Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109.
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PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around beta-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer. (+info)
CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration.
(10/4803)
CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis. (+info)
Role of proteases in implantation.
(11/4803)
Implantation of the embryo into the endometrium is a critical step in the establishment of pregnancy and the failure of embryos to implant is a major limiting factor in the success of reproductive technologies. Furthermore, one or more of the molecules of importance at implantation could provide a suitable target for post-coital contraception. While there is considerable species variation in the extent to which the trophoblast invades the maternal endometrium and makes contact with the maternal blood supply, many of the molecular mechanisms are conserved among species. Three families of protease are involved in the matrix degradation required for implantation: the cysteine, serine and matrix metalloproteinases. Other proteases are required for the activation of regulatory molecules. Although trophoblast from all species appears to have a high invasive potential, this is limited by the presence of partner protease inhibitors, the presence of which provides restraint to this invasion. It is the balance between the proteases and their inhibitors at any focal point that determines the site and extent of trophoblast invasion. This review examines the literature regarding proteases and their inhibitors at early implantation sites across a range of species with very different forms of placentation and evaluates their common features and their dissimilarities. (+info)
Arterial damage induced by cryopreservation is irreversible following organ culture.
(12/4803)
OBJECTIVES: The aim of the present study was to investigate the changes which occur to the arterial wall following cryopreservation and thawing and to determine whether these changes are reversible after a week of culture in an organ bath. MATERIALS AND METHODS: Rat iliac arterial segments were cryopreserved. Once thawed, the arterial segments were cultured for a period of 0, 1, 2, 4 or 7 days. Freshly isolated rat iliac vessels cultured for 7 days served as the control group. Evaluation was made of ultrastructural changes, the expression of metalloproteinase activity (MMP-1, MMP-3 and MMP-9) and the apoptotic state of cells. RESULTS: The freezing-thawing process induced damage to the arterial segments compared to fresh control vessels. After 1 week of culture, arteries showed a high degree of tissue degeneration. Only a few individual endothelial cells remained on the luminal surface. There was a gradual increase in the proportion of apoptotic cells. The sequential expression of MMP-1 during the first 2 days and subsequent expression of MMP-3 and MMP-9 were of most significance. CONCLUSIONS: Cryopreservation induced damage to the vessels which could not be reversed by organ culture. The changes observed in the expression of metalloproteinases may be indicative of the degenerative process which occurs in the extracellular matrix. (+info)
Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes.
(13/4803)
PURPOSE: Retinal neovascularization is one of the leading causes of blindness. A crucial event in this process is the remodeling and penetration of the capillary basement membrane by migrating endothelial cells. This process requires proteolysis of basement membrane components by a variety of proteinases. The objective of the present study was to determine the expression of proteinases in human retinal tissues showing active neovascularization. METHODS: Epiretinal neovascular membranes surgically removed from patients with proliferative diabetic retinopathy were analyzed by zymography, and the types and amounts of proteinases present in the tissues were determined. Retinas from nondiabetic donor eyes served as control specimens. RESULTS: Both the high- (54 kDa) and low- (33 kDa) molecular-weight forms of urokinase were present at significantly higher levels in neovascular membranes than in normal retinas. The pro forms of the matrix metalloproteinases (MMP) MMP-2 and MMP-9 were significantly elevated in the neovascular membranes in comparison with levels in normal retinas. In addition, the active forms of these enzymes were present in the membranes, whereas there was no detectable level of the active forms in normal retinas. CONCLUSIONS: Human diabetic neovascular membranes contain high levels of urokinase and MMP. The increased activity of proteinases in the final common pathway of retinal neovascularization indicates that inhibition of these enzymes may be a useful therapeutic target as an alternative approach in the management of proliferative retinopathies. (+info)
Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.
(14/4803)
Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease. (+info)
Endothelin-1 and its mRNA in the wall layers of human arteries ex vivo.
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BACKGROUND: The participation of endothelin-1 (ET-1) in the control of vascular tone in humans has been questioned, on the basis of the finding of subthreshold immunoreactive (ir) ET-1 plasma levels. However, because most ET-1 is secreted abluminally, it might attain a higher concentration in the tunica media than in plasma. Furthermore, evidence indicates that vascular smooth muscle cells (VSMCs) can synthesize ET-1 on stimulation in vitro. We therefore looked for irET-1 in the different layers of the wall of human arteries, including renal, gastric, and internal thoracic artery wall, obtained ex vivo from consenting patients with coronary artery disease and/or high blood pressure undergoing surgery, as well as from young organ donors. METHODS AND RESULTS: We performed immunohistochemistry with specific anti-ET-1 and anti-vWF antibodies followed by detection with an avidin-biotin complex ultrasensitive kit. The presence of preproET-1 and human endothelin-converting enzyme-1 (hECE-1) mRNA was also investigated by reverse transcription-polymerase chain reaction in homogenates of vessel wall, including preparations deprived of both endothelium and adventitia, and in isolated VSMCs. We detected irET-1 in the endothelium of all arteries and in the tunica media of internal thoracic artery from most patients with coronary artery disease. PreproET-1 and hECE-1 mRNA was also detected in VSMCs isolated from these vessels. irET-1 and irvWF staining in endothelium and tunica media was measured by use of microscope-coupled computer-assisted technology. Significant correlations between the amount of irET-1 in the tunica media and mean blood pressure (P<0.05), total serum cholesterol (P<0.05), and number of atherosclerotic sites (P<0.001) were found. Thus, in organ donors, irET-1 was detectable almost exclusively in endothelial cells, whereas in patients with coronary artery disease and/or arterial hypertension, sizable amounts of irET-1 were detectable in the tunica media of different types of arteries. In addition, VSMCs isolated from these vessels coexpressed the preproET-1 and hECE-1 genes. CONCLUSIONS: Collectively, these findings are consistent with the contention that endothelial damage occurs in most patients with atherosclerosis and/or hypertension and that ET-1 is synthesized in VSMCs of these patients. (+info)
Suppression of experimental abdominal aortic aneurysms by systemic treatment with a hydroxamate-based matrix metalloproteinase inhibitor (RS 132908).
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BACKGROUND: Abdominal aortic aneurysms (AAAs) are associated with chronic inflammation, disruption of medial elastin, and increased local production of elastolytic matrix metalloproteinases (MMPs). The purpose of this study was to investigate how treatment with a hydroxamate-based MMP antagonist (RS 132908) might affect the development of experimental AAAs. METHODS: Male Wistar rats underwent intraluminal perfusion of the abdominal aorta with 50 units of porcine pancreatic elastase followed by treatment for 14 days with RS 132908 (100 mg/kg/day subcutaneously; n = 8) or with vehicle alone (n = 6). The external aortic diameter (AD) was measured in millimeters before elastase perfusion and at death, with AAA defined as an increase in AD (DeltaAD) of at least 100%. Aortic wall elastin and collagen concentrations were measured with assays for desmosine and hydroxyproline, and fixed aortic tissues were examined by light microscopy. RESULTS: AAAs developed in all vehicle-treated rats, with a mean AD (+/- SE) that increased from 1.60 +/- 0.03 mm before perfusion to 5.98 +/- 1.02 mm on day 14 (DeltaAD = 276.4 +/- 67.7%). AAAs developed in only five of eight animals (62.5%) after MMP inhibition, with a mean AD that increased from 1.56 +/- 0.05 mm to 3.59 +/- 0.34 mm (DeltaAD = 128.1 +/- 18.7%; P <.05, vs vehicle). The overall inhibition of aortic dilatation attributable to RS 132908 was 53.6 +/- 6.8%. Aortic wall desmosine fell by 85.4% in the vehicle-treated rats (1210.6 +/- 87.8 pmol/sample to 176.7 +/- 33.4 pmol/sample; P <.05) but only by 65.6% in the animals treated with RS 312908 (416.2 +/- 120.5 pmol/sample). In contrast, hydroxyproline was not significantly affected by either elastase perfusion or drug treatment. Microscopic examination revealed the preservation of pericellular elastin and a greater degree of fibrocollagenous wall thickening after MMP inhibition, with no detectable difference in the extent of inflammation. CONCLUSIONS: Systemic MMP inhibition suppresses aneurysmal dilatation in the elastase-induced rodent model of AAA. Consistent with its direct inhibitory effect on various MMPs, RS 132908 promotes the preservation of aortic elastin and appears to enhance a profibrotic response within the aortic wall. Hydroxamate-based MMP antagonists may therefore be useful in the development of pharmacologic approaches to the suppression of AAAs. (+info)