Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.     

Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta

BACKGROUND: Abdominal aortic aneurysms (AAAs) are characterized by degradation of collagen and elastin resulting from increases in matrix metalloproteinase (MMP) activity. Previous authors have identified isolated increases in expression of specific MMPs in AAAs, but none have compared relative levels of expression of particular MMPs to one another or to those of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). This study proposes to quantify relative mRNA levels for interstitial collagenase (MMP-1), 72 kd type IV collagenase (MMP-2), 92 kd type IV collagenase (MMP-9), TIMP-1, and TIMP-2 in normal aorta (NA) and AAA to provide insight as to the relative importance of each in aneurysm formation. METHODS: Competitive polymerase chain reactions (PCRs) with gene-specific external standards and cDNA derived from AAAs (n = 8; mean age, 67.4 years) and NA (n = 5; mean age, 40.6 years) were used to quantify mRNA levels. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, determined by means of competitive PCR, and compared by means of Mann-Whitney statistics. RESULTS: Significant increases in MMP mRNA expression in AAA over NA were observed for MMP-1 (3.64 versus 0.3, p = 0.007), MMP-9 (78.03 versus 3.35, p = 0.003), TIMP-1 (835.32 versus 477.2, p = 0.027), and TIMP-2 (18.09 versus 4.14, p = 0.003). The ratio of MMP to TIMP mRNA levels was higher in AAA than NA (0.135 versus 0.045, p = 0.018). CONCLUSIONS: Increases in expression of MMP-1, MMP-9, and MMP/TIMP ratios may result in increased proteolysis and matrix degradation, which characterize AAAs. MMP-9 appears to be the predominant metalloproteinase expressed in AAA, because its mRNA levels were more than 20 times and 2 times higher than those of MMP-1 and MMP-2, respectively. TIMP-1 mRNA levels were in molar excess to those of any of the metalloproteinases studied.     

Comparison of elastolytic activity between experimental aneurysm and experimental diabetes mellitus

In order to clarify the degradation of elastin under abnormal conditions, we examined the aortic elastolytic activity in rat experimental diabetes mellitus induced by treatment with streptozotocin and in rat experimental aneurysm induced by treatment with an inhibitor of lysyloxidase (beta-aminopropionitrile: BAPN). Measurement of the aortic elastolytic activity used 14C-labeled elastin as the substrate, and the determined value was compared with the aortic lysosomal enzyme (acid phosphatase) activity. In the case of experimental diabetes, the aortic elastolytic activity was not changed, but the aortic acid phosphatase activity was significantly increased compared with the control. In the case of the experimental aneurysm, the aortic elastolytic activity measured after 2 and 3 weeks was increased compared with each control. There was a negative correlation (r=-0.435, n=36) between the elastolytic activity and the cross-linking (desmosine) content in the aorta. The ratio of elastolytic activity to desmosine content was significantly increased compared with the control. Therefore, the degradation of aortic elastin in the experimental aneurysm was caused by elastase, not by lysosomal enzymes. We concluded that an elastase-like enzyme mainly contributed to the degradation of elastin in the experimental aneurysm since the inhibitory pattern of the elastolytic activity in the experimental aneurysm was similar to that of pancreatic elastase.     

The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions

Matrix metalloproteinases (MMPs) are a family of enzymes which, in concert, are capable of degrading collagen. We investigated whether human MMPs could participate in the degradation of dentin organic matrix after demineralization. We performed Western blot analyses using MMP-specific antibodies to identify MMPs in human dental caries lesions. Enzymography and functional activity assays, with 125I-labeled gelatin as substrate or quantitating the degradation of type I collagen, were used to determine the activity of purified and salivary gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8) enzymes with and without acid-activation in pHs relevant to caries. Respective analyses were done with caries-related bacteria. We performed electron microscope analyses to assess the degradative activity of sterilized salivary host MMPs on demineralized human dentin. Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The latent purified forms of these enzymes were activated at low pH (4.5), followed by neutralization, mimicking the conditions during caries progression. Incubation of human saliva at low pH followed by neutralization resulted in a four-fold increase in the gelatinolytic activity. No gelatinolytic or collagenolytic activity was observed in bacterial samples. The activated enzymes in saliva degraded demineralized dentin organic matrix in vitro. These results demonstrate the pH-dependent activation mechanism of MMPs, which may have a distinct role in different physiological and pathological conditions. They further demonstrate that host MMPs, activated by bacterial acids, have a crucial role in the destruction of dentin by caries.     

A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sj?gren's syndrome contain increased latent and active gelatinase-B levels

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys/IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly/IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sj?gren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 +/- 1.9 x 10(4) cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 +/- 2.5 U/mg, controls 1.0 +/- 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 +/- 9.8 U/mg, controls 16.5 +/- 2.6 U/mg (p = 0.01). This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.     

Identification and partial characterization of three calcium- and zinc-independent gelatinases constitutively present in human circulation

Three constitutive gelatinases in human plasma were identified and characterized relative to known matrix metalloproteinase (MMP) gelatinases: MMP-2 (fibroblast 72-kDa) and MMP-9 (neutrophil 92-, 130-, and 225-kDa). Substrate gel electrophoresis (gelatin zymography) revealed an apparent Mw of 78-, 82-, and 89-kDa for these gelatinases. Densitometry revealed that MMP-9 and MMP-2 were highly calcium sensitive requiring 50-150 microM and 500 microM calcium for half-maximal activity, respectively. Of the new gelatinases, only the 89-kDa form demonstrated slight calcium activation. The three gelatinases were unaffected by known MMP inhibitors: EDTA (5 mM), 1,10-phenanthroline (2 mM), and pepstatin (18 microM). Serine and thiol protease inhibitors (leupeptin, aprotinin, PMSF, TLCK, TPCK, antichymostatin, antipain) were also ineffective. Solution-phase IEF revealed that the 78- and 82-kDa forms focused at neutral pI 6.72-7.95 whereas the 89-kDa focused at an acidic pI 4.89-5.18 (similar to neutrophil and fibroblast forms). The data indicate that these gelatinases are not MMPs or partially activated MMPs. Their role in normal and pathological conditions is not known.     

Pharmacologic suppression of experimental abdominal aortic aneurysms: acomparison of doxycycline and four chemically modified tetracyclines

BACKGROUND: Matrix metalloproteinases (MMPs) likely contribute to the degradation of medial elastin in abdominal aortic aneurysms (AAAs), and tetracycline antibiotics exhibit MMP-inhibiting properties. The purpose of this study was to compare the effects of doxycycline and several non-antibiotic chemically modified tetracyclines (CMTs) in a rat model of elastase-induced AAA. METHODS: Fifty-two male Wistar rats underwent intraluminal perfusion of the abdominal aorta with porcine pancreatic elastase. The rats then were treated for 7 days with subcutaneous injections of saline solution, different doses of doxycycline, or 1 of 4 different CMTs. The aortic diameters were measured with microcalipers, and the fixed tissues were examined by means of light microscopy. Gelatin zymography was used to assess the MMP activity in the aortic tissue extracts. RESULTS: The mean aortic diameter in the control group increased by 126% +/- 14% on day 7 (from 1.57 +/- 0.04 mm to 3.54 +/- 0.27 mm; P <.05), and 5 of 6 animals (83%) had AAAs. Doxycycline appeared to inhibit aortic dilatation in a dose-dependent manner, and AAAs did not develop in any animals. Half-maximal effects were observed at a dose of approximately 6 mg/kg/day, and maximal effects were noted at greater than 30 mg/kg/day. No AAAs were observed in the animals that were treated with CMTs at 15 mg/kg/day. Each of the following CMTs exhibited an efficacy that was similar to that of doxycycline (percent inhibition of aortic dilatation vs control; all P <.05): CMT-3 (47.6%), CMT-4 (38.9%), CMT-7 (47.6%), CMT-8 (54.0%), and doxycycline (51.6%). Tissues from saline solution-treated controls exhibited a transmural inflammatory response and marked destruction of the medial elastic lamellae. Tetracycline derivatives limited the disruption of medial elastin without appearing to alter either the inflammatory response or the rat aortic wall production of metallogelatinases. CONCLUSION: Tetracycline derivatives suppress the development of AAAs after elastase-induced aortic injury in the rat. The aneurysm-suppressing effects of doxycycline appear to be dose-dependent and distinct from its antibiotic activities, and they coincide with the structural preservation of medial elastin fibers. Further studies are needed to explore the potential of MMP-inhibiting tetracyclines as a novel pharmacologic strategy for the suppression of aortic aneurysms.     

Identification of matrix metalloproteinases (MMPs) in synovial fluid from patients with temporomandibular disorder

Proteolytic enzymes with gelatinolytic activity in the synovial fluid (SF) of temporomandibular joint (TMJ) arthropathies were assayed by gelatin-impregnated gel enzymography. SF samples were collected from 10 TMJs in patients with closed lock (CL) condition and 5 TMJs from asymptomatic healthy volunteers. Two proteinases with gelatinolytic activities at 92 kDa and 72 kDa were detected in both the normal and the diseased TMJs. Also detected were weak bands at molecular weights of 83 kDa and 66 kDa. All of these proteinase activities were inhibited by EDTA and tissue inhibitor of metalloproteinases (TIMP), required Ca2+ for activation, and were detected with gelatin but not casein as substrate, suggesting that these enzymes were matrix metalloproteinases (MMPs). The 72 kDa and 66 kDa bands further reacted with anti-MMP-2 antibody by Western blot analysis, and the proteinases in the TMJ-SF could cleave type IV collagen in vitro without any activation. These four activities identified by enzymography were, therefore, identified as 92 kDa-gelatinase (proMMP-9), 83 kDa-gelatinase (active MMP-9), 72 kDa-gelatinase (proMMP-2) and 66 kDa-gelatinase (active MMP-2). Densitometric analyses of these bands revealed higher levels of the active form of MMP-9 in the CL patients compared to controls. These findings suggest that MMP-2 and -9 could be dominant proteinases in the TMJ-SF and possibly reflect TMJ pathology.     

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans

Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.     

In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta

Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n = 8), AOD (n = 8), and NL (n = 7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to alpha-tubulin. Mean values +/- SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n = 5), AOD (n = 2), and NL (n = 2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855 +/- 0.180) than NL (0.046 +/- 0.23) (P < .02), but differences between AOD (0.406 +/- 0.196) and AAA or AOD and NL were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased amounts of collagenase and gelatinase in porcine myocardium following ischemia and reperfusion

We studied the presence of collagen degrading enzymes (matrix metalloproteinases, MMPs) in porcine myocardium following ischemia and late reperfusion. In nine pigs, left anterior descending coronary artery was occluded for 6 h followed by reperfusion for 3 h. Six pigs without coronary occlusion served as controls. After the reperfusion period, transmural biopsies from the anterior (ischemic zone) and posterior wall (non-ischemic myocardium) in the left ventricle were obtained and extracted. Heparin-Sepharose isolated components in extracts were analysed for collagenase (triple-helical collagen degradation) and gelatinase activity (zymography). Immunohistochemistry using anti-human (MMP-1, MMP-2, MMP-9, and fibronectin) antibodies was performed on additional biopsies. Collagenase (MMP-1) and gelatinases (MMP-2, MMP-9) could be demonstrated in the extracts of non-ischemic myocardium from ischemic/reperfused as well as control pigs and MMP-1 and MMP-9 activity was found to be increased in ischemic/reperfused myocardium compared with non-ischemic myocardium. In ischemic/reperfused myocardium from live pigs investigated, myocyte necrosis could be confirmed by fibronectin immunoreaction in myocytes and MMP-1 and MMP-9 immunoreactions were increased. MMP-9 was present in cells likely to be infiltrating leukocytes in a patchy distribution throughout the ischemic myocardium. Quite coincident with MMP-9 positive cells, MMP-1 immunoreaction appeared in necrotic myocytes, in addition to reactions observed in vessel walls, endo- and epicardium, and extracellular matrix in non-ischemic myocardium. Thus, the results showed increased amounts of collagenase (MMP-1) and gelatinase (MMP-9) in ischemic/ reperfused myocardium, indicating the appearance of increased amounts of collagen degrading enzymes very early following ischemia and late reperfusion.     

Relationship between matrix metalloproteinase expression and tumor angiogenesis in human breast carcinoma

Intratumoral proteases are known to be involved in not only tumor cell invasion but also a variety of stromal reactions including neovascularization. In this study, we have examined the expression of matrix metalloproteinases (MMPs) by gelatin gel zymography and compared its expression with angiogenesis activities including the expression of several endothelial growth regulators and intratumoral microvessel density (MVD) in human breast cancer tissues. There was a significant correlation between activated MMP-2 expression and vascular endothelial growth factor (VEGF) expression (p=0.045). In addition, the expression of activated MMP-9 expression was significantly correlated with thymidine phosphorylase (TP) expression (p=0.0044). Pro MMP-9 expression tended to correlated with the increment of MVD (p=0.063). MMP-2 and MMP-9 expressions were frequently co-upregulated with endothelial growth regulators in human breast cancer tissues, which underlines the cooperative function of MMPs in neovascularization.     

Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.     

Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis

A critical outcome of periodontal disease is degradation of the collagenous periodontal ligament that connects teeth to bone in the dental arch. Periodontal diseases occur in response to bacterial colonization of the teeth, but their molecular pathogenesis is still speculative. One family of enzymes, known as the matrix metalloproteinases (MMPs), has been implicated in the degradation of the periodontal ligament. MMPs, which are also suspected to play a role in many other physiologic and pathologic remodeling processes, can be secreted by epithelial cells surrounding the teeth and are found in relative abundance in tissues and fluids near periodontally diseased sites. Since most MMPs are secreted as inactive zymogens which may be activated by limited proteolysis, it has been suggested that proteinases expressed by the infecting periodontal pathogens might activate latent host MMPs to initiate or accelerate degradation of the collegenous periodontal ligament. The aim of this work was to examine interactions between purified host MMPs and bacterial proteinase. In this article, we demonstrate that a proteinase isolated from the periodontopathogen Porphyromonas gingivalis can activate MMP-1, MMP-3, and MMP-9 and can catalyze the superactivation of MMP-1 by MMP-3. Activation of these MMPs is demonstrated to result from initial hydrolysis within their propeptide. Also, for MMP-1 and MMP-9, the P. gingivalis proteinase cleaves the MMP propeptide following a lysine residue at a previously unreported site which, for both MMPs, is one residue NH2-terminal to the known autocatalytic cleavage site. These data describe a mode of virulence for the periodontopathogen Porphyromonas gingivalis that involves activation of host-degradative enzymes.     

Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases

In interstitial lung diseases, deposition of extracellular matrix (ECM) in alveoli and degradation of ECM lead to pulmonary structural remodeling. The changes in ECM and the localization of matrix metalloproteinases (MMPs) and a tissue inhibitor of metalloproteinases (TIMP) in the lung tissues of patients with bronchiolitis obliterans organizing pneumonia (BOOP) and idiopathic pulmonary fibrosis (IPF) were investigated. Immunohistochemical analysis for the detection of fibronectin, collagen-I, -III, and -IV, smooth muscle actin, MMP-1 (interstitial collagenase), -2 (gelatinase A), and -9 (gelatinase B), and TIMP-2, and in situ hybridization for the detection of MMP-9 mRNA were performed. Western blotting of lung tissue homogenates was performed for MMP-2 and MMP-9. The gelatinolytic activities of the homogenates were also determined using gelatin zymography. Fibronectin and collagen-I, -III, and -IV were detected in the intra-alveolar fibrosis in addition to the interstitium of these diseases. MMP-1, MMP-2, MMP-9, and TIMP-2 were detected in the regenerated epithelial cells covering intra-alveolar fibrosis. Myofibroblasts in intra-alveolar fibrosis in BOOP showed predominant reaction for MMPs, and they ultrastructurally appeared to be phagocytosing collagen fibrils, and those of IPF showed a predominant reaction for TIMP-2. New vascularization in intra-alveolar fibrosis was exclusively observed in cases of BOOP, and the endothelial cells were positive for MMP-2. Western blotting showed the existence of a latent form of MMP-9 and latent and active forms of MMP-2, and gelatin zymography revealed that the ratio of active/latent forms of MMP-2 in BOOP is significantly larger than that in the control lungs. Predominant MMPs in BOOP may constitute the mechanism of reversibility of fibrotic changes in this disease. TIMP-2 in myofibroblasts in IPF may contribute to the stable ECM deposition and the irreversible pulmonary structural remodeling.     

Increased production of gelatinase B (matrix metalloproteinase-9) and interleukin-6 by activated rat microglia in culture

Activated macrophages produce several matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM)-degrading enzymes, during wound healing and in other inflammatory states. In response to brain injury, brain microglia become "activated," in a way similar to peripheral tissue macrophages, a process which includes differentiation and probably invasion and proliferation. Little is known about the ECM-degrading MMPs that are secreted by microglia upon activation. Thus, it was of interest to determine whether activated microglia secrete MMPs. Conditioned media samples obtained from cultured microglia that were stimulated with various activating agents were subjected to gelatin-substrate zymography. Microglia constitutively express low levels of a 94-kDa gelatinase (GLase) activity. Treatment with LPS, zymosan, and fixed Staphylococcus aureus for 24 hr stimulated the activity of the 94-kDa GLase, 4-20-fold, in a dose-dependent manner. Addition of INF gamma inhibited the LPS-stimulated activity of MMP-9. LPS, zymosan, and fixed Staphylococcus aureus also stimulated the secretion of IL-6 from microglia in a dose-dependent manner. The 94-kDa GLase activity was Ca++ dependent, it was inhibited by 1,10-phenanthroline, and it was activated by organomercurial compounds. When immunoblots were performed using specific antisera against the 94-kDa gelatinase B (MMP-9) with untreated and LPS-stimulated conditioned medium samples, a 94-kDa immunopositive band was observed. Thus, it appears that the 94-kDa GLase is gelatinase B (MMP-9). These results indicate that activators of peripheral macrophages are potent secretagogues for the MMPs in cultured microglia. The ability of activated microglia to secrete MMPs suggests that these enzymes may play an important function in the brain parenchyma during inflammatory states.     

Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells

PURPOSE: To identify matrix metalloproteinases (MMPs) released by ciliary smooth muscle cells in vitro and to determine whether MMP release is altered by exposure to prostaglandins (PGs). METHODS: Human ciliary smooth muscle cells were grown to confluence in monolayer cultures and treated with PGF2 alpha, 11-deoxy-PGE1, or PhXA85 (the nonesterified analogue of PhXA41) for 12 to 72 hours. The activity of MMP in the medium was assayed using gelatin and casein zymography. Identification of the specific MMP associated with each band was made by Western blot analysis. Band intensity, which reflects activity, was measured with a scanning laser densitometer. RESULTS: Three major bands appeared in the gelatin zymographs at positions corresponding to molecular weights of 62 kDa, 68 kDa, and 97 kDa. A single band at 50 kDa predominated in the casein zymograms. Substitution of EDTA for calcium and zinc in the development solution eliminated the appearance of these bands, indicating that they reflect MMP activity. Immunoblotting, using MMP-specific antibodies, confirmed that the three bands in the gelatin zymographs were MMP-1, MMP-2, and MMP-9, respectively; the single band in the casein zymographs was MMP-3. Treatment with 200 nM PGF2 alpha, 11-deoxy-PGE1, or PhXA85 for 72 hours increased the combined density scores for MMP-1 and MMP-2 by 37%, 64%, and 27%; the density scores for MMP-9 by 268%, 253%, and 125%; and the density scores for MMP-3 by 35%, 71%, and 22%, respectively. CONCLUSIONS: These results indicate that ciliary smooth muscle cells can secrete MMP-1, MMP-2, MMP-3, and MMP-9. In addition, exposure to PGF2 alpha, 11-deoxy-PGE1, or PhXA85 increases production of all four MMPs. These observations support the hypothesis that increased MMP production by ciliary muscle cells has a role in increasing uveoscleral outflow facility after topical PG administration.