High Levels Of Matrix Metalloproteinases Research Articles

Abstract 5270: Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer

Abstract Histone deacetylases (HDACs) are associated with the development and progression of cancer, but which HDAC isoforms play important roles in breast cancer metastasis is not known. Here, we identify the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) than MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5270. doi:10.1158/1538-7445.AM2011-5270

Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.

Stromal cell-derived factor-1 and vascular endothelial growth factor as biomarkers for lymph node metastasis and poor cancer-specific survival in prostate cancer patients after radical prostatectomy

ObjectivesThis study aims to analyze the clinicopathologic significance of stromal cell-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9) expression in human prostate cancer (CaP), and their involvement in the prognosis of CaP. Materials and methodsThe expression of SDF-1, VEGF, and MMP-9 were measured using immunohistochemistry in 148 CaP patients who underwent radical prostatectomy for clinically localized disease and in 10 samples of benign prostatic hyperplasia (BPH). ResultsIn the CaP group, VEGF and MMP-9 were more strongly expressed in the tumor cells compared with the BPH group. High intensity SDF-1, VEGF, and MMP-9 stains in tumor areas strongly correlated with lymph node metastasis, pathologic stage, and differentiation. Univariate and multivariate analysis showed that SDF-1, VEGF, and lymph node metastasis were independent prognostic factors for prostate cancer-specific survival. High levels of MMP-9, pathologic stage, and differentiation were associated with prostate cancer-specific survival in univariate analysis but the risk estimate was not significant in multivariate analysis. ConclusionsHigh expression levels of SDF-1, VEGF, and MMP-9 are more correlated with lymph node metastatic prostate carcinoma compared with non-lymph-node metastatic cancer. High expression levels of SDF-1 and VEGF strongly predict the biochemical progression in CaP patients after radical prostatectomy.

Cyanobacterial Microcystis aeruginosa Lipopolysaccharide Elicits Release of Superoxide Anion, Thromboxane B2, Cytokines, Chemokines, and Matrix Metalloproteinase-9 by Rat Microglia

Microcystis aeruginosa (M. aeruginosa) is a cosmopolitan Gram-negative cyanobacterium that may contaminate freshwater by releasing toxins, such as lipopolysaccharide (LPS) during aquatic blooms, affecting environmental and human health. The putative toxic effects of cyanobacterial LPS on brain microglia, a glial cell type that constitutes the main leukocyte-dependent source of reactive oxygen species in the central nervous system, are presently unknown. We tested the hypothesis that in vitro concentration- and time-dependent exposure to M. aeruginosa LPS strain UTCC 299 would activate rat microglia and the concomitant generation of superoxide anion (O₂⁻). After a 17-h exposure of microglia to M.aeruginosa LPS, the following concentration-dependent responses were observed: 0.1-100 ng/ml M. aeruginosa LPS enhanced O₂⁻ generation, with limited inflammatory mediator generation; 1000-10,000 ng/ml M. aeruginosa LPS caused thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and macrophage inflammatory protein-2 (MIP-2/CXCL2) release, concurrent with maximal O₂⁻ generation; 100,000 ng/mL M. aeruginosa LPS deactivated O₂⁻ production but maintained elevated levels of TXB₂, MMP-9, tumor necrosis factor-α (TNF-α), interleukin 1-α (IL-1α), and interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α/CCL3), and MIP-2/CXCL2, with concomitant lactic dehydrogenase release. Although M. aeruginosa LPS was consistently less potent than Escherichia coli LPS, with the exception of O₂⁻, TXB₂, and MCP-1/CCL2 generation, it was more efficacious because higher levels of MMP-9, TNF-α, IL-1α, IL-6, MIP-1α/CCL3, and MIP-2/CXCL2 were produced. Our in vitro studies suggest that one or more of the inflammatory mediators released during M. aeruginosa LPS stimulation of microglia may play a critical role in the subsequent ability of microglia to generate O₂⁻. To our knowledge, this is the first experimental evidence that LPS isolated from a M. aeruginosa strain, can activate brain microglia in vitro, as well as the release of O₂⁻, and other inflammatory mediators hypothesized to be involved in neuroinflammation and neurodegeneration.

Mesenchymal stem cells inhibit both endogenous and exogenous MMPs via secreted TIMPs

Mesenchymal stem cells (MSCs) have been shown to be perivascular, occupying a prime location for regulating vessel stability. Here, we focused on the MSC-contribution of key regulators of the perivascular niche, the matrix metalloproteinases (MMPs) and their inhibitors, the TIMPs. Despite secretion of active forms of MMPs by MSCs, MMP enzyme activity was not detected in MSC-conditioned medium (MSC-CM) due to TIMP-mediated inhibition. By means of bifunctional-crosslinking to probe endogenous MMP:TIMP interactions, we showed MMP-2-inhibition by TIMP-2. MSCs also inhibited high levels of exogenous MMP-2 and MMP-9 through TIMP-2 and TIMP-1, respectively. Furthermore, MSC-CM protected vascular matrix molecules and endothelial cell structures from MMP-induced disruption. MSCs remained matrix-protective when exposed to pro-inflammatory cytokines and hypoxia, countering these stresses with increased TIMP-1 expression and augmented MMP-inhibition. Thus, MSCs are revealed as robust sources of TIMP-mediated MMP-inhibition, capable of protecting the perivascular niche from high levels of MMPs even under pathological conditions.

Serum matrix metalloproteinases and tympanosclerosis

To investigate levels of matrix metalloproteinases 2 and 9, and of their tissue inhibitor (i.e. tissue inhibitor matrix metalloproteinase 1), in the serum of patients with tympanosclerosis. We included 40 patients (age range 13-63 years) who had undergone surgery in the ENT department of Izmir Atatürk Training and Research Hospital between 2002 and 2007. Twenty had uncomplicated chronic otitis media and 20 had tympanosclerosis. We also included as the control group 20 individuals with no history of previous otic complaints or systemic or infectious disease. Serum levels of serum matrix metalloproteinases 2 and 9 and of tissue inhibitor matrix metalloproteinase 1 were measured in all subjects and compared. Significantly higher levels of serum matrix metalloproteinases 2 and 9 were found in the tympanosclerosis group, compared with the chronic otitis media and control groups. There was no statistically significant difference in tissue inhibitor matrix metalloproteinase 1 level between the three groups. Tympanosclerosis surgery has poor success rates, since the pathological process is still active. We suggest that high levels of matrix metalloproteinases may play a role in the continuation of the disease process.

In Vivo Evaluation of a New Magnetic Resonance Imaging Contrast Agent (P947) to Target Matrix Metalloproteinases in Expanding Experimental Abdominal Aortic Aneurysms

Abdominal aortic aneurysm (AAA) rupture is a devastating event, and development of noninvasive methods to detect AAA at risk is needed. Matrix metalloproteinases (MMPs) play a major role in AAA growth and their subsequent rupture. This study was aimed to evaluate the ability of P947, a recently developed magnetic resonance imaging (MRI) contrast agent, to target MMPs in vivo in expanding experimental AAAs. AAAs were induced in Wistar rats (n = 18) by perfusion of a segment of the abdominal aorta with porcine elastase. After 5 or 6 days of elastase perfusion, when the aortic segment was expanding and showed inflammation with high MMP levels, rats were injected either with P947 (n = 6), P1135, a scramble form of P947 (n = 6), or with the reference contrast agent Gadolinium-DOTA (Gd-DOTA) (n = 3). Sham-operated rats (n = 3) were injected with P947 as controls. Imaging was performed on the animals using a 1.5T MRI scanner before and at different times after injection of contrast agents (100 μmol/kg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography of culture media conditioned by incubation with perfused aortic segment or control TA from elastase-perfused rats (n = 3) was performed to determine levels of MMP2 and MMP9. In addition, in situ gelatin zymography was used to localize these active MMPs on frozen histologic sections. The normalized signal enhancement determined on MRI images was higher in the perfused aortic segment of rats injected with P947 (162%) than in rats injected with P1135 (100%) or Gd-DOTA (117%) (P < 0.01 using the Friedman test) from 5 to 125 minutes after injection. The area of contrast enhancement on MRI images colocalized with the fluorescence generated by MMPs in the AAA inflammatory area, as detected by in situ zymography on histologic sections. Our data showed that MRI using P947 allows detection of MMP activity within the inflammatory wall of experimental AAAs, thus representing a potential noninvasive method to detect AAAs with a high risk of rupture.

Epigenetic Repression of Matrix Metalloproteinases in Myofibroblastic Hepatic Stellate Cells through Histone Deacetylases 4: Implication in Tissue Fibrosis

Matrix metalloproteinases (MMPs), which are highly expressed in acute injury, are progressively repressed or silenced in fibrotic liver, favoring extracellular matrix accumulation, while the underlying mechanism is largely unknown. Similarly, normal/quiescent hepatic stellate cells (HSCs) express high levels of MMPs in response to injury signals, such as interleukin-1. After transdifferentiation, the myofibroblastic HSCs are incapable of expressing many MMPs; however, the major signaling pathways required for MMP expression are intact, indicating that repression is at the level of the chromatin. Indeed, both the MMP9 and MMP13 genes are inaccessible to transcription factors and RNA polymerase II, in association with impaired histone acetylation in their promoters. In accordance with impaired histone acetylation at the cellular level, histone deacetylase-4 is accumulated during HSC transdifferentiation. Furthermore, ectopic expression of histone deacetylase-4 in quiescent HSCs results in repression of MMP promoter activities as well as endogenous MMP9 protein expression. Thus, our findings suggest that a histone deacetylase-4-dependent mechanism underlies the epigenetic silencing of MMP genes during tissue fibrogenesis.

Matrix metalloproteinase-9 measured in urine from bladder cancer patients is an independent prognostic marker of poor survival

Introduction. Matrix metalloproteinase 9 (MMP-9) is an endopeptidase involved in various cellular processes, such as tumour development and metastatic spread. In biological samples, MMP-9 can occur as pro-MMP-9 and active MMP-9, or these factors complexed with the inhibitor TIMP-1. An assay, which can measure active and total MMP-9 in biological samples, has been used on the urine from bladder cancer patients and demonstrated a significant correlation between MMP-9 and clinical parameters. The prognostic value of these measurements has never been investigated. Using this assay we have investigated the prognostic influence of total and active MMP-9 in urine from bladder cancer patients. Material and methods. Fresh voided urines from 188 consecutive patients diagnosed with bladder cancer were collected and frozen at diagnosis. After 15 years follow-up 13 patients were still alive, and 175 patients had died. MMP-9 was measured with an immunocapture activity assay. Results. Median MMP-9total was 173.7 units/10 g creatinine (range 0–34 792), and median MMP-9active was 14 units/10g creatinine (range, 0–294 757). The two factors were correlated (Spearman´s rho 0.74, p<0.0001). High MMP-9total and MMP-9active were significantly correlated with large tumour size and poor malignancy grade. Increasing tertiles of MMP-9total and MMP-9active were associated with poor overall survival (p<0.0001 and p=0.003, respectively). A Cox multivariate analysis using death as endpoint identified high tertiles of MMP-9total as independent prognostic markers with a relative risk 2.25 (95% confidence interval, 1.53–3.30). Conclusion. MMP-9 measured in urine from bladder cancer patients was a strong independent prognostic marker of poor survival. This is the first time high levels of MMP-9 measured in urine from bladder cancer patients have been linked to poor prognosis. This may reflect MMP-9 playing a role in tumour invasion and metastasis. It may be possible to non-invasively measure tumour response to therapy and identify possible tumour recurrence in an early phase.

CD10 + Pancreatic Stellate Cells Enhance the Progression of Pancreatic Cancer

Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer by producing extracellular matrix and soluble factors. However, the functional heterogeneity of PSCs has not been identified until now. Detailed characterization of the PSCs in human pancreatic cancer would provide a set of potential targets for stroma-directed therapy. We isolated PSCs from fresh pancreatic ductal adenocarcinoma tissue and sorted them by flow cytometry according to cell surface expression of CD10, which is a stromal prognostic marker for various tumors. We analyzed the functional differences between CD10(+) PSCs and CD10(-) PSCs. Immunohistochemical analysis showed that the frequency of CD10 expression by PSCs was markedly higher in tumor tissue than in normal tissue (33.7% vs 0%, respectively, P = .028). In pancreatic ductal adenocarcinoma, CD10 expression by PSCs was associated with positive nodal metastases (P = .011) and a shorter survival time (P < .001). In vitro coculture experiments showed that CD10(+) PSCs promoted the invasiveness of pancreatic cancer cell lines, SUIT-2 and Panc-1 cells more intensively than CD10(-) PSCs. CD10(+) PSCs significantly increased the tumor growth and invasiveness of SUIT-2 cells in a murine cotransplantation model. CD10(+) PSCs secreted higher levels of matrix metalloproteinase 3 than CD10(-) PSCs, and knockdown of matrix metalloproteinase 3 in cocultured PSCs reduced the invasion of SUIT-2 and Panc-1 cells. CD10(+) PSCs enhance the progression of pancreatic cancer cells. CD10(+) PSCs may be a candidate for selective therapeutic targeting in the treatment of pancreatic cancer.

Microarray-based Transcriptional and Epigenetic Profiling of Matrix Metalloproteinases, Collagens, and Related Genes in Cancer

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Persistent Chronic Inflammation and Infection by Chikungunya Arthritogenic Alphavirus in Spite of a Robust Host Immune Response

Alphaviruses, including Chikungunya virus (CHIKV), produce a transient illness in humans, but severe forms leading to chronic incapacitating arthralgia/arthritis have been reported by mechanisms largely ill-characterized. The pathogenesis of CHIKV was addressed in a prospective cohort study of 49 hospitalized patients from Reunion Island subsequently categorized into two distinct groups at 12 mo postinfection. Comprehensive analyses of the clinical and immunological parameters throughout the disease course were analyzed in either the "recovered" or the "chronic" groups to identify prognostic markers of arthritis-like pathology after CHIKV disease. We found that the chronic group consisted mainly of more elderly patients (>60 y) and with much higher viral loads (up to 10(10) viruses per milliliter of blood) during the acute phase. Remarkably, a rapid innate immune antiviral response was demonstrated by robust dendritic/NK/CD4/CD8 cell activation and accompanied by a rather weak Th1/Th2 cytokine response in both groups. Interestingly, the antiviral immune response witnessed by high levels of IFN-alpha mRNA in PBMCs and circulating IL-12 persisted for months only in the chronic group. CHIKV (RNA and proteins) was found in perivascular synovial macrophages in one chronic patient 18 mo postinfection surrounded by infiltrating NK and T cells (CD4(++) but rare cytotoxic CD8). Fibroblast hyperplasia, strong angiogenesis, tissue lesions given the high levels of matrix metalloproteinase 2, and acute cell death [high cleaved poly(ADP-ribose) polymerase staining] were observed in the injured synovial tissue. These observed cellular and molecular events may contribute to chronic arthralgia/arthritis targeted by methotrexate used empirically for effective treatment but with immunosuppressive function in a context of viral persistence.

Adjuvant glycerol is not beneficial in experimental pneumococcal meningitis

BackgroundBacterial meningitis in children causes high rates of mortality and morbidity. In a recent clinical trial, oral glycerol significantly reduced severe neurological sequelae in paediatric meningitis caused by Haemophilus influenzae type b, and a tendency towards a benefit of adjunctive glycerol was seen in pneumococcal meningitis.MethodsHere we examined the effects of glycerol in pneumococcal meningitis of infant rats and adult mice. All animals received ceftriaxone, and glycerol or placebo. Brain damage, hearing loss, and inflammatory parameters were assessed.ResultsClinically and by histopathology, animals treated with glycerol or placebo did not differ. While both groups showed equally high levels of matrix metalloproteinase-9 at 24 h after infection, a significant difference in favour of glycerol was observed at 40 h after infection. However, this difference in matrix metalloproteinase-9 in late disease did not result in an improvement of histopathologic parameters.ConclusionNo benefit of adjunctive glycerol was found in these models of pneumococcal meningitis.

The essential roles of matrix metalloproteinase-2, membrane type 1 metalloproteinase and tissue inhibitor of metalloproteinase-2 in the invasive capacity of acute monocytic leukemia SHI-1 cells

The frequency of extramedullary infiltration (EMI) in acute myeloblastic leukemia (AML) is reported up to 40% and most prevalent in myelo-monoblastic and monoblastic subtypes of AML (M4 and M5 according to FAB classification). The majority of patients with EMI suffered poor prognosis. To explore mechanism underlying EMI, we analyzed SHI-1 cells, a highly invasive human acute monocytic leukemia cell line, and found their strong expression of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2). SHI-1 cells showed higher invasive ability to traverse reconstituted basement membranes (Matrigel) and stronger activation of proMMP-2 than other leukemia cell line such as NB4, K562, U937 and THP-1 cells. When co-cultured with bone marrow stromal cells (BMSCs), the invasive capacity and proMMP-2 activation of SHI-1 cells enhanced remarkably. Furthermore, the inhibition of MMP-2, MT1-MMP, or TIMP-2 by small interfering RNA (siRNA) substantially impaired SHI-1 cells invasion and decreased proMMP-2 activation. In the contrast, up-regulated expression of TIMP-2 for 2–3 folds level increased cell invasion and proMMP-2 activation. These results demonstrated that constitutively high expression of MMP-2, MT1-MMP and TIMP-2 in SHI-1 cells facilitated cell invasion by promoting proMMP-2 activation. Moreover, up-regulation of TIMP-2 exhibited not a repressive but an activating effect on SHI-1 cells invasion. Our study indicated that increasing TIMP-2 in AML patients with EMI may potentially cause adverse effects, particularly in patients containing high levels of MMP-2 and MT1-MMP.

Matrix metalloproteinase‐9 in fulminant hepatic failure†

We read with interest the article by Chen et al.,1 who found altered expression of several tight junction (TJ) proteins in cultured brain endothelial cells and brain from mice with acute liver failure (ALF) and relate these abnormalities to the activation of matrix metalloproteinase-9 (MMP-9). The results are in accordance to prior data in the same animal model of toxic liver injury (azoxymethane).2 Their findings led to the proposal that MMP-9 released by the necrotic liver could alter the expression of TJ proteins and cause blood-brain barrier (BBB) leakage and brain swelling.3 The hypothesis is interesting because it could result in new treatments for this severe complication of fulminant hepatic failure (FHF). To further explore the relevance of this hypothesis, we determined the plasmatic levels of MMP-9 (via enzyme-linked immunosorbent assay) in 32 patients with FHF and compared the values to those obtained in 11 patients with acute hepatitis A and 20 patients with advanced (Child class B/C) hepatic cirrhosis with or without hepatic encephalopathy. During the follow-up of patients with FHF, intracranial hypertension was diagnosed in 14 patients (confirmed by intracranial pressure monitor in 13). We found high levels of MMP-9 compared to normal reference values in all group of patients (Fig. 1). Patients with acute liver injury (FHF or acute hepatitis A) showed higher values than those with chronic liver failure (cirrhosis). There was no association between MMP-9 and intracranial hypertension. Plasmatic levels of MMP-9 as determined by an enzyme-linked immunosorbent assay. FHF, fulminant hepatic failure; HC, hepatic cirrhosis; HE, hepatic encephalopathy; ICH, intracranial hypertension. *Compared to controls. #Compared to HC with or without HE. The presence of high levels of MMP-9 in our patients can be explained by remodeling liver parenchyma during acute and chronic liver injury.4 The lack of relationship with intracranial hypertension does not invalidate that MMP-9 can cause disturbances of TJ proteins. However, our results are in accordance with a series of data indicating that brain edema in FHF is mostly secondary to cytotoxic mechanisms. In vasogenic edema, brain swelling relates to leakage of the BBB and develops in the extracellular compartment. In comparison, cytotoxic edema develops secondary to osmotic differences across the BBB or energy failure and causes accumulation of water in the intracellular compartment. Most available data indicate that the BBB is grossly intact in FHF.5 Magnetic resonance shows a decrease in the apparent diffusion coefficient in humans6 and in rats,7 which is in accordance with an increase of water in the intracellular compartment. Our findings indicate that MMP-9 is increased in the plasma of patients with FHF, but does not participate in the pathogenesis of brain edema. MMPs are big molecules that must cross the BBB to exert their function in brain tissue. In patients and experimental models of stroke, the effects of MMPs are associated with neutrophil infiltration that may carry some of those MMPs in their tertiary granules.8 In previous neuropathological studies, no signs of neutrophil infiltration were detected in patients with FHF.5 Recently, Bémeur et al.9 performed a study in mice with azoxymethane-induced ALF, where they strictly controlled the temperature (a factor that affects the outcome of this model) and could not detect immunoglobulin G extravasation, in accordance with BBB integrity. Lluis Palenzuela Ph.D.* , Antoni Mas M.D. §, Joan Montaner M.D. , Juan Cordoba M.D.* §, * Liver Unit and Institut de Recerca, Hospital Universitari Vall d'Hebron, Barcelona, Spain, Neurovascular Research Laboratory, Hospital Universitari Vall d'Hebron, Barcelona, Spain, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, Madrid, Spain, § Liver Unit and Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clinic i Provincial, Barcelona, Spain.

Matrix Metalloproteinases in the Urine and Tissue of Patients With Juvenile Polyps: Potential Biomarkers for the Presence of Polyps

Juvenile polyps are the most common type of pediatric gastrointestinal polyp and are typically characterized as hamartomatous overgrowths. Juvenile polyps are highly vascularized tissues and display a markedly increased mucosal microcirculation. Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that play an essential role in the physiological degradation of the extracellular matrix in normal development, tumor invasion, and metastasis, as well as angiogenesis. We hypothesized that the presence of these enzymes in urine may serve as clinical biomarkers of juvenile polyps. In this preliminary study, we analyzed 32 urine samples collected prospectively from 16 subjects with known or suspected juvenile polyps who presented to the endoscopy unit for colonoscopic evaluation and 16 age- and sex-matched controls. Urinary MMPs were analyzed by zymography and their localized tissue expression was assayed via immunohistochemistry of tissue sections. MMPs were detected in the urine of patients with juvenile polyps with significantly higher frequency when compared with urine of control subjects. In addition, immunohistochemistry demonstrated that high levels of MMPs were localized in the epithelium and lamina propria of polyp tissue when compared with colonic tissue collected from healthy control subjects. These data are the first to demonstrate that MMPs are present in the urine and tissue of patients with juvenile polyps, and these enzymes have the potential to serve as surrogate markers for the presence of polyps.

Evaluation of metalloproteinase 2 and 9 levels and their inhibitors in combined dyslipidemia

To evaluate the distribution of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and their specific inhibitors in a sample of patients affected by mild dyslipidemia but not yet treated with antihyperlipidemic drugs. One hundred and sixty-eight Caucasian patients aged >or=18 yr of either sex with combined dyslipidemia and who had never previously taken lipid-lowering medications were evaluated. As a control population, we enrolled 179 Caucasian healthy subjects, aged >or=18 yr of either sex. We evaluated body mass index (BMI), fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostasis model assessment (HOMA index), systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), triglycerides (Tg), lipoprotein(a) Lp(a), plasminogen activator inhibitor-1 (PAI-1), homocysteine (Hct), fibrinogen (Fg), high sensitivity C-reactive protein (Hs-CRP), adiponectin (ADP), MMP-2, MMP-9, tissue inhibitors of metalloproteinase-1 (TIMP-1), and tissue inhibitors of metalloproteinase-2 (TIMP-2). TC, Tg, and LDL-C were higher (P < < 0.05, P < < 0.01 and P < < 0.05, respectively) in the dyslipidemic group, while HDL-C levels were lower (P < < 0.01) compared with the control group. Increases of PAI-1, Hct, Fg, and Hs-CRP (P < < 0.01, P < < 0.05, P < < 0.05, and P < < 0.05, respectively) were present in the dyslipidemic group, while ADP level was lower (P < < 0.01) in the dyslipidemic patients compared with controls. MMP-2, MMP-9, TIMP-1, and TIMP-2 levels were higher (P < < 0.0001) in the dyslipidemic group. Combined hyperlipidemic patients have increased levels of prothrombotic and microinflammatory parameters and higher levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 than control subjects. The prognostic importance of this observation has to be evaluated in adequately designed prospective studies.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate differences in MMP and TIMP levels in the gingival crevicular fluid (GCF) at the resorption and apposition sides of orthodontically moved teeth, and to compare these with control teeth. GCF samples were collected from eight orthodontic patients wearing fixed appliances with superelastic nickel-titanium coil springs. The samples were analysed by gelatin zymography, which allows detection of both active and latent MMPs, and reverse zymography for analysis of TIMPs. Western blotting was performed to confirm the identity of MMPs. The data were analysed using either the one-way analysis of variance or the Kruskal-Wallis test. In general, higher levels of MMPs and TIMPs were found at both the resorption and apposition sides compared with the control teeth. Remarkably, partially active MMP-1 was found in GCF from both the resorption and the apposition side but was barely present at the control teeth. TIMP-1 was strongly increased at the apposition side. Gelatinases were mainly present at the resorption side, while gelatinolytic fragments were exclusively detected at the apposition side. MMP-9, which is known to be involved in bone degradation, and a 48 kDa gelatinase were increased at the resorption side. The small increase in TIMP-1 at the resorption side might stimulate bone resorption, whereas the large increase at the apposition side reduces bone resorption. The analysis of MMPs and TIMPs may contribute to the improvement of orthodontic treatment regimens.

Macrophage fusion, giant cell formation, and the foreign body response require matrix metalloproteinase 9

Macrophages undergo fusion to form multinucleated giant cells in several pathologic conditions, including the foreign body response (FBR). We detected high levels of matrix metalloproteinase (MMP)-9 during macrophage fusion in vitro and in foreign body giant cells (FBGCs) in vivo. Wild-type (WT) bone marrow-derived macrophages were induced to fuse with IL-4 in the presence of MMP-9 function-blocking antibodies and displayed reduced fusion. A similar defect, characterized by delayed shape change and abnormal morphology, was observed in MMP-9 null macrophages. Analysis of the FBR in MMP-9 null mice was then pursued to evaluate the significance of these findings. Specifically, mixed cellulose ester disks and polyvinyl alcohol sponges were implanted s.c. in MMP-9 null and WT mice and excised 2-4 weeks later. Histochemical and immunohistochemical analyses indicated equal macrophage recruitment between MMP-9 null and WT mice, but FBGC formation was compromised in the former. In addition, MMP-9 null mice displayed abnormalities in extracellular matrix assembly and angiogenesis. Consistent with a requirement for MMP-9 in fusion, we also observed reduced MMP-9 levels in MCP-1 null macrophages, previously shown to be defective in FBGC formation. Collectively, our studies show abnormalities in MMP-9 null mice during the FBR and suggest a role for MMP-9 in macrophage fusion.

Endothelial cell/macrophage cocultures as a model to studyStreptococcus suis-induced inflammatory responses

Endothelial cells and macrophages are thought to play a critical role in the inflammatory response that contributes to meningitis. To investigate the interactions between Streptococcus suis and these two cell types, we developed a coculture model composed of brain microvascular endothelial cells and macrophage-like cells, and characterized the production of proinflammatory cytokines, chemokines, prostaglandin E(2) (PGE(2)), and matrix metalloproteinase 9 (MMP-9) following a challenge with bacteria. Streptococcus suis cells stimulated the secretion of all the inflammatory mediators as well as MMP-9 in the coculture model. Responses to S. suis infection were influenced by endothelial cell/macrophage ratios and were dependent on the multiplicity of infection. Except for IL-6, significantly higher amounts of inflammatory mediators and MMP-9 were produced with the coculture model at an endothelial cell/macrophage ratio of 1 : 10 than at a ratio of 1 : 1. When infected with S. suis, endothelial cells and macrophages acted in synergy to increase the secretion of IL-6 and PGE(2). Using a model that more closely reproduces the in vivo situation, we showed that S. suis can induce the secretion of high levels of inflammatory mediators and MMP-9, which may contribute to the development of meningitis.