Pioglitazone Attenuates Lupus Nephritis Symptoms in Mice by Modulating miR-21-5p/TIMP3 Axis: the Key Role of the Activation of Peroxisome Proliferator-Activated Receptor-γ.

Lupus nephritis (LN) is a severe symptom of systemic lupus erythematosus and miR-21-5p is upregulated during LN. In the current study, the effects of pioglitazone (Pg), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, on LN development were assessed and explained by focusing miR-21-5p/TIMP3 axis. The expressions of miR-21-5p and PPARγ in LN mice were detected and then the mice were treated with pioglitazone to evaluate the anti-LN effects of agent. The miR-21-5p level was induced in MRL/lpr mice to confirm the central role of miR-21-5p inhibition in the protective effects of Pg against LN. The level of miR-21-5p was upregulated, while the level of PPARγ was downregulated in MRL/lpr mice. Pg inhibited miR-21-5p in renal tissues, which induced the expression of TIMP3. The changes in miR-21-5p/TIMP3 axis led to the improvements in renal structure and function, and inhibited autoimmune response. The induction of miR-21-5p impaired the effects of Pg, along with the suppression of TIMP3. The expression of miR-21-5p was associated with the progression of LN, contributing to the suppression of TIMP3 and development of LN. The inhibition of the miR-21-5p by Pg would restore the structure and function of kidneys in LN mice via the activation of PPARγ.

Resveratrol inhibits the tumor migration and invasion by upregulating TET1 and reducing TIMP2/3 methylation in prostate carcinoma cells.

BACKGROUND: Recently, resveratrol (Res) has been suggested to suppress the migration and invasion of prostate cancer (PCa). In the present study, we aimed to investigate the effects of Res on genomic DNA methylation, as well as the migration and invasion of PCa cells. METHODS: The suppression by Res of the growth of PCa cells was verified through a cytotoxicity assay. In addition, the effects of Res on 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and ten-eleven translocation 1 (TET1) levels were assessed, and the cell migration and invasion were also determined. The expressions of TET1, tissue inhibitor of metalloproteinases (TIMP) 2, TIMP3, MMP2, and MMP9 were detected through Western blot analysis. Afterward, TET1 was silenced using lentiviral short hairpin RNA to examine the effect of TET1 on the Res-triggered inhibition of migration and invasion of PCa cells. RESULTS: Our results showed that Res upregulated the 5hmC and TET1 levels and downregulated the 5mC level. Moreover, Res also inhibited the migration and invasion of PCa cells, promoted the demethylation of TIMP2 and TIMP3 to upregulate their expressions, and suppressed the expressions of MMP2 and MMP9. The silencing of TET1 in the presence of Res showed that Res could exert its effect through TET1. CONCLUSIONS: Our findings indicated that Res inhibited the migration and invasion of PCa cells via the TET1/TIMP2/TIMP3 pathway, which might potentially serve as a target for the treatment of PCa.

Comparative analysis of the expression of metalloproteases (MMP-2, MMP-9, MMP-11 and MMP-13) and the tissue inhibitor of metalloprotease 3 (TIMP-3) between previous negative biopsies and radical prostatectomies. / Análisis comparativo de la expresión de metaloproteasas (MMP-2, MMP-9, MMP-11 y MMP-13) y del inhibidor tisular de la metaloproteasa 3 (TIMP-3) entre las biopsias previas negativas y las prostatectomías radicales.

Metalloproteases (MMPs) and tissue inhibitor of metalloprotease-3 (TIMP-3) have been associated to the risk of having cancer and tumor aggressiveness. When facing the difficulties of prostate cancer diagnosis, the expression of MMPs and TIMP-3 in negative biopsies could be helpful to evaluate a diagnostic suspicion. Our objective is to carry out a comparative study of the expression of MMPs and TIMP-3 in previous negative biopsies and radical prostatectomies (RP). MATERIAL AND METHODS: Retrospective analysis of a hospital-based cohort including 21 patients with suspicion of prostate carcinoma, whose expressions of MMP-2, 9, 11 and 13 and TIMP-3 were evaluated by immunohistochemistry in the tumor area from previous negative biopsies and RP. RESULTS: Immunohistochemical staining values (Score) for MMPs (-11 and -13) and TIMP-3 showed no significant differences when comparing the areas of negative biopsies where tumors subsequently developed with those of the RP. However, we did observe a significant difference in the increased expression of MMP-2 (P=.002) and MMP-9 (P=.001) in the tumor area of the RP with respect to the corresponding area of the previous negative biopsy. CONCLUSIONS: Our data indicate a higher overall expression of MMP-2 and MMP-9 in the tumor area of the RP compared to the corresponding areas of the negative previous biopsy, which seems to be associated to the process of malignant transformation.

Transcriptomic expression levels of the VHL, TIMP-3, and RASSF1A genes in renal tumors.

OBJECTIVE: In this study, we aimed to investigate the relation between the mRNA expression levels of VHL, TIMP-3 and RASSF1A genes, and the histopathological and clinical characteristics of patients with renal tumors. PATIENTS AND METHODS: Radical nephrectomy specimens of cases presented without neoadjuvant treatment were confirmed to be cancerous, non-cancerous, benign, and healthy after removal from separate localizations. A total of 69 patients with kidney tumors (138 tissue samples) were included in the study group. RNA isolation, reverse transcriptase PCR (RT-PCR), and quantitative real time PCR (qPCR) were performed, and the GAPDH gene was used to normalize mRNA levels. RESULTS: In the RCC cancerous tissue, TIMP-3 levels increased 1.3 times and RASSF1A levels increased 1.4 times compared to the corresponding levels in non-cancerous tissues, and there was no statistically significant difference in these values. On the other hand, VHL gene expression levels in cancerous tissue were 2.8 times higher than in matched adjacent non-cancerous tissues (p < 0.05). In the case of oncocytomas, TIMP-3 levels were found to be 3.2 times higher, RASSF1A levels 3.8 times higher, and VHL levels 2.2 times lower than the corresponding levels in healthy tissues (p < 0.05). CONCLUSIONS: The roles of VHL, TIMP-3, and RASSF1A mRNA expression in contributing to the development of renal tumors could not be clearly established. Further studies are therefore required to elucidate the mechanisms underlying renal tumors.

Differential expression and localization of human tissue inhibitors of metalloproteinases in proliferative diabetic retinopathy.

PURPOSE: Tissue inhibitors of metalloproteinases (TIMPs) block the catalysis by matrix metalloproteinases (MMPs) and have additional biologic activities, including regulation of cell growth and differentiation, apoptosis, angiogenesis and oncogenesis. We investigated the expression levels of all the four human TIMPs and correlated these levels with those of MMP-9 and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 38 PDR and 21 nondiabetic control patients and epiretinal membranes from 14 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR) were studied by enzyme-linked immunosorbent assay, Western blot analysis and immunohistochemistry. RESULTS: Tissue inhibitor of metalloproteinases-1, TIMP-4, MMP-9 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic controls (p < 0.0001 for all comparisons), whereas TIMP-2 and TIMP-3 levels did not differ significantly. TIMP-1, TIMP-4, MMP-9 and VEGF levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p < 0.0001, 0.001, 0.013, 0.004, respectively). Significant positive correlations existed between levels of TIMP-1 and levels of TIMP-4 (r = 0.37; p = 0.004), MMP-9 (r = 0.65; p < 0.0001) and VEGF (r = 0.59; p < 0.0001), between levels of TIMP-4 and levels of MMP-9 (r = 0.61; p < 0.0001) and VEGF (r = 0.62; p < 0.0001) and between levels of MMP-9 and VEGF (r = 0.62; p < 0.0001). TIMP-1 and TIMP-3 were expressed in vascular endothelial cells in PDR epiretinal membranes and in myofibroblasts and leucocytes in PDR and PVR epiretinal membranes. CONCLUSION: The differential expression of TIMPs in PDR suggests that among the 4 TIMPs, TIMP-1 and TIMP-4 may be possible biomarkers of disease activity.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc.

Myocardial overexpression of TIMP3 after myocardial infarction exerts beneficial effects by promoting angiogenesis and suppressing early proteolysis.

Myocardial infarction (MI) results in loss of cardiomyocytes, adverse extracellular matrix (ECM) and structural remodeling, and left ventricular (LV) dilation and dysfunction. Tissue inhibitors of metalloproteinase (TIMPs) inhibit matrix metalloproteinases (MMPs), the main regulators of ECM turnover. TIMPs also have MMP-independent functions. TIMP3 levels are reduced in the heart within 24 h of MI in mice. We investigated if overexpression of TIMP3 post-MI limits adverse remodeling and LV dilation and dysfunction. MI was induced by left anterior descending coronary artery ligation in 10- to 12-wk-old male C57BL/6J mice, and adenoviral constructs expressing human (h)TIMP3 (Ad-hTIMP3) or no TIMP (Ad-Null) were injected in the peri-infarct zone (5.4 × 107 plaque-forming units/heart, 5 injections/heart). Cardiac function assessed by echocardiography showed improved LV physiology and reduced LV dilation after TIMP3 overexpression compared with the Ad-Null-MI group. Post-MI adverse remodeling was attenuated in the Ad-hTIMP3-MI group, as assessed by greater cardiomyocyte density, less infarct expansion, and ECM disruption. TIMP3 overexpression blunted the early rise in proteolytic activities post-MI. A higher density of coronary arteries and a greater number of proliferating endothelial cells were detected in the infarct and peri-infarct regions in the Ad-hTIMP3-MI group compared with the Ad-Null-MI group. In vitro three-dimensional angiogenesis assay confirmed that recombinant TIMP3 promotes angiogenesis in human endothelial cells, although biphasically and in a dose-dependent manner. Intriguingly, overexpression of Ad-hTIMP3 at 10-fold higher concentration had no beneficial effects, consistent with antiangiogenic effects of TIMP3 at higher doses. In conclusion, optimal overexpression of TIMP3 can be a promising therapeutic approach to limit adverse post-MI remodeling by dually inhibiting early proteolysis and promoting angiogenesis.NEW & NOTEWORTHY Here, we report that tissue inhibitor of metalloproteinase 3 overexpression after myocardial infarction improves myocardial structural remodeling and function by promoting angiogenesis and inhibiting early proteolysis. This demonstrates the therapeutic potential of preserving the local balance of tissue inhibitor of metalloproteinase 3 in the heart given its diverse functions in modulating different processes involved in the adverse postmyocardial infarction remodeling.

Comparison of Selected Protein Levels in Tumour and Surgical Margin in a Group of Patients with Oral Cavity Cancer.

Oral cavity cancer belongs to head-and-neck squamous cell carcinoma group. The purpose of the study was to assess the levels of certain proteins in a tumour and surgical margin in a group of patients with oral cavity cancer. The levels of DAPK1, MGMT, CDH1, SFRP1, SFRP2, RORA, TIMP3, p16, APC and RASSF1 proteins were measured by ELISA in tissue homogenates. The protein levels of DAPK1, MGMT, CDH1, SFRP2 and RASSF1 were significantly higher in tumour tissue than in the margin, contrary to TIMP3 which was lower in the tumour itself. DAPK1 level in the tumour was significantly higher in females than in males, the MGMT and p16 levels were lower in the tumours with lymph node metastasis (N1 + N2) than in N0 samples. The CDH1 expression was higher in a group with smoking habits, whereas TIMP3 was lower in this group. Changes in the levels of proteins in tumour and surgical margin may be either reflective of tumour occurrence and development, or they might be also responsible for the progress and reoccurrence of the disease. Levels of the studied proteins might be good prognostic factors; however, further studies are required.

Long-Term Selenium-Deficient Diet Induces Liver Damage by Altering Hepatocyte Ultrastructure and MMP1/3 and TIMP1/3 Expression in Growing Rats.

The effects of selenium (Se)-deficient diet on the liver were evaluated by using growing rats which were fed with normal and Se-deficient diets, respectively, for 109 days. The results showed that rats fed with Se-deficient diet led to a decrease in Se concentration in the liver, particularly among male rats from the low-Se group. This causes alterations to the ultrastructure of hepatocytes with condensed chromatin and swelling mitochondria observed after low Se intake. Meanwhile, pathological changes and increased fibrosis in hepatic periportal were detected by hematoxylin and eosin and Masson's trichrome staining in low-Se group. Furthermore, through immunohistochemistry (IHC) staining, higher expressions of metalloproteinases (MMP1/3) and their tissue inhibitors of metalloproteinases (TIMP1/3) were observed in the hepatic periportal of rats from the low-Se group. However, higher expressions of MMP1/3 and lower expressions of TIMP1/3 were detected in hepatic central vein and hepatic sinusoid. In addition, upregulated expressions of MMP1/3 and downregulated expressions of TIMP1/3 at the messenger RNA (mRNA) and protein levels also appeared to be relevant to low Se intake. In conclusion, Se-deficient diet could cause low Se concentration in the liver, alterations of hepatocyte ultrastructure, differential expressions of MMP1/3 and TIMP1/3 as well as fibrosis in the liver hepatic periportal.

Potentially positive ageing-related variations of medial smooth muscle cells in the saphenous veins used as aortocoronary bypass grafts.

INTRODUCTION: Currently, elderly people constitute a large proportion of patients undergoing coronary artery bypass grafting (CABG). Activated smooth muscle cells in the tunica media of saphenous vein (SV) grafts are thought to play a key role in the formation of neointima and development of occluding atherosclerotic plaques. The aim of this study was to identify ageing-related variations in the expression of the smooth muscle cells pro-teins that may impact on patency rate of the grafts and the CABG outcomes. MATERIAL AND METHODS: The study involved 216 consecutive patients with the mean of 62.7 ± 8.4 years who underwent isolated CABG with at least one SV aortocoronary bypass graft. Expression of a-smooth muscle actin (a-SM actin), smooth muscle-myosin heavy chain (SM-MHC), calponin (CALP), cytokeratin 8 (CK-8), metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-2 and -3 (TIMP-2, TIMP-3) in the SV wall was assessed by immunohistochemistry and correlated with the age of patients. RESULTS: Calponin and a-SM actin were expressed in all studied SV transplants. SM-MHC immunoreactivity was observed in SV segments in 68.5% of patients, whereas MMP-2a and TIMPs expression was found in 75% of cases. In more than 50% of analyzed SV transplants, no expression of cytokeratin-8 was found. Moderate correlations between preexisting expressions of either cytoskeletal or hemostatic proteins in the tunica media of the SV grafts and the age of CABG patients were demonstrated. They were positive for SM-MHC (r = 0.494), CALP (r = 0.548), TIMP-2 (r = 0.413) and TIMP-3 (r = 0.406) whereas negative for CK-8 (r = -0.528) and MMP-2 (r = -0.417). CONCLUSIONS: Age-dependent decreases in the expression of MMP-2 and CK-8 accompanied by increases in expression of SM-MHC, TIMP-2 and TIMP-3 may promote SV graft patency and, thus, suggest a rationale for common use of SV grafts in the elderly.

Sulfated Hyaluronan Alters the Interaction Profile of TIMP-3 with the Endocytic Receptor LRP-1 Clusters II and IV and Increases the Extracellular TIMP-3 Level of Human Bone Marrow Stromal Cells.

Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.

KDM1A promotes tumor cell invasion by silencing TIMP3 in non-small cell lung cancer cells.

Epigenetic regulation plays an important role in tumor metastasis. KDM1A is a histone demethylase specific for H3K4me2/me1 demethylation, and has been found to be overexpressed in many cancers, including non-small cell lung cancer (NSCLC). However, the role of KDM1A in lung cancer remains unclear. Here, we show that KDM1A promotes cancer metastasis in NSCLC cells by repressing TIMP3 (tissue inhibitor of metalloproteinase 3) expression. Consistently with this, overexpression of TIMP3 inhibited MMP2 expression and JNK phosphorylation, both of which are known to be important for cell invasion and migration. Importantly, knockdown of TIMP3 in KDM1A-deficient cells rescued the metastatic capability of NSCLC cells. These findings were also confirmed by pharmacological inhibition assays. We further demonstrate that KDM1A removes H3K4me2 at the promoter of TIMP3, thus repressing the transcription of TIMP3. Finally, high expression of KDM1A and low expression of TIMP3 significantly correlate with a poor prognosis in NSCLC patients. This study establishes a mechanism by which KDM1A promotes cancer metastasis in NSCLC cells, and we suggest that KDM1A may be a potential therapeutic target for NSCLC treatment.

Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma.

BACKGROUND: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. METHODS: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. RESULTS: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). CONCLUSION: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma.

In vitro evaluation of anticancer properties of exopolysaccharides from Lactobacillus acidophilus in colon cancer cell lines.

The present work aims at studying the effect of exopolysaccharides (EPS) from Lactobacillus acidophilus on the colon cancer cell lines in vitro. Initial analysis showed that EPS has antioxidative properties. EPS was also found to induce cytotoxicity in two colon cancer cell lines, viz. HCT15 and CaCo2 under normoxia and hypoxia. The membrane integrity was also found to be affected in EPS-treated cells. Once the toxic concentration was determined (5 mg/ml), the effect of EPS on the messenger RNA (mRNA) expression of various genes was studied by quantitative real-time (RT)-PCR under both normoxic and hypoxic conditions. The results suggest that EPS downregulated the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) and upregulated the expression of tissue inhibitor of metalloproteinases-3 (TIMP-3), hypoxia-inducible factor-2α (HIF-2α), and hemeoxygenase-1 (HO-1). An increase in plasminogen activator inhibitor-1 (PAI-1) was also observed. These results show that EPS may inhibit the expressions of genes involved in tumor angiogenesis and survival. Increase in the expression of HO-1 also shows that EPS have antioxidative properties.

Effect of Xianziyizhen Recipe Capsule on PGI2-PPARδ Signaling Pathway in Embryo Implantation Dysfunction Mice.

PROBLEM: We investigated the effect of Xianziyizhen recipe capsule (XRC), a kidney-tonifying herb, on the PGI2-PPARδ signaling pathway at the maternal-fetal interface in embryo implantation dysfunction (EID) mice. METHOD OF STUDY: Intragastric administration of Progynova (estradiol) or XRC was performed in EID mouse model, following experimental induction of kidney deficiency by co-treatment with chemotherapy drug hydroxyurea and antiprogesterone mifepristone. The PPARδ and IL-11 mRNA expression in endometrium were detected by real-time relative reverse transcription-polymerase chain reaction (RT-PCR). Further, the protein expression of COX-2, PGI2, MMP-9, and TIMP-3 was detected in endometrial glandular epithelium and in stromal cells by immunohistochemical (IHC) assay. RESULTS: The results showed that hydroxyurea and mifepristone-induced EID were associated with significantly lower PPARδ and IL-11 mRNA levels in endometrium and reduced COX-2, PGI2, MMP-9, and TIMP-3 levels in endometrial glandular epithelium, compared with normal controls. However, XRC and Progynova treatment reversed these effects, leading to significant increases in PPARδ and IL-11 mRNA expression, and COX-2, PGI2, MMP-9 and TIMP-3 protein levels, when compared with the levels observed in EID mice. CONCLUSION: These results strongly suggested that XRC is beneficial in EID treatment and that XRC may mediate its effects through regulation of the PGI2-PPARδ signaling pathway.

Tumor progression driven by pathways activating matrix metalloproteinases and their inhibitors.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is still a problem worldwide. In some publications interactions between the expression of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, and their tissue inhibitors (TIMPs) implicated during cancer progression were suggested. METHODS: The immunohistochemical staining using primary antibody against MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were performed. The research group consists of primary N(0) LSCC (20 cases), primary N(+) LSCC (17 cases), and 18 cases of normal mucosa. RESULTS: Studied MMPs and TIMPs were localized in tumor cells and tumor stroma compartment. MMP-2 expression was higher in stroma compared to tumor cells. MMP-9, TIMP-1, TIMP-2, and TIMP-3 expression was higher in tumor cells than in tumor stroma (P < 0.05). In tumor stroma MMP-2, MMP-9, TIMP-1, and TIMP-3 expression, in LSCC N(0) vs. LSCC N(+) was significantly higher (P < 0.05). The ratios between MMP-2 and TIMP-3 expression were statistically significant (N(0) vs. N(+); P = 0.012). The analyses using classification trees predicted the probability of metastases according to TIMP-3/MMP-14/MMP-2 and MMP-9/TIMP-1 expression levels. CONCLUSIONS: The presence of MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3 expression in tumor cells and in tumor stroma, and additionally different expression according to lymph node involvement suggested of their impact during cancer progression. The significant correlation between TIMP-3 expression and the presence of lymph node metastases and MMP-2 expression might suggest the importance of TIMP-3 as a prognostic factor during tumor progression. The evaluation of molecular markers which participate in MMP-2 activation pathway have a major impact during metastasis.

The angiogenic factor PlGF mediates a neuroimmune interaction in the spleen to allow the onset of hypertension.

Hypertension is a health problem affecting over 1 billion people worldwide. How the immune system gets activated under hypertensive stimuli to contribute to blood pressure elevation is a fascinating enigma. Here we showed a splenic role for placental growth factor (PlGF), which accounts for the onset of hypertension, through immune system modulation. PlGF repressed the expression of the protein Timp3 (tissue inhibitor of metalloproteinases 3), through the transcriptional Sirt1-p53 axis. Timp3 repression allowed costimulation of T cells and their deployment toward classical organs involved in hypertension. We showed that the spleen is an essential organ for the development of hypertension through a noradrenergic drive mediated by the celiac ganglion efferent. Overall, we demonstrate that PlGF mediates the neuroimmune interaction in the spleen, organizing a unique and nonredundant response that allows the onset of hypertension.

A novel action mechanism for MPT0G013, a derivative of arylsulfonamide, inhibits tumor angiogenesis through up-regulation of TIMP3 expression.

Tissue inhibitors of metalloproteinases 3 (TIMP3) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), acting as potent antiangiogenic proteins. In this study, we demonstrated that the arylsulfonamide derivative MPT0G013 has potent antiangiogenic activities in vitro and in vivo viainducing TIMP3 expression. Treatments with MPT0G013 significantly inhibited endothelial cell functions, such as cell proliferation, migration, and tube formation, as well as induced p21 and cell cycle arrest at the G0/G1 phase. Subsequent microarray analysis showed significant induction of TIMP3 gene expression by MPT0G013, and siRNA-mediated blockage of TIMP3 up-regulation abrogated the antiangiogenic activities of MPT0G013 and prevented inhibition of p-AKT and p-ERK proteins. Importantly, MPT0G013 exhibited antiangiogenic activities in in vivo Matrigel plug assays, inhibited tumor growth and up-regulated TIMP3 and p21 proteins in HCT116 mouse xenograft models. These data suggest potential therapeutic application of MPT0G013 for angiogenesis-related diseases such as cancer.

Interaction of ERK1/2 and Smad2/3 signaling pathways in TGF-ß1-induced TIMP-3 expression in rat chondrocytes.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is an important natural inhibitor of matrix metalloproteinases (MMPs) and of a disintegrin and metalloproteinase with thrombospondin motif (ADAMTs), which can cleave cartilage extracellular matrix components to cause cartilage degradation. In this study, our data suggest TGF-ß1 induces TIMP-3 expression through activations of both the ERK1/2 and Smad2/3 signaling pathways. TGF-ß1-stimulated TIMP-3 expression was significantly inhibited by SB525334 (TGF-ß receptor I kinase inhibitor), accompanied by a reduction in ERK1/2 and Smad3 phosphorylation. We used PD98059 (MEK inhibitor) and SIS3 (inhibitor of Smad3 phosphorylation) to investigate the respective roles of ERK1/2 and Smad2/3 signaling pathways in TGF-ß1-induced TIMP-3 expression. The results show PD98059 treatment significantly suppressed TGF-ß1-induced ERK1/2 phosphorylation and TIMP-3 expression. Under these conditions, the degree of Smad3 phosphorylation correlated with ERK1/2 activation, which suggests that ERK1/2 may activate Smad3 phosphorylation. SIS3 significantly inhibited TGF-ß1-induced Smad3 phosphorylation and TIMP-3 expression. ERK1/2 phosphorylation alone had no effect on TGF-ß1-induced TIMP-3 expression, which suggests ERK1/2 via Smad3 phosphorylation regulates TGF-ß1-induced TIMP-3 expression. Here, we demonstrate that ERK1/2 may be capable of activating the Smad2/3 signaling pathway to result in TGF-ß1-induced TIMP-3 up-regulation.

Altered microRNA expression after infection with human cytomegalovirus leads to TIMP3 downregulation and increased shedding of metalloprotease substrates, including MICA.

Proteolytic shedding of ligands for the NK group 2D (NKG2D) receptor is a strategy used by tumors to modulate immune recognition by NK cells and cytotoxic T cells. A number of metalloproteases, especially those of the A disintegrin and metalloprotease (ADAM) family, can mediate NKG2D ligand cleavage and this process can be modulated by expression of the thiol isomerase ERp5. In this article, we describe that an increased shedding of the NKG2D ligand MICA is observed postinfection with several strains of human CMV due to an enhanced activity of ADAM17 (TNF-α converting enzyme) and matrix metalloprotease 14 caused by a reduction in the expression of the endogenous inhibitor of metalloproteases tissue inhibitors of metalloproteinase 3 (TIMP3). This decrease in TIMP3 expression correlates with increased expression of a cellular miRNA known to target TIMP3, and we also identify a human CMV-encoded microRNA able to modulate TIMP3 expression. These observations characterize a novel viral strategy to influence the shedding of cell-surface molecules involved in immune response modulation. They also provide an explanation for previous reports of increased levels of various ADAM17 substrates in the serum from patients with CMV disease. Consistent with this hypothesis, we detected soluble MICA in serum of transplant recipients with CMV disease. Finally, these data suggest that it might be worthwhile to prospectively study ADAM17 activity in a larger group of patients to assay whether this might be a useful biomarker to identify patients at risk for development of CMV disease.