Matrix Metalloproteinase-9 Antibody Research Articles

Correlation of MMP-9 (Matrix Metalloproteinase 9) and SMA (Smooth Muscle Actin) expression with Basal Cell Carcinoma’s risk of recurrence

Basal Cell Carcinoma (BCC) is the most common cancer in many countries, with its incidence steadily rising. Data from the Indonesian Pathologists Association show that among 1,530 skin cancer cases, BCC was the most frequent (39.93%), followed by Squamous Cell Carcinoma (23%) and Malignant Melanoma (7.9%). According to the 2018 WHO classification, BCC is divided into Low-Risk and High-Risk groups based on the risk of recurrence. BCC invades surrounding tissues through extracellular matrix degradation and enhanced cell motility, driven by the expression of MMP-9 (Matrix Metalloproteinase 9) and SMA (Smooth Muscle Actin). This study aims to evaluate the expression of MMP-9 and SMA in BCC using immunohistochemistry and analyze their correlation with the risk of recurrence. A total of 40 paraffin blocks from BCC patients diagnosed between 2019 and 2021 at Dr. Saiful Anwar General Hospital in Malang, Indonesia, were sampled. The blocks were sectioned, stained with MMP-9 and SMA antibodies, and examined for positive expression indicated by brown staining in the cytoplasm of tumor cell and peritumoral stroma. Spearman correlation showed r = 0.811 for MMP-9 and r = 0.857 for SMA (p < 0.05, CI: 95%), indicating a significant relationship between these markers and the risk of recurrence. Higher MMP-9 and SMA expression corresponded to a higher risk of BCC recurrence, suggesting their potential as prognostic biomarkers. However, further research is needed to assess recurrence risk more accurately and determine the most appropriate therapy.

Comparison of MMP-9 Density Between Triple Negative and HER2 Enriched Breast Carcinoma Subtypes

Background
 Matrix metalloproteinase-9 (MMP-9) is a protease that plays an important role in various biological processes of breast cancer. This marker may be useful in determining the prognosis and become a promising therapy, especially in breast carcinoma with an aggressive subtype including HER2 enriched and triple negative. Both subtypes have high MMP-9 expression, but it is necessary to differentiate them because they have different biological and molecular aspects. This study aims to compare the density of MMP-9 between triple negative and HER2 enriched breast carcinoma subtypes and their relationship with clinicopathological characteristics.
 
 Methods
 This is a cross sectional study using 32 samples of paraffin blocks and slides of breast carcinoma patients with triple negative and HER2 Enriched subtypes at the Department of Anatomical Pathology Faculty of Medicine Sriwijaya University/Dr. Mohammad Hoesin Hospital Palembang for the period January 1, 2016 to December 31, 2019. Each sample was stained with MMP-9 antibodies immunohistochemistry (polyclonal, Abcam). Comparative analysis of MMP-9 density between the two subtypes was carried out using the median value based on the Mann-Whitney analysis. MMP-9 density was categorized as high and low based on the ROC curve and analyzed by using chi-square test to assess its association with clinicopathological characteristics.
 
 Results
 Mann-Whitney test showed that the MMP-9 density was higher in the HER2 enriched subtype (median 12.0) than triple negative (median 8.8) with a p value of 0.632. The relationship distribution of high MMP-9 density was found in the category of age group >40 years old (60.0%), positive lymphovascular invasion (86.7%) and histological grade III (80.0%) with p values of 0.798, 0.402 and 0.691.
 
 Conclusion
 MMP-9 density in HER2 enriched subtype was higher than triple negative subtype, but statistically not significant.

Matrix Metalloproteinase-9 Expression is Enhanced by Ischemia and Tissue Plasminogen Activator and Induces Hemorrhage, Disability and Mortality in Experimental Stroke

Matrix metalloproteinase-9 (MMP-9) degrades collagen and other cellular matrix proteins. After acute ischemic stroke, increased MMP-9 levels are correlated with hemorrhage, lack of reperfusion and stroke severity. Nevertheless, definitive data that MMP-9 itself causes poor outcomes in ischemic stroke are limited. In a model of experimental ischemic stroke with reperfusion, we examined whether ischemia and recombinant tissue plasminogen activator (r-tPA) therapy affected MMP-9 expression, and we used specific inhibitors to test if MMP-9 affects brain injury and recovery. After stroke, MMP-9 expression increased significantly in the ischemic vs. non-ischemic hemisphere of the brain (p < 0.001). MMP-9 expression in the ischemic, but not the non-ischemic hemisphere, was further increased by r-tPA treatment (p < 0.001). To determine whether MMP-9 expression contributed to stroke outcomes after r-tPA treatment, we tested three different antibody MMP-9 inhibitors. When compared to treatment with r-tPA and saline, treatment with r-tPA and MMP-9 antibody inhibitors significantly reduced brain hemorrhage by 11.3 to 38.6-fold (p < 0.01), brain swelling by 2.8 to 4.3-fold (p < 0.001) and brain infarction by 2.5 to 3.9-fold (p < 0.0001). Similarly, when compared to treatment with r-tPA and saline, treatment with r-tPA and an MMP-9 antibody inhibitor significantly improved neurobehavioral outcomes (p < 0.001), decreased weight loss (p < 0.001) and prolonged survival (p < 0.01). In summary, both prolonged ischemia and r-tPA selectively enhanced MMP-9 expression in the ischemic hemisphere. When administered with r-tPA, specific MMP-9 inhibitors markedly reduced brain hemorrhage, swelling, infarction, disability and death, which suggests that blocking the deleterious effects of MMP-9 may improve outcomes after ischemic stroke.

The Type IV Collagenase Inhibitor, BiPS, Alters Keratin Expression in Sulfur Mustard Exposed Mouse Skin

Sulfur mustard (SM) is a potent chemical vesicant targeting many tissues including skin. The SM induced cutaneous toxicity is associated with edema, epidermal erosions, inflammation, and prolonged wound repair. Earlier studies have shown that overexpression of matrix metalloproteinase 9 (MMP9) degrades extracellular matrix proteins and leads to the separation of epidermis from dermis in skin exposed to SM. We have found that MMP9, a type IV collagenase, disrupts collagen IV (Col IV) a major component of the basement membrane (BM) zone in SM induced blisters. Col IV in the BM network has recently been reported to be a regulator of epidermal differentiation and keratin expression during wound repair. The type IV collagen (Col IV) BM network may regulate epidermal differentiation and keratin expression during development and wound repair. In this present study, we evaluated the MMP9 inhibitor BiPS [N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide] in an epidermal wound healing model following SM exposure. Time course (24, 72, and 168 hrs post‐SM exposure) and compound evaluation studies (BiPS as a single pretreatment) were performed in a mouse ear vesicant model (MEVM) using histopathological changes, degree of hyperplasia and edema, and expression of type IV collagen, MMP9, keratin 10 (K10, differentiation marker), and keratin 6 (K6, wound‐inducible marker) for comparison. MMP9 mRNA and protein expression increased with time. Dual immunofluorescence studies using Col IV α1 and MMP9 antibodies showed BM zone breakdown of Col IV and apparent MMP9 expression in the epidermis and the adjacent dermal matrix of the SM injured skin. Dual immunofluorescent studies using K6 and K10 showed strong K6 and weak K10 expression in the hyperplastic epidermis. In the compound evaluation study, the BiPS pretreatment group demonstrated significant reduction in edema with 25% reduction of epidermal thickness, and improvement in overall tissue structure by 72 hr post‐SM exposure. mRNA expression of MMP9 was significantly downregulated in the BiPS treated group by 168 hr post exposure. Immunofluorescent studies showed that significant reduction of MMP9 expression in the skin and a continuous expression pattern of Col IV; similar to the unexposed control skin. Dual immunofluorescent studies show reduced K6 and increased K10 expression in hyperplastic epidermis when compared to SM treated samples. These results suggest that BiPS treatment may effectively improve BM integrity by the inhibition of MMP9 in SM exposed skin. This may in turn modulate the epidermal keratin expression and promote keratinocyte differentiation for wound repair. Targeting MMP9 for the restoration of BM may be an effective strategy for countering SM‐induced cutaneous injury.Support or Funding InformationGrant funding supported by ES005022, T32ES007148, and NIAMS U54AR055073.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Noninvasive detection of matrix metalloproteinase-9 in atherosclerotic lesions using technetium-99m-labeled single-photon emission computed tomography in vivo

Previous studies have suggested that matrix metalloproteinase (MMP) inhibitor uptake may offer a precise estimation of MMP activity in atherosclerotic lesions. In this study, we explored the feasibility of noninvasive detection of MMP-9 activity using technetium-99m-labeled matrix metalloproteinase-9 antibody (Tc-McAb) in vivo. ApoE-deficient (ApoE) atherosclerosis mice models (n=10) were induced through a high-cholesterol diet following ligation of their left common carotid artery. After 4 weeks, the models were verified through proton density-weighted and T2-weighted images obtained by MRI. C57BL/6 sham mice (n=8) were used as controls. In addition, normal mice (n=20) were used to characterize blood clearance. After radiolabeled McAb administration, single-photon emission computed tomography (SPECT) was performed. Subsequently, left common carotid arteries were harvested for ex-vivo autoradiograph imaging. Then, morphology and activity assays of MMP-9 were histologically and immunohistochemically examined. MRI showed higher signal intensities in the left common carotid arteries with irregular stenoses in the lumen of blood vessels in atherosclerosis mice models in vivo. Atherosclerotic lesions on left common carotid artery specimens were also clearly visualized using SPECT 2 h after Tc-McAb administration in vivo. Note that the radiochemistry purity of the Tc-McAb used was 85-95%. Biodistribution studies have shown that the clearance of Tc-McAb from blood was rapid. In addition, atherosclerotic lesions were clearly visualized on radioautography film shadows ex vivo. MMP-9 activities within the atherosclerotic lesions were noninvasively detected using Tc-labeled SPECT in vivo.

Chrysin Inhibits Tumor Promoter-Induced MMP-9 Expression by Blocking AP-1 via Suppression of ERK and JNK Pathways in Gastric Cancer Cells

Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients’ survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

Lipocalin-2 and matrix metalloproteinase-9 expression in high-grade endometrial cancer and their prognostic value.

To demonstrate lipocalin-2 (LCN-2) immunohistochemical expression together with matrix metalloproteinase-9 (MMP-9) protein in high-grade endometrial cancer and determine their correlations with FIGO (International Federation of Gynecology and Obstetrics) stage, histological subtype, presence of vascular invasion, patient age and overall and disease-free survival. Immunohistochemical staining was performed using LCN-2 and MMP-9 antibodies on high-grade endometrial cancer (n=85) diagnosed at Split University Hospital Centre during 1998-2010. Immunohistochemical expression was determined on archived paraffin-embedded samples and scored semiquantitatively. Survival time was analysed using the Kaplan-Meier method, and the log-rank test was used to assess between-group differences. The Cox proportional hazard regression model was used on multivariate survival analysis. Patients were followed from the time of primary surgery until death or last follow-up until December 2012. LCN-2 and MMP-9 were highly expressed in high-grade endometrial cancer. Univariate analysis showed positive immunohistochemical staining for LCN-2 and MMP-9 to be associated with shorter survival in patients with high-grade endometrial cancer. Multivariate analysis showed LCN-2 overexpression to be associated with shorter overall and disease-free survival in high-grade endometrial cancer. Our findings suggest that LCN-2 expression may be an important independent indicator of shorter survival in patients with high-grade endometrial cancer.

Transforming growth factor β1 and extracellular matrix protease expression in the uterosacral ligaments of patients with and without pelvic organ prolapse.

This study was undertaken to evaluate the expression of transforming growth factor β1 (TGF-β1) and matrix metalloproteinase 9 (MMP-9), key regulators of the extracellular matrix composition, in the uterosacral ligaments (USLs) of women with pelvic organ prolapse (POP) compared with controls. Under an institutional review board approval, USL samples were obtained from women undergoing vaginal hysterectomy for stage 2 or greater POP (cases, n = 21) and from women without POP undergoing vaginal hysterectomy for benign indications (controls, n = 19). Hematoxylin and eosin and trichrome staining were performed on the USL sections, and the distribution of smooth muscle and fibrous tissue were quantified. Immunohistochemical staining was performed using anti-TGF-β1 and anti-MMP-9 antibodies. The expressions of TGF-β1 and MMP-9 were evaluated by the pathologist, who was blinded to all clinical data. Transforming growth factor β1 expression positively correlated with MMP-9 expression (R = 0.4, P = 0.01). The expressions of TGF-β1 and MMP-9 were similar in subjects with POP versus controls. There was a significant increase in fibrous tissue (P = 0.008) and a corresponding decrease in smooth muscle (P = 0.03), associated with increasing age. The TGF-β1 expression, but not MMP-9 expression, also significantly increased with age (P = 0.02). Although our study uncovered age-related alterations in USL composition and TGF-β1 expression, there was no difference in the expression of TGF-β1 or MMP-9 in the subjects with POP versus controls.

The role of MMP-2 and MMP-9 as prognostic markers in the early stages of tongue squamous cell carcinoma.

Tongue cancer is the most common intra-oral malignancy with a high rate of morbidity and mortality owing to its increased propensity for tumor invasion and metastasis. These processes require a controlled degradation of the extracellular matrix. Matrix Metalloproteinase-2 (MMP-2) and Matrix Metalloproteinase-9 (MMP-9) are known to be important regulators of matrix lysis and play a significant role in the metastasis of malignancies. To study the expression of MMP-2 and MMP-9 in the early stages of tongue squamous cell carcinoma and find the association between their expression and local recurrence, metastasis, and survival rates of the subjects. Fifty-nine tumor biopsy samples of tongue squamous cell carcinoma in T1 N0 M0 and T2 N0 M0 stages were immunostained with MMP-2 and MMP-9 antibodies. The immunohistochemical expression was compared with the patient characteristics and outcome. Cytoplasmic expression of MMP-2 correlated with that of MMP-9 (r = 0.716, P < 0.001). Greater expression of MMP-2 and MMP-9 was observed in patients who subsequently developed local recurrence (P = 0.044 and P < 0.001, respectively), regional and/or distant metastasis (P < 0.001 and P = 0.001, respectively) of the tumor. Further, a higher expression of these biomarkers was associated with shorter survival. MMP-9 was found to have better specificity for local recurrence, metastasis and survival. Our results showed that these biomarkers may serve as indicators of a patient's risk potential for poor prognosis and presage the need for more aggressive treatment measures.

Matrix metalloproteinase-9 overexpression is closely related to poor prognosis in patients with colon cancer

BackgroundMatrix metalloproteinase-9 (MMP-9) is an important member of the matrix metalloproteinase family and is considered to be involved in the invasion and metastasis of cancer cells. This study analyzed the expression of MMP-9 in colon cancer patients and the relationship between this expression and clinicopathological features and survival.MethodsWe immunohistochemically investigated 68 specimens of colon cancer tissues and corresponding distal normal mucosa tissues using MMP-9 antibody. Then, the correlation between MMP-9 expression and clinicopathological features and its prognostic relevance were determined.ResultsThe expression rate of MMP-9 in colon cancer tissues was significantly higher than that in distal normal mucosa (69.1% versus 2.9%, P < 0.001). Significant correlations were only found between high levels of MMP-9 expression and metastasis of lymph nodes and Dukes’ stage. Overexpression of MMP-9 was associated with shorter survival times in univariate analysis. Multivariate analysis confirmed that MMP-9 expression was an independent prognostic factor.ConclusionsMMP-9 is correlated with the metastasis of lymph nodes, and its elevated expression may be an adverse prognostic indicator for the patients of colon cancer.

Expression and prognostic relevance of Cyclophilin A and matrix metalloproteinase 9 in esophageal squamous cell carcinoma

AimsTo guide clinicians in selecting treatment options for esophageal squamous cell carcinoma (ESCC) patients, reliable markers predictive of clinical outcome are desirable. This study analyzed the correlation of cyclophilin A (CypA) and matrix metalloproteinase 9 (MMP9) in ESCC and their relationships to clinicopathological features and survival.MethodsWe immunohistochemically investigated 70 specimens of ESCC tissues using CypA and MMP9 antibodies. Then, the correlations between CypA and MMP9 expression and clinicopathological features and its prognostic relevance were determined.ResultsSignificant correlations were only found in high level of CypA and MMP9 expression with tumor differentiation and lymph node status. Significant positive correlations were found between the expression status of CypA and that of MMP9. Overexpression of CypA and metastasis were significantly associated with shorter progression free survival times in univariate analysis. Multivariate analysis confirmed that CypA expression was an independent prognostic factor.ConclusionsCypA might be correlated with the differentiation, and its elevated expression may be an adverse prognostic indicator for the patients of ESCC. CypA/MMP9 signal pathway may be attributed to the malignant transformation of ESCC, and attention should be paid to a possible target for therapy.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1166551968105508.

Abstract 128: Serum Anti-MAA Antibody Titers are Associated with Acute Myocardial Infarction and MMP-9

Introduction Atherosclerotic plaque rupture is the leading cause of acute myocardial infarction (AMI). Malondialdehyde acetaldehyde (MAA) modified proteins have recently been implicated in this process by their ability to initiate inflammatory responses. Matrix metalloproteinase 9 (MMP-9) is increased following AMI and has been associated with the disease due to its ability to degrade the fibrotic cap that forms on some plaques. Objective The purpose of this study was to evaluate the relationship between MMP-9 and anti-MAA antibody isotypes in patients experiencing acute myocardial infarction (AMI). Methods Serum samples from AMI patients (n=10) were collected and tested for the presence of anti-MAA antibody isotypes, peak CK, peak troponin, and MMP-9 levels. Samples were collected at the time of AMI, 24 and 48 hours post event. Data was correlated to determine relationships with antibody isotypes directed against MAA protein antigens. Results MMP-9 increased significantly from the initial presentation at 24 hours post-AMI (p=0.006) and 48 hours post-AMI (p= 0.044). Anti-MAA IgM decreased significantly (p= 0.021) between initial and 24 hours post AMI. A similar significant decrease was observed for anti-MAA IgG (p= 0.006). Anti-MAA IgA levels did not change significantly post-AMI. Significant correlations were demonstrated between peak CK and anti-MAA IgG 24 hours post AMI (r= -0.819, p= 0.013), between peak troponin and anti-MAA IgG 24 hours post AMI (r= -0.860, p= 0.006) and between MMP-9 levels 24 hours post AMI and anti-MAA IgM levels 24 hours post MI ( r= 0.0712, p=0.021). Conclusions The negative correlation of anti-MAA IgG to cardiac enzymes is suggestive that myocardial infarction and tissue necrosis results in the release of the MAA antigen from infarcted myocardium and clearance of anti-MAA antibodies. Additionally, anti-MAA IgM and MMP-9 are positively correlated with each other 24 hours post-AMI, suggesting that anti-MAA IgM titers may correlate to the severity of the AMI and thus CAD. Implications Anti-MAA antibodies might serve as important biomarkers of impending atherosclerotic plaque rupture and subsequent AMI and warrants further investigation in a larger cohort of patients.

Abstract PR2: Stroma-derived matrix metalloproteinase 9 (MMP9) confers resistance to anti-EGFR therapy through cell-extrinsic activation of ERBB2/ERK/JUN pathway

Abstract Matrix metalloproteinases (MMPs) have been studied historically for their role in tissue invasion and metastasis. The tumor microenvironment also produces MMPs that may influence other tumor behaviors, as recent mouse models suggest in the intestine and other sites. We examined MMP9 expression in 642 cases of human colorectal cancer (CRC) in relation to clinicopathological features and mutations. MMP9 antibody (Ab) unambiguously stained mesenchymal cells in 104 cases (16.2%) and gave weaker stromal staining in another 12.8%. High stromal MMP9 expression correlated most strongly with the CpG island methylator phenotype (CIMP-high) in tumors (multivariate odds ratio – OR – 2.45, 95% confidence interval – CI – 1.41 to 4.27, p=0.0015) and also with absence of tumor KRAS mutations (multivariate OR 0.54, 95% CI 0.33 to 0.87, p=0.011). The latter association suggested KRAS allelic status as one reason for heterogeneity in CRC cell line responses to MMP9. Four out of 6 KRASWT cell lines proliferated significantly more in the presence of exogenous MMP9, while 4 of 5 KRASMut lines failed to respond. This led us to ask if stromal MMP9 expression might predict the outcome of treatment with anti-EGFR mAb cetuximab, allowing KRASWT tumors to bypass mAb blockade of EGFR-KRAS pathway activation. We retrospectively evaluated MMP9 expression by immunohistochemistry on primary tumors and progression-free survival (PFS) in 86 patients who had received chemotherapy combined with cetuximab as first-line or salvage therapy. Tumors from 29 patients (33.8%) harbored mutant KRAS. PFS of patients with MMP9high;KRASWT tumors (median 3.5 months) was lower than that of patients with low or no stromal MMP9 expression and KRASWT tumors (median 5.8 months, 95% CI for the hazard ratio 1.135-2.179). Patients with KRASMut tumors showed no significant difference in PFS with respect to MMP9 expression (2.55 months and 2.25 months in the MMP9high and MMP9neg/low subgroups, respectively). Thus, low stromal MMP9 expression in KRASWT tumors predicted a better outcome with cetuximab treatment than high MMP9 expression. In CRC cell lines that grow faster in response to MMP9, this factor activates ERBB2 and ERK/JUN signaling. Together, these findings suggest that stroma-derived MMP9 may help tumors bypass common mutational mechanisms for constitutive growth factor pathway activation and confer resistance to anti-EGFR therapy through cell-extrinsic activation of the ERBB2/ERK/JUN pathway. Stromal MMP9 expression may thus predict cetuximab response in KRASWT CRCs and have value in selecting patients for treatment. This proffered talk is also presented as Poster A26.

An immunoaffinity liquid chromatography–tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

An immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap–release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.

Prognostic Significance of Matrix Metalloproteinase-9 (MMP-9) in Stage II Colorectal Carcinoma

Approximately 30% of all colorectal cancer patients are diagnosed with stage II disease. Adjuvant therapy is not widely recommended. However, it is well-established that a subgroup of patients with stage II is at high risk for recurrence within their life time and should be considered for adjuvant chemotherapy. The present work was designed to assess the value of matrix metalloproteinase-9 (MMP-9) as a predictor of disease outcome in a series of 202 stage II colorectal cancer (CRC) patients with long-term follow-up. The present study comprises a series of 202 patients who underwent bowel resection for stage II CRC at Turku University Hospital. Archival paraffin-embedded CRC tissue samples were used to prepare tissue microarray blocks for immunohistochemical staining with MMP-9 antibody. Forty-eight percent of all CRC samples were positive for MMP-9. There was no significant correlation between MMP-9 expression and age, depth of invasion, and lymph node status. However, MMP-9 expression was significantly related to histological grade (p = 0.03) and location of the tumor (p = 0.01), therefore, being lower in high-grade tumors and most intense in carcinomas of the descending colon and rectum. Tumors with high MMP-9 expression showed a higher recurrence rate than tumors with low expression (p = 0.02). MMP-9 negative tumors had a more favorable disease-free survival (DFS) than those expressing MMP-9 (p = 0.03). The same was true with disease-specific survival (DSS; p = 0.02) as well, high expression of MMP-9 being associated with shorter survival rates. In multivariate (Cox) survival analysis, MMP-9 expression proved to be an independent predictor of DFS, but not DSS, which was predicted by age and sex only. Quantification of MMP-9 expression seems to provide valuable prognostic information in stage II CRC, particularly, in selecting the patients at high risk for recurrent disease who might benefit from adjuvant therapy.

Osteoclast differentiation and recruitment during early stages of experimental tooth movement in rats

Osteoclasts are derived from macrophage-lineage precursors. ED1 is an antibody that can recognize this lineage of cells. Matrix metalloproteinase 9 (MMP9) is essential for the migration of osteoclasts and their precursors during osteoclastogenesis. The aim of this research was to investigate differentiation and recruitment of osteoclasts during the early phase of experimental tooth movement in rats. The upper three molars of Wistar rats at one side were moved mesially, using Ni-Ti coil springs of 10 cN, for 6, 12, 24, 36, 48, 72, 96, and 120 h. The contralateral sides served as controls. Immunohistochemical staining using ED1 and MMP9 antibodies was performed. ED1(+) and MMP9(+) mononuclear and multinuclear cells were counted and statistically analysed. After force application, the number of ED1(+)/MMP9(+) multinuclear cells first increased in the bone marrow. At compressed areas, the number of ED1(+) mononuclear cells decreased; this was followed by an increase in the number of ED1(+/)MMP9(+) mononuclear and multinuclear cells. At tension areas, the number of ED1(+)/MMP9(+) multinuclear cells decreased while the number of ED1(+) mononuclear cells remained stable. It was concluded that force application induces osteoclast differentiation within the bone marrow. These osteoclasts probably migrate subsequently into the compressed PDL. Pre-existing osteoclasts disappear at the tension areas while the number of mononuclear macrophage-lineage cells remains stable.

Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-κB

Neutrophil gelatinase-associated lipocalin (NGAL) has recently emerged as an important modulator of cell homeostasis. Elevated plasma NGAL levels, possibly because of activation of blood leukocytes, are associated with atherosclerosis. However, little is known about induction of NGAL expression in blood vessels. Using a rat carotid artery injury model, we found that NGAL was highly induced in the intima after angioplasty but was attenuated by adenovirus-mediated expression of a dominant-negative mutant of inhibitor of nuclear factor (NF)-kappaB kinase beta (dnIKKbeta). Expression of NGAL mRNA and protein was also up-regulated in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells (SMCs) in response to interleukin-1beta stimulation. Rat SMC-produced NGAL was present as mono- and homomeric forms in the cytosol and in a complex containing matrix metalloproteinase-9 (MMP-9) after secretion. In agreement with levels of NGAL, proteolytic activity of MMP-9 was markedly high in the intima of injured vessels and in the culture supernatant of activated intimal SMCs but was reduced in the vessels transduced with dnIKKbeta. The present study reveals a previously unrecognized vascular response to an-gioplastic injury, characterized by NF-kappaB-dependent expression of NGAL in vascular SMCs. Further-more, SMC-produced NGAL interacts with MMP-9, a mechanism by which NGAL may modulate MMP-9 proteolytic activity in the vascular repair process.

MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by α4β1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration

AbstractB-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FN-H89, VCAM-1, or TNF-α–activated human umbilical vein endothelial cells (HUVECs) up-regulated MMP-9 production, measured by gelatin zymography. This effect was mediated by α4β1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also up-regulated MMP-9, independently of α4β1 and involving ERK1/2 but not Akt activity. Accordingly, α4β1 engagement activated the PI3-K/Akt/NF-κB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti–MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with 3 different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVECs. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1, or fibronectin; MMP-9 localized to podosomes in a PI3-K–dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by α4β1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.

TNFα-induced MMP-9 promotes macrophage recruitment into injured peripheral nerve

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced hours after injury to peripheral nerve. This study shows that MMP-9 gene deletion and neutralization with MMP-9 antibody reduce macrophage content in injured wild-type nerves. In mice with delayed Wallerian degeneration (Wld S), MMP-9 and tumor necrosis factor alpha (TNFα) decline in association with the reduced macrophage recruitment to injured nerve that characterizes this strain of mice. We further determined that TNFα acts as an MMP-9 inducer by establishing increased MMP-9 levels after TNFα injection in rat sciatic nerve in vivo and primary Schwann cells in vitro. We found reduced MMP-9 expression in crushed TNFα knockout nerves that was rescued with exogenous TNFα. Finally, local application of MMP-9 on TNFα−/− nerves increased macrophage recruitment to the lesion. These data suggest that TNFα lies upstream of MMP-9 in the pathway of macrophage recruitment to injured peripheral nerve.

Differential Regulation of Platelet Aggregation by Matrix Metalloproteinases-9 and -2

We have recently found matrix metalloproteinase-2 (MMP-2) in human platelets and reported that the release of this enzyme during platelet activation stimulates aggregation. We have now identified matrix metalloproteinase-9 (MMP-9) in human platelets and resistance-sized (approximately 200 microm) arteries. Resting platelets released small quantities of pro-MMP-9. Maximal release of MMP-9 was detected during partial (appr. 30% maximum) aggregation with thrombin. However, maximal release of MMP-2 was associated with maximal aggregation. MMP-9 antibodies induced aggregation of resting platelets and potentiated aggregation of platelets induced by thrombin and collagen. Moreover, MMP-9 microisolated from arteries as well as recombinant human MMP-9 (0.1-30 ng/ml) inhibited thrombin and collagen-induced aggregation. We conclude that MMP-9 is an inhibitor of aggregation and in this action opposes the effects of MMP-2. The MMP-2/MMP-9 system may play an important role in the regulation of platelet-platelet and platelet-vessel wall interactions.