A gelatin in situ-overlay technique localizes brain matrix metalloproteinase activity in experimental focal cerebral ischemia.

To determine the activity of matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, which play an important role in ischemic stroke and intracerebral hemorrhage, we adapted a simple and rapid method for localizing gelatinase activity to a gelatin film in situ-overlay technique previously used in cancer research. Ten micrometer cryosections of rat brain from controls and animals subjected to 3 h of ischemia and 48 h of reperfusion (suture model for transient cerebral ischemia) were used. After thawing, a gelatin film with a polyester base was put on the slide, incubated for 24 h at 37 degrees C, stained with Ponceau S, and then discolored in bi-distilled water. Non-staining areas on the film corresponded to lysis zones, caused by activated MMPs. This was proven by MMP incubation at various concentrations on the plain gelatin film and pretreatment with EDTA (an MMP inhibitor), which prevents lysis zones in normal and ischemic brains. As confirmatory tests, SDS-PAGE zymography was used to define MMP activity, and also MMP-2 immunohistochemistry to detect the possibly cellular origin of MMPs. Normal rat brain exhibited a low background activity, which was visible as a light halo-like lysis zone over and around the brain. Areas in normal brain with medium MMP activity were within the white matter (corpus callosum, anterior commissure, and cerebellum). Ischemic brain exhibited high activity lysis zones within the infarcted area (detected by microtubuli associated protein-2 staining). These zones consisted of microscopically small lysis holes with a diameter of about 10-20 microm. Immunohistochemistry showed that especially microvessels expressed MMP antigen. SDS-PAGE zymography differentiated between a high level of activated MMPs in the ischemic area and a low level in the non-ischemic basal ganglia. The gelatin film in situ-overlay technique is able to localize MMP activity in ischemic rat brain tissue on a microscopic level.

Localization of gelatinolytic activity can be detected in breast cancer tissues by film in situ zymography.

BACKGROUND: Extracellular matrix-degrading proteinases secreted by malignant tumor cells have been thought to play an essential role in the processes of invasion and metastasis. However, existence and localization of gelatinase activity in breast cancer tissues have not been clarified. METHODS: We developed a novel film for highly reproducible detection and the localization of gelatinolytic activity. This film has a gelatin layer with a constant thickness 7 microm, and adequate crosslinking to control the speed of degradation by proteases. Cryosections of several breast cancer tissues were put on this gelatin film and incubated for 16 hrs at 37 degrees C. After staining with ponceau 3R dye, the digested area was evaluated under light microscopy. RESULTS: Digestion of gelatin was detected in more than 90%of breast cancer specimens, although it varied in degree and area for each case. In most cases, the gelatinolytic activity was located within cancer nests, and was not detected in stromal cells surrounding cancer cells. The gelatinolytic activity was inhibited by 1,10-phenanthroline, an inhibitor of matrix metalloproteinases (MMPs). CONCLUSIONS: In this study, the localization of net MMP activity was confirmed in breast cancer nest using film in situ zymography. Detailed analysis on the relationship between the strength or distribution of MMP activity and malignancy are anticipated in the future.

Clarification of the active gelatinolytic sites in human ovarian neoplasms using in situ zymography.

Tissues from 26 human ovarian common epithelial tumors were examined to determine where and how gelatinolytic activity was present, in relation to tumor-stromal interaction and histologic types. For this purpose, we used in situ zymography, a newly developed technique using gelatin-coated film. Gelatinolytic activity was evident in ovarian carcinomas and in borderline tumors. Benign tumors had no or only weak activity. Four tissue localization patterns of gelatinolysis were identified: pattern A, tumor cytoplasm; pattern B, tumor-stromal junction; pattern C, stroma; and pattern D, cystic fluid. Mucinous cystadenocarcinomas showed A and/or D patterns. One mucinous and one serous adenocarcinoma and one mucinous borderline tumor had a B pattern. Most serous and all clear cell adenocarcinomas showed strong gelatinolysis of C pattern, especially in the desmoplastic stroma, an area where the tumor cells were dispersed. Immunohistochemically in 12 adenocarcinomas and 3 borderline tumors, the tumor cytoplasm was positive for matrix metalloproteinases (MMP-2) (5 cases), MMP-7 (9 cases), and MMP-9 (6 cases). Stromal components were positive for MMP-2 in 5 cases and for MMP-9 in 3 cases, but they were not positive for MMP-7. MMP antigens were mostly distributed in an almost identical pattern consistent with that seen with in situ zymography. In situ zymography clarified the cellular localization of active gelatinolysis in human ovarian neoplasms, a finding which supports the view that interaction between tumor and stroma is critical for tumor growth. This newly developed method contributes to a better understanding of biologic features of ovarian malignancies.

Inhibition of gelatinolytic activity in tumor tissues by synthetic matrix metalloproteinase inhibitor: application of film in situ zymography.

Inhibition of gelatinolytic activity in implanted tumor tissues by oral administration of N-biphenyl sulfonyl-phenylalanine hydroxamic acid (BPHA), a selective matrix metalloproteinase (MMP) inhibitor, was demonstrated by means of film in situ zymography (FIZ). Active-MMP-2 but not pro-MMP-2 showed gelatinolytic activity in FIZ, whereas both forms of MMP-2 were found to be active in conventional zymography. A mixture of either tissue inhibitors of metalloproteinase-2 or BPHA with active-MMP-2 resulted in inhibition of gelatinolytic activity in FIZ but not in zymography. Thus, FIZ, but not zymography, could detect net MMP activity in tumor tissues. When a specimen from Ma44 human lung cancer xenograft was subjected to FIZ, gelatinolytic activity was markedly detected with precise localization in the tumor tissues. The gelatinolytic activity detected in Ma44 tumor tissues was found to be mainly derived from MMPs because the gelatin-degrading activity was inhibited by pretreatment of the tumor specimen with MMP inhibitors. Oral administration of BPHA but not (-)BPHA, an enantiomer of BPHA lacking MMP inhibitory activity, successfully inhibited the MMP activity localized in Ma44 tumor tissues in both a dose-dependent and time-dependent manner. The data presented in this report showed for the first time that oral administration of synthetic MMP inhibitor could inhibit the net activity of MMP activity in tumor tissues, suggesting the usefulness of the FIZ technique for determining the effective dose of MMP inhibitor in clinical studies.

Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in rheumatoid synovium.

In vitro, membrane-type matrix metalloproteinases (MT-MMP) are known to activate the zymogen of MMP-2 (proMMP-2, progelatinase A), which is one of the key MMP in joint destruction in rheumatoid arthritis. In the present study, we examined the production and activation of proMMP-2, and the expression of MT1-MMP, MT2-MMP, and MT3-MMP, their correlation with proMMP-2 activation, and their localization in rheumatoid synovial tissue. Using sandwich enzyme immunoassay and gelatin zymography techniques, proMMP-2 production levels and activation ratios were found to be significantly higher in rheumatoid synovium compared with normal synovium (p < 0.01). Quantitative RT-PCR analyses demonstrated that MT1-MMP and MT3-MMP were expressed in all rheumatoid synovial tissue (30 of 30 cases), but that the mean expression level of MT1-MMP was approximately 11-fold higher than MT3-MMP. Significant correlation was found between the mRNA expression level of MT1-MMP and the activation ratio of proMMP-2 (p < 0.01). In situ hybridization indicated that the hyperplastic lining cells of rheumatoid synovium expressed MT1-MMP. Immunohistochemistry demonstrated that MT1-MMP was co-localized with MMP-2 and with a tissue inhibitor of metalloproteinase-2, and was mainly located in the rheumatoid synovial lining cells. In situ zymography of rheumatoid synovium showed gelatinolytic activity, predominantly in the lining cell layer. This activity was blocked when incubated with BB94, a specific MMP inhibitor. These results demonstrate that MT1-MMP plays an important role in the activation of proMMP-2 in the rheumatoid synovial lining cell layer, and suggest that its activity may be involved in the cartilage destruction of rheumatoid arthritis.

Increased expression of matrix metalloproteinase 7 and 9 and membrane type 1-matrix metalloproteinase in esophageal squamous cell carcinomas.

BACKGROUND: The 5-year survival rate of patients with submucosal (sm) esophageal carcinoma continues to be much worse than that with intramucosal (m) lesions. The reasons are unclear, but matrix metalloproteinases (MMPs) and membrane type matrix metalloproteinases (MT-MMPs) are known to be involved in degradation of extracellular matrix macromolecules associated with tumor cell invasion. Expression of these enzymes may have some relation to the difference in survival between patients with sm and m esophageal carcinomas. METHODS: Surgical specimens from 148 primary esophageal squamous cell carcinomas were examined for expression of MMP-1, 2, 3, 7, and 9; tissue inhibitor of MMP (TIMP)-1 and 2; MT1-MMP; and MT2-MMP by immunohistochemical staining. The results were compared with tumor progression and other clinicopathologic parameters. Localization of gelatinase activity was also confirmed in carcinoma cells by in situ zymography. RESULTS: Expression of MMP-7 and 9, expression of MT1-MMP, and gelatinase activity were elevated in the carcinomas, correlating with depth of tumor invasion. Percentages of positive sm cases were 44.0%, 51.9%, and 52.0%, respectively, significantly higher than for the m cases. Percentages of venous invasion positive cases were 61.9%, 65.1%, and 65.1%, respectively. CONCLUSIONS: Expression of MMP-7 and 9 and MT1-MMP is closely associated with invasion depth and venous invasion in esophageal squamous cell carcinomas.

FP-21399 blocks HIV envelope protein-mediated membrane fusion and concentrates in lymph nodes.

The identification of fusin and other chemokine receptors as coreceptors for HIV-1 has renewed the interest in agents that may prevent viral entry. Polyanionic compounds such as dextran sulfate, curdian sulfate, and suramin act on the V3 loop of the viral envelope and may prevent its interaction with fusin. These agents show activity against a wide range of HIV-1 strains, but have undesirable circulating half-life, bioavailability, and toxicity. We have developed a small molecule inhibitor of HIV-1 that has several advantages over these other agents. FP-21399 is a novel compound of the bis(disulfonaphthalene) dimethoxybenzene class that blocks entry of HIV into CD4+ cells and blocks fusion of infected and noninfected CD4+ cells. This compound only weakly inhibits binding of CD4 and gp120, at concentrations much greater than are required to block viral entry. Furthermore, FP-21399 can block the interaction between gp120 and antibodies directed against the V3 loop, but does not block binding of antibodies directed against the V4 loop. Animal studies demonstrate that FP-21399 is concentrated in lymph nodes, making it a promising compound for anti-HIV therapy. In SCID mice reconstituted with human immune cells, maintenance of HIV-1 infection was blocked by a 5-day treatment with low doses of FP-21399, suggesting that lymph node accumulation may contribute to antiviral activity. Finally, attempts to generate drug-resistant virus in cell culture resulted in only weakly resistant variants with IC90 values that are much lower than concentrations of FP-21399 found in lymph nodes.