Proteinase induction by endothelial cells during wound repair.
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Animals↗
Biomedical subjects
M J Banda
Publications and source records attributed to M J Banda.
Interaction of mouse macrophage elastase with native and oxidized human alpha 1-proteinase inhibitor.
Native and oxidized alpha 1-proteinase inhibitor (alpha 1-PI) were compared as substrates for the metalloproteinase macrophage elastase. At substrate concentrations at which native alpha 1-PI was readily degraded by macrophage elastase, oxidized alpha 1-PI was hardly degraded at all. Incubation of macrophage elastase with oxidized alpha 1-PI before the addition of native alpha 1-PI showed that oxidized alpha 1-PI was not an inhibitor of macrophage elastase. Competition experiments with up to twofold excess oxidized alpha 1-PI did not interfere with the degradation of native alpha 1-PI by macrophage elastase. Sequence analysis of amino acids in degraded native alpha 1-PI showed that macrophage elastase attacked a single peptide bond between Pro-357 and Met-358, the latter representing the P1 reactive-site residue of alpha 1-PI. In oxidized alpha 1-PI, Met-358 was converted to methionine sulfoxide and macrophage elastase hydrolyzed the bond between Phe-352 and Leu-353. These data suggest that methionine may be the primary cleavage site for macrophage elastase and not leucine, as previously thought.
Amino Acid Sequence↗
Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity.
We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.
Animals↗
Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors.
Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.
Animals↗
Some protease inhibitors are also inhibitors of poly(ADP-ribose) polymerase.
The low-molecular-weight peptide protease inhibitors, tosyl-lysine-chloromethyl ketone, antipain and leupeptin, inhibited poly(ADP-ribose) [poly(ADP-Rib)] polymerase in permeable cells. The concentrations required for 50% inhibition were 3.6, 5 and 29 mM, respectively. Two peptides without protease inhibitor activity, fibrinopeptide A and phenylalanine-leucine-(glutamine)2-leucine, also inhibited poly (ADP-Rib) synthesis; doses required for 50% inhibition were 0.37 and 11.2 mM, respectively. These concentrations lie within a range bracketed by the 50% inhibition concentrations of the strong and weak poly(ADP-Rib) synthesis inhibitors, 3-amino-benzamide (0.15 mM) and caffeine (greater than 100 mM), respectively. N-Ethylmaleimide also inhibited poly(ADP-Rib) synthesis, at a 50% inhibitory dose of 0.3 mM, in the absence of exogenous thiol reagents. High-molecular-weight protease inhibitors, such as soybean (including Bowman-Birk reagent) and lima bean trypsin inhibitors and human alpha 1-protease inhibitor, had no effect on poly(ADP-Rib) synthesis up to 2 mg/ml. Interference with transformation and other cellular effects that have been reported in carcinogen-damaged cells treated with low-molecular-weight peptide protease inhibitors may therefore involve common mechanisms with poly(ADP-Rib) inhibitors. Similar effects of high-molecular-weight protease inhibitors presumably involve different mechanisms.
Antipain↗
Effect of lactate, pyruvate, and pH on secretion of angiogenesis and mitogenesis factors by macrophages.
Macrophages secrete substances that stimulate angiogenesis and mitogenesis at wound sites. To test whether metabolic characteristics of the wound such as high lactate concentration and low pH may regulate the expression of these substances, macrophages were cultured in lactalbumin hydrolysate and then in increasing concentrations of lactate, pyruvate, and hydrogen ions, and cell-free supernatants were collected. Cultures incubated in concentrations of lactate approximating those observed in wounds secreted an angiogenesis factor or factors, but cultures incubated in equivalent concentrations of pyruvate or at pH 6.2 did not. These observations seem to be specific to macrophages because capillary endothelial cells and fibroblasts failed to secrete an angiogenesis factor when cultured under hypoxic conditions or in high concentrations of lactate. None of these conditions changed the expression of macrophage mitogens.
Angiogenesis Inducing Agents↗
Cell interactions in post-traumatic fibrosis.
Models of reparative fibrosis, or wound healing, disclose a basic spatial relationship between inflammatory cells, fibroblasts, dividing fibroblasts and angiogenic endothelial cells. Many components of the extracellular fluid, especially steep gradients of PO2, PCO2, pH, glucose and lactate, have been measured. After components of coagulation play out their role in the first few days after injury, macrophages sense excitatory substances and signs of respiratory distress, and secrete chemoattractants for endothelial cells and fibroblasts. If excitatory substances (such as silica) are eliminated, active fibrosis ends with the restoration of the energy supply. Some of the mitogens and chemoattractants acting as intercellular messengers between these cells have been isolated and identified.
Animals↗
Wound fluid angiogenesis factor stimulates the directed migration of capillary endothelial cells.
During wound healing, new capillaries grow into the wound site. An angiogenesis factor isolated from wound fluid stimulates the movement of capillary endothelial cells in a filter migration assay. Experiments were carried out to determine whether the movement seen in the assay was chemokinetic or chemotactic. Capillary endothelial cells were plated onto a collagen-coated coverslip and inverted over a visualization apparatus. Cells exposed to a constant concentration of wound fluid angiogenesis factor (WAF) were more mobile than cells not exposed to WAF, and this movement was chemokinetic. When exposed to a gradient of WAF, the cells translocated toward the higher concentration; this directional movement was chemotactic. Cells in a gradient of WAF morphologically aligned with the gradient. These data support the idea that wound healing angiogenesis is regulated by the chemotaxis of capillary endothelial cells.
Angiogenesis Inducing Agents↗
Regulation of alpha 1 proteinase inhibitor function by rabbit alveolar macrophages. Evidence for proteolytic rather than oxidative inactivation.
Rabbit alveolar macrophages were cultured in an environment conducive to the secretion of both reactive oxygen and proteinases, so that the relative importance of proteolytic and oxidative inactivation of alpha 1-proteinase inhibitor by alveolar macrophages could be evaluated. The inactivation of alpha 1-proteinase inhibitor was proportional to its proteolysis, and there was no detectable inactivation in the absence of proteolysis. Although the live macrophages were capable of secreting reactive oxygen, they did not inactivate alpha 1-proteinase inhibitor by oxidation. The inactivation of alpha 1-proteinase inhibitor by proteolysis was proportional to the secretion of elastinolytic activity by the alveolar macrophages. The inability of the alveolar macrophages to oxidize alpha 1-proteinase inhibitor was attributed to the methionine in the macrophages, in secreted proteins, and in the culture medium competing for oxidants. The data suggest that proteolytic inactivation of alpha 1-proteinase inhibitor may be important in vivo and that the methionine concentration in vivo may protect alpha 1-proteinase inhibitor from significant oxidative inactivation.
Animals↗
Oxygen tension regulates the expression of angiogenesis factor by macrophages.
When cultured in a hypoxic environment similar to that found in the center of a wound, macrophages secreted active angiogenesis factor into the medium. Under conditions similar to those of well-oxygenated tissue, macrophages did not secrete active angiogenesis factor. Macrophages that secreted the factor at hypoxic conditions stopped secreting it when returned to room air. Thus the control of angiogenesis in wound healing may be the result of macrophages responding to tissue oxygen tension without the necessity of interacting with other cell types or biochemical signals.
Angiogenesis Inducing Agents↗
Selective proteolysis of immunoglobulins by mouse macrophage elastase.
Mouse macrophage elastase, a metalloproteinase secreted by inflammatory macrophages, catalyzed the limited proteolysis of selected subclasses of mouse immunoglobulins, including monomeric IgG2a, IgG3, and some forms of IgG2b. Mouse IgG1 was resistant to elastase degradation; however, human IgG1 was degraded. IgG3 in immune complexes was cleaved in a manner similar to that of monomeric IgG3. Degradation by macrophage elastase was limited to the heavy chain, resulting in products that did not compete for binding to the macrophage Fc receptor. Macrophage elastase usually produced a pepsin-like rather than a papain-like pattern of proteolysis, resulting in the release of F(ab')2 and Fc' subfragments. This degradation of IgG differed from the papain-like cleavage of IgG by granulocyte elastase. Macrophage elastase degraded papain-generated Fc fragments of IgG2a into multiple fragments. Therefore, macrophage elastase at concentrations found in culture medium has the potential to regulate some aspects of cellular events associated with immunoglobulins.
Animals↗
12-O-tetradecanoylphorbol-13-acetate (TPA) induces sister-chromatid exchanges and delays in cell progression in Chinese hamster ovary and human cell lines.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a 20-45% increase in sister-chromatid exchange (SCE) frequency in Chinese hamster ovary cells (CHO) and in 2 SV40-transformed human fibroblast cell lines (GM637 and XP12RO) at concentrations up to 1 microgram/ml. The increase was independent of the time at which the cells were fixed after treatment and was not due to an impurity in the TPA preparation or to increased incorporation of bromodeoxyuridine into the DNA. There was no synergistic effect on SCE induction when CHO cells were simultaneously exposed to TPA and the carcinogens mitomycin C or ultraviolet light, but there was when TPA and benzo[a]pyrene were used. In addition to its weak SCE-inducing effects in CHO cells, TPA caused slight delays in cell cycle progression and greatly enhanced the cell cycle delay induced by benzo[a]pyrene.
Animals↗
Isolation of a nonmitogenic angiogenesis factor from wound fluid.
Angiogenesis, or new capillary growth, is essential to normal growth and wound healing. It is also active in several pathologic states, including the growth of malignant tumors. An extracellular, nonneoplastic angiogenesis factor has been isolated from cell-free rabbit wound fluid by pore-limit dialysis and chromatography on a size-exclusion HPLC column. The isolated angiogenesis factor was purified 9,600-fold with a yield of 81% and has a molecular weight between 2,000 and 14,000. Wound fluid angiogenesis factor was completely separated from the mitogenic activity of wound fluid; it did not increase the number of capillary endothelial cells in vitro or stimulate [3H]thymidine uptake by these cells. The isolated angiogenesis factor stimulated endothelial cell migration in vitro, and less than 200 ng of the factor stimulated angiogenesis in vivo in the corneal implant assay.
Angiogenesis Inducing Agents↗
Partitioning of bone marrow into stem cell regulatory domains.
To examine the hypothesis that bone marrow consists of discrete stem cell regulatory volumes or domains, we studied spleen colony-forming unit (CFU-S) population growth kinetics in unirradiated WBB6F1-W/Wv mice receiving various doses of +/+ bone marrow cells. Assay of femoral marrow CFU-S content in the eight recipient dose groups revealed a family of growth curves having an initial dose-independent exponential phase and a subsequent dose-dependent deceleration phase. CFU-S content at the growth transition (inflection point) was not a simple linear function of inoculum dose but was shown rather to reflect a random distribution of initially seeded donor CFU-S in discrete volumes of recipient bone marrow. The inoculum dose resulting in a mean of 1 CFU-S per bone marrow sampling unit was estimated to be 17 x 10(6) bone marrow cells, corresponding to a total marrow uptake of approximately 5100 CFU-S (based on a seeding efficiency factor of 10%). If we assume single-hit kinetics, it follows that the recipient W/Wv bone marrow may contain approximately 5100 domains in which stem cell proliferation is geared to the density of the stem cell population. When the various inocula were corrected for multiple seeding in a given domain, the mean inflection point per domain was similar and indicative of five or so divisions before departure from exponential growth at approximately 20% of final CFU-S content 8 days after bone marrow injection. The partitioning of bone marrow into highly localized functional units is consistent with the putative regulatory role of short-range interactions between stem cells and essential stromal elements.
Animals↗
Elastases and elastin degradation.
The metabolic turnover of mature elastin fibers in adult animals is relatively slow. Although only small amounts of elastin are degraded normally, increased degradation and fragmentation of elastic fibers may play a significant role in disease processes. Elastinolytic enzymes are found in microorganisms, snake venoms, and in a number of mammalian cells and tissues, including pancreas, polymorphonuclear leukocytes, and macrophages. Elastinolytic enzymes fall into all 4 classes of proteinases (aspartic, cysteine, serine, and metallo) and show a spectrum of different specificities. All elastases studied to date have catalytic activity against protein and peptide substrates other than elastin. The presence of elastase activity is a virulence factor associated with the pathogenicity of Pseudomonas and other bacteria, dermatophytic fungi, and necrosis by rattlesnake venoms. Only elastinolytic enzymes are capable of inducing experimental pulmonary emphysema. Elastin degradation mediated by living macrophages and trophoblasts is confined to the immediate pericellular environment. Destruction of mature elastin by other mammalian elastases is probably the result of an imbalance in the normal inhibitor-proteinase ratio. The major plasma inhibitors contributing to the regulatory balance are alpha 1-proteinase inhibitor and alpha 2-macroglobulin.
Animals↗
Mouse macrophage elastase. Purification and characterization as a metalloproteinase.
Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.
Animals↗
Limited proteolysis by macrophage elastase inactivates human alpha 1-proteinase inhibitor.
Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit human granulocyte elastase, a serine proteinase implicated in the pathogenesis of emphysema. Macrophage elastase apparently does not degrade human granulocyte elastase-alpha 1-proteinase inhibitor complexes or release active granulocyte elastase from these complexes. The ability of macrophage elastase to degrade alpha 1-proteinase inhibitor is inhibited by EDTA and alpha 2-macroglobulin.
Granulocytes↗