Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone

There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.     

Activation of Gelatinase A (72-kDa Type IV Collagenase) Induced by Monensin in Normal Human Fibroblasts

In monolayer culture, fibroblasts secrete all matrix metalloproteinases, including gelatinase A (72-kDa type IV collagenase), as inactive zymogens. Whereas limited proteolysis by plasmin or other matrix metalloproteinases (MMPs) can accomplish the extracellular activation of other proenzymes in this family, gelatinase A proenzyme is uniquely refractory to cleavage by such proteinases. Previously it has been shown that fibroblasts cultured in the presumably more physiologic culture milieu of a type I collagen lattice can be induced to secrete active gelatinase A. In monolayer culture, however, the plant lectin concanavalin A will induce gelatinase A activation. Here we show that in monolayer culture activation of gelatinase A by normal fibroblasts is also induced by the sodium ionophore monensin. The monensin response is dose-dependent, time-dependent, requires protein synthesis, and is specific to gelatinase A among the secreted matrix metalloproteinases. The activator appears to be associated with cell membranes and may be membrane-type matrix metalloproteinase 1(MT-MMP1). Both mRNA and immunodetectable protein of MT-MMP1 are increased with monensin treatment while message for the protein inhibitor of gelatinase A, TIMP-2, is unchanged. The monensin-induced signal transduction pathway leading to gelatinase activation in monolayer culture appears to be different from the integrin-mediated pathway operative in the collagen lattice system. The tyrosine kinase inhibitor genistein blocks monensin activation of gelatinase A in monolayer culture. In contrast, genistein has no effect on proenzyme activation in the collagen lattice. Likewise, the cyclooxygenase inhibitor indomethacin abrogates the monensin effect in monolayer culture and can be reversed by addition of exogenous prostaglandin E₂(PGE₂). Neither indomethacin nor PGE₂affects activation of gelatinase A in the collagen lattice.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Dynamics of matrix metalloproteinases activity of primary murine embryonic fibroblasts during cultivation

Embryonic cells regulate the expression of matrix metalloproteinases (MMP) providing remodulation of extracellular matrix, which in turn provides the changes in cell adhesion and migration during the cell development and differentiation. In present work we studied the changes of gelatinases (MMP-2 and MMP-9) and collagenases (MMP-1 and MMP-8) activities in the process of cultivating the primary murine embryonic fibroblasts (MEF). Cultivation was continued for 6 passages, after that the culture died in time. According to gelatin and collagen zymography results, drastic changes of all MMPs activities occurred during the third passage of cell cultivation. The MMP-1 and MMP-9 activity appears in the middle of cultivation and then disappeared at the end. The most important event MEF cultivation is appearance and subsequent reservation of collagenase MMP-8 and active form of gelatinase MMP-2.     

Complex role of matrix metalloproteinases in angiogenesis

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a significant role in regulating angiogenesis,the process of new blood vessel formation.Interstitial collagenase (MMP-1),72kDa gelatinase A/type IV collagenase (MMP-2),and 92 kDA gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and promote angiogenesis.TIMP-1,TIMP-2,TIMP-3,and possibly,TIMP-4 inhibit neovascularization.A new paradign is emerging that matrilysin (MMP-7),MMP-9,and metalloelastase (MMP-12) may block angiogenesis by converting plasminogen to angiostatin,which is one of the most potent angiogenesis antagonists.MMPs and TIMPs play a complex role in regulating angiogenesis.An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide important information to allow the control of angiogenesis,e.g.the stimulation of angiogenesis for coronary collateral circulation formation;while the inhibition for treating arthritis and cancer.     

Immortalization of human aortic smooth muscle cells with origin-minus simian virus 40 DNA.

We established two cell lines of human smooth muscle cells (SMC) by transfection of cells from the aortic intima and aortic media with origin-minus simian virus 40 (ori-minus SV40) DNA. Ori-minus SV40 DNA very efficiently immortalized human smooth muscle cells in culture. Proteins that these cell lines produced included type I, III, IV, and V collagens, fibronectin, and human matrix metalloproteinases (MMP)-1 (tissue collagenase), -2 ("type IV collagenase"), and -3 (stromelysin). The protein production in these cell lines generally mimicked that of normal SMC, but the immortalization stimulated the cell line of medial SMC to produce excessive MMP-2 and to secrete MMP-9 (92-kDa gelatinase). However, since these cell lines did not show a fully malignant phenotype, we concluded that, in addition to the degradation of extracellular matrix macromolecules, including basement membrane components by MMP-2, -3, and/or -9, some additional factors must be involved for the malignancy of fully transformed cells and that these immortalized human aortic SMC, which share many characteristics with normal SMC, will prove useful to study the role(s) of metalloproteinases in atherosclerosis.     

Collagen type I, III and V differently modulate synthesis and activation of matrix metalloproteinases by cultured rabbit periosteal fibroblasts.

In the present study we investigated whether the collagen types I, III and V affect the activity of fibroblasts obtained from rabbit periosteum. The cells were cultured on plates either or not coated with different amounts of collagen type I, III or V and analyzed for their attachment, DNA synthesis and the expression and activity of matrix metalloproteinases (MMPs). Our data show that the three collagen types promoted attachment and spreading of the cells and stimulated DNA synthesis when used in relatively low concentrations. High concentrations of type V-but not of type I or III-proved to inhibit thymidine incorporation. The expression and activity of matrix metalloproteinase 1 (MMP-1; interstitial collagenase) decreased under the influence of relatively low amounts of collagen (<40 microg/well), whereas higher levels increased its release. Matrix metalloproteinase 2 (MMP-2; gelatinase A) was up-regulated by the different types of collagen; the active fraction of stromelysin-1 (MMP-3) decreased. Accordingly, the mRNA expression of MMP-1 and -3 were reduced. The expression of MMP-2 mRNA, however, proved to be unaffected. Blocking antibodies to beta(1)-integrin or echistatin increased the level of MMP-1 but had no effect on MMP-2. All parameters tested were similarly affected by type I and III collagen, whereas the effect of type V was always less. We conclude that the collagen types I, III and V provide different sets of signals for fibroblasts that differently modulate their proliferation and MMP expression.     

Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens.

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.     

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

Matrix metalloproteinase induction in fibrosis and fibrotic nodule formation due to silica inhalation

Matrix metalloproteinases (MMPs) are the principle enzymes that initiate degradation of collagen. We examined the role of MMPs during alveolar wall fibrosis and fibrotic nodule formation from silica exposure. Rats were exposed to filtered air or 15 mg/m(3) silica by inhalation for 5 days/wk, 6 h/day. Lungs were preserved by intratracheal instillation of fixative at 20, 40, 60, 79, and 116 days of exposure. Additional groups were fixed after 20, 40, and 60 days of exposure followed by 36 days of recovery. The number of nodules, defined by a collagenous core and a bounding cell layer detached from the alveolar wall, was determined by morphometry. Lungs showed increased alveolar wall collagen and fibrotic nodules at 79 and 116 days of exposure with increased collagenase and gelatinase activity. The number of nodules per lung in exposed groups increased from 619 +/- 447 at 40 days to 13,221 +/- 1,096 at 116 days (means +/- SE, n = 5). No nodules were seen in control lungs. Silica-exposed rats with a 36-day recovery in filtered air showed enhanced MMP activity over exposure to silica for the same duration with no recovery. MMP-2 and MMP-9 were significantly elevated in alveolar macrophages after 40-day exposure. Stromelysin expression was demonstrated in alveolar macrophages and cells within fibrotic nodules. TIMP-1 expression was not significantly altered. In summary, MMP activity was upregulated at 40 days of silica exposure and progressively increased during ensuing fibrotic responses. Early expression of stromelysin was found in fibrosing alveolar walls and fibrotic nodules.     

Collagen breakdown in soft connective tissue explants is associated with the level of active gelatinase A (MMP-2) but not with collagenase.

Recent data suggest that gelatinase A (matrix metalloproteinase-2, MMP-2) plays an important role in the degradation of collagen of soft connective tissues. In an attempt to investigate its participation in more detail we assessed the digestion of collagen in cultured rabbit periosteal explants and compared this with the level of active MMP-2 and collagenases. The data demonstrated that both collagen degradation and MMP activity increased with time. Conditioned medium obtained from explants cultured for 72 h showed that the level of active MMP-2 correlated with collagen degradation (r = 0.80, d.f. = 23, P < 0.0001). Such a relationship was not found with collagenase activity (r = -0.08, d.f. = 21, NS). The possible involvement of MMP-2 in collagen degradation was investigated further by incubating explants with selective gelatinase inhibitors (CT1166, CT1399 and CT1746). In the presence of these compounds breakdown of collagen was almost completely abolished (approximately 80%). Finally we assessed whether periosteal fibroblasts had the capacity to degrade collagen type I that conferred resistance to collagenase activity. Breakdown of this collagen did not differ from degradation of normal collagen. Taken together, our data provide support for the view that MMP-2 plays a crucial role in collagen degradation of soft connective tissue.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.     

Expression of matrix metalloproteinase activity in idiopathic dilated cardiomyopathy: A marker of cardiac dilatation

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM), ventricular systolic dysfunction and chamber dilatation are accompanied by architectural remodeling, wall thinning and cardiac myocyte slippage. Recent work has demonstrated an association between collagen degradation and an increased expression of matrix metalloproteinases (MMPs). Accordingly, we have sought to correlate (a) collagen degradation with MMP elevations and, (b) assay the neutralizing potential of a known inhibitor of MMP, tetracycline on MMPs in DCM. METHODS: Assessment of LV volume and shape by 2-D echocardiography was performed. Light microscopic assessment of histopathology in picrosirius red stained biopsy samples of 11 DCM patients and six post-transplant patients was performed. Zymographic estimation of MMP activity and influence of tetracycline on MMP activity was assessed. RESULTS: Small amount of interstitial collagen was noted in the control group, whereas in the DCM hearts, chamber dilatation was associated with areas of scanty myocyte necrosis, islands of excess collagen, and focal areas of absent or scanty collagen with intact myocytes. In cardiomyopathic tissue, collagenase activity was markedly elevated at 63% compared with 8% in post-transplant tissue. Tetracycline at a concentration of 285+/-10 microM (IC50) inhibited collagenase activity by 50% in cardiomyopathic tissue. CONCLUSIONS: Areas of focal interstitial collagen accumulation were accompanied by collagen fiber lysis and increased collagenase activity in dilated cardiomyopathy. This enhanced collagenolytic activity found in endomyocardial biopsy tissue was inhibited by tetracycline. The non-antibiotic property of tetracycline may be of potential value in the prevention of ventricular dilatation in idiopathic dilated cardiomyopathy.     

Degradation of type I collagen films by mouse osteoblasts is stimulated by 1,25 dihydroxyvitamin D3 and inhibited by human recombinant TIMP (tissue inhibitor of metalloproteinases)

Mouse calvarial osteoblasts grown on native type I collagen films degrade collagen in response to 1,25 (OH) 2vitD3. Collagen degradation is accompanied by increased latent collagenase and gelatinase secretion and by a reduction in free TIMP. Exogenous human recombinant TIMP abolished 1,25 (OH) 2vitD3 stimulated collagen degradation and inhibited background collagenolysis. No active metalloproteinases were detectable in the culture medium suggesting sequestration of active enzyme at the site of action or inhibition by residual TIMP. Chondrocytes could not mimic osteoblasts in this system.     

Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling

Summary Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator). Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated. Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membranebound receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding noncollagenous proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases, such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines, including transforming growth factor β and interleukin 1α.     

In silico scaffold evaluation and solid phase approach to identify new gelatinase inhibitors

Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents.     

Matrix metalloproteinase‐2 and ‐9 activities in human keloids,hypertrophic and atrophic scars: a pilot study

Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP‐9 activity was undetectable in gelatin zymography analysis. Pro‐MMP‐2 levels (median) were highest in normal skin group 53.58 (36.40–75.11) OD µg^?1 protein, while active MMP‐2 levels were highest in keloid group 52.53 (42.47–61.51) OD µg^?1 protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two‐phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of doxycycline on sulfur mustard-induced respiratory lesions in guinea pigs

Respiratory tract lesions induced by the chemical warfare agent sulfur mustard (SM) are characterized by epithelial damages associated with inflammatory cell infiltration. Here we evaluated the imbalance between gelatinase and tissue inhibitors of metalloproteinases (TIMPs), and we tested pretreatment with the protease inhibitor doxycycline. Guinea pigs were intoxicated intratracheally with SM and evaluated 24 h after exposure. Matrix metalloproteinase (MMP) gelatinase activity of bronchial lavage (BL) fluid from SM-exposed guinea pigs was high compared with controls, as shown by both zymography and biotinylated substrate degradation, whereas TIMP-1 and -2 levels by immunoblotting were similar. Extensive areas of lysis were evidenced by in situ zymography, indicating imbalance between gelatinases and inhibitors towards net proteolytic activity. Doxycycline pretreatment resulted in 1) decreased gelatinase activity (zymography, free gelatinase activity assay, and in situ zymography); 2) decreased inflammation (BL fluid cellularity and protein level); and 3) dramatic decrease in histological epithelial lesions. Our results suggest inadequate levels of TIMP to counteract increased gelatinase activity and further support a role for MMP gelatinases in SM-induced respiratory lesions. They also suggest that doxycycline may hold promise as a therapeutic tool.