Presence Of Elastase Research Articles

Insect cell production of a secreted form of human α 1-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities

α 1-proteinase inhibitor (API) is a potential therapeutic agent in all diseases in which elastase released by neutrophils has to be effectively neutralized. We ligated the cDNA of human API to the C-terminal section of an insulin-like growth factor II analogue (BOMIGF), known to be properly folded and secreted in insect cells using the baculovirus expression system. The BOMIGF-API chimera was recovered from the incubation medium of the infected cells. It shared the properties of both IGFs and API. It inhibited neutrophil elastase and formed SDS-stable complexes with the enzyme. The attachment of the large API protein to the C-terminal end of the 10 kDa IGF analogue did not destroy the IGF-mediated stimulation of thymidine incorporation into bovine fetal erythroid cells. We tested the capacity of the chimera to affect fibronectin-dependent TF-1 cell migration. BOMIGF-API significantly restored TF-1 cell migration in the presence of elastase, which is the enzyme of burn wound fluid most probably involved in fibronectin degradation. Some of the beneficial uses for this chimera may include all instances for which inhibition of elastase-mediated extracellular matrix destruction as well as stimulation of cell migration and proliferation are required for tissue repair.

Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture.

Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% (P < 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% (P < 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% (P < 0.01). alpha(1)-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema.

Activity of pancreatic endopeptidases towards luteinizing hormone-releasing hormones

LHRH and its analogues have low oral bioavailability; this is in part due to their degradation by peptidases present in the intestinal lumen. To determine the appropriate inhibitors to co-administer with LHRH oral formulations, the peptidases involved in their digestion have to be identified. Human (hLHRH) and salmon (sLHRH) LHRH analogues contain a number of potential cleavage sites for the lumenal pancreatic secreted serine endopeptidases: chymotrypsin, trypsin and elastase. The rate of LHRH degradation by equimolar concentrations of chymotrypsin, trypsin and elastase were examined separately in vitro , at pH 8.0, 15°C. At a molar ratio of 1:1000 (enzyme:LHRH), both LHRH analogues were rapidly hydrolysed by α-chymotrypsin with half-lives of 2.5±0.3 and 2.7±0.4 min (mean±S.D., n=3), respectively, whereas in the presence of elastase both LHRH analogues were slowly hydrolysed with half-lives of 90±15 and 114±21 min (mean±S.D., n=3), respectively. Trypsin had no activity towards either LHRH analogues after 2 h incubation. The degradation of the LHRH analogues by elastase is likely to be a property of the chymotrypsin impurity. It is concluded that protection of the LHRH analogues from α-chymotrypsin is a requirement for the development of oral absorbable product.

Elastosis in breast--correlation with epithelial proliferation in benign disease and carcinomatous growth.

Elastosis in the breast is an unusual phenomenon and its morphogenesis has not yet been fully ascertained. The degree of elastosis in the breast associated with benign diseases, including fibroadenoma and fibrocystic disease as well as with breast carcinoma, was examined with special reference to the correlation between the degree of epithelial proliferation and elastosis. Using the immunohistochemical method, the presence of elastase (EL) and alpha 1-antichymotrypsin (ACT), one of the protease inhibitors, in these epithelial cells was also investigated to elucidate the role of an imbalance in these enzymes in the morphogenesis of the elastosis. Consequently, it was shown that there is a tendency in fibroadenoma and fibrocystic disease, for epithelial proliferation to be related to the degree of elastosis, and that the lack of EL in proliferated epithelial cells might play a role in the occurrence of elastosis, although ACT has no significant correlation. On the contrary, in our study noninvasive carcinoma showed marked periductal elastosis but no stromal elastosis, while invasive carcinoma showed various degrees of periductal and stromal elastosis. In invasive carcinoma, especially scirrhous carcinoma, the degree of ACT in cancer cells correlated well with stromal elastosis, although there was no correlation with EL. These findings suggest that an imbalance of the protease-antiprotease system, produced by epithelial cells of the breast, contribute to the morphogenesis of elastosis, although the physiological event, aging, is only marginally related to elastosis. Further investigation of the cells producing elastin and regulatory factors may be necessary.

Risk factors for the degradation of lung elastic fibers in the ventilated neonate. Implications for impaired lung development in bronchopulmonary dysplasia.

In order to evaluate the risk for proteolytic destruction of lung parenchymal elastic fibers in ventilated premature infants, the concentrations of elastase were determined in tracheal aspirates of 65 infants from whom we obtained a total of 327 sequential samples. Elastase was detected at least once in 39 of the 65 infants studied. Eleven of these infants were ventilated with greater than 60% oxygen for greater than 5 days. In 19 infants, the presence of elastase was associated with positive bacterial and/or viral cultures and/or elevated ratios (greater than 0.22) of immature neutrophils to total neutrophils. Elastase was not detected in the lung secretions of 26 infants ventilated with greater than 60% oxygen for less than 3 days, suggesting minimal risk for elastic fiber destruction in the intubated infant who neither has pneumonia nor requires prolonged hyperoxic ventilation. The risk for elastic fiber destruction was further evaluated by analyzing sequential urine and tracheal aspirate samples for the presence of an elastolytic degradation product of elastin (desmosine). The biochemical data indicated a potential risk for proteolytic destruction of elastic fibers in association with infection and/or prolonged hyperoxic exposure. In addition, autopsy specimens obtained from three of the infants revealed structurally abnormal lung parenchymal elastic fibers. Because elastic fibers are believed to provide the structural support for alveolar septal development, proteolytic degradation of these fibers may be a significant factor in the impaired lung development that occurs in infants with bronchopulmonary dysplasia.

In vivo inactivation of antithrombin III is promoted by heparin during cardiopulmonary bypass.

To study the in vivo effect of heparin on antithrombin III (AT3) when elastase is elevated, the blood of 20 patients undergoing cardiopulmonary bypass (CPB) was assayed for elastase and AT3. The model was chosen because CPB is known to increase plasma elastase and the patients were heparinized. The blood of 20 patients undergoing cardiac surgery was assayed for elastase and AT3 one day preoperatively, every half hour during CPB, and one day postoperatively. Elastase increased significantly and AT3 decreased significantly during CPB. There was a direct correlation between the rise in elastase and decrease in AT3. AT3 decreased even further when elastase was elevated and patients were heparinized (AT3/elastase = 0.04 + 0.07), compared with the drop with elevated elastase alone (AT3/elastase = 0.11 + 0.14) (P less than .0015). These data indicate that (1) CPB is associated with an increase in plasma elastase, (2) elevated plasma elastase is associated with a reduction in AT3, and (3) heparin promotes the inactivation of AT3 when serum elastase is increased. These data confirm the in vitro observation that heparin accelerates the inactivation of AT3 in the presence of elastase.

Elastase-induced experimental aneurysms in rats.

An experimental in vivo model of aortic aneurysm was established by perfusing an isolated segment of rat abdominal aorta with pancreatic elastase. Ten rats were used in each protocol. Saline-perfused aortas developed no aneurysmal dilations. Elastase-perfused aortas contained aneurysms in the perfused area and a total loss of elastic tissue. Control aortas contained no elastic tissue lesions. There was a quantitative relation between the amount of elastase perfused and aneurysm formation: 1-2 units induced neither macroscopic nor microscopic lesions; 3-6 units induced microscopic elastic tissue damage without macroscopic aneurysm; and more than 6 units produced aneurysmal dilation in all cases. In situ elastase secretion by macrophages was induced by perfusing rat aortas with thioglycollate-activated macrophages or with thioglycollate alone. There was aortitis without true aneurysm and a total loss of elastic tissue in the vicinity of activated macrophages within the aortic media. Perfusion of infra-aneurysmal amount of elastase (1 or 2 units) or thioglycollate plus plasmin (2 units) always induced a large aneurysm, whereas plasmin alone induced neither macroscopic nor microscopic lesions. These morphological results were supported by the significantly elevated elastolytic activity within the aortic wall of animals perfused with thioglycollate plus plasmin 9 days, after perfusion (207.6 +/- 54.8 micrograms elastin-rhodamine lysed/18 hr; control rats, 25.43 +/- 11.13). The results suggest that the presence of elastase within the aortic media leads to aneurysm formation. Activated macrophages within the aortic media may be responsible for elastase secretion and elastic tissue destruction. Plasmin may enhance elastase activity and aggravate the aneurysmal lesion.

Polymeric prodrugs, 6. Synthesis and examination of 6‐purinethiol bound to poly(1‐vinyl‐2‐pyrrolidone‐co‐maleic acid)

AbstractA low‐molecular‐weight disulfide derivative of the antileukemic agent 6‐purinethiol (2) was used for coupling with a macromolecular carrier, poly(1‐vinyl‐2‐pyrrolidone‐co‐maleic acid) via spacer groups. The in vitro hydrolytic stabilities of the polymeric prodrugs were examined at pH 7,2 and 8,8. An enhanced cleavage of the disulfide bridge yielding 6‐purinethiol was observed. Enzymatic hydrolysis of the prodrug containing an enzyme‐sensitive spacer was also done in the presence of elastase.

Estimation and characterization of hamster leukocyte elastase

A method is described for the preparation of granule extract from hamster polymorphonuclear leukocytes. The presence of elastase in this extract has been ascertained by the dual criteria of elastin solubilization and synthetic elastase substrate hydrolysis. The extract is also able to cleave one out of three cathepsin-G substrates. The amount of elastase in the granule extract has been estimated by titration with human plasma α 1-proteinase inhibitor, an experiment from which it has been concluded that one hamster leukocyte contains 0.1 pg elastase or less. This quantity is more than 30 times lower than that found in human leukocytes. Investigations with synthetic substrates and inhibitors indicated that hamster and human leukocyte elastases have very similar catalytic properties: they bind very tightly to trifluoroacetylated inhibitors, and the S 1 subsite of their active center binds preferentially a valine residue. In addition, the activity of both elastases is enhanced by ionic strength and hydrophobic solvents. The elastase inhibitory capacity of hamster serum has been assessed with pure human leukocyte and porcine pancreatic elastases and compared to that of human serum. It was found that hamster serum contains about four times less inhibitor(s) on a molar basis than human serum. This inhibitory capacity is not low enough to compensate for the low levels of leukocyte elastase. Hence, it is unlikely that chronic emphysema may be induced in hamsters with the use of leukoattractants.

Presence of elastase in teleost islet tissue.

Presence of elastase in teleost islet tissue.