Cellular Distribution of Endothelin-converting Enzyme-1 in Human Tissues

Reed

Journal of Biological Chemistry

2001

317

245

Deposition of ␤-amyloid (A␤) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of A␤ is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of A␤. Much less, however, is known about the mechanisms responsible for A␤ removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel A␤-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular A␤ concentration that appears to be due to inhibition of intracellular A␤ degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular A␤ concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic A␤40 and A␤42 in vitro at multiple sites.

Section: Fig 4 Removal Of Exogenous Synthetic A␤ By Ece-overexpressmentioning

confidence: 99%

“…The first identified, ECE-1, is abundantly expressed in the vascular endothelial cells of all organs and is also widely expressed in nonvascular cells of tissues including lung, pancreas, testis, ovary, and adrenal gland (17)(18)(19). A comprehensive analysis examining both ECE activity and expression in human brain has not been reported.…”

mentioning

confidence: 99%

Degradation of the Alzheimer's Amyloid β Peptide by Endothelin-converting Enzyme

Reed

Journal of Biological Chemistry

2001

317

245

Laboratory Investigation

“…Sections were washed in PBS, incubated with primary antibodies: human anti-CD31 and anti-CD68 (Dako, Zug, Switzerland), anti-␣-SMA (Sigma, Buchs, Switzerland), monoclonal anti-ET-1 (ABR; Affinity Bioreagents, Alexis Corporation, Läufelingen, Switzerland), polyclonal anti-ET-1 (Peninsula, Brunschwig, Basel, Switzerland), anti-ECE-1 (antiserum 473-17-A; (Korth et al, 1999), or to Ulex Europaeus (Vector, Alexis Corporation) and subsequently exposed either to avidin-biotin complex (ABC; Dako) or to peroxidase-conjugated secondary immunoglobulins, according to the manufacturers' instructions. Peroxidase activity was visualized using 0.035% diaminobenzidine (Fluka, Buchs, Switzerland) as a chromogen and slides were counterstained with hematoxylin.…”

Section: Immunohistochemistrymentioning

confidence: 99%

The Endothelin System in Human Glioblastoma

Egidy

Eberl

Valdenaire

et al. 2000

Self Cite

SUMMARY:Endothelin-1 (ET-1) is a powerful mitogenic and/or anti-apoptotic peptide produced by many cancer cells. To evaluate the potential role of the endothelin system in glioblastoma we first determined the cellular distribution of the mRNA and proteins of the components of the endothelin system, preproendothelin-1 (PPET-1), endothelin-converting enzyme-1 (ECE-1), and ET A and ET B receptors in human glioblastoma tissue and glioblastoma cell lines. PPET-1, ECE-1, and ET A receptor were highly expressed in glioblastoma vessels and in some scattered glioblastoma areas whereas ET B receptor was mainly found in cancer cells. This suggests that glioblastoma vessels constitute an important source of ET-1 that acts on cancer cells via the ET B receptor. Four human glioblastoma cell lines expressed mRNA for all of the components of the ET-1 pathway. Bosentan, a mixed ET A and ET B receptor antagonist, induced apoptosis in these cell lines in a dose-dependent manner. Apoptosis was potentiated by Fas Ligand (APO-1L, CD95L), a pro-apoptotic peptide, only in LNZ308 cells, corresponding to the known functional Fas expression in these cell lines. LNZ308 cells also expressed the long and short forms of the cellular FLICE/caspase-8 inhibitory protein (FLIP). Bosentan and a protein kinase C inhibitor down-regulated short FLIP in these cells. ET-1 induced transient phosphorylation of extracellular signal-regulated kinase but did not induce long-term thymidine incorporation in LNZ308 glioblastoma cells. These results suggest that, in glioblastoma cells, ET-1, mainly acting via the ET B receptor, is a survival/antiapoptotic factor produced by tumor vasculature, but not a proliferation factor, involving protein kinase C and extracellular signal-regulated kinase pathways, and stabilization of the short form of FLIP. (Lab Invest 2000, 80:1681-1689.

“…ECE-1, however, is the most physiologically implicated because of its wide action and expression. 18,19 ET-1 binds 2 different receptor subtypes: endothelin A (ET A ) receptors, which have a higher affinity for ET-1 than ET-3, and endothelin B (ET B ) receptors, which have equal affinity for ET-1 and ET-3. 20,21 The ET A receptors are located on VSMCs and mediate vasoconstriction.…”

mentioning

confidence: 99%

Tissue-Engineered Human Vascular Media With a Functional Endothelin System

et al. 2005

Background-Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor's hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel. Methods and Results-Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ET A receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ET A but not the ET B receptor was present in these human tissue-engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media. Key Words: endothelin Ⅲ muscle contraction Ⅲ myocytes, smooth muscle C oronary artery disease is the most common cause of morbidity and mortality in Western countries. 1 Treatment options include drugs, balloon angioplasty, and CABG surgery. Most artery bypass graft surgery involves harvesting of the long saphenous vein or mammary artery. However, in about a third of patients, this surgery proves inadequate or unsuitable. 2 Surgeons have therefore turned to the use of prosthetic materials like polyethylene terephthalate (Dacron), polytetrafluoroethylene, expanded polytetrafluoroethylene, and polyurethane. [2][3][4][5][6][7][8] However, the results obtained with these prosthetic materials prove to be inferior to those obtained with autologous conduits, especially when the blood vessel diameter is Ͻ5 mm. The problems that can arise when these types of materials are used include increased risk of thrombosis and infection, limited durability, lack of compliance both of the graft and around the anastomosis, 9 and failure as a result of restenosis. These problems necessitate further interventions. 10 -12 One approach to the elaboration of a vascular prosthesis would be to construct one that would very closely resemble a natural healthy blood vessel in both composition and characteristics. This prosthesis would be composed of human cells only (devoid of exogenous synthetic material) and would have the capacity to contract and relax in response to vasoactive agents. We have recently developed a tissueengineering approach to produce a completely biological blood vessel from...