Therapeutic potential of endothelin inhibitors in canine hemangiosarcoma.

AIMS: Hemangiosarcoma (HSA) that originates from vascular endothelial cells is the most common splenic malignant neoplasm in dogs, as it accounts for approximately 20% of all canine soft tissue sarcomas. In this study, inhibitory effects of endothelin receptor antagonists on the growth of HSA cells were examined using cell lines established from canine HSA. MAIN METHODS: The preproendothelin-1 (PPET-1), endothelin type A receptor (ETA) and endothelin type B receptor (ETB) mRNA expression levels in HSA cell lines (n=5) were analyzed quantitatively by real-time RT-PCR. These levels were compared with those in HSA tissues (n=11) and those in normal splenic tissues (n=6). ETA and ETB protein expression was examined by western blot. The production and secretion of endothelin-1 (ET-1) and big ET-1 by cell lines were analyzed by measuring the levels in the culture medium by ELISA. The inhibitory effects of endothelin receptor antagonists (ambrisentan, BQ788 and bosentan) on cell growth were evaluated by WST-8 assay. KEY FINDINGS: The PPET1 and ETA mRNA expression levels were elevated in HSA tissues and HSA cell lines compared with normal tissues. In cell lines, the production of ET-1 and big ET-1 peptide as well as the expression of ETA protein were detected, but the levels of ETB were not measured. Ambrisentan and bosentan inhibited growth activity in cell lines. Ambrisentan was more effective than bosentan. SIGNIFICANCE: These findings demonstrate the importance of the ETA axis in canine HSA as well as the potential of ETA inhibitors in the treatment of canine HSA.

Big endothelin-1 as a tumour marker for canine haemangiosarcoma.

Haemangiosarcoma (HSA) is an important malignant neoplasm of dogs that originates from vascular endothelial cells. This study explored the suitability of using serum big endothelin-1 (ET-1) as a tumour marker for canine spontaneous HSA. Serum big ET-1 was measured in dogs with splenic HSA (n = 14), splenic malignant tumours other than HSA (n = 10), benign splenic lesions (n = 11) and normal healthy dogs (n = 17) by ELISA. Serum big ET-1 levels in dogs with HSA were significantly (P < 0.01) higher than in other dogs. High sensitivity (100%, 95% confidence interval 86-100%) and specificity (95%, 95% confidence interval 86-95%) for HSA diagnosis were obtained using a cut-off of 17 pg/mL according to receiver operating characteristic (ROC) curves (area under ROC curve 0.93). PPET1, ETA, VEGF and Hif1-α mRNA expression, measured by real-time PCR, were elevated in HSA compared with normal tissues. These findings suggest that elevated serum big ET-1 could be used as a diagnostic marker for canine HSA.

Effect of feeding behavior on circadian regulation of endothelin expression in mouse colon.

AIMS: The function, regulation and gene expression of the endothelin (ET) system in the intestine is not well understood. We investigated the dependence on feeding schedule and biological clock of the regulation of ET-1 gene expression in mouse colon. MAIN METHODS: Mice were fed freely, fasted for 48 h and re-fed after fasting. KEY FINDINGS: Where indicated ET-1 gene expression was highest in the colon compared with other tissues examined in fasted mice. Fasting increased the level, while maintaining the rhythmicity, of ET-1 gene expression in epithelial colonic tissue. Re-feeding, however, decreased ET-1 gene expression and suppressed rhythmic oscillation, and the rhythmicity also changed for gene expression for circadian clocks, period-1 and period-2 (Per1 and Per2). Furthermore, the decrease in ET-1 gene expression induced by re-feeding was blocked by pre-treatment with hexamethonium and atropine. The daily change in ET-1 gene expression in colon, which depends on feeding schedule via the autonomic nervous system, is synchronized with peripheral circadian oscillators under conditions of free feeding and fasting but not re-feeding. The decrease in ET-1 gene expression in the proximal colon induced by re-feeding occurs via the nervous system. SIGNIFICANCE: ET-1 plays an important physiological role, which is dependent on feeding behavior.

Endothelin system in intestinal villi: A possible role of endothelin-2/vasoactive intestinal contractor in the maintenance of intestinal architecture.

The endothelin system consists of three ligands (ET-1, ET-2 and ET-3) and at least two receptors (ETA and ETB). In mice ET-2 counterpart is a peptide originally called "vasoactive intestinal contractor" (VIC) for this reason, this peptide is frequently named ET-2/VIC. In intestinal villi, fibroblasts-like cells express endothelin's receptors and response to ET-1 and ET-3 peptides, changing their cellular shape. Several functions have been attributed to these peptides in the "architecture" maintenance of intestinal villi acting over sub-epithelial fibroblasts. Despite this, ET-2/VIC has not been analyzed in depth. In this work we show the intestine gene expression and immunolocalization of ET-1, ET-2 and the ETA and ETB receptors from duodenum to rectus and in the villus-crypt axis in mice, allowing a complete analysis of their functions. While ET-1 is expressed uniformly, ET-2 had a particular distribution, being higher at the bottom of the villi of duodenum, ileum and jejunum and reverting this pattern in the crypts of colon and rectus, where the higher expression was at the top. We postulated that ET-2 would act in a cooperative manner with ET-1, giving to the villus the straight enough to withstand mechanical stress.

Regulation of endothelin-1 expression and function by nutrient stress in mouse colon epithelia.

OBJECTIVE: The endothelin (ET) system is influenced by a variety of stress conditions in many tissues. However, the effects of nutrient stress conditions on ET expression and its function are not well understood in the intestinal tract, while ET-1 gene expression and peptide were found in the intestinal tract. The aim of this study was to investigate the effect of feeding and fasting on the expression of ET-1 and short-circuit current (Isc) induced by ET-1 in mouse colon. MATERIAL AND METHODS: Mice were fed freely, fasted for 48 h, and re-fed after fasting, respectively. ET-1 mRNA levels and peptide concentrations were analyzed using real-time polymerase chain reaction (PCR) and sandwich ELISA, respectively. Isc of epithelial tissue was measured under short-circuit conditions using a Ussing chamber. RESULTS: ET-1 mRNA expression and peptide concentrations in epithelial colonic tissue were significantly increased 48 h after fasting, and decreased within 2 h of re-feeding after a 48-h fast. Furthermore, the addition of ET-1 to the serosal but not the mucosal side increased Isc in colonic epithelia. An increase in Isc was caused by chloride ion (Cl(-)) secretion because Isc induced by ET-1 was blocked by bumetanide and Cl(- -) free conditions. In addition, an increase in Isc induced by ET-1 in colon excised from fasted mice was much lower than that obtained from free-fed mice. CONCLUSIONS: Gene expression, peptide concentration, and the function of ET-1 in mouse colonic epithelia are regulated by nutrient stress.

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.

Endothelin-2/vasoactive intestinal contractor via ROCK regulates transglutaminase 1 on differentiation of mouse keratinocytes.

We previously found that endothelin-2/vasoactive intestinal contractor (ET-2/VIC) greatly increased in mouse epidermis after birth. In the present study, we evaluated whether ET-2/VIC expression was associated with the calcium-induced differentiation of cultured mouse keratinocytes. The differentiation induction was revealed by morphological change, cornified envelope (CE) formation, and involucrin and transglutaminase 1 (TG 1) expressions. ET-2/VIC gene expression and peptide production subsequently increased in the induction of the differentiation. We also found that Y-27632, a Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor, suppressed up-regulation of ET-2/VIC gene expression, the induction of morphological change, the CE formation, and TG 1 expression, but not involucrin expression. These results indicate new three findings, (1) ET-2/VIC expression increases and has potential as a differentiation marker, (2) ET-2/VIC expression is mediated by ROCK, and (3) the ROCK regulated TG 1 expression, on the calcium-induced differentiation of mouse keratinocytes.

High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis.

We examined the expression profiles of vasoactive intestinal contractor/endothelin-2 (VIC/ET-2) at both gene and peptide level in skin irradiated with different ultraviolet wavelengths. We found that VIC/ET-2 gene expression is sensitive only to ultraviolet-C (UVC) irradiation and has an immediate response. These results provide direct evidence that high doses of UVC irradiation induce an increase in gene expression and protein production of VIC/ET-2 and endothelin (ET) receptors in a dose-dependent manner in epidermal keratinocytes. We suggest that VIC/ET-2 can play an essential role in the maintenance, protection and hyperpigmentation of the epidermis exposed to UVC irradiation from artificial or natural sources.

cDNA cloning and sequence analysis of preproendothelin-1 (PPET-1) from salmon, Oncorhynchus keta.

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).

Primary structure of cat preproendothelin-2 and cat renal mRNA expression of preproendothelin-1 and preproendothelin-2 in naturally occurring renal failure.

Endothelin (ET)-1 is involved in the pathophysiology of various renal disorders, promoting renal cellular proliferation and extracellular matrix protein accumulation, and, thus, diminishing fundamental renal function, including filtration. To determine whether ET-1 and ET-2 play a role in feline chronic renal failure, we analyzed the messenger RNA (mRNA) expression of the prepro-ET (PPET )-1 and PPET-2 genes in affected cat kidney after molecular cloning of full-length PPET-2 complementary DNA (cDNA). Conceptual analysis of the primary structure of cat PPET-2 based on the cloned sequence demonstrated that the putative regions corresponding to a mature peptide and peptidase processing sites are present in cat PPET-2. Homology analysis showed that the similarity of the cat PPET-2 amino acid sequence with those from human, mouse, rat, rabbit, dog, ferret, cow, and horse was 73.0%, 68.6%, 69.1%, 76.4%, 81.2%, 83.1%, 76.3%, and 79.2%, respectively. Analysis of PPET-1 and PPET-2 mRNA in cat by reverse transcription polymerase chain reaction showed upregulated expression of both genes in kidneys affected by renal failure.

Rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction.

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Expression of preproendothelin-2 splice variant in cat.

Previous studies on human uterine and placental tissues have found variants, derived from alternatively spliced mRNAs, of preproendothelin-2 (PPET2) that lack a post-translational proteolytic site essential for normal processing. Here we report a splice variant of cat PPET2 mRNA expressed in the stomach. After cloning the full-length cDNA of cat PPET2, organ distribution analysis of the transcript by reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. In addition to the fragment with a size predicted based on the cDNA sequence obtained by cloning, an additional PCR fragment of smaller size was detected in stomach tissue. Subsequent cloning and sequence analysis of the smaller PCR product demonstrated that it derives from a splice variant with full-length deletion of a region corresponding to exon 4 of the PPET2 gene.

Endothelin-2/vasoactive intestinal contractor: regulation of expression via reactive oxygen species induced by CoCl2, and Biological activities including neurite outgrowth in PC12 cells.

This paper reviews the local hormone endothelin-2 (ET-2), or vasoactive intestinal contractor (VIC), a member of the vasoconstrictor ET peptide family, where ET-2 is the human orthologous peptide of the murine VIC. While ET-2/VIC gene expression has been observed in some normal tissues, ET-2 recently has been reported to act as a tumor marker and as a hypoxia-induced autocrine survival factor in tumor cells. A recently published study reported that the hypoxic mimetic agent CoCl2 at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced intracellular reactive oxygen species (ROS) increase and neurite outgrowth in neuronal model PC12 cells. The ROS was generated by addition of CoCl2 to the culture medium, and the CoCl2-induced effects were completely inhibited by the antioxidant N-acetyl cysteine. Furthermore, interleukin-6 (IL-6) gene expression was up-regulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by CoCl2-induced ROS may be associated with neuronal differentiation through the regulation of IL-6 expression. CoCl2 acts as a pro-oxidant, as do Fe(II, III) and Cu(II). However, some biological activities have been reported for CoCl2 that have not been observed for other metal salts such as FeCl3, CuSO4, and NiCl2. The characteristic actions of CoCl2 may be associated with the differentiation of PC12 cells. Further elucidation of the mechanism of neurite outgrowth and regulation of ET-2/VIC expression by CoCl2 may lead to the development of treatments for neuronal disorders.

Elevated endothelin-1 expression in dogs with heartworm disease.

We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9+/-2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4+/-0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.

Differential expression of endothelin-2 along the mouse intestinal tract.

Endothelin (ET)-2, an ET family peptide, is highly expressed in intestine. However, the specific distribution and function of ET-2 remain unknown. We elucidated the expression profile and localization of ET-2 in mouse gastrointestinal tract. Real-time PCR analysis revealed that ET-2 gene expression in the gastrointestinal tract of healthy animals was relatively high in the colon. Immunohistochemical analysis revealed ET-2-like immunoreactivity mainly in epithelial cells of the mucosa throughout the intestinal tract of healthy animals. Intracellularly, ET-2 was concentrated close to the basement membrane of intestinal epithelial cells. A weak ET-2-like immunoreactivity was also localized to some neurofibers and the myenteric plexus of the muscle layer, coexpressing with vasoactive intestinal peptide. ET-2-like immunoreactivity was also detected at Brunner's glands of the duodenum and follicle-associated epithelium of Peyer's patch. In contrast, ET-1-like immunoreactivity was uniformly distributed in epithelial cells. In dextran sulfate sodium (DSS)-induced colitis, colonic ET-2 was upregulated during the late stage of DSS treatment. These results suggest that in intestinal epithelial cells ET-2 could be secreted into the lamina propria and the dome region in Peyer's patch, and that it might modulate immune cells in these sites for mucosal defense.

Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor.

We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 microM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 microM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells.

Differential circadian expression of endothelin-1 mRNA in the rat suprachiasmatic nucleus and peripheral tissues.

The vasoconstrictor endothelin-1 (ET-1) is implicated in normal neuronal functions. Here we show the circadian expression of ET-1 mRNA in the rat suprachiasmatic nucleus (SCN) that is considered to be the location of the central circadian pacemaker, as well as in peripheral tissues including the brain, heart, and lungs. The expression of ET-1 in the SCN oscillated with a peak at Zeitgeber time (ZT) 4 under light-dark conditions. A significant number of cells in the SCN was stained with ET-1 probe during circadian time (CT) 6, but there was no significant staining at CT18 by mRNA in situ hybridization. The circadian rhythm of ET-1 mRNA in the whole brain also oscillated, but peaked at ZT20. Endothelin-1 expression in the lungs and heart peaked at ZT12 and ZT20, respectively. The results are the first description of the circadian expression of ET-1 mRNA. The diversity of rhythmic expressions among the SCN, whole brain, lungs and heart suggests that ET-1 has different functions in these tissues.

Complete cDNA sequence and mRNA expression of dog preproendothelin-3.

The full-length cDNA of dog preproendothelin-3 (PPET3) was cloned from lung tissue using RT-PCR and rapid amplification of cDNA ends. Aside from the poly (A) tail, the full-length cDNA was 1976 bp. A polyadenylation signal sequence and one copy of a consensus sequence, ATTTA, which is related to mRNA turnover, was found in the 3' noncoding region. The cDNA had a 594-bp open reading frame encoding a 198-amino acid polypeptide. Regions corresponding to a bioactive mature ET3 peptide, an intermediate form known as big-ET3, and an ET3-like peptide were observed in dog PPET3. Expression of PPET3 mRNA was detected throughout the organs examined, which included heart, lung, liver, kidney, spleen, stomach, pancreas, duodenum, colon, uterus, ovary and testis.

Molecular cloning and sequence analysis of the porcine precursor of endothelin-2.

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.

Structure of the precursor of Xenopus laevis endothelin-3 and phylogenetic analysis.

Endothelin (ET)-related receptors homologous to mammalian receptors have been cloned from Xenopus laevis, indicating that ET-related ligands may be present in this species. Here we cloned cDNAs encoding preproendothelin-3 (PPET-3) from the X. laevis intestinal cDNA library. X. laevis ET-3 cDNA encodes 201 amino acids, including a 20-amino-acid putative signal sequence, as well as mature ET-3, big ET-3, and ET-3-like sequences. X. laevis ET-3 differs by one amino acid from mammalian ET-3, and is identical to frog ET-3 recently purified from Rana ridibunda. This sequence together with other published PPET sequences were used to analyze the phylogenetic relationship among all ET family genes. This is the first report of the cDNA encoding the precursor protein of ET-3 in a non-mammalian species.