Secretion of matrix metalloproteinases and their inhibitors (tissue inhibitor of metalloproteinases) by human prostate in explant cultures: reduced tissue inhibitor of metalloproteinase secretion by malignant tissues.

Unregulated secretion of matrix metalloproteinases (MMPs) or their endogenous protein inhibitors (tissue inhibitor of metalloproteinases, TIMPs) has been implicated in tumor invasion and metastasis. Species of MMPs and TIMPs secreted by epithelial cultures of normal, benign, and malignant prostate were identified and their levels were compared. Fragments of fresh tissue were cultured in a serum-free medium that supported the outgrowth of prostatic epithelial cells. Biochemical analysis of the conditioned media by gelatin zymography and enzyme assays showed that both normal and neoplastic tissues secreted latent and active forms of both M(r) 72,000 type IV collagenase (MMP-2) and M(r) 92,000 gelatinase (MMP-9). However, conditioned media from malignant prostate explants contained a higher proportion of the active form of MMP-2. Significant amounts of free TIMPs were secreted by normal juvenile and adult prostates, but they were either markedly reduced or not detectable in conditioned media from neoplastic tissues. These findings suggest that there is an imbalance of secretion between MMPs and TIMPs in prostatic carcinoma.

Modulation by propranolol of the lysyl cross-links in aortic elastin and collagen of the aneurysm-prone turkey.

dl-Propranolol (propranolol) fed to immature and mature aneurysm-prone turkeys (Broad-Breasted White, BBW) for 6 weeks significantly raised the tensile strength of tissue rings from the abdominal aorta. The drug-mediated increase in tensile strength values was dose-related and independent of its heart rate- and arterial pressure-lowering effects. Propranolol acts, in part, by (a) stimulating lysyl oxidase to produce greater amounts of reactive aldehydes for intermolecular cross-links, (b) enhancing the progression of chemically unstable to stable forms of intermolecular elastin cross-links (lysinonorleucine and the desmosines), and (c) reducing the density of the age-related intermolecular cross-linking of collagen (pyridinoline). These propranolol effects on the lysyl cross-links were demonstrated in both the immature and mature animals and suggest a heretofore unrecognized potential for this widely used cardiovascular drug.

Overexpression of collagenase (MMP-1) and stromelysin (MMP-3) by pterygium head fibroblasts.

BACKGROUND: The balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) determines the extent of connective tissue degradation and remodeling. OBJECTIVE: To determine whether pterygium, characterized by fibrovascular invasion into the cornea, may in part be mediated by an increased activity of MMPs. MATERIALS AND METHODS: Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured human pterygium head, body, and subconjunctival fibroblasts, and normal corneal and conjunctival fibroblasts were determined by Northern hybridization, enzyme-linked immunosorbent assay, Western blotting, zymography, and quantitative collagenase assay, respectively. RESULTS: Compared with normal conjunctival fibroblasts from 6 subjects, the expression of MMP-1 and MMP-3 transcripts was dramatically increased in pterygium head fibroblasts of 8 patients, but not in pterygium body fibroblasts of 6 patients. The protein levels and collagenolytic and caseinolytic activities of MMP-1 and/or MMP-3 were also markedly increased in pterygium head fibroblasts. The MMP-1 and MMP-3 proteins and activity decreased in order from pterygium head to body to subconjunctival fibroblasts. There was no difference in the transcript and protein expression of MMP-2, TIMP-1, TIMP-2, and uPA among these groups. CONCLUSION: Pterygium head fibroblasts express increased mRNA, protein, and activities of MMP-1 and MMP-3. CLINICAL RELEVANCE: Overexpression of MMP-1 and MMP-3, a phenotype previously linked with UV exposure in dermal fibroblasts to explain the pathologic finding of elastotic degeneration, suggests that pterygium head fibroblasts might be intrinsically altered by UV, which might be responsible for corneal invasion.

Role of metalloproteinases in human osteoarthritis.

Extracts of human articular cartilage have been examined for the presence of metalloproteinases that degrade proteoglycans and collagen of the extracellular matrix. Two enzymes (stromelysin and acid metalloproteinase) that degrade proteoglycan are elevated at least 150% in osteoarthritis (OA), whereas the tissue inhibitor of metalloproteinases (TIMP) shows only a small increase. We postulate an imbalance of enzyme over inhibitor that leads to net matrix destruction in OA. Stromelysin is shown to have an acid pH optimum of about 5.5. At this pH it can become spontaneously active and is less susceptible to TIMP inhibition than at pH 7.5. We postulate that the chondrocytes may secrete acid into the pericellular space, leading to localized enzyme activation and proteoglycan digestion.

An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples.

An enzyme-linked immunosorbent assay method has been developed for the determination of desmosine. The method is based on an inhibition immunoassay (under nonequilibrium conditions) and uses rabbit antisera directed against a desmosine-bovine serum albumin conjugate and microtiter plates coated with desmosine-gelatin conjugate. The assay quantitates desmosine in the range 2.5-50 pmol in tissue and urine samples. Important applications of this rapid and sensitive assay are in studying elastin metabolism and in screening for monoclonal antibodies against desmosine. Methods are described for obtaining a constant level of substitution of desmosine per molecule of bovine serum albumin and for preparing a desmosine-gelatin coating antigen. Five different antibody preparations directed against desmosine exhibit 15-20% cross-reactivity toward pyridinoline (3-hydroxypyridinium), a nonreducible collagen crosslinking compound also present in urine and many tissue samples.

Content of the collagen and elastin cross-links pyridinoline and the desmosines in the human uterus in various reproductive states.

During pregnancy the collagen content of the human uterus increases sevenfold and the elastin content increases fourfold to fivefold. The stable pyridinoline cross-link is found in uterine collagen at a level of 0.11 mol per mole of collagen. The same ratio, or a higher one, is found at the end of pregnancy, indicating that pyridinoline synthesis keeps pace with the rapid synthesis of collagen. This cross-link would participate in the maintenance of high mechanical strength of the uterus needed during parturition. Uterine elastin contains 2.4 residues of desmosine plus isodesmosine in 1000 residues of amino acids. This value falls to 0.95 at term, indicating that synthesis of desmosines does not keep pace with the synthesis of elastin. Therefore, desmosine measurements do not provide an accurate index of elastin changes in pregnancy. Collagen and elastin contents in nongravid uteri increase with successive pregnancies; the cross-links remain constant during this change.

Activation of cartilage stromelysin-1 at acid pH and its relation to enzyme pH optimum and osteoarthritis.

Stromelysin-1 was purified from human articular cartilage and compared to synovial fibroblast enzyme and to recombinant enzyme. If the latent enzyme was incubated at pH 5.5 with substrates such as aggrecan, it spontaneously became active. Incubation of latent zymogen alone at pH 5.5 gave increasing activation over a period of at least 5 hours. However, this activation process was not accompanied by any shift in molecular weight even after continued incubation for 18 hours. Maximum activity observed was 45-60% of that seen with APMA activation. Stromelysin-1 has a pH optimum of 5.5-6.5 on various macromolecular and peptide substrates. Interaction with TIMP is reduced at pH 5.5 relative to that at 7.5. The hypothesis is presented that osteoarthritis may be initiated by acid activation of stromelysin-1.

Desmosines in human urine. Amounts in early development and in Marfan's syndrome.

Desmosines from 24 h human urine samples were isolated, characterized and quantified. The desmosines are in peptidyl form (1000--1500 molecular weight), and their amount is decreased by two-thirds between 7 and 25 years of age. Patients with Marfan's syndrome have significantly lower urinary amounts of desmosines than do comparable controls during the early development period.

Collagen cross-linking compounds in human urine.

We report the isolation and chemical characterization of collagen cross-linking compounds, 3-hydroxypyridinium and dihydroxylysinonorleucine, from human urine.

Changes in desmosine and pyridinoline crosslinks during rapid synthesis and degradation of elastin and collagen in the rat uterus.

The wet weight of the rat uterus increased 8-fold during pregnancy and fell by 70% within 5 days postpartum. Uterine collagen increased about 5-fold during pregnancy and also fell by 70% within 5 days. The crosslink pyridinoline remained constant at 0.28 mole/mole collagen at every time point, with the possible exception of 11-12 days of pregnancy. The pyridinoline link can therefore form within the short time span of a few days, a feature presumed to be necessary to maintain the full mechanical strength of the uterus during labor. Uterine elastin increased about 8-fold during pregnancy, but the desmosines did not keep pace and fell from a normal value of 1.43 mole/mole elastin to a low of 0.89 at term. Moreover, elastin content reached a maximum several days prior to parturition and then declined continuously to 5 days postpartum. During this decline there was a selective loss of the poorly crosslinked elastin. The desmosines cannot be used as a direct measure of uterine elastin content, because of their continuously changing levels. Desmosines and pyridinoline were measured both by ELISA and by the amino acid analyzer. The two methods gave almost identical results when elastin and collagen were first separated from each other.

Purification of the neutral proteoglycan-degrading metalloproteinase from human articular cartilage tissue and its identification as stromelysin matrix metalloproteinase-3.

The 'neutral' proteoglycan-degrading metalloproteinase of human articular cartilage was purified 3,500-fold by use of an anti-(matrix metalloproteinase-3) immunoglobulin G affinity column. Molecular masses of the latent and multiple active forms and specificity of action on casein, transferrin, gelatin and fibronectin were identical with those of authentic stromelysin (matrix metalloproteinase-3) from cultured human rheumatoid synovial fibroblasts. The optimum pH of this proteinase on proteoglycan monomer was pH 5.5, and on Azocoll, 6.2; digestion of fibronectin and gelatin was more extensive at pH 5.5 than at 7.5.

Two pools of glycogen in Saccharomyces.

The effect of different extraction procedures on the yields of water-soluble and water-insoluble glycogen fractions from a number of Saccharomyces strains was studied by using a specific method for glycogen determination. The similarity of the yields obtained by the different procedures showed that neither form of glycogen is an artifact, and variations in the relative amounts of glycogen in the two fractions during cell growth and in different yeast strains suggest that they represent different pools of storage material with specific roles in cell development and differentiation. A proportion of the water-insoluble glycogen fraction, solubilized by mechanical agitation, was shown to be strongly associated with a beta-glucan-like polysaccharide that may be a cell wall component.

A possible role for dehydrodihydroxylysinonorleucine in collagen fibre and bundle formation.

The concentrations of NaB3H4-reducible collagen cross-links were determined at the time when collagen fibres and bundles are observed in electron micrographs of connective tissue developing around the implanted Ivalon sponge in adult male rats. The highest radioactivity occurs with hydroxylysinonoreleucine and histidinohydroxymerodesmosine, and the lowest with lysinonorleucine, the reducible amounts of these cross-links remaining relatively constant as fibres and bundles appear. On the other hand, dihydroxylysinonorleucine amounts are low during the initial stages of connective-tissue formation and rise sharply as collagen fibres and bundles develop and collagen matures, as shown by increased resistance of insoluble collagen to digestion with bacterial collagenase. The bulk of hydroxylysinonorleucine and dihydroxylysinonorleucine is glycosylated, the former with galactosyl or glucosylgalactosyl residues and the latter with glucosylgalactosyl residues. The changing relationships between the amounts of 3H-labelled hydroxylysinonorleucine, glucosylgalactosyldihydroxylysinonorleucine and non-glycosylated dihydroxylysinonorleucine as fibres and bundles appear suggest three post-translational steps involving lysyl-derived cross-links in the organization of collagen into fibres and bundles.

Remodeling of human myocardial collagen in idiopathic dilated cardiomyopathy. Role of metalloproteinases and pyridinoline cross-links.

A major contribution to the mechanical strength of the heart is provided by a continuous fibrillar collagen network embracing individual myocytes and forming an interstitial and perivascular framework. This study explores the possibility that idiopathic dilated cardiomyopathy may involve extensive changes in this collagenous framework. Idiopathic dilated cardiomyopathy hearts were obtained at transplant and compared with control hearts from autopsy. Idiopathic dilated cardiomyopathy showed a doubling of collagen concentration and a quadrupling of the total collagen per heart, whereas the stable mature cross-link, pyridinoline, diminished from 2.07 mol/mol of collagen to 1.0. Neutrophil-type collagenase activity is elevated approximately 30-fold as is the activity of gelatinase. Tissue inhibitor of metalloproteinase activity falls to negligible levels in idiopathic dilated cardiomyopathy, whereas alpha 2-macroglobulin is high. It is postulated that collagen critical to mechanical stability of the heart is degraded by metalloproteinase activity and is replaced by fibrous intercellular deposits of poorly cross-linked collagen. These changes contribute to weakening and dilatation of the ventricular wall.

Chondroprotective effect of betamethasone in lapine pyogenic arthritis.

The chondroprotective effect of betamethasone was examined to determine if corticosteroids can decrease articular cartilage injury caused by inflammatory exudate in Staphylococcus aureus gonarthritis in rabbits. Three experimental groups of antibiotic-treated rabbits were created, comparing parenteral versus low-dose intraarticular routes of betamethasone administration. Rabbits that received ceftriaxone plus supplemental parenteral betamethasone (group 2) demonstrated significantly less articular cartilage proteoglycan loss than did rabbits treated with antibiotics alone (group 1). Supplemental intraarticular betamethasone (group 3) was somewhat less effective in this regard, possibly reflecting the smaller steroid dosage. This animal study introduces histologic and biochemical evidence that betamethasone, administered early and in conjunction with appropriate systemic antibiotics, may help protect infected articular cartilage from proteolytic degradation. Further study is needed to prove safety and efficacy of corticosteroids before recommending their clinical use in the treatment of septic arthritis.

Role of fascial collagen in stress urinary incontinence.

OBJECTIVES: Our purpose was to determine whether collagen of the pubocervical fasciae that support the urethrovesical junction undergoes alterations that might contribute to incontinence. STUDY DESIGN: Pubocervical fascia was collected as a residual tissue in 82 patients, aged 25 to 73 years, during surgical treatment of cystocele (n = 26, no incontinence) or of stress urinary incontinence (n = 56). Measurements were made of collagen content, solubility, and cross-linking and of collagenase activity. RESULTS: Patients treated for incontinence had the same mean age and parity as the control cystocele group. There was a highly significant (20%, P <.0005) decrease in collagen content in fascial tissue from incontinent women. There was no difference in the percentage of acid-soluble (0.7%) and pepsin-soluble (17%) collagen in the 2 groups of patients; this agrees with the lack of significant change in the degree of collagen cross-linking by pyridinoline. Collagenase activity was significant in fascia but did not change in incontinence. Incontinent women had an increased body mass index. CONCLUSIONS: The pubocervical fasciae of incontinent women show a diminished content of collagen, but this is not accompanied by changes in collagen solubility or cross-linking or in collagenase activity. This decrease in collagen may contribute to the weakening of support of the bladder neck.

Role of metalloproteinases in the development and healing of acetic acid-induced gastric ulcer in rats.

BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) are believed to be active in connective tissue remodeling associated with various physiological processes and in pathological conditions such as cancer and arthritis. However, the role of MMPs in gastrointestinal ulceration has not been clearly established. Therefore, the aim of this study was to examine the role of collagenase and gelatinases A and B in the development and healing of acetic acid-induced gastric ulcer in rats. METHODS: Gastric ulcer was induced by injecting 20 microliters glacial acetic acid into gastric wall of rat stomachs. To examine whether changes in the ulcer formation and healing phase correlate with MMP activity, Triton X-100/CaCl2 and Tris/CaCl2 (60 degrees C) extracts of stomachs were prepared from controls and animals killed 24 h (formation phase) and 7 days (healing phase) after acetic acid administration. Total collagenase and gelatinase activities were measured using (H3)labeled-acetylated type I collagen or gelatin as substrate, respectively, prepared from rat skin. RESULTS: Twenty-four hours after administration of acetic acid, the mean area of ulcer crater was 51.2 mm2. By day 7, the mean size of ulcer crater was reduced to 35.9 mm2. The mean activity of collagenase in gastric tissue from controls animals was 0.007 U/g tissue. In acetic acid-treated rats, this activity increased to 2.18 U/g at 24 h and declined to 0.69 U/g by day 7. Similarly, total gelatinase activity increased from 20.5 U/g tissue (controls) to 28.8 U/g at 24 h and declined to 23.9 U/g at day 7. Gelatinzymography revealed that gelatinase B levels were greatly increased at 24 h and declined by day 7, whereas the gelatinase A levels remained constant. CONCLUSIONS: The data showed that the formation of acetic acid-induced ulcer in rats is accompanied by an elevation of collagenase and gelatinase B that gradually tend to return to control values during the healing phase.

Intraarticular injections with methylprednisolone acetate reduce osteoarthritic lesions in parallel with chondrocyte stromelysin synthesis in experimental osteoarthritis.

OBJECTIVE: To examine the effect of intraarticular injections of methylprednisolone acetate (MA) on osteoarthritic lesions and chondrocyte stromelysin synthesis in experimental osteoarthritis (OA). METHODS: In 15 mongrel dogs, the anterior cruciate ligament of the right knee was sectioned by a stab wound. Eight dogs received intraarticular injections of MA (20 mg) at the time of surgery and 4 weeks later; 7 had no treatment. The dogs were killed 8 weeks after surgery. Five normal dogs were used as controls. Macroscopic evaluation of the lesions, including measurements of osteophytes and areas of surface lesions on the condyles and plateaus, was conducted, along with histologic evaluation of the severity of lesions. Immunohistochemical analysis was carried out using a rabbit polyclonal antibody against stromelysin, followed by evaluation of matrix and chondrocyte staining using morphometric analysis. RESULTS: Treatment with MA significantly reduced the incidence (P < 0.0004) and size (P < 0.0001) of osteophytes. The histologic grading of cartilage lesions was also significantly reduced both on condyles (P < 0.01) and on plateaus (P < 0.002). Immunohistochemical studies revealed, for OA cartilage, a marked increase (P < 0.002) in the percentage of chondrocytes positive for stromelysin and in the intensity of staining throughout all the layers of the cartilage, as well as specific matrix staining (P < 0.005). Treatment with MA reduced staining at both the chondrocyte (P < 0.002) and the matrix (P < 0.01) levels toward normal. CONCLUSION: These findings provide additional evidence for the protective effect of corticosteroid injections on OA lesions, and indicate that the effect of this drug may be mediated through the suppression of stromelysin synthesis.

Matrix metalloproteinase-3 (stromelysin-1). Identification as the cartilage acid metalloprotease and effect of pH on catalytic properties and calcium affinity.

Human pro-MMP-3 (pro-matrix metalloproteinase-3) was purified from three sources: articular cartilage and conditioned media from synovial fibroblasts and Chinese hamster ovary cells expressing recombinant pro-MMP-3. All three preparations reacted with two monoclonal antibodies specific for human fibroblast pro-MMP-3. Each preparation of active MMP-3 possessed properties identical to those previously reported for the cartilage acid metalloproteinase (MMP-6; Azzo and Woessner, J. F., Jr. (1986) J. Biol. Chem. 261, 5434-5441): an acid pH optimum of 5.3-5.5 for digestion of cartilage aggrecan; digestion of oxidized insulin B-chain at Ala14-Leu15 and Tyr16-Leu17 in a ratio of 3:1; and heat stability at neutral pH. Further characterization of MMP-3 establishes that the acid pH optimum for cartilage aggrecan is not due to substrate denaturation since the same optimum is found by viscosity assay, by SDS-polyacrylamide gel electrophoresis assay of G1 domain, and by digestion of aggrecan in fresh cartilage fragments in vitro. Fibronectin was also digested optimally at pH 5.5 and NH2-terminal sequence analysis revealed no pH change in a major proteolytic site of cleavage at the Pro689-Leu690 bond. The specificity constant kcat/Km is maximal at pH 5.5 as determined in a quenched fluorescence peptide assay. This is due to an increase in kcat at pH 5.5 without any substantial effect on Km. The affinity of MMP-3 for calcium is decreased about 10-fold at pH 5.3 compared to neutral pH. Finally, the neutral cartilage metalloproteinase is identified as 72-kDa pro-MMP-2 based on M(r), specificity of insulin B-chain cleavage, and reactivity with a specific polyclonal antibody to human MMP-2.

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo.

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.