Construction, expression and characterization of a soluble form of human endothelin-converting-enzyme-1.

Endothelin-converting-enzyme-1 (ECE-1) belongs to the family of zinc metallopeptidases and is responsible for generating endothelin (ET) peptides from their inactive precursors the big endothelins (bigET). The enzyme is a type II integral membrane protein consisting of a short amino-terminal cytosolic domain of 56 amino acids, a single transmembrane domain and a large putative extracellular domain containing the catalytic site. Recombinant and native ECE-1 are expressed as a dimer. We have constructed a soluble form of ECE, named sECE*, by fusing the cleavable signal peptide of pro-opiomelanocortin in frame to the complete extracellular domain of human ECE-1. Stable expression of this construct in CHO cells resulted in the secretion of a fully active enzyme. In contrast to membrane-bound ECE, sECE* was expressed as a monomer, highly glycosylated, as assessed by gel filtration and Western blot. However, recombinant sECE* converted bigET-1 with similar specific activity as ECE-1a. This activity was completely inhibited by phosphoramidon, but not by thiorphan and captopril. sECE* was active in a broad range of pH, showing an optimum of 6.6-6.8 for bigET-1. Thus, the extracellular domain alone is sufficient for conferring full ECE-1 activity, inhibitors recognition and substrate specificity.

Cellular distribution of endothelin-converting enzyme-1 in human tissues.

Endothelin-converting enzyme-1 (ECE-1) is the key enzyme of endothelin biosynthesis, catalyzing the final processing step. As shown by the targeted disruption of the ECE-1 gene, mature endothelins must be produced at specific sites for normal embryonic development. Therefore, it is important to know the exact pattern of ECE-1 gene expression. In this study we investigated the cellular distribution of ECE-1 in a variety of human tissues by in situ hybridization and immunohistochemistry. Widespread expression of the ECE-1 gene was noted, with a similar distribution pattern for mRNA and protein in normal human tissues, suggesting a major biological role for ECE-1. ECE-1 levels were particularly high in the cardiovascular, reproductive, and endocrine systems. There was strong and consistent labeling for ECE-1 in the vascular endothelial cells of all organs examined and in various nonvascular cells, especially some glandular cells. A large amount of ECE-1 protein and mRNA was detected in the Leydig cells of the testis and in the granulosa and theca cells of the ovary. In the adrenal gland, ECE-1 was detected in the cortex and medulla, with the strongest labeling in the zona glomerulosa. Therefore, ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. These results point out the potential side effects of ECE-1 inhibitors that are currently under development for treatment of cardiovascular diseases. (J Histochem Cytochem 47:447-461, 1999)

The endothelin system in normal human colon.

Endothelin (ET)-1 is a potent vasoconstrictor and mitogenic peptide that has a variety of biological effects in noncardiovascular tissues. The precise cellular distribution of the ET-1 system in the wall of the normal human colon was studied to identify the physiological role of ET in the gut. In situ hybridization revealed ET-converting enzyme-1 (ECE-1) mRNA in all vessels, the colon epithelium, and macrophages. Prepro-ET-1 (PPET-1) mRNA had a similar distribution except for a scattered signal in mucosal microvessels. ET(A) and ET(B) receptor mRNAs were mainly in the lamina propria, pericryptal myofibroblasts, microvessels, and mononuclear cells, with ET(A) mRNA more abundant than ET(B) mRNA. (125)I-ET-1 binding showed ET(B) along the crypts and in nerve fibers descending from the ganglionic plexus that contained PPET-1, ECE-1, and ET(B) transcripts, whereas glia contained ET(A) receptors. The finding of the entire ET system in the normal mucosa suggests its implication in some characteristic functions of the colon and its secretion as both a neuroactive and a vasoactive peptide.

Expression of the endothelin-converting enzyme-1 isoforms in endothelial cells.

The transformed human endothelial cell line EA.hy926 is commonly used for studying in vitro different aspects of endothelial cell biology such as signal transduction, expression or angiogenesis. These cells have the ability to process big endothelin (big-ET) into endothelin (ET), and express the endothelin-converting enzyme ECE-1. Several isoforms of ECE-1 which differ only in their N-terminal part (i.e. the end of the cytosolic domain) have now been identified. We could detect the co-expression of all four isoforms. Recent works have shown that the variable cytosolic domain is responsible for the differential intracellular localization of ECE-1 isoforms. Using antibodies directed against ECE-1a and ECE-1b/c/d, we have characterized the intracellular distribution of these isoforms in EA.hy926 cells by immunofluorescence. Electron microscopy allowed us to identify further the intracellular compartment that contains ECE-1 as multivesicular bodies, a compartment involved in the endocytic pathway. In addition, using an antibody directed against the catalytic domain, we could demonstrate that no monomeric ECE-1 is present at the plasma membrane. Indeed, detection of ECE-1 immunoreactivity at the cell surface of living cells required a dithiothreitol (DTT) treatment. Altogether, these results demonstrate that the EA.hy926 cell line is a helpful model for studying the regulation of the production of endothelin by ECE.

Hemorrhage after pancreatoduodenectomy.

OBJECTIVE: The authors reviewed the hemorrhagic complications of patients who underwent pancreatoduodenectomies between 1972 and 1996. SUMMARY BACKGROUND DATA: Although recent studies have demonstrated a reduction in the mortality of pancreatic resection, morbidity is still high. Bleeding is a close second to anastomotic dehiscence in the list of dangerous postoperative complications. METHODS: The medical records from a prospective data bank of 559 patients who underwent pancreatic resection at the Surgical Clinic of Mannheim (Heidelberg University) were analyzed in regard to postoperative hemorrhagic complications. Differences were evaluated with the Fisher exact test. RESULTS: The overall mortality rate was 2.7%. Postoperative bleeding occurred in 42 patients (7.5%), with 6 episodes ending fatally (14.3%). Erosive bleeding after pancreatic leak was noted in 11 patients (26.2%), 4 of whom died. Gastrointestinal hemorrhage occurred in 22 patients, and operative field hemorrhage was present in 20 cases. Relaparotomy was necessary in 29 patients. An angiography with interventional embolization for recurrent bleeding was performed in three patients. Seven hemorrhages (4.6%) occurred after pancreatectomy for chronic pancreatitis and 35 episodes of bleeding (8.6%) were encountered after pancreatectomy for malignant disease. Obstructive jaundice was present in 359 patients (63.9%). In this group of patients, 32 (8.9%) postoperative hemorrhages occurred. Preoperative biliary drainage did not influence the type and mortality rate of postoperative hemorrhage in jaundiced patients. CONCLUSION: The prevention of these bleeding complications depends in the first place on meticulous hemostatic technique. Preoperative biliary drainage does not lower postoperative bleeding complications in jaundiced patients. Continuous, close observation of the patient in the postoperative period, so as to detect complications in time, and expeditious hemostasis are paramount.

Modulation of human colon tumor-stromal interactions by the endothelin system.

Tumor neovascularization is considered to be a critical step in the development of a malignant tumor. Endothelin (ET)-1 is a powerful vasoconstrictor and mitogenic peptide that is produced by many cancer cell lines. The cellular distribution of the ET components was evaluated in human colon tumors and compared to normal colon. There was more of the ET components (preproET-1, endothelin-converting enzyme-1, and ETA and ETB receptors) in adenomas and adenocarcinomas than in the normal colon. There was overproduction of preproET-1 and endothelin-converting enzyme-1 in carcinoma cells and stromal vessels, suggesting that they are a local source of ET-1. ETA receptors were present in stromal myofibroblasts of neoplastic tissue, and there were large amounts of ETB receptors in the endothelium and myofibroblasts. There was also a redistribution of alpha-smooth muscle actin-positive cells in the vascular structures of tumors. An experimental rat model of induced colon cancer treated for 30 days with bosentan, a mixed antagonist of both ET receptors, confirmed the morphological changes observed during the tumor vascularization. Our data suggest that ET-1 and its receptor play a role in colon cancer progression, with ET-1 functioning as a negative modulator of the stromal response.

The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).