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.

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.

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.

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.

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.

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.

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.

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.

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.

Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration.

The angiogenic potential of solid tumors, or the ability to initiate neovasculature development from pre-existing host vessels, is facilitated by soluble factors secreted by tumor cells and involves breaching of extracellular matrix barriers, endothelial cell (EC) proliferation, migration and reassembly. We evaluated the angiogenic potential of human melanoma cell lines differing in their degree of aggressiveness, based on their ability to regulate directionally persistent EC migration. We observed that conditioned-medium (CM) of the aggressive melanoma cell line BLM induced a high effective migratory response in ECs, while CMs of Mel57 and 1F6 had an inhibitory effect. Further, the melanoma cell lines exhibited a varied expression profile of tissue inhibitor of metalloproteinase-3 (TIMP3), detectable in the CM. TIMP3 expression inversely correlated with aggressiveness of the melanoma cell line, and ability of the respective CMs to induce directed EC migration. Interestingly, TIMP3 expression was found to be silenced in the BLM cell line, concurrent with its role as a tumor suppressor. Treatment with recombinant human TIMP3 and CM of modified, TIMP3 expressing, BLM cells mitigated directional EC migration, while CM of TIMP3 silenced 1F6 cells induced directed EC migration. The functional implication of TIMP3 expression on tumor growth and angiogenic potential in melanoma was evaluated in vivo. We observed that TIMP3 expression reduced tumor growth, angiogenesis and macrophage infiltration of BLM tumors while silencing TIMP3 increased tumor growth and angiogenesis of 1F6 tumors. Taken together, our results demonstrate that TIMP3 expression correlates with inhibition of directionally persistent EC migration and adversely affects the angiogenic potential and growth of melanomas.

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.

Multivalent pseudopeptides targeting cell surface nucleoproteins inhibit cancer cell invasion through tissue inhibitor of metalloproteinases 3 (TIMP-3) release.

Blockage of the metastasis process remains a significant clinical challenge, requiring innovative therapeutic approaches. For this purpose, molecules that inhibit matrix metalloproteinases activity or induce the expression of their natural inhibitor, the tissue inhibitor of metalloproteinases (TIMPs), are potentially interesting. In a previous study, we have shown that synthetic ligands binding to cell surface nucleolin/nucleophosmin and known as HB 19 for the lead compound and NucAnt 6L (N6L) for the most potent analog, inhibit both tumor growth and angiogenesis. Furthermore, they prevent metastasis in a RET transgenic mice model which develops melanoma. Here, we investigated the effect of N6L on the invasion capacity of MDA-MB-435 melanoma cells. Our results show that the multivalent pseudopeptide N6L inhibited Matrigel invasion of MDA-MB-435 cells in a modified Boyden chamber model. This was associated with an increase in TIMP-3 in the cell culture medium without a change in TIMP-3 mRNA expression suggesting its release from cell surface and/or extracellular matrix. This may be explained by our demonstrated N6L interaction with sulfated glycosaminoglycans and consequently the controlled bioavailability of glycosaminoglycan-bound TIMP-3. The implication of TIMP-3 in N6L-induced inhibition of cell invasion was evidenced by siRNA silencing experiments showing that the loss of TIMP-3 expression abrogated the effect of N6L. The inhibition of tumor cell invasion by N6L demonstrated in this study, in addition to its previously established inhibitory effect on tumor growth and angiogenesis, suggests that N6L represents a promising anticancer drug candidate warranting further investigation.

Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex.

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Methyl deficient diet aggravates experimental colitis in rats.

Inflammatory bowel diseases (IBD) result from complex interactions between environmental and genetic factors. Low blood levels of vitamin B12 and folate and genetic variants of related target enzymes are associated with IBD risk, in population studies. To investigate the underlying mechanisms, we evaluated the effects of a methyl-deficient diet (MDD, folate, vitamin B12 and choline) in an experimental model of colitis induced by dextran sodium sulphate (DSS), in rat pups from dams subjected to the MDD during gestation and lactation. Four groups were considered (n = 12-16 per group): C DSS(-) (control/DSS(-)), D DSS(-) (deficient/DSS(-)), C DSS(+) (control/DSS(+)) and D DSS(+) (deficient/DSS(+)). Changes in apoptosis, oxidant stress and pro-inflammatory pathways were studied within colonic mucosa. In rat pups, the MDD produced a decreased plasma concentration of vitamin B12 and folate and an increased homocysteine (7.8 ± 0.9 versus 22.6 ± 1.2 µmol/l, P < 0.001). The DSS-induced colitis was dramatically more severe in the D DSS(+) group compared with each other group, with no change in superoxide dismutase and glutathione peroxidase activity, but decreased expression of caspase-3 and Bax, and increased Bcl-2 levels. The mRNA levels of tumour necrosis factor (TNF)-α and protein levels of p38, cytosolic phospolipase A2 and cyclooxygenase 2 were significantly increased in the D DSS(+) pups and were accompanied by a decrease in the protein level of tissue inhibitor of metalloproteinases (TIMP)3, a negative regulator of TNF-α. MDD may cause an overexpression of pro-inflammatory pathways, indicating an aggravating effect of folate and/or vitamin B12 deficiency in experimental IBD. These findings suggest paying attention to vitamin B12 and folate deficits, frequently reported in IBD patients.

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.

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.

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

TIMP-3: a novel target for glucocorticoid signaling at the blood-brain barrier.

Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood-brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.

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.

Upregulation of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases in rapid atrial pacing-induced atrial fibrillation.

Remodeling of atrial extracellular matrix (ECM) in atrial fibrillation (AF) involves changes in the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). The contributions of MMPs and TIMPs to the pathogenesis of AF development have not been clearly defined. This study evaluated the in situ activity and expression of gelatinases (MMP-2 and MMP-9) and their relationship with TIMP-1 or TIMP-3 in atria undergoing rapid atrial pacing for the induction of AF (4 weeks' pacing followed by 2 weeks of maintained AF) in pigs. In AF atria, in situ gelatinase activity was mainly localized in the interstitium of atrial myocardium, and was significantly larger than that of sinus rhythm control (i.e., sham control). The significant increase of MMP-9 in its pro-form and mRNA level, but not MMP-2, was shown to be responsible for the increased gelatinase activity in atria with AF. The inhibitory activities of glycosylated TIMP-1 and TIMP-3, but not TIMP-2, in AF tissues were markedly elevated and also localized in the atrial interstitium. TIMP-1 was found to be mostly colocalized with gelatinase activity over the AF tissues, implying the coexistence of gelatinase activity and TIMP-1, but TIMP-3 appeared only partially colocalized and discontinued the gelatinase activity surrounding the cardiomyocytes. TIMP-1 and TIMP-3 may play differential roles in the inhibition of gelatinase activity in vivo. Together with the survey of several MMPs transcripts and the level of transforming growth factor-beta1 (TGF-beta1), we proposed that the increased activity of gelatinase (i.e., MMP-9), TIMP-1 and TIMP-3 and their interaction may contribute to atrial ECM remodeling of AF.