstration of common expression in human right atrial tissue.

The endothelins (ET) are a group of three vasoactive peptides also known to be involved in vascular remodeling. ET1 is the most extensively studied, but recent evidence has highlighted the role of the little investigated ET2 gene as a potential candidate gene in regulating blood pressure. To allow the future role of this gene to be studied the structure of human ET2 was characterized and intron/exon boundaries were determined. With this structural information and using reverse-transcriptase PCR technology we show that the ET2 gene is commonly expressed in human right atrial tissue. This work will allow a more detailed assessment of the role of this physiologically important gene in human essential hypertension.

Selective expression of endothelin 1 mRNA in rat retina.

Endothelin is a potent vasoactive agent and three isoforms--endothelin 1 (ET-1), endothelin 2 (ET-2) and endothelin 3 (ET-3)-- have been found in ocular tissues. However its source has not been determined. Therefore we have investigated the ET mRNA in the rat retina using reverse transcriptase with polymerase chain reaction. For ET-1, three retina samples were positive. For ET-2 and ET-3, all samples were negative. ET-1 mRNA was abundantly expressed compared with ET-2 and ET-3 mRNA (p < 0.05). Our study provides direct evidence that ET-1 is abundantly synthesized in the rat retina. The ET-1 within the retina could contribute to the regulation of the retinal circulation through vessel contraction.

Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice.

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.

Vasoactive intestinal contractor/endothelin-2 gene expression in the murine central nervous system.

Vasoactive intestinal contractor (VIC) is a member of the endothelin (ET) family. We have investigated the regional distribution of VIC/ET-2 and of ET-1 gene expression in the adult murine brain and pituitary gland. We used real-time quantitative reverse transcription-linked polymerase chain reaction. VIC/ET-2 gene expression was observed at high levels in the pituitary gland and medulla oblongata in both the mouse and rat. Moderate to low levels of expression were observed in other brain regions. On the contrary, ET-1 gene expression was quite low in the pituitary gland in comparison with the levels observed in the cerebral cortex, striatum, and midbrain. Cold injury to the mouse cerebral cortex caused a significant decrease in VIC/ET-2 gene expression in this structure, whilst expression of the ET-1 gene was increased. These results suggest that VIC/ET-2 may have certain physiological roles that differ from those of ET-1 in the brain and pituitary gland.