Matrix metalloproteinase (MMP-2 and MMP-9) and steroid receptor expressions in feline mammary tumors

We evaluated the presence of estrogen (ER) and progesterone (PR) receptors, and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) enzymes in 18 feline mammary tubulopapillary carcinomas. Immunohistochemistry was performed to localize ER, PR, MMP-2 and MMP-9 in situ. Western blotting and zymographic analyses also were performed to investigate the presence and activities of MMP-2 and MMP-9 enzymes in fresh tissue homogenates. ER immune expression was detected in five samples (27.7%) and PR was positive in sixteen (88.8%) samples. Diffuse cytoplasmic staining of MMP-2 and MMP-9 in neoplastic mammary epithelial cells, stromal fibroblasts and inflammatory cell was evident. MMP-2 and MMP-9 staining was observed also in metastasizing neoplastic cells within lymphatic vessels. MMP-2 and MMP-9 enzymes and their activities in fresh tumor homogenates were demonstrated by zymography. Comparison of MMP-9 gelatinolytic bands from tumor samples and controls revealed a statistically significant difference. We demonstrated elevated MMP-9 and MMP-2 levels in tumor samples by Western blotting; analysis of protein bands revealed 1.9-to-3 fold increase in MMP-9 in tumor samples and the difference was statistically significant. Our results suggest that the expression of MMP-9 can be an important indicator for tumor progression and the possible metastatic nature of feline tubulopapillary carcinomas.     

Matrix metalloproteinase (MMP)-2 and MMP-9 polymorphisms and haplotypes as disease biomarkers

Chaudhary and colleagues observed associations of matrix metalloproteinase (MMP)-2 (-1306C/T) and MMP-9 (-1562C/T) promoter polymorphisms with head and neck squamous cell carcinoma (HNSCC), but not with oral submucous fibrosis (OSMF) in an Indian population. We suggest that they could carry out a haplotype analysis with their data on MMP-2 genotypes (-1306C/T and -168G/T) and that they consider genotyping the microsatellite -90 (CA)_14–24 in the MMP-9 promoter region in order to perform haplotype analysis in combination with their data on MMP-9 (-1562C/T) polymorphism. These suggestions could provide additional information with clinical relevance to cancer susceptibility.     

Immunocharacterization of matrix metalloproteinase-2 and matrix metalloproteinase-9 in canine transmissible venereal tumors

Matrix metalloproteases (MMPs) are endogenous proteases that are responsible for degradation of extracellular matrix (ECM) proteins and cell surface antigens. The breakdown of ECM participates in the local invasion and distant metastases of malignant tumors. Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round cell neoplasm of dogs that affects mainly the external genitalia of both sexes. CTVT generally is a locally invasive tumor, but distant metastases also are common in puppies and immunocompromised dogs. We investigated the immune expressions and activities of MMP-2 and MMP-9 in CTVT. The presence of these enzymes in tumor cells and tissue homogenates was demonstrated by immunohistochemistry and western blotting. We used gelatin substrate zymography to evaluate the activities of MMP-2 and MMP-9 enzymes in tumor homogenates. We found that tumor cells expressed both MMP-2 and MMP-9. Electrophoretic bands corresponding to MMP-9 and MMP-2 were identified in immunoblots and clear bands that corresponded to the active forms of MMP-2 and MMP-9 also were detected in gelatin zymograms. Our study is the first detailed documentation of MMPs in CTVT.     

Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis

Pulmonary fibrosis has an aggressive course and is usually fatal an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. Matrix metalloproteinases (MMPs) are a major group of proteinases known to regulate the ECM remodeling and so they are hypothesized to be important in the process of lung fibrosis. These led to the concept that modulation of airway remodeling including excessive proteolytic damage of the tissue may be of interest for future treatment. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could argue in favor of antiprotease treatments. Moreover, these observations emphasize that effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of expensive lung destruction and fibrosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

ER和PRmRNAs在内异症子宫内膜表达的变化

目的 :探讨雌激素受体 (ER)和孕激素受体 (PR)在子宫内膜异位症 (内异症 )子宫内膜的表达。方法 :利用大鼠内异症动物模型 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测子宫内膜ER和PRmRNAs的表达情况。结果 :内异症模型组大鼠异位内膜ER、PRmRNAs的表达低于在位内膜和对照组正常子宫内膜 ,与后两者比较差异有显著性意义 (P <0 .0 1) ;而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著性意义 (P >0 .0 5 )。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜和正常子宫内膜ER/PRmRNA比值 (P <0 .0 1)。结论 :内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。     

Epidermal growth factor receptor (EGF-R) localization in the apical membrane of the enterocytes of rat duodenum

The maintenance of gastrointestinal epithelium integrity requires a fine balance between proliferation and differentiation as well as protection against gastric acid secretion. Some growth factors, such as epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), bind to epidermal growth factor receptor (EGF-R) to exert these functions. The exact location of EGF-R within the duodenal area is controversial and still not completely known. Immunohistochemical and Western blot techniques using a monoclonal anti-EGF-R antibody were performed on the adult rat duodenum. The duodenal enterocytes expressed EGF-R in the apical membrane and in the supranuclear area along the length of the villuos. The Lieberkhüm crypts and Brunner's glands also showed a positive immunostaining. By Western blot analysis we identified in the duodenal scrape a band with an apparent molecular weight of 175 kDa. Our results suggest a functional role for the luminal EGF and/or TGF-alpha in the establishment and maintenance of the epithelial renewal, probably by stimulation of cell proliferation, differentiation and migration.     

Matrix metalloproteinase (MMP)-2 and MMP-9 polymorphisms and haplotypes as disease biomarkers

Chaudhary and colleagues observed associations of matrix metalloproteinase (MMP)-2 (-1306C/T) and MMP-9 (-1562C/T) promoter polymorphisms with head and neck squamous cell carcinoma (HNSCC), but not with oral submucous fibrosis (OSMF) in an Indian population. We suggest that they could carry out a haplotype analysis with their data on MMP-2 genotypes (-1306C/T and -168G/T) and that they consider genotyping the microsatellite -90 (CA)(14-24) in the MMP-9 promoter region in order to perform haplotype analysis in combination with their data on MMP-9 (-1562C/T) polymorphism. These suggestions could provide additional information with clinical relevance to cancer susceptibility.     

Expression of matrix metalloproteinase (MMP-2) and extracellular matrix metalloproteinases inducer (EMMPRIN) in benign and advanced breast cancer tissue samples

Tumor cell derived matrix metalloproteinases are a family of enzymes associated with the tumor invasion and metastasis. Extracellular matrix metalloproteinases inducer (EMMPRIN) stimulates synthesis of gelatinase A (MMP-2) in peritoneal fibroblasts. In the present study the role of MMP-2 and EMMPRIN in the progression of breast cancer has been investigated. Gelatinase-A and EMMPRIN were analyzed in benign as well as in stage II and stage III breast cancer tissue samples by gelatin zymography assay, immunoprecipation analysis and Western blot analysis with a monoclonal primary antibody specific for EMMPRIN. Our results showed over expression of EMMPRIN in advanced stages of breast cancer tissues compared with benign tumor tissue samples. The expression of MMP-2, the active and latent forms of the enzyme increased with tumor progression from Stage II to Stage III of breast cancer and it was not expressed in benign tissues. The expression MMP-2 correlates with tumor progression. This observation obviously indicates that EMMPRIN and MMP-2 are the major determinants of malignancy in cancers.     

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.     

Tissue microarray-based immunohistochemical study can significantly underestimate the expression of HER2 and progesterone receptor in ductal carcinoma in situ of the breast

Abstract

The accuracy of immunohistochemical (IHC) analysis on tissue microarray (TMA)-based studies largely depends on the uniformity of the staining pattern for a given antibody and minimal intratumor heterogeneity of a given tumor. Our study was designed to investigate the concordance of expression in TMA and whole sections of estrogen receptor (ER), progesterone receptor (PR) and HER2 using IHC analysis for ductal carcinoma in situ (DCIS) of the breast. Seventy-five consecutive cases of DCIS were retrieved, reviewed and used to construct the TMA. IHC analysis of the expression of ER, PR, and HER2 were performed on TMA and whole sections of the corresponding cases, and the results were compared. The specificity and sensitivity for TMA-based assays were 87.0, 75.9, 90.6 and 90.4%, and 76.1, 27.3 for ER, PR and HER2, respectively. The concordance and discordance were 89.3, 76.0 and 72.0%, and 6.7, 13.3 and 16.0% for ER, PR, HER2, respectively. The kappa values were 0.83, 0.89 and 0.42 for ER, PR and HER2, respectively. The non-concordance rates were inversely related to core number, with 46.67, 22.67 and 11.56% for one core, two cores, and three cores, respectively, per marker per case (p < 0.001), but not associated with tumor size. Our results showed that the intratumor heterogeneity and the number of cores have a great impact on the results of TMA-based studies. Increasing the number of tissue cores per case may help improve the accuracy and concordance with whole section results. Although TMA remains an effective tool for translational research, we should be cautious in our interpretation of these results.     

Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

Matrix metalloproteinase (MMP) 9 induced in skin and subcutaneous tissue by implanted chitin in rats

Chitin was found to induce matrix metalloproteinases (MMPs) activity in rat skin and subcutaneous tissue. Sponge type chitin (22.5 mg) was implanted in subcutaneous tissue of 8-week-old rats by skin incision. MMPs activity was more pronounced in the chitin-treated group than only incision group until on day 2.5 postoperatively. Gelatin zymography revealed that the induced MMPs had a molecular mass of 92 and 82 kDa corresponding to MMP-9 and pro MMP-9, respectively. We here discuss the mechanism of MMP induction by chitin.     

Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.     

Identification of a novel gelatinolytic metalloproteinase (GMP) in the body wall of sea cucumber (Stichopus japonicus) and its involvement in collagen degradation

Body wall that mainly consists of collagen and polysaccharides is the edible part of sea cucumber and is easy to go autolysis, while the proteinase(s) responsible for autolysis remains unclear. In the present study, a gelatinolytic metalloproteinase (GMP) from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatographic steps including DEAE-Sephacel, Sephacryl S-200, Q-Sepharose and Phenyl-Sepharose. The molecular mass of GMP as estimated by SDS-PAGE and gelatin zymography was 45 kDa. The enzyme revealed high activity at a slightly alkaline pH range (8.0–9.0) and the optimal temperature was at 40–45 °C. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the activity, whereas other proteinase inhibitors did not show any effect. Peptide mass fingerprinting of the enzyme obtained 3 peptide fragments with a total of 58 amino acid residues, which was 91.4% identical to an alkaline metalloprotease from Pseudomonas fluorescens, strongly suggesting it is a metalloproteinase. Divalent metal ion Ca²⁺ is essential for its activity, indicating it is a calcium-dependent metalloproteinase. Furthermore, GMP hydrolyzed collagen effectively at 37 °C and gradually even at 4 °C, suggesting its involvement in the autolysis of sea cucumber.     

中枢神经系统感染患者脑脊液和血清中MMP-2、MMP-9、MCP-1表达的研究

目的探讨中枢神经系统感染患者脑脊液和血清中MMP-2(基质金属蛋白2)、MMP-9(基质金属蛋白9)、MCP-1(单核细胞趋化蛋白-1)表达的意义。方法选取2012年12月至2014年5月我院收治的中枢神经系统感染患者60例,其中结核性脑膜炎组(TBM组)、化脓性脑膜炎组(PM组)、真菌性脑膜炎组(CM组)和病毒性脑膜炎组(VM组)各15例;另外选取同期健康体检者15例作为对照组。治疗前、治疗后检测5组患者脑脊液常规及生化,采用ELISA法检测其脑脊液及血清中MMP-2、MMP-9、MCP-1的表达,并对患者进行格拉斯哥昏迷评分(GCS),分析其内在联系。结果治疗前、治疗后TBM组、PM组、CM组和VM组的脑脊液细胞数、蛋白水平均高于对照组,且TBM组、PM组的脑脊液细胞数、蛋白水平均高于CM组和VM组(P0.05);治疗前、治疗后TBM组、PM组、CM组和VM组的脑脊液糖水平、GCS评分低于对照组,治疗前TBM组、PM组、CM组、VM组的脑脊液氯化物水平低于对照组(P0.05);治疗后四组与对照组在脑脊液氯化物水平以及GCS评分等方面的差异无统计学意义(P0.05);治疗前、治疗后TBM组、PM组、CM组、VM组的脑脊液和血清中MMP-2、MMP-9、MCP-1水平均显著高于对照组,且治疗前TBM组、PM组的脑脊液和血清中MMP-2、MMP-9、MCP-1水平均显著高于CM组、VM组以及对照组(P0.05);治疗后PM组、CM组、VM组的脑脊液和血清中MMP-2、MMP-9、MCP-1水平的差异无统计学意义(P0.05);经Pearson进行相关性分析,BM组、PM组、CM组以及VM组的脑脊液和血清中MMP-2、MMP-9、MCP-1表达均与GCS评分负相关(相关系数r=-0.859~-0.574,P0.05)。结论中枢神经系统感染患者脑脊液和血清中MMP-2、MMP-9、MCP-1表达均与GCS评分相关,动态检测脑脊液和血清中MMP-2、MMP-9、MCP-1表达均对中枢神经系统感染的诊断和判断预后有积极意义。