Tissue Inhibitor of Metalloproteinases-2 Mediates Kidney Injury during Sepsis.

INTRODUCTION: Urinary tissue inhibitor of metalloproteinases (TIMP)-2 has been identified as a predictive marker for acute kidney injury (AKI), including sepsis-associated AKI (S-AKI). Whether TIMP-2 might be causally related to AKI and hence represent a viable drug target is unclear. OBJECTIVE: The aim of this study was to evaluate whether suppression of TIMP-2 attenuates S-AKI. METHODS: Balb/c mice were randomized to sham or cecal ligation and puncture surgery and treated with or without a TIMP-2-neutralizing antibody. Animals were followed for 48 h and then sacrificed for analysis of TIMP-2 expression, cell cycle, and histology. RESULTS: Anti-TIMP-2 resulted in decreased lumen TIMP-2 expression which markedly increased cell cycle progression and attenuated epithelial cell injury by histology. CONCLUSIONS: TIMP-2 mediates S-AKI and appears to be a viable drug target.

Matrix Metalloproteinases System and Types of Fibrosis in Rat Heart during Late Pregnancy and Postpartum.

Background and objectives: Cardiac remodeling in pregnancy and postpartum is poorly understood. The aim of this study was to evaluate changes in cardiac fibrosis (pericardial, perivascular, and interstitial), as well as the expression of matrix metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (Tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-4) during late pregnancy and postpartum in rat left ventricle. Materials and Methods: Female Sprague-Dawley rats were used for this study. Rats were divided three groups: non-pregnant, late pregnancy, and postpartum. The heart was weighed and cardiac fibrosis was studied by conventional histological procedures. The expression and transcript level of target proteins were evaluated using immunoblot techniques and quantitative PCR. Results: The experiments showed an increase of perivascular, pericardial, and interstitial fibrosis in heart during pregnancy and its reversion in postpartum. Moreover, in late pregnancy, MMP-1, MMP-2, and MMP-9 metalloproteinases were downregulated and TIMP-1 and TIMP-4 were upregulated in left ventricle. Conclusions: Our data suggest that the metalloproteinases system is involved in the cardiac extracellular matrix remodeling during pregnancy and its reversion in postpartum, this improves the knowledge of the adaptive cardiac remodeling in response to a blood volume overload present during pregnancy.

Exogenous Tissue Inhibitor of Metalloproteinase-2 Affects Matrix Metalloproteinase-2 Expression in Conjunctival Filtering Blebs and Bleb Scarring in Rats.

OBJECTIVE: To examine the effect of tissue inhibitor of metalloproteinase-2 (TIMP-2) on conjunctival filtering bleb scarring. METHODS: A model of conjunctival filtering bleb was established whereby rats were injected with saline, blank adenoviral vector, or adenoviral vector carrying TIMP-2 into the bleb. Filtration bleb formation and matrix metalloproteinase-2 (MMP-2) expression were examined. RESULTS: All operated eyes formed obvious elevated blebs on day 1. In the normal saline group, empty plasmid group, and gene transfection group maintenance time of filtrating blebs was 5-14, 5-14, and 6-16 days, and average survival time was 8.24, 8.16, and 9.44 days, respectively. MMP-2 expression increased slightly in the gene transfection group at 3 and 5 days after surgery, reached a peak after 14 days, and then gradually decreased. MMP-2 expression was weakly positive in the normal conjunctival epithelium, but was hardly detected in the lamina propria. Seven days after surgery, the epithelium and lamina propria of the conjunctival filtering bleb exhibited strong positive expression in the empty plasmid group but only weak expression in the adenovirus group. CONCLUSION: Exogenous TIMP-2 interfered with local MMP-2 expression, delaying peak expression of MMP-2 and slowing the scarring of filtering blebs during wound healing of subconjunctival tissue.

New insights into the natural course of knee osteoarthritis: early regulation of cytokines and growth factors, with emphasis on sex-dependent angiogenesis and tissue remodeling. A pilot study.

OBJECTIVE: This study was conducted to identify cytokine profiles associated with radiographic phenotypes of knee osteoarthritis (rKOA) with a focus on early stage of the disease. METHODS: The pilot population study involved 60 middle-aged patients (mean age 50 ± 7.3y.). Standardized weight-bearing anteroposterior and axial radiographs were used to assess rKOA severity in tibiofemoral (TFJ) of patellofemoral joint (PFJ) by grading system (grades 0-3). Luminex (xMAP®) technology was used to simultaneously assess 60 biomarkers (BMs). RESULTS: Several pathways of angiogenic (CXCL10/IP-10, FGF1/2, PDGF-AA/BB, ANG1, RANTES), tissue remodeling/fibrosis (MMP1/3, TIMP2/3/4, TGFß), and fat tissue (leptin) BMs associated with rKOA severity already in very early phase (grade 1). We identified several sets of cytokines as key markers of early knee osteoarthritis (KOA) predicting radiographic features in logistic-regression models (AUC = 0.80-0.97). Marked sex-specificity of rKOA course was detected: upregulation of angiogenesis dominated in females, whereas the activation of tissue remodeling was dominant in males. Several of these shifts, e.g., decrease of CXCL10/IP-10, took place only in grade 1 KOA and disappeared or reversed in later stages. OA of different knee-joint compartments has distinct profiles of cytokines. A broad list of BMs (TIMP2/3/4, MMP1/3, TGFß1/2, vWF-A2, sE-selectin and leptin) associated with OA in the PFJ. CONCLUSION: Our results demonstrate that substantial and time-limited shifts in the angiogenic and TIMP/MMP systems occur in the early stage of KOA. Our study findings highlight the sex-, grade- and compartment-dependent shifts in above processes. The data may contribute to the individualized prevention of KOA in the future.

Involvement of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the regression of chicken postovulatory follicles.

The study was undertaken to examine mRNA expression and localization of selected matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and the activity of MMPs in chicken postovulatory follicles (POFs) during their apoptotic regression. Apoptotic cells and apoptosis-related caspase expression and activity were examined as well. Chickens were sacrificed 2 h and 21 h after ovulation, and five POFs (POF1 to POF5) were isolated from the ovaries. It was found that the number of apoptotic cells (TUNEL-positive) increased along with follicle regression. The relative expression (RQ) of caspase-2, -3, -8 and -9 mRNA increased (P < 0.05) in POF5, while the activity of all examined caspases elevated gradually (approximately 80-150%) reaching the highest level in POF3, and then slowly decreased to the value noted in POF1 (P < 0.05 - P < 0.001). Real-time polymerase chain reaction revealed different expression of MMP-2, -7, -9 and TIMP-2 and -3 on mRNA levels, and activity assay showed the changes in activity of MMP-2 and -9 in the POFs. Regression of the follicles was accompanied predominantly by an increase in the relative expression of MMP-2, and a decrease in TIMP-2 and -3 mRNAs (P < 0.05 - P < 0.001). The activity levels of MMP-2 and -9 showed pronounced changes during the examined period. During follicle regression elevated activity of MMP-2 and -9 was found (P < 0.05 - P < 0.001). Immunohistochemistry demonstrated tissue- and follicle-dependent immunoreactivity of the examined members of the MMP system. In summary, the results showing the apoptotic regression-related changes as well as tissue-dependent differences in the expression of selected MMPs and TIMPs, and activity of MMP-2 and MMP-9, point to the significance that these molecules might participate in the complex orchestration of chicken POF regression.

Gelatinases and their tissue inhibitors are associated with oxidative stress: a potential set of markers connected with male infertility.

The expression and activity of matrix metalloproteinases (MMPs) may be regulated by oxidative stress in various pathophysiological processes; therefore, the aim of the present study was to analyse the associations between the expression of the gelatinases MMP-9 and MMP-2 and their tissue inhibitors TIMP-1, TIMP-2 and levels of total antioxidant capacity (TAC) and advanced oxidation protein products (AOPP) in seminal plasma prepared for artificial insemination. Levels of MMPs and TIMPs were evaluated using ELISA, whereas TAC and AOPP in the seminal plasma of 131 childless men and 38 fertile volunteers were determined spectrophotometrically. Seminal MMP-9 expression was higher in childless men than in fertile subjects, whereas there was no significant differences in MMP-2 expression between the analysed seminal groups. TIMP-1 and TIMP-2 expression was similar in all groups. However, TAC expression was significantly higher in infertile normozoospermic and oligozoospermic men and AOPP expression was higher in astheno-, oligo- and normozoospermic infertile patients than in fertile men. High AOPP, together with an increased MMP-9:TIMP-1 ratio alters the oxidative-antioxidative balance of the ejaculate, thereby reducing male fertility, and therefore these parameters may serve as additional diagnostic markers of semen quality and male reproductive potential.

Oncogenic miR-20a and miR-106a enhance the invasiveness of human glioma stem cells by directly targeting TIMP-2.

Emerging evidence has shown that cancer stem cells (CSCs) are the cellular determinants to promote cancer invasion and metastasis. However, the mechanism underlying CSC invasion remains unknown. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression, and their expressions are often dysregulated in cancers. In the present study, we demonstrated that two functionally related microRNAs, miR-20a and -106a (miR-20a/106a), were capable of enhancing the invasiveness of CD133(+) glioma stem cells (GSCs) isolated from both glioblastoma cell line U87 and primary human glioma specimens. We found that the level of miR-20a/106a in GSCs was significantly higher than that in the committed CD133(-) glioma cells, and correlated with the invasive capability of GSCs. By bioinformatic analysis, we identified tissue inhibitor of metalloproteinases-2 (TIMP-2) as one of the miR-20a/106a-targeted genes. TIMP-2 level correlated inversely with miR-20/106 expression. Directly targeting by miR-20a/106a on 3'-untranslation region (3'-UTR) of TIMP-2 mRNA was confirmed by 3'-UTR dual-luciferase reporter assay. Knockdown of miR-20a/106a in GSCs increased endogenous TIMP-2 protein abundance, thereby inhibiting GSC invasion. We also found that Nordy, a synthetic lipoxygenase inhibitor, inhibited GSC invasiveness by elevating the expression of TIMP-2 via downregulation of miR-20a/106a. Our results indicate that miR-20a/106a has a key role in GSC invasion and may serve as targets for treatment of glioblastoma.

Antiangiogenic activity of trabectedin in myxoid liposarcoma: involvement of host TIMP-1 and TIMP-2 and tumor thrombospondin-1.

Trabectedin is a marine natural product, approved in Europe for the treatment of soft tissue sarcoma and relapsed ovarian cancer. Clinical and experimental evidence indicates that trabectedin is particularly effective against myxoid liposarcomas where response is associated to regression of capillary networks. Here, we investigated the mechanism of the antiangiogenic activity of trabectedin in myxoid liposarcomas. Trabectedin directly targeted endothelial cells, impairing functions relying on extracellular matrix remodeling (invasion and branching morphogenesis) through the upregulation of the inhibitors of matrix metalloproteinases TIMP-1 and TIMP-2. Increased TIMPs synthesis by the tumor microenvironment following trabectedin treatment was confirmed in xenograft models of myxoid liposarcoma. In addition, trabectedin upregulated tumor cell expression of the endogenous inhibitor thrombospondin-1 (TSP-1, a key regulator of angiogenesis-dependent dormancy in sarcoma), in in vivo models of myxoid liposarcomas, in vitro cell lines and primary cell cultures from patients' myxoid liposarcomas. Chromatin Immunoprecipitation analysis showed that trabectedin displaced the master regulator of adipogenesis C/EBPß from the TSP-1 promoter, indicating an association between the up-regulation of TSP-1 and induction of adipocytic differentiation program by trabectedin. We conclude that trabectedin inhibits angiogenesis through multiple mechanisms, including directly affecting endothelial cells in the tumor microenvironment--with a potentially widespread activity--and targeting tumor cells' angiogenic activity, linked to a tumor-specific molecular alteration.

Matrix metalloproteinase-2 of human carotid atherosclerotic plaques promotes platelet activation. Correlation with ischaemic events.

Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p<0.05). Moreover, the platelet aggregation-potentiating effect, the active-MMP-2 content and the active MMP-2/pro-MMP-2 ratio of plaque extracts were significantly higher in plaques from patients who developed a subsequent major cardiovascular event. In conclusion, atherosclerotic plaques exert a prothrombotic effect by potentiating platelet activation due to their content of MMP-2; an elevated MMP-2 activity in plaques is associated with a higher rate of subsequent ischaemic cerebrovascular events.

Serum levels and tissue expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinases 2 (TIMP-2) in colorectal cancer patients.

The objective of the study was the assessment of serum levels and tissue expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in patients with colorectal cancer (CRC). The study included 72 CRC patients and 68 healthy subjects. The serum levels of MMP-2 and TIMP-2 were measured using enzyme-linked immunosorbent assay (ELISA) method, whereas tissue expression of MMP-2 and TIMP-2 in cancer cells, interstitial inflammatory cells, and adjacent normal colorectal mucosa were examined by immunohistochemical staining of tumor samples. The serum levels of MMP-2 and TIMP-2 in cancer patients were significantly lower than those in control group, but the percentage of positive immunoreactivity of these proteins were higher in malignant and inflammatory cells as compared to normal tissue. There was a significant correlation between MMP-2 immunoreactivity in inflammatory cells and the presence of distant metastases and between TIMP-2 expression in inflammatory cells and tumor size, nodal involvement, and distant metastases. Area under receiver operating characteristic (ROC) curve (AUC) for serum MMP-2 was higher than for serum TIMP-2. Moreover, positive tissue expression of MMP-2 was a significant prognostic factor for CRC patients' survival. Our findings suggest that MMP-2 and TIMP-2 might play a role in the process of colorectal cancer invasion and metastasis, but the significance of their interactions with tumor stroma and interstitial inflammatory infiltration in colorectal neoplasia require further elucidation.

Effects of exercise on markers of venous remodeling in lungs of horses.

OBJECTIVE: To determine the effects of 2 weeks of intense exercise on expression of markers of pulmonary venous remodeling in the caudodorsal and cranioventral regions of the lungs of horses. ANIMALS: 6 horses. PROCEDURES: Tissue samples of the caudodorsal and cranioventral regions of lungs were obtained before and after conditioning and 2 weeks of intense exercise. Pulmonary veins were isolated, and a quantitative real-time PCR assay was used to determine mRNA expression of matrix metalloproteinase-2 and -9, tissue inhibitor of metalloproteinase-1 and -2, collagen type I, tenascin-C, endothelin-1, platelet-derived growth factor, transforming growth factor (TGF)-ß, and vascular endothelial growth factor (VEGF). Protein expression of collagen (via morphometric analysis) and tenascin-C, TGF-ß, and VEGF (via immunohistochemistry) was determined. RESULTS: Exercise-induced pulmonary hemorrhage was detected in 2 horses after exercise. The mRNA expression of matrix metalloproteinase-2 and -9, tissue inhibitor of metalloproteinase-2, TGF-ß, and VEGF was significantly lower in pulmonary veins obtained after exercise versus those obtained before exercise for both the caudodorsal and cranioventral regions of the lungs. Collagen content was significantly higher in tissue samples obtained from the caudodorsal regions of the lungs versus content in samples obtained from the cranioventral regions of the lungs both before and after exercise. Exercise did not alter protein expression of tenascin-C, TGF-ß, or VEGF. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study indicated 2 weeks of intense exercise did not alter expression of marker genes in a manner expected to favor venous remodeling. Pulmonary venous remodeling is complex, and > 2 weeks of intense exercise may be required to induce such remodeling.

TIMP-2 mutant decreases MMP-2 activity and augments pressure overload induced LV dysfunction and heart failure.

Pressure overload induces cardiac extracellular matrix (ECM) remodelling and results in heart failure. ECM remodelling by matrix metalloproteinases (MMPs) is primarily regulated by their target inhibitors, tissue inhibitor of matrix metalloproteinases (TIMPs). It is known that TIMP-2 is highly expressed in myocardium and is required for cell surface activation of pro-MMP-2. We and others have reported that imbalance between angiogenic growth factors and anti-angiogenic factors results in transition from compensatory cardiac hypertrophy to heart failure. We previously reported the pro-angiogenic role of MMP-2 in cardiac compensation, however, the specific role of TIMP-2 during pressure overload is yet unclear. We hypothesize that genetic ablation of TIMP-2 exacerbates the adverse cardiac matrix remodelling due to lack of pro-angiogenic MMP-2 and increase in anti-angiogenic factors during pressure overload stress and results in severe heart failure. To verify this, ascending aortic banding (AB) was created to mimic pressure overload, in wild type C57BL6/J and TIMP-2-/- (model of MMP-2 deficiency) mice. Left ventricular (LV) function assessed by echocardiography and pressure-volume loop studies showed severe LV dysfunction in TIMP-2-/- AB mice compared to controls. Expression of MMP-2, vascular endothelial growth factor (VEGF) was decreased and expression of MMP-9, anti-angiogenic factors endostatin and angiostatin was increased in TIMP-2-/- AB mice compared with wild type AB mice. Connexins (Cx) are the gap junction proteins that are widely present in the myocardium and play an important role in endothelial-myocyte coupling. Our results showed that expression of Cx 37 and 43 was decreased in TIMP-2-/- AB mice compared with corresponding wild type controls. These results suggest that genetic ablation of TIMP-2 decrease the expression of pro-angiogenic MMP-2, VEGF and increases anti-angiogenic factors that results in exacerbated abnormal ventricular remodelling leading to severe heart failure.

MMP-8 overexpression and persistence of neutrophils relate to stress-impaired healing and poor collagen architecture in mice.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are critical for tissue remodeling during wound repair. Psychological stress has been found to impair wound healing in humans and animals. The objective of this study was to assess MMP and TIMP gene expression during stress-impaired healing. Female SKH-1 mice (n=299) were divided into control and stress groups (13h restraint/day for 3days prior to and 5days post-wounding). Two 3.5mm cutaneous full-thickness wounds were placed on the dorsum of each mouse and wound measurements were performed daily. RT-PCR for gene expression of MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 was performed at days 1, 3 and 5. Immunohistochemical analyses of the healed wounds were performed at days 15 and 28. As expected, wounds healed more slowly in restraint-stressed mice compared to controls. Stressed mice exhibited MMP-8 overexpression and lower TIMP-1 levels during healing, and poorer collagen organization once healed. MMP-8 overexpression may have stemmed from a higher level of neutrophils, observed in wound tissue on days 3 and 5. These findings implicate higher neutrophil numbers, MMP-8 overexpression, and TIMP-1 under-expression, as mechanisms that may compromise wound outcomes such as scarring under conditions of stress.

TIMP2 deficient mice develop accelerated osteoarthritis via promotion of angiogenesis upon destabilization of the medial meniscus.

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.

Seizure activity in dogs is associated with enhanced TIMP-2 expression of microglia.

In the pathogenesis of epilepsy aberrant synaptic plasticity plays an important role. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are responsible for nervous tissue remodelling resulting in synaptic plasticity in the central nervous system (CNS) and might therefore be crucially involved in epileptogenesis. To assess the potential pathogenetic role of microglial MMPs and TIMPs in seizure induction, twenty-four dogs suffering from different intracranial diseases with and without seizure activity were comparatively examined. Microglial cells were isolated by density gradient centrifugation and their expression profiles of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) were examined via quantitative real-time PCR (qPCR). Interestingly, a significant up-regulation of TIMP-2 expression was found for the first time in dogs suffering from seizures. In conclusion, microglial TIMP expression might be involved in seizure generation.

Biochemical comparison of osteoarthritic knees with and without effusion.

BACKGROUND: Several symptom-relieving interventions have been shown to be efficacious among osteoarthritis (OA) patients with knee effusion; however, not every symptomatic knee OA patient has clinical effusion. Results may be over-generalized since it is unclear if effused knees represent a unique pathological condition or subset compared to knees without effusion. The primary purpose of this study was to determine if biochemical differences existed between OA knees with and without effusion. METHODS: The present cross-sectional study consisted of 22 volunteers (11 with knee effusion, 11 without knee effusion) with confirmed late-stage radiographic knee OA (Kellgren-Lawrence score ≥ 3). Synovial fluid samples were collected and analyzed using a custom multiplex enzyme-linked immunosorbent assay to determine eight specific biomarker concentrations (e.g., catabolic, anabolic). RESULTS: Matrix metalloproteinase (MMP)-3, tissue inhibitor of MMPs (TIMP)-1, TIMP-2, and interleukin-10 were significantly higher in the knees with effusion than in the knees without effusion. CONCLUSIONS: The biochemical differences that existed between knees with and without effusion provide support that OA subsets may exist, characterized by distinct biochemical characteristics and clinical findings (e.g., effusion).

Endogenous angiogenesis inhibitor blocks tumor growth via direct and indirect effects on tumor microenvironment.

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) belongs to a small family of endogenous proteins that inhibits a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP-dependent and -independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, and enzyme-linked immunosorbent assay (ELISA), we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intratumoral microvascular density, and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and immunohistochemical analyses of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.

Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation.

Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover, these findings, together with our previous reports and the emerging data from in vivo studies of TIMP inhibition, provide strong evidence that TIMP-1 is mechanistically central to liver fibrosis and an important potential therapeutic target.

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention.

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-maternal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF(2α) analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stroma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle.