Reverse Zymography Research Articles

Up-Regulation of Matrix Metalloproteinase Expression and Activation Following Cyclical Compressive Loading of Articular Cartilage in Vitro

Osteoarthritis (OA) results in articular cartilage degeneration and subchondral bone remodeling. Excessive or abnormal loading of the joint may contribute to matrix destruction by creating an imbalance between proteinases and their inhibitors. This study investigates whether cyclical loading regulates expression and/or activation of metalloproteinases 2 and 9 (MMPs) in articular cartilage explants. Gelatin zymography, reverse zymography, and MMP activity assays of mechanically loaded bovine cartilage explants (0.5 MPa, 1 Hz, 3 h) showed increased expression and activation of MMPs 2 and 9, whereas expression of the tissue inhibitors of MMPs was unaffected. This shows, for the first time that mechanical loading can influence tissue homeostasis generating an imbalance of proteinases and their inhibitors inducing turnover and/or catabolic events in cartilage.

Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro

The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines. Corneal lesions and MMP inductionin vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa. Effects of pseudomonal virulence factors [elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)], tumor necrosis factor (TNF)-α and interleukin (IL)-1β on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting. Corneal ulcers with typical ring abscesses were observed 12–24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas. Pseudomonal elastase caused the most extensive damage to both cell types. RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9. Exotoxin A had no effect on MMP expression. Both IL-1β and TNF-α augmented MMP-9 expression in HT1080 cells. Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells. In conclusion, corneal destruction seen with P. aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase.

A unique substrate recognition profile for matrix metalloproteinase-2.

The catalytic domains of the matrix metalloproteinases (MMPs) are structurally homologous, raising questions as to the degree of distinction, or overlap, in substrate recognition. The primary objective of the present study was to define the substrate recognition profile of MMP-2, a protease that was historically referred to as gelatinase A. By cleaving a phage peptide library with recombinant MMP-2, four distinct sets of substrates were identified. The first set is structurally related to substrates previously reported for other MMPs. These substrates contain the PXX/X(Hy) consensus motif (where X(Hy) is a hydrophobic residue) and are not generally selective for MMP-2 over the other MMPs tested. Two other groups of substrates were selected from the phage library with similar frequency. Substrates in group II contain the L/IXX/X(Hy) consensus motif. Substrates in group III contain a consensus motif with a sequence of X(Hy)SX/L, and the fourth set of substrates contain the HXX/X(Hy) sequence. Substrates in Group II, III, and IV were found to be 8- to almost 200-fold more selective for MMP-2 over MMP-9. To gain an understanding of the structural basis for substrate selectivity, individual residues within substrates were mutated, revealing that the P(2) residue is a key element in conferring selectivity. These findings indicate that MMP-2 and MMP-9 exhibit different substrate recognition profiles and point to the P(2) subsite as a primary determinant in substrate distinction.

Differential expression of extracellular matrix components in the bovine oviduct during the oestrous cycle

Components of the extracellular matrix take part in tissue rebuilding as well as activating surface-bound growth factors. In the present study, expression and selected activities of urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), their inhibitors (plasminogen activator inhibitor 1 (PAI-1) and tissue inhibitors of metalloproteinases (TIMPs)) were examined in bovine oviducts by RT--PCR, ribonuclease protection assay and activity assays. A high content of mRNA encoding for uPA was detected before ovulation with a three-fold decrease after ovulation. In contrast, PAI-1 expression appeared to be stable during the oestrous cycle. Oviductal flushings produced caseinolytic zones in zymograms containing plasminogen at approximately 50 kDa and 28 kDa. An activity assay for uPA showed highest net activity during the early to mid-luteal phase. Increased TIMP-1 and MMP-2 mRNA concentrations were found around the time of ovulation compared with the luteal phase. In contrast, MMP-1 mRNA transcripts were enriched during the early to mid-luteal phase. Gelatin zymograms detected a 70--72 kDa protease activity showing an oestrous cycle-dependent activity with highest activity before ovulation. Reverse zymography detecting TIMPs revealed proteins between 21 kDa and 24 kDa. Only for the smallest (21 kDa) protein were amounts increased around the time of ovulation compared with the luteal phase. The observation that several extracellular matrix components were regulated distinctly in bovine oviducts indicates that local interactions between these components, growth factors, gametes and the embryo are possible and may influence fertilization and early embryonic development.

Novel in vitro assays for assessing the haemorrhagic activity of snake venoms and for demonstration of venom metalloproteinase inhibitors

Standard methods used for assessing the haemorrhagic toxicity of snake venoms and the effectiveness of antivenoms are laborious, expensive and involve the use of large numbers of laboratory animals. This paper examined the feasibility of using a gelatin degradation ELISA for preliminary screening of snake venom metalloproteinases (MPs). Potent gelatinolytic activity was observed in venoms from snakes of the family Viperidae and, as expected, little or no activity was evident in the venoms of snakes that induce neurotoxic pathology (most elapids). A reverse gelatin zymography assay was used on a variety of venoms to demonstrate a number of inhibitors of MP activity, the first such demonstration of its kind.

Selective induction of tissue inhibitor of metalloproteinase-1 in bleomycin-induced pulmonary fibrosis.

Tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that have the capacity to modify cellular activities and to modulate matrix turnover. We demonstrate that TIMP-1 messenger RNA (mRNA) and protein expression are selectively and markedly increased in a murine model of bleomycin-induced pulmonary fibrosis. Northern analysis showed that lung steady-state TIMP-1 mRNA levels increased 14-fold after bleomycin administration compared with control mice. Expression of the genes for TIMP-2, TIMP-3, and interstitial collagenase (matrix metalloproteinase-13) was unaltered in the injured lung. In situ hybridization demonstrated that TIMP-1 gene induction was spatially restricted to areas of lung injury. Metalloproteinase inhibitory activity of relative molecular mass of ~ 21 to 28 kD, corresponding to the molecular weights for TIMP-1 and TIMP-2, was identified in lung extracts of bleomycin-injured mice by reverse zymography. Western analysis demonstrated that TIMP-1 protein levels in bronchoalveolar lavage fluid (BALF) of bleomycin-treated mice increased 220- and 151-fold at Days 4 and 28, respectively, compared with control mice. TIMP-2 immunoreactive protein in the BALF increased 20- and 103-fold relative to controls at Days 4 and 28, respectively. These results demonstrate that TIMP-1 gene expression is selectively increased, and that the expression of TIMP-1 and TIMP-2 is differentially regulated in bleomycin-induced pulmonary fibrosis. The profound and durable increase in TIMP-1 and TIMP-2 proteins suggests an important regulatory role for these antiproteases in the inflammatory and fibrotic responses to bleomycin-induced lung injury.

Monocyte/mesangial cell interactions in high-glucose co-cultures.

Monocytes bind to human mesangial cells (HMC) in a co-culture model of leukocyte/ glomerular cell interactions. Since monocytic infiltration has been demonstrated in the early stages of diabetic glomerulopathy, we examined whether co-culture with myelomonocytes of the U937 cell line in media mimicking the diabetic microenvironment modulated phenotype, growth, and extracellular matrix production patterns of HMC. HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcription-polymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG. U937 adhesion at 1-3 h was increased in HG (from 54.9+/-6.6 to 87.1+/-5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993+/-505 micro(2) with 1.2+/-0.1 hillocks/high-power field, which increased to 13 651+/- 1114 micro(2) with 0.5+/-0.2 hillocks/high-power field in HG (P<0.05). TUNEL+HMC were nearly identical (4.9+/-1.7 vs 4.2+/-0.4% in HG, P=NS). Enhanced transcription and secretion of urokinase (uPA, +656%), COL IV (+137%), TGF-beta1 (+590%) were observed in co-cultures in HG. COL IV and TGF-beta1, but not uPA, were also increased in HMC alone, exposed to HG for 5 days. MMP-2/TIMP-2 ratio was decreased while MMP-1/TIMP-1 was increased in HG co-cultures. In both NG and HG, U937 adhesion reduced HMC number and hillocks at 24 h, with constant apoptosis. The effects of U937 were no longer detectable at 48 h, when apoptosis was 2.1+/-0.6 vs 4.0+/-0.4% in HG, and cell counts returned above basal, possibly due to a delayed proliferative response. High glucose medium increases U937 cell adhesion to HMC. In turn, monocytes modulate number and spatial distribution of HMC, which are also markedly affected by ambient glucose levels. These interactions may be relevant to leukocyte infiltration, mesangial expansion, and glomerulosclerosis in diabetes.

Secretion of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases into follicular fluid during follicle development in equine ovaries

Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.

Secretion of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases into follicular fluid during follicle development in equine ovaries

Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in cerebrospinal fluid differ in multiple sclerosis and Devic's neuromyelitis optica.

Matrix metalloproteinases (MMPs) are increased in the CSF of patients with multiple sclerosis. Devic's neuromyelitis optica (DNO) is a demyelinating syndrome that involves the optic nerve and cervical cord but differs pathologically from multiple sclerosis. Therefore, we hypothesized that the type of inflammatory reaction that causes MMPs to be elevated in multiple sclerosis would be absent in patients with DNO. CSF was collected from 23 patients with relapsing-remitting or secondary progressive multiple sclerosis, all of whom were experiencing acute symptoms, from seven patients with DNO, and from seven normal volunteers. Diagnoses were made according to current criteria on the basis of clinical manifestations, imaging results and CSF studies. IgG synthesis was increased in the CSF of multiple sclerosis patients but not in that of DNO patients. Zymography, reverse zymography and ELISA (enzyme-linked immunosorbent assay) were used to measure gelatinase A (MMP-2), gelatinase B (MMP-9) and tissue inhibitors of metalloproteinases (TIMPs). Zymograms showed that multiple sclerosis patients had elevated MMP-9 compared with DNO patients and controls (P: < 0.05). TIMP-1 and TIMP-2 levels were similar in all three groups. We conclude that multiple sclerosis patients have higher MMP-9 levels in the CSF than patients with DNO, which supports the different pathological mechanisms of these diseases.

Zymography and reverse zymography for detecting MMPs, and TIMPs.

Zymography is the electrophoretic separation of proteins through a polyacrylamide gel containing a proteolytic substrate. After denaturing (but nonreducing) electrophoresis, proteins are renatured and incubated in an appropriate buffer for proteolytic activity. Clear zones of lysis in the stained gel indicated active proteinases. Reverse zymography is a similar technique to detect proteinase inhibitors. After renaturing of proteins, the gel is incubated with metalloproteinases which digest the substrate incorporated into the gel. Inhibitors are shown as dark zones of inhibition against a clear background upon staining.

Tissue inhibitor of metalloproteinase-4 instigates apoptosis in transformed cardiac fibroblasts.

Tumor cells become malignant, in part, because of their activation of matrix metalloproteinases (MMPs) and inactivation of tissue inhibitor of metalloproteinases (TIMPs). Myocardial tumors are rarely malignant. This raises the possibility that the MMPs and TIMPs are differentially regulated in the heart compared to other tissues. Therefore, we hypothesized that a tissue specific tumor suppressor exists in the heart. To test this hypothesis we prepared cardiac tissue extracts from normal (n = 4), ischemic cardiomypathic (ICM) [n = 5], and dilated cardiomyopathic (DCM) [n = 8] human heart end-stage explants. The level of cardiospecific TIMP-4 was determined by SDS-PAGE and Western-blot analysis. The results suggested reduced levels of TIMP-4 in ICM and DCM as compared to normal heart. TIMP-4 was purified by reverse phase HPLC and gelatin-sepharose affinity chromatography. Collagenase inhibitory activity of chromatographic peaks was determined using fluorescein-conjugated collagen as substrate and fluorescence spectroscopy. The activity of TIMP-4 (27 kDa) was characterized by reverse zymography. The role of TIMP-4 in cardiac fibroblast cell migration was examined using Boyden chamber analysis. The results suggested that TIMP-4 inhibited cardiac fibroblast cells migration and collagen gel invasion. To test whether TIMP-4 induces apoptosis, we cultured cardiac normal and polyomavirus transformed fibroblast cells in the presence and absence of TIMP-4. The number of cells were measured and DNA laddering was determined. The results suggested that TIMP-4 controlled normal cardiac fibroblast transformation and induced apoptosis in transformed cells. Cardiospecific TIMP-4 plays a significant role in regulating the normal cell phenotype. The reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse extracellular matrix degradation (remodeling), cardiac hypertrophy and failure. This study suggests a possible protective role of TIMP-4 in other organs which are susceptible to malignancy.

Peritonitis Increases MMP-9 Activity in Peritoneal Effluent from CAPD Patients

Patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD) sometimes experience ultrafiltration failure. Mesothelial basement membrane thickening and the accumulation of submesothelial fibrotic tissue are common features of the diseased peritoneum. Peritonitis can lead to ultrafiltration failure, but the precise mechanism is not clear. The key enzymes in extracellular matrix (ECM) remodeling, namely matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are produced by human peritoneal mesothelial cells. Using peritoneal effluent from 13 CAPD patients with peritonitis and 7 noninfected CAPD control individuals, we examined MMP and TIMP activities by gelatin and reverse zymography. Latent and activated types of MMP-2 and -9, and TIMP-1 and -2 were identified in peritoneal effluent (from all CAPD patients). Levels of latent and activated type MMP-9, as well as of TIMP-1 activities were higher at the onset of peritonitis than either during the recovery phase of peritonitis and/ or in control individuals. Activated MMP-9 activity positively correlated with leukocyte numbers and IL-6 levels in peritoneal effluent. Activities of MMP-2 and TIMP-2 in peritoneal effluent did not change between the onset of peritonitis and recovery. We concluded that increased MMP-9 and TIMP-1 levels might be associated with peritoneal ECM remodeling during peritonitis.

Expression of Matrix Metalloproteinases 2 and 9 and Tissue Inhibitors of Metalloproteinase 1 and 2 in Inflammation-Induced Corneal Neovascularization

Purpose: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. Methods: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. Results: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. Conclusion: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.

Inhibition of matrix metalloproteinases by over-expression of tissue inhibitor of metalloproteinase-2 inhibits the growth of experimental hemangiomas.

Inhibitors of proteases prevent tumor-associated matrix degradation, affecting tumor growth, angiogenesis and metastasis. Our study was designed to investigate the effect of inhibition of matrix metalloproteinases (MMPs) on the growth of experimental hemangiomas, using the model of murine endothelioma eEnd.1 cells. In nude mice, these cells generate hemangiomas, consisting mostly of host-recruited endothelial cells, whose growth requires the activity of MMPs. In vitro, eEnd.1 cells produce factors that recruit endothelial cells and stimulate them to release MMPs. Over-expression of TIMP-2, following retrovirus-mediated gene transfer, decreased tumor growth in vivo. The infected clone CR1, which produces high levels of TIMP-2 (as assessed by Northern blot, ELISA and reverse zymography), formed slow-growing tumors that did not grow beyond 0.4 g, while clone 1H, which produces little TIMP-2, grew not dissimilarly to mock-infected cells and parental e.End.1 cells. Histologically, control tumors presented the features of cavernous hemangiomas, while CR1 tumors had a more solid pattern, showing foci of apoptotic cells. In vitro, TIMP-2 over-expression had no autocrine anti-proliferative effect on endothelioma cells but reduced their ability to recruit endothelial cells. CR1 cells lacked the capacity of mock-infected or parental eEnd.1 cells to stimulate endothelial cell motility and invasiveness. Antibodies against TIMP-2 restored the ability of CR1 to induce endothelial cell invasion. We conclude that, in this model, genetic increase of TIMP-2 inhibits tumor growth, apparently by affecting the recruitment and organization of host endothelial cells by the transformed cells.

Inhibition of matrix metalloproteinases by over-expression of tissue inhibitor of metalloproteinase-2 inhibits the growth of experimental hemangiomas

Inhibitors of proteases prevent tumor-associated matrix degradation, affecting tumor growth, angiogenesis and metastasis. Our study was designed to investigate the effect of inhibition of matrix metalloproteinases (MMPs) on the growth of experimental hemangiomas, using the model of murine endothelioma eEnd.1 cells. In nude mice, these cells generate hemangiomas, consisting mostly of host-recruited endothelial cells, whose growth requires the activity of MMPs. In vitro, eEnd.1 cells produce factors that recruit endothelial cells and stimulate them to release MMPs. Over-expression of TIMP-2, following retrovirus-mediated gene transfer, decreased tumor growth in vivo. The infected clone CR1, which produces high levels of TIMP-2 (as assessed by Northern blot, ELISA and reverse zymography), formed slow-growing tumors that did not grow beyond 0.4 g, while clone 1H, which produces little TIMP-2, grew not dissimilarly to mock-infected cells and parental e.End.1 cells. Histologically, control tumors presented the features of cavernous hemangiomas, while CR1 tumors had a more solid pattern, showing foci of apoptotic cells. In vitro, TIMP-2 over-expression had no autocrine anti-proliferative effect on endothelioma cells but reduced their ability to recruit endothelial cells. CR1 cells lacked the capacity of mock-infected or parental eEnd.1 cells to stimulate endothelial cell motility and invasiveness. Antibodies against TIMP-2 restored the ability of CR1 to induce endothelial cell invasion. We conclude that, in this model, genetic increase of TIMP-2 inhibits tumor growth, apparently by affecting the recruitment and organization of host endothelial cells by the transformed cells. © 2001 Wiley-Liss, Inc.

MMP-2- and MMP-9-Linked Gelatinolytic Activity in the Sputum from Patients with Asthma and Chronic Obstructive Pulmonary Disease

Background: The course of asthma and chronic obstructive pulmonary disease (COPD) is associated with bronchial morphological changes. Metalloproteinases are thought to play a role in these structural changes. Methods: We studied the gelatinolytic activity present in the induced sputum from 20 patients with asthma, 20 with COPD and 19 healthy controls. The assessment of gelatinolytic activity was performed by quantitative zymography, and gelatinolytic species were identified by Western blot analysis. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was detected by reverse zymography and ELISA. Results: From zymography, we found significantly higher gelatinolytic activity linked to pro-matrix metalloproteinase-9 (pro-MMP-9) in the sputum from asthmatics (p < 0.0001) and COPD patients (p < 0.0001) compared to the control group. Furthermore, the activated form of MMP-9 (85 kD) was found in the sputum from 60% of asthmatics and 85% of COPD patients, but was absent in that of control subjects (p < 0.0001). Importantly, although less frequently detectable than pro-MMP-9, pro- MMP-2 (72 kD) was found more frequently in asthmatics (50%) than in control subjects (5%) (p < 0.005). We also described two unusual gelatinolytic species of 45 and 120 kD and showed that they derived from MMP-9 according to their ability to bind gelatin and anti-MMP-9 antibody. Levels of TIMP-1 were higher in asthmatics (p < 0.05) and COPD patients (p < 0.05) than in controls. Conclusion: Asthmatics and COPD patients display an increased gelatinolytic activity linked to MMP-2 and MMP-9 and higher levels of TIMP-1 in their sputum.

Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines.

As stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and interleukin-8 (IL-8) are implicated in the homing and mobilization of human hematopoietic progenitors (HPC), we hypothesized that these chemokines mediate the migration of HPC across subendothelial basement membranes by regulating production of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs). Assays for migration across reconstituted basement membrane (Matrigel) and chemotaxis were carried out using CD34(+) cells derived from normal human bone marrow (BM) and mobilized peripheral blood (PB). Secretion of MMPs and TIMPs was evaluated by zymography and reverse zymography and gene expression by RT-PCR. We found that an SDF-1 gradient increases the chemotaxis of BM and PB CD34(+) cells across Matrigel (BM > PB), which is blocked by inhibitors of MMPs (o-phenanthroline, rhTIMP-1, rhTIMP-2, and anti-MMP-9 and anti-MMP-2 antibodies) but enhanced by tumor necrosis factor-alpha (TNF-alpha), a strong stimulator of MMPs. Preincubation of these cells with SDF-1 stimulated the secretion of MMP-2 and MMP-9 in BM and PB CD34(+) cells but of TIMP-1 and TIMP-2 only in PB CD34(+) cells. Preincubation with MIP-1alpha and IL-8 also stimulated the secretion of MMP-9 and MMP-2 (BM > PB), but with respect to TIMPs, the effect was reversed (PB > BM), resulting in trans-Matrigel migration of BM but not of PB CD34(+) cells. We therefore propose that MMPs and TIMPs are involved in 1) SDF-1-induced chemotaxis of human HPC across subendothelial basement membranes, and 2) MIP-1alpha- and IL-8-stimulated migration of HPC.

Evidence That the Serum Inhibitor of Hyaluronidase May Be a Member of the Inter-α-inhibitor Family

A study of the uncharacterized serum inhibitors of hyaluronidase, first described half a century ago, was undertaken. Activity was measured against bovine testicular hyaluronidase using a microtiter-based assay and reverse hyaluronan substrate gel zymography. The predominant inhibitory activity was magnesium-dependent and could be eliminated by protease or chondroitinase digestion and by heat treatment. Kinetics of inhibition were similar against hyaluronidases from testis and snake and bee venoms. The inhibitor had no effect on Streptomyces hyaluronidase, indicating that inhibition was not through protection of the hyaluronan substrate. Inhibition levels in serum were increased in mice following carbon tetrachloride or interleukin-1 injection, inducers of the acute-phase response. Reverse zymography identified a predominant band of 120-kDa relative molecular size, with two bands of greater and one of smaller size. The predominant protein was tentatively identified as a member of the inter-alpha-inhibitor family. Inhibition was also observed using either purified inter-alpha-inhibitor or an inter-alpha-inhibitor-related 120-kDa complex. Inter-alpha-inhibitor, found in the hyaluronan-rich cumulus mass surrounding mammalian ova and the coat of fibroblasts and mesothelial cells, may function to stabilize such matrices by protecting against hyaluronidase degradation. Turnover of circulating hyaluronan is extraordinarily rapid, with a half-life of 2-5 min. Prompt increases in levels of serum hyaluronan occur in patients with shock, septicemia, or massive burns, increases that can be attributed, in part, to suppression of degradation by these acute-phase reactants, the inhibitors of hyaluronidase.

Temporal changes in collagen composition and metabolism during rodent palatogenesis

Cleft lip and palate is a common craniofacial malformation in man. The aetiology is multifactorial and not known. Since collagen is a major structural component of the developing palate, we studied its composition and metabolism during palate shelf formation and elevation in the rat. Palatal shelves were harvested at embryonic days (E) 15, 16 and 17 as well as post-partum. Palatal collagen increased threefold from E15 to E17 and tenfold from E17 to 5-day-old pups. Palatal calcification was seen in the main, post-partum. Collagen cross-linking, which may be important in shelf elevation and union, varied. The concentration of hydroxylysyl-pyridinolone cross-links was greatest prior to shelf elevation, declining thereafter. Similarly, the highest concentration of dihydroxylysinononorleucine was seen at E16 and this supports the concept of a compliant mesenchymal shelf responding to an intrinsic elevating force. We then determined if enzymes responsible for matrix degradation, matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMPs) altered over the same time periods. MMP-2, and TIMP-1 and TIMP-2 were identified by gelatin zymography and reverse zymography, respectively. MMP-3 activity was determined with a fluorogenic substrate assay. TIMP-1, TIMP-2 and MMP-3 levels remained constant from E15 to E17. The MMP-2 levels showed a significant elevation from E15 to E16 and E16 to E17. This suggests the regulation of extracellular matrix is likely to be of importance in palate morphogenesis.