Heparin-enhanced zymographic detection of matrilysin and collagenases.

Unlike the gelatinases (MMP-2 and -9), matrilysin (MMP-7) and collagenases (MMP-1 and -13) are difficult to detect at low levels in conventional casein or gelatin zymography. In this report, heparin was used to enhance the zymographic assays for MMP-7, -1, and -13. With the addition of heparin to the enzyme sample, MMP-7 can be detected at a level of 30 pg in transferrin zymography and MMP-1 and -13 can be detected at a level of 0.2 ng in gelatin zymography. Carboxymethylated transferrin is used instead of casein as a substrate for assaying rat MMP-7. This substrate does not require a prerun step or substrate cross-linking to give uniform staining and clear band formation. It is necessary for heparin to run to the same region of the gel as the enzyme to produce its enhancing effect. For MMP-7 movement of heparin and enzyme is almost equal; for the collagenases it is necessary to add heparin to each well after the electrophoretic run is underway. Possible mechanisms of activity enhancement are discussed.

Moderation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors.

OBJECTIVE: To determine the effect of matrix metalloproteinase (MMP) inhibitors in mono-iodoacetate-induced arthritis in rats. DESIGN: The ability of compounds to inhibit MMPs in vitro was assessed kinetically using a quenched fluorescent substrate. Rats were injected with iodoacetate intraarticularly in one knee joint and damage to the tibial plateau was evaluated from digitized images captured using an image analyser and by histology. Collagenase and gelatinase activity in cartilage from iodoacetate injected knees were evaluated using(3)H-rat type I collagen and gelatin zymography, respectively. RESULTS: Collagenase and gelatinase activity significantly increased in the knee cartilage of rats injected with iodoacetate with peak activity by day 7. Three MMP inhibitors were examined for their efficacy in the rat iodoacetate-induced arthritis model. Significant (P< 0.05) inhibition of cartilage damage was observed in animals treated orally with 35 mg/kg b.i.d. of the three different MMP inhibitors. Inhibition of cartilage damage by the MMP inhibitors ranged from 36-42%. CONCLUSION: MMP inhibitors are partially protective against cartilage and subchondral bone damage induced by iodoacetate. These results support an important role for MMPs in mediating the joint damage in this model of arthritis.

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.

TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix.

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B beta-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.

Induction of pulmonary matrilysin expression by combustion and ambient air particles.

The molecular mechanism(s) by which chemically complex air pollution particles mediate their adverse health effects is not known. We have examined the ability of combustion and ambient air particles to induce pulmonary matrilysin expression due to the well-documented role of matrix metalloproteinases in tissue injury and repair responses. Rats were exposed to saline, residual oil fly ash (2.5 mg/rat), or ambient air particles (2.5 mg/rat) via intratracheal instillation and examined 3-72 h after exposure. Saline-exposed animals had low levels of matrilysin mRNA, whereas the animals exposed to either complex particle showed an early induction of pulmonary matrilysin gene expression as well as of the 19-kDa activated form of matrilysin. Immunocytochemistry and in situ hybridization analyses identified the alveolar macrophages and monocytes as primary sources of air pollution particle-induced matrilysin expression. Matrilysin gene induction and protein activation by combustion and ambient air particles correlated with the early histopathological changes produced by these particles. These results demonstrate the ability of combustion and ambient air particles to induce pulmonary matrilysin expression and suggest a role for this matrix metalloproteinase in the initiation of lung injury produced by these particles.

Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7).

Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound. The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane. This is established by extraction experiments and confocal microscopy. The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment. The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion. Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle. The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; the K(D) is 5-10 nM. Zymographic measurement of MMP-7 activity is greatly enhanced by heparin. Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands. Evidence is also found for the binding of MMP-2, -9, and -13. Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels. Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins. Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis.

Matrix metalloproteinase inhibition. From the Jurassic to the third millennium.

A brief historical introduction to the matrix metalloproteinase (MMP) field, which began in 1962, is followed by an overview of the inhibition of these proteases by natural inhibitors such as alpha 2 macroglobulin and the TIMPs (tissue inhibitors of metalloproteinases) and by synthetic inhibitors, which are largely chelating agents. The latter include thiol, alkylcarbonyl, phosponamidate and hydroxamate compounds, as well as the tetracyclines. A review of the most recent progress concludes with prognostications as to where the field may be going next.

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.

Matrilysin activity in the rat uterus during the oestrous cycle and implantation.

The objective of this study was to follow changes in the activity of the small matrix metalloproteinase matrilysin (MMP-7) in the rat uterus during the oestrous cycle and embryo implantation. Matrilysin was extracted from rat uteri, partially purified and separated into active and latent forms. The two forms of the enzyme were quantified at all stages of the oestrous cycle and after oestradiol and progesterone treatment. The activity was also measured during the first 7 days of pregnancy. Both latent and active forms of MMP-7 reached a peak during the pro-oestrous stage of the cycle; the concentrations were three times higher than at dioestrus and metoestrus. In rats treated with 0.1 mg oestradiol at metoestrus, both latent and active forms of the enzyme increased by more than two-fold after 24 h. In rats treated at pro-oestrus with 0.4 mg progesterone, there was a 70% increase in latent MMP-7, but no change in the active form. The highest concentrations of MMP-7 were observed on the first day of pregnancy. Between days 3 and 7 of pregnancy, the concentrations were relatively constant and comparable to the low concentrations at dioestrus. Enzyme activities were not different at implantation sites compared with remote sites.

Role of matrix proteases in processing enamel proteins.

This article reviews the current status of research on proteases of the enamel layer that are capable of processing and degrading proteins of the enamel matrix. Following a brief survey of the historical development of this discipline, a summary is presented of the current status. Two proteases have recently been cloned: EMSP-1 (enamel matrix serine protease-1), a serine protease, and enamelysin, a metalloprotease. These two are placed into their appropriate families: the chymotrypsin family S1 of clan SA of the serine protease class and the matrixin family or matrix metalloproteinase family, M10 of clan MB (the metzincins) of the metalloprotease class. The major features of these two families are outlined. The article concludes with some suggested areas for future research--identifying further proteases and characterizing those now known.

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.

Onapristone and prostaglandin E2 induction of delivery in the rat in late pregnancy: a model for the analysis of cervical softening.

OBJECTIVES: Our purpose was to develop a method to induce premature delivery in rats and to use this method to identify biochemical changes that are critical to cervical dilatation by comparison to changes that occur during term delivery. STUDY DESIGN: Rats were treated with antiprogestational agents onapristone or lilopristone in combination with prostaglandin E2 or estradiol on day 19 of pregnancy to induce delivery before term. Mechanical and biochemical changes of the isolated cervix were compared with changes found at term and in 20-day controls. RESULTS: Rats treated with a combination of onapristone and prostaglandin E2 were consistently delivered 25 hours after treatment began. The physical characteristics of the cervix of treated rats changed to match those of term cervices. The ratio of the small sulfated proteoglycan (decorin) to collagen changed on induction to match the ratio found at term. CONCLUSIONS: This induction protocol can be used to advance the time of delivery in rats, with the further advantage that the time of delivery can be accurately predicted. The data strengthen a proposed model in which the interaction of decorin and collagen is an important determinant of the biomechanical properties of the cervix during pregnancy.

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.

Regulation of matrilysin in the rat uterus.

Matrilysin was first discovered in the involuting rat uterus; it has also been known as uterine metalloproteinase, putative metalloproteinase (Pump-1), and matrix metalloproteinase 7 (MMP-7). It is the smallest member (28 kDa) of a family of 15 MMPs that together are able to degrade most of the macromolecules of the extracellular matrix. This family is briefly reviewed; all members are zinc metalloproteinases that occur in zymogen form with the active site zinc blocked by cysteine. Matrilysin can degrade a wide range of gelatins, proteoglycans, and glycoproteins of the matrix and can activate several other MMPs including collagenase. With respect to the uterus, matrilysin is localized to epithelial cells and varies in amount with the estrus cycle and is found in high levels during postpartum involution. There is evidence for a role in the last stage of cervical ripening and immediately postpartum. Induction of premature delivery by onapristone and prostaglandin E2 advances these changes in matrilysin. Regulation of the enzyme levels in the uterus are considered from four viewpoints: control of protein synthesis (particularly in response to hormones), activation of the proenzyme to functional protease, retention of enzyme by binding to matrix components such as heparan sulfate, and inhibition by natural inhibitors such as tissue inhibitor of metalloproteinases (TIMPs) and alpha 2-macroglobulin.

Expression and physicochemical characterization of human proliferating cell nuclear antigen.

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-200, and hydroxylapatite chromatography. Approximately 20 mg of PCNA was isolated from E. coli cells derived from 2 L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase delta activity at least 25-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 102,000 and a Stokes radius of 37 Angstrum by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96,800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a dimeric species at low dilution. Cross-linking experiments revealed the presence of PCNA dimers which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a dimer at low protein concentrations.

Characterization of rat uterine matrilysin and its cDNA. Relationship to human pump-1 and activation of procollagenases.

A small uterine metalloproteinase of the rat has been shown by amino acid and cDNA sequencing to be orthologous to human pump-1. Both proteinases are now designated as matrilysin or matrix metalloproteinase 7. The properties of purified uterine metalloproteinase and recombinant pump-1 were compared. Their specificities on substrates (gelatins, fibronectin, transferrin, elastin, Azocoll, and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3,[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2) are similar and distinct from those of the stromelysins and gelatinases. The two matrilysins have similar sensitivity to hydroxamate and pseudopeptide inhibitors. Rat matrilysin selectively cleaves the alpha 2(I) chain of rat gelatin, producing major cuts at Gly713-decreases-Ile714, Gly775-decreases-Leu776, and Gly809-decreases-Ile810. Rat matrilysin produces maximum activation of latent human interstitial collagenase 1 (pro-matrix metalloproteinase 1) when added in the presence of 4-aminophenylmercuric acetate (APMA) by cleaving the Gln80-decreases-Phe81 bond. Rat and human matrilysin do not directly activate latent rat collagenase 3 (matrix metalloproteinase 13) and do not enhance its activation when added together with APMA. Autoactivation of collagenase 3 in the presence of APMA results in cleavage at Val81-decreases-Tyr82 corresponding to the Gln80-decreases-Phe81 cleavage in collagenase 1. Thus collagenase 3 is capable of maximal autoactivation, whereas collagenase 1 is dependent upon another matrix metalloproteinase in order to be activated to its full potential.