Inosine, not adenosine, initiates endothelial glycocalyx degradation in cardiac ischemia and hypoxia.

Ischemia/reperfusion and hypoxia/reoxygenation of the heart both induce shedding of the coronary endothelial glycocalyx. The processes leading from an oxygen deficit to shedding are unknown. An involvement of resident perivascular cardiac mast cells has been proposed. We hypothesized that either adenosine or inosine or both, generated by nucleotide catabolism, attain the concentrations in the interstitial space sufficient to stimulate A3 receptors of mast cells during both myocardial ischemia/reperfusion and hypoxia/reoxygenation. Isolated hearts of guinea pigs were subjected to either normoxic perfusion (hemoglobin-free Krebs-Henseleit buffer equilibrated with 95% oxygen), 20 minutes hypoxic perfusion (buffer equilibrated with 21% oxygen) followed by 20 minutes reoxygenation, or 20 minutes stopped-flow ischemia followed by 20 minutes normoxic reperfusion (n = 7 each). Coronary venous effluent was collected separately from so-called transudate, a mixture of interstitial fluid and lymphatic fluid appearing on the epicardial surface. Adenosine and inosine were determined in both fluid compartments using high-performance liquid chromatography. Damage to the glycocalyx was evident after ischemia/reperfusion and hypoxia/reoxygenation. Adenosine concentrations rose to a level of 1 µM in coronary effluent during hypoxic perfusion, but remained one order of magnitude lower in the interstitial fluid. There was only a small rise in the level during postischemic perfusion. In contrast, inosine peaked at over 10 µM in interstitial fluid during hypoxia and also during reperfusion, while effluent levels remained relatively unchanged at lower levels. We conclude that only inosine attains levels in the interstitial fluid of hypoxic and postischemic hearts that are sufficient to explain the activation of mast cells via stimulation of A3-type receptors.

Shedding of the coronary endothelial glycocalyx: effects of hypoxia/reoxygenation vs ischaemia/reperfusion.

BACKGROUND: Vascular endothelium is covered by a glycocalyx. Damage to the glycocalyx after systemic inflammation or ischaemia/reperfusion contributes to increased vascular permeability and leucocyte adhesion. The underlying mechanisms leading to ischaemia/reperfusion-induced glycocalyx shedding are incompletely understood, in terms of lack of oxygen, absence of flow, or return of oxygen. METHODS: Isolated guinea pig hearts perfused with Krebs-Henseleit buffer at 37°C underwent 20 min of either stopped-flow ischaemia or hypoxic perfusion with subsequent reperfusion/reoxygenation (n = 6 each). Hearts perfused with normoxic buffer served as time controls. Epicardial transudate was collected to assess coronary net fluid filtration, colloid extravasation, and histamine release by mast cells. Syndecan-1 and heparan sulphate were measured in coronary effluent, together with lactate, purines, and the release of mast-cell tryptase ß. Additional hearts were perfusion-fixed to visualize the glycocalyx. RESULTS: Both ischaemia and hypoxia with reperfusion/reoxygenation resulted in significant increases in net fluid filtration (P < 0.05) and release of syndecan-1 and heparan sulphate in coronary effluent. These effects were already seen with the onset of hypoxic perfusion. Histamine was released during hypoxia and reoxygenation and also reperfusion, as was tryptase ß, and high concentrations of adenosine (>1 µmol litre⁻¹, hypoxia group) and inosine (> 7 µmol litre⁻¹, ischaemia group) were measured in effluent (P < 0.05). Damage to the coronary glycocalyx was evident upon electron microscopy. CONCLUSIONS: Both ischaemic and hypoxic hypoxia initiate glycocalyx degradation, promoting an increase in permeability. A contributing mechanism could be purine-mediated degranulation of resident mast cells, with liberated tryptase ß acting as potential 'sheddase'.

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2.

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.

Matrix metalloproteinases (MMPs) in breast cancer cell lines of different tumorigenicity.

In the present study we investigated the presence, amount and activity of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8, -9, -10, -11 and -13 and TIMP-1 in three well-defined breast cancer cell lines with different biological behaviour; i.e. poorly-invasive MCF-7 cells, invasively growing MDA-MB-231 cells and invasive and highly-metastatic MDA-MB-435 cells. The parallel immunocytochemical determination of the degree of cellular differentiation, as monitored by the immunocytochemical expression of cytokeratins (CK), confirmed differences in the tumor cell differentiation. Thereby, MCF-7 cells expressed more glandular CKs than MDA-MB-231 cells, while MDA-MB-435 cells were only labelled by pancytokeratin markers, but neither by glandular nor by squamous epithelial CKs. Conditioned media were analyzed for the presence of MMPs and TIMP-1 using Western blot with specific polyclonal antibodies and for gelatinolytic and caseinolytic activity by zymography. In addition, the cellular pool of several MMPs was investigated by immunocytochemistry. An enhanced cytoplasmatic staining for MMP-3 and -9, MMP-1, -10 and -11 was seen in the highly metastatic cells at almost equal levels, while MMP-2 revealed only a minor intracellular staining in all three cell lines. Western blots of conditioned media showed enhanced amounts of MMP-1, -3, -7, -10 and -11 in media of the two metastatic cell lines. Casein zymography correlated with the results of the MMP-1 Western blots. By means of gelatin zymography, MMP-2 and -9 were detectable in cell culture supematants of all the three cell lines, while gelatinolytic activity was elevated in the media of the more malignant MDA-MB-435 cells. Separate addition of EDTA or Pefa bloc SL partially inhibited the gelatinoltic activity indicating the presence of metallo- and serine proteinases, respectively; combined application of both inhibitors resulted in a complete suppression of activity. We provide evidence that the deviation expression in secretion of various MMPs in breast cancer cell lines of different tumorigenicity correlates with the biological behaviour of these cells, ie. the more malignant cells synthesize more MMPs than the less malignant ones. In addition, the secretion of MMP-1, -3, -7, -10 and -11 was enhanced in the malignant MDA-MB-231 and -435 cells when compared to the corresponding intracellular pool. This analysis confirms previous results obtained in a keratinocyte tumor cell model and provides evidence for a more general biological association between MMP-expression and tumor cell growth.

Mast cells in an angiosarcoma complicating xeroderma pigmentosum in a 13-year-old girl.

A cutaneous angiosarcoma, a rare tumor that occurs almost exclusively in sun-exposed skin of individuals older than 50 years, developed in an adolescent with Xeroderma pigmentosum (XP). As this is the second report of a child with angiosarcoma and XP, ultraviolet-induced DNA damage may be involved in the pathogenesis of this tumor. Strongly increased numbers of mast cells were found, particularly in the peripheral tumor area, which may reflect with the requirement of mast cells for the growth of vascular structures or a role for mast cells in the antitumor immune response.

Epoxysuccinyl peptide-derived affinity labels for cathepsin B.

Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes. Selectively targeting labels of this enzyme are important tools to gain more detailed understanding of its specific roles. Starting from our recently developed irreversible epoxysuccinyl-based inhibitor (R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH, R=OMe), we have synthesized two affinity labels, R=NH-(CH(2))(6)-NH-rhodamine B and R=NH-(CH(2))(6)-NH-biotin. Using MCF-7 cells, the labeled inhibitors were shown to be virtually non-cell-permeant. Moreover, affinity blot analysis with the biotinylated inhibitor allowed a highly sensitive and selective non-radioactive detection of active cathepsin B.

Beta-cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells.

Beta-cyclodextrin is known to form inclusion complexes with hydrophobic drugs. Several tumor cell lines are known to secrete and/or contain membrane-associated cathepsin B which is possibly involved in invasion and metastasis. Based on these information, our recently developed endo-epoxysuccinyl peptide inhibitor MeO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH for cathepsin B was conjugated with beta-cyclodextrin to obtain a site-directed drug carrier system. Furthermore, the conjugate, was shown to form an inclusion complex with the cytotoxic drug methotrexate.

[Tryptase, a marker for the activation and localization of mast cells]. / Tryptaseryptase, ein Marker für die Aktivierung und Lokalisation von Mastzellen.

The serine protease tryptase (ECNr. 3.4. 21.59), which is almost exclusively expressed in mast cells, is released by mast cell degranulation in an enzymatically active form together with other mediators, e.g. histamine, into the extracellular space and the circulation. The capability of the enzyme to directly stimulate several cell types as well as to cleave polypeptide hormones and to activate pro-enzymes suggests a role for tryptase in inflammatory and tissue-remodeling processes. Therefore, in the skin, a role of tryptase is suggested not only in mastocytosis and immediate type hypersensitivity reactions, but also in other inflammatory diseases, degenerative or neoplastic conditions as well as in wound healing, where an accumulation and/or activation of mast cells is found. Extracellular tryptase may be superior to histamine as a parameter for the onset and course of immediate type reactions and as an indicator for the activation of mast cells in other conditions. Its absence during histamine-liberating reactions may suggest basophil activation. In addition, tryptase has been shown to be a sensitive and specific marker for the localization of mast cells in tissues.

LEKTI, a novel 15-domain type of human serine proteinase inhibitor.

Proteinase inhibitors are important negative regulators of proteinase action in vivo. We have succeeded in isolating two previously unknown polypeptides (HF6478 and HF7665) from human blood filtrate that are parts of a larger precursor protein containing two typical Kazal-type serine proteinase inhibitor motifs. The entire precursor protein, as deduced from the nucleotide sequence of the cloned cDNA, exhibits 15 potential inhibitory domains, including the Kazal-type domains, HF6478, HF7665, and 11 additional similar domains. An inhibitory effect of HF7665 on trypsin activity is demonstrated. Because all of the 13 HF6478- and HF7665-related domains share partial homology to the typical Kazal-type domain but lack one of the three conserved disulfide bonds, they may represent a novel type of serine proteinase inhibitor. The gene encoding the multidomain proteinase inhibitor, which we have termed LEKTI, was localized on human chromosome 5q31-32. As shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis, it is expressed in the thymus, vaginal epithelium, Bartholin's glands, oral mucosa, tonsils, and the parathyroid glands. From these results, we assume that LEKTI may play a role in anti-inflammatory and/or antimicrobial protection of mucous epithelia.

A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis. Isolation, sequence analysis, cDNA cloning, recombinant expression, and characterization.

A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.

A new structural class of serine protease inhibitors revealed by the structure of the hirustasin-kallikrein complex.

BACKGROUND: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS: The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

Classical second messengers are not involved in proteinase-induced degranulation of airway gland cells.

Neutral serine proteinases such as mast cell chymase, cathepsin G, and neutrophil elastase are far more potent secretagogues for airway gland serous cells than all other agonists studied (e.g., histamine and bradykinin). To determine the mechanism of proteinase-induced secretion, we investigated the stimulation-secretion coupling in cultured bovine serous cells. Histamine stimulates degranulation of serous cells via adenosine 3', 5'-cyclic monophosphate-, protein kinase C-, and intracellular Ca2+ concentration ([Ca2+]i)-dependent pathways. Similarly, bradykinin-induced secretion involves inositol phosphates, protein kinase C, and [Ca2+]i. Degranulation caused by both agonists also depends on the activity of an endogenous metalloprotease, which is required in a late step of stimulation-secretion coupling, i.e., after Ca2+ entry. On the basis of the effect of different inhibitors, this metalloprotease is a Zn(2+)- and Ca(2+)-dependent enzyme similar to a gelatinase A synthesized by serous cells. In marked contrast to other secretagogues, degranulation induced by chymase, cathepsin G, and neutrophil elastase neither involves the classical second messengers nor the activity of the endogenous metalloprotease. These observations suggest that exogenous proteinases such as chymase, cathepsin G, and elastase may substitute for or mimic the action of an endogenous metalloprotease and directly activate degranulation, bypassing the signal transduction mechanisms necessary for secretion caused by other agonists.

Mast cell-derived tryptase in odontogenic cysts.

Inflammatory and developmental cysts of the jaws are relatively common bone destructive lesions in the human maxillofacial skeleton but their pathogenesis is still poorly understood. In this study the role of mast cells (MC), and mast cell tryptase in particular, was evaluated in the pathophysiology of bone resorption and jaw cyst formation in different types of cysts. The distribution of MC and the amount of tryptase in histological tissue sections were determined by immunohistochemistry using monoclonal antihuman tryptase antibodies and the results were quantitated by using an image analyzing system. The amount of tryptase was further studied by Western-blotting and measurement of trypsin-like activity from the neutral salt extracts obtained from different types of jaw cysts. In contrast to control tissue, high trypsin-like activities and abundant immunoreactive tryptase were observed in the extracts of all types of cysts studied (radicular, dentigerous and keratocyst). In tissue sections the highest amount of tryptase-positive staining was observed in radicular cysts (mean 6.2% of reference area) and the lowest amount in keratocysts (mean 2.1% of reference area, P < 0.01). MC were found to be located in inflammatory cell-rich tissue areas and just beneath the cyst epithelium. Importantly, MC located at the border of bone were observed to be degranulated, indicating high activity of MC and release of tryptase at the regions of early bone destruction. Based on previous findings addressing the role of mast cell tryptase in proteolytic cascades, and the known association of MC with osteoporosis, we suggest that mast cells and mast cell tryptase may contribute significantly to jaw cyst tissue remodelling during growth of a cyst, and to the destruction of the surrounding bone, resulting in jaw cyst expansion.

Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors.

A Kazal-type inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis.

Human tryptase, a tetrameric proteinase expressed by mast cells, is virtually unique among the serine proteinases as it is not inhibited by any proteinaceous inhibitor tested so far. We have now isolated, sequenced, and characterized an inhibitor of human tryptase from the medical leech Hirudo medicinalis. LDTI (Leech-Derived Tryptase Inhibitor) was purified to apparent homogeneity by cation exchange and affinity chromatography. Amino acid sequencing of the protein consisting of 46 residues (M(r) 4738) revealed a high degree of similarity to the non-classical Kazal-type inhibitors bdellin B-3 and rhodniin, inhibitors isolated from the medical leech and the insect Rhodnius prolixus, respectively. LDTI is a tight-binding and relatively specific inhibitor of human tryptase; it inhibits only trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with similar affinities. Inhibition studies using small chromogenic substrates revealed that LDTI inhibits the amidolytic activity of tryptase by approximately 50%, suggesting that most likely due to steric hindrance LDTI binds to and inhibits only 2 of 4 active sites of tryptase. LDTI appears useful as a prototype of inhibitors of human tryptase and as a pharmacological tool for the investigation of the role of tryptase in health and disease.

Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis.

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.

Mast cell proteoglycans modulate the secretagogue, proteoglycanase, and amidolytic activities of dog mast cell chymase.

Chymase, a potent secretagogue for airway gland serous cells, is stored in secretory granules and released from mast cells together with proteoglycans. To investigate the hypothesis tha tproteoglycans modulate chymase-induced effects, we studied the influence of proteoglycans purified from dog mastocytoma cells on chymase-induced secretion from cultured bovine airway gland serous cells. Heparin proteoglycans reduced the chymase-induced secretory response, whereas glycosaminoglycans and chondroitin sulfate proteoglycans had less of an effect. Chymase released together with proteoglycans from activated mast cells caused secretion comparable to that caused by purified chymase reconstituted with purified proteoglycans. Despite partial inhibition by exocytosed proteoglycans, the secretagogue activity of chymase remains substantial compared to that of histamine. However, proteoglycans virtually abolished chymase-induced degradation of the products of serous cell secretion. Although the secretagogue and proteoglycanase activities of chymase are inhibited by most classes of mast cell granule-associated glycans, the amidolytic activity of chymase toward tripeptide 4-nitroanilide substrates is augmented. These findings suggest that mast cell proteoglycans modulate the secretagogue, proteoglycanase, and peptidase activity of chymase, and the results predict that the extent of this modulation in vivo depends on the nature of the proteoglycans with which chymase is released from mast cells.

Inhibition of human neutrophil elastase by ICI 200,355.

To examine the pathogenetic role of neutrophil elastase in airway hypersecretion, we have studied the novel inhibitor of this enzyme, [4-(4-bromophenylsulfonylcarbamoyl)benzoyl-L-valyl-L-proline 1 (RS)-(1-trifluroacetyl-2-methylprolyl)amide] (ICI 200, 355). This compound was a potent (Ki = 0.6 +/- 0.22 nM) inhibitor of human neutrophil elastase and a much weaker inhibitor of other hydrolases. ICI 200,355 also inhibited the ongoing destruction of insoluble elastin by human neutrophil elastase. ICI 200,355 produced a concentration-dependent inhibition of the secretory response induced by human neutrophil elastase (10(-8) M), with an IC50 of 1.6 x 10(-8) M. ICI 200,355 had no effect on baseline secretion or on the secretory response to chymase, cathepsin G or Pseudomonas aeruginosa elastase. Thus, ICI 200,355 appears to be a useful tool for investigating the role of human neutrophil elastase in inflammatory disorders associated with hypersecretion, such as cystic fibrosis, chronic bronchitis, and asthma.