The present study aims at investigating the mechanism by which membrane-type 4 matrix metalloproteinase (MT4-MMP), a membrane-anchored MMP expressed by human breast tumour cells promotes the metastatic dissemination into lung. We applied experimental (intravenous) and spontaneous (subcutaneous) models of lung metastasis using human breast adenocarcinoma MDA-MB-231 cells overexpressing or not MT4-MMP. We found that MT4-MMP does not affect lymph node colonization nor extravasation of cells from the bloodstream, but increases the intravasation step leading to metastasis. Ultrastructural and fluorescent microscopic observations coupled with automatic computer-assisted quantifications revealed that MT4-MMP expression induces blood vessel enlargement and promotes the detachment of mural cells from the vascular tree, thus causing an increased tumour vascular leak. On this basis, we propose that MT4-MMP promotes lung metastasis by disturbing the tumour vessel integrity and thereby facilitating tumour cell intravasation.

The expression of tissue inhibitor metalloproteinase-1 (TIMP-1) significantly increased after acute cerebral ischaemia and involved in neurodegeneration. The purpose was to prospectively investigate the relationship between serum TIMP-1 with post-stroke cognitive impairment. Our participants were from an ancillary study of China Antihypertensive Trial in Acute Ischemic Stroke. 598 ischaemic stroke patients from seven participating hospitals were included. Cognitive impairment was evaluated using Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) at 3 months. 316 (52.84%) or 384 (64.21%) participants had cognitive impairment according to MMSE or MoCA, respectively. Compared with the first quartile of TIMP-1, the multivariate-adjusted odds ratios (95% confidence intervals) for the highest quartile were 1.80 (1.09-2.97) for cognitive impairment defined by MMSE and 2.55 (1.49-4.35) by MoCA. Multiple-adjusted spline regression models showed linear associations between TIMP-1 concentrations and cognitive impairment (P value for linearity < 0.01). The addition of TIMP-1 to models including conventional factors improved reclassification for cognitive impairment, as shown by net reclassification index or integrated discrimination improvement (P < 0.05). Participants with both higher TIMP-1 and matrix metalloproteinase-9 levels simultaneously had highest risk of cognitive impairment. Higher serum TIMP-1 levels were associated with increased risk of cognitive impairment after acute ischaemic stroke, independently of established risk factors.

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase-13 (MMP-13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP-13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N-nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild-type (WT) and MMP-13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF-β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP-13 knockout mice. Protein and mRNA levels of CTGF, TGF-β1, α-SMA and type I collagen and the levels of MMP-2, MMP-9 and cleaved products of CTGF were markedly increased in NDMA-treated WT mice compared to the MMP-13 knockout mice. Blocking of MMP-13 with CL-82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full-length CTGF and its C-terminal fragments and active TGF-β1. The data demonstrate that MMP-13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP-13 and CTGF has potential therapeutic implications to arrest liver fibrosis.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common histological types of head and neck cancer. Epiberberine is a potent antitumour agent for several types of cancer. This study is aimed at investigating the regulatory and molecular mechanism of epiberberine on HNSSC cell metastasis. The results showed that epiberberine inhibited the motility of Ca9-22 and FaDu cell lines at nontoxicity doses. Moreover, the epithelial-mesenchymal transition (EMT)-related proteins, vimentin, snail and slug, were found suppressing after epiberberine treatments. In addition, the JNK signalling cascade and the metalloproteinase 13 (MMP-13) expression were also found downregulated by epiberberine. In conclusion, the present study demonstrates that epiberberine suppresses cell migration and invasion by regulating the JNK pathway and MMP-13. These results suggest that epiberberine could be a potential antimetastatic agent in HNSCC cells.

Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain. Polydatin (PD) has been shown to exert multiple pharmacological effects on different diseases; here, we test the therapeutic potential of PD for IVDD. In in-vitro experiments, we confirmed PD is nontoxic to nucleus pulposus cells (NPCs) under the concentration of 400 μmol/L. Furthermore, PD was able to decrease the level of senescence in TNF-α-treated NPCs, as indicated by β-gal staining as well as senescence markers p53 and p16 expression. In the aspect of extracellular matrix (ECM), PD not only reduced metalloproteinase 3 (MMP-3), metalloproteinase 13 (MMP-13) and a disintegrin-like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) expression, but also increased aggrecan and collagen II levels. Mitochondrion is closely related to cellular senescence and ECM homeostasis; mechanistically, we found PD may rescue TNF-α-induced mitochondrial dysfunction, and it may also promote Nrf2 expression and activity. Silencing Nrf2 partly abolished the protective effects of PD on mitochondrial homeostasis, senescence and ECM homeostasis in TNF-α-treated NPCs. Correspondingly, PD ameliorated IVDD in rat model by promoting Nrf2 activity, preserving ECM and inhibiting senescence in nucleus pulposus cells. To sum up, our study suggests that PD exerts protective effects in NPCs against IVDD and reveals the underlying mechanism of PD on Nrf2 activation in NPCs.

This present study is aimed to investigate the role of microRNA-365 (miR-365) in the development of intervertebral disc degeneration (IDD). Nucleus pulposus (NP) cells were transfected by miR-365 mimic and miR-365 inhibitor, respectively. Concomitantly, the transfection efficiency and the expression level of miRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Meanwhile, NP cells apoptosis was measured through propidium iodide (PI)-AnnexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit. Subsequently, immunofluorescence (IF) staining was performed to assess the expression of collagen II, aggrecan and matrix metalloproteinase 13 (MMP-13). In addition, bioinformatic prediction and Luciferase reporter assay were used to reveal the target gene of miR-365. Finally, we isolated the primary NP cells from rats and injected NP-miR-365 in rat IDD models. The results showed that overexpression of miR-365 could effectively inhibit NP cells apoptosis and MMP-13 expression and upregulate the expression of collagen II and aggrecan. Conversely, suppression of miR-365 enhanced NP cell apoptosis and elevated MMP-13 expression, but decreased the expression of collagen II and aggrecan. Moreover, the further data demonstrated that miR-365 mediated NP cell degradation through targeting ephrin-A3 (EFNA3). In addition, the cells apoptosis and catabolic markers were increased in NP cells when EFNA3 upregulated. More importantly, the vivo data supported that miR-365-NP cells injection ameliorated IDD in rats models. miR-365 could alleviate the development of IDD by regulating NP cell apoptosis and ECM degradation, which is likely mediated by targeting EFNA3. Therefore, miR-365 may be a promising therapeutic avenue for treatment IDD through EFNA3.

The anti-inflammatory effect of sinapic acid (SA) has been reported in several studies. However, whether SA has the same effect on osteoarthritis (OA) has yet to be clearly elucidated. We designed a series of in vitro and in vivo procedures to verify the above conjecture. Compared with controls, SA-pretreated human chondrocytes showed lower levels of interleukin (IL)-1β-induced IL-6, prostaglandin E2 (PGE2), nitric oxide (NO) and tumour necrosis factor-α (TNF-α) in vitro. Meanwhile, SA could also reverse the degradation of type II collage and aggrecan, as well as the overproduction of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and a disintegrin and metalloproteinase thrombospondin motifs (ADAMTS)-5. Furthermore, activation of nuclear factor κB (NF-κB), which was induced by IL-1β, was also inhibited by SA through the pathway of nuclear factor-erythroid 2-related factor-2 (Nrf2)/heme oxygenase 1. In vivo, SA could delay the progress of mice OA models. We propose that SA may be applied as a potential therapeutic drug in OA treatment.

Bladder cancer (BC) is the second most common urological tumour in Western countries. Approximately, 80% of patients with BC will present with non-muscle invasive bladder cancer (NMIBC), whereas a quarter will have muscle invasive disease (MIBC) at the time of BC diagnosis. However, patients with NMIBC are at risk of BC recurrence or progression into MIBC, and an MIBC prognosis is determined by the presence of progression and metastasis. Matrix metalloproteinase 2 (MMP2), a type of matrix metalloproteinase (MMP), plays a major role in tumour invasion and is well-characterized in BC prognosis. In BC, the mechanisms regulating MMP2 expression, and, in turn, promote cancer invasion, have hardly been explored. Thrombospondin-4 (THBS4/TSP4) is a matricellular glycoprotein that regulates multiple biological functions, including proliferation, angiogenesis, cell adhesion and extracellular matrix modelling. Based on the results of a meta-analysis in the Gene Expression Profiling Interactive Analysis 2 database, we observed that TSP4 expression levels were consistent with overall survival (OS) rate and BC progression, with the highest expression levels observed in the advanced stages of BC and associated with poor OS rate. In our pilot experiments, incubation with recombinant TSP4 promoted the migration and invasion in BC cells. Furthermore, MMP2 expression levels increased after recombinant TSP4 incubation. TSP4-induced-MMP2 expression and cell motility were regulated via the AKT signalling pathway. Our findings facilitate further investigation into TSP4 silencing-based therapeutic strategies for BC.

Chondrocytes' hypertrophy includes metabolic changes, matrix remodelling, proliferation and apoptosis, characteristics associated with the progression of osteoarthritis. We investigated a possible association among Runt-related transcription factor 2 (RUNX-2), SOX-9 and fibroblast growth factor (FGF)-23 mRNA expressions in articular chondrocytes in order to elucidate their contribution in the osteoarthritic hypertrophic cartilage. SOX-9, FGF-23, RUNX-2 and matrix metalloproteinase (MMP)-13 mRNA expressions were evaluated in osteoarthritic and normal chondrocytes by real-time PCR whereas MMP-13 protein expression by immunofluorescense. RUNX-2, FGF-23 and SOX-9 were down-regulated using small interfering RNA technology and transfection with liposomes. The effect of human recombinant FGF-23 (hrFGF-23) on SOX-9 and RUNX-2 expression was tested in normal chondrocytes. We found higher expression of RUNX-2 and FGF-23 and a decreased expression of SOX-9 mRNA in osteoarthritic chondrocytes compared to normal (P < 0.0001). RUNX-2 down-regulation resulted in reduced MMP-13 expression in osteoarthritic chondrocytes and inhibition of SOX-9 in increased RUNX-2 and MMP-13 mRNA expression in normal chondrocytes, whereas inhibition of FGF-23 resulted in reduced RUNX-2 mRNA expression in osteoarthritic chondrocytes (all P < 0.0001). Silencing of RUNX-2 or FGF-23 did not affect SOX-9 mRNA levels in osteoarthritic chondrocytes. Moreover simultaneous down-regulation of SOX-9 and up-regulation of FGF-23 mRNA expressions in normal chondrocytes resulted in additive up-regulation of RUNX-2 mRNA expression. Treatment of normal chondrocytes with hrFGF-23 resulted in increased RUNX-2 mRNA expression, whereas it had no effect on SOX-9 mRNA expression. We demonstrated convincing associations among RUNX-2, SOX-9 and FGF-23 in relation to MMP-13 expression in osteoarthritic chondrocytes, contributing to a better understanding of the abnormal gene expression and cartilage degeneration processes associated with osteoarthritis.

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL-1β/NF-κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10-week-old male C57BL/6J mice. ANE was then intra-articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin-1β (IL-1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE -treated mice compared with vehicle-treated mice. ANE decreased the expressions of matrix metalloproteinase-13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL-1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL-1β/NF-κB pathway activation.

We evaluated the influence of aerobic training on cardiac remodeling in untreated spontaneously hypertensive rats (SHR). Four experimental groups were used: sedentary (W-SED, n=27) and trained (WEX, n=31) normotensive Wistar rats, and sedentary (SHR-SED, n=27) and exercised (SHR-EX, n=32) hypertensive rats. At 13 months old, trained groups underwent treadmill exercise five days a week for four months. Statistical analysis: ANOVA or Kruskal-Wallis. Exercised groups had higher physical capacity. Hypertensive groups presented left ventricular (LV) concentric hypertrophy with impaired function. Left atrium diameter, LV posterior wall thickness and relative thickness, and isovolumetric relaxation time were lower in SHR-EX than SHR-SED. Interstitial collagen fraction and Type I-Type III collagen ratio were higher in SHR-SED than W-SED. In SHR-EX these parameters had intermediate values between W-EX and SHRSED with no differences between either group. Myocardial matrix metalloproteinase-2 activity, evaluated by zymography, was higher in SHR-SED than W-SED and SHR-EX. TIMP-2 was higher in hypertensive than normotensive groups. In conclusion, low intensity aerobic exercise reduces left atrium dimension and LV posterior wall thickness, and improves functional capacity, diastolic function, and metalloproteinase-2 activity in adult SHR.

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low-density lipoprotein receptor-related protein 1 (LRP1) and MMP-9 and MMP-2 spatiotemporal expression after MI. Real-time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri-infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri-infarct and infarct areas. LRP1 also colocalized with proline-rich tyrosine kinase 2 (pPyk2) and MMP-9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP-9 activity in fibroblasts, without significant changes in MMP-2 activity. MMP-9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP-9 up-regulation associated with ventricular remodelling after MI.

Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-gamma induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro. We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.

Rosmarinic acid (RosA) is a water-soluble polyphenol, which can be isolated from many herbs such as orthosiphon diffuses and rosmarinus officinalis. Previous studies have shown that RosA possesses various biological properties. In this study, we investigate the anti-osteoarthritic effects of RosA in rat articular chondrocytes. Chondrocytes were pre-treated with RosA, followed by the stimulation of IL-1β. Real-time PCR and Western blot were performed to detect the expression of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13. Nitric oxide and PGE2 production were measured by Griess reagent and enzyme-linked immunosorbent assay (ELISA). The expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) was also investigated by Western blot analysis. We found that RosA down-regulated the MMPs expression as well as nitric oxide and PGE2 production in IL-1β-induced chondrocytes. In addition, RosA inhibited p38 and JNK phosphorylation as well as p65 translocation. The results suggest that RosA may be considered a possible agent in the treatment of OA.

Osteosarcoma, the most common bone malignancy, has a high morbidity rate and poor prognosis. Krüppel-like factor 5 (KLF5) is a key transcriptional regulator of cellular proliferation whose overexpression is observed in osteosarcoma cell lines (U2OS, 143B, MG63 and SAOS2). ML264, a small-molecule inhibitor of KLF5, exerts antiproliferative effects in colorectal cancer; however, its function in osteosarcoma remains unknown. Here, we explored the possible antitumour effects of ML264 on 143B and U2OS cell lines and murine tumour xenograft model. ML264 suppressed proliferation and clonogenic ability of osteosarcoma cells in a dose-dependent manner. Moreover, ML264 induced G0/G1 cell cycle arrest, with no influence on apoptosis, and inhibited the migratory and invasive abilities of osteosarcoma cells, as demonstrated by wound-healing and Transwell assays. Exposure to ML264 reduced the mRNA and protein levels of molecules associated with epithelial-mesenchymal transition phenotype, including N-cadherin, vimentin, Snail, matrix metalloproteinase (MMP) 9 and MMP13. Inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation and Wnt signalling was also observed. In the murine model of osteosarcoma, tumour growth was efficiently suppressed following a 10-day treatment with ML264. Collectively, our findings demonstrate the potential value of ML264 as a novel anticancer drug for osteosarcoma.

Osteoarthritis has become one of the main diseases affecting the life of many elderly people with high incidence of disability, and local chronic inflammation in the joint cavity is the most crucial pathological feature of osteoarthritis. Astilbin is the main active component in a variety of natural plants such as Hypericum perforatum and Sarcandra glabra, which possess antioxidant and anti-inflammatory effects. At present, there is no study about the protective effect of Astilbin for osteoarthritis. The purpose of this study was to investigate the effect of Astilbin in human OA chondrocytes and mouse OA model, which was established by surgery-mediated destabilization of the medial meniscus (DMM). In vitro, we found that Astilbin pre-treatment inhibited lipopolysaccharide (LPS)-induced overproduction of inflammation-correlated cytokines such as nitric oxide (NO), prostaglandin E2 (PGE2), tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6), and suppressed overexpression of inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Astilbin, on the other hand, prevented the LPS-induced degradation of extracellular matrix (ECM) by down-regulating MMP13 (matrix metalloproteinases 13) and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5). Moreover, by inhibiting the formation of the TLR4/MD-2/LPS complex, Astilbin blocked LPS-induced activation of TLR4/NF-κB signalling cascade. In vivo, Astilbin showed the chondro-protective effect in the surgical-induced OA mouse models. In conclusion, our findings provided evidence that develops Astilbin as a potential therapeutic drug for OA patients.

Intervertebral discs (IVD) degeneration, which is caused by ageing or mechanical stress, leads to IVD disease, including back pain and sciatica. The cytokine interleukin (IL)-17A is elevated in NP cells during IVD disease. Here we explored the pharmacotherapeutic potential of IL-17A for the treatment of IVD disease using small-molecule inhibitors that block binding of IL-17A to the IL-17A receptor (IL-17RA). Treatment of NP cells with IL-17A increased expression of cyclooxygenase-2 (COX-2), IL-6, matrix metalloproteinase (MMP)-3 and MMP-13. These increases were suppressed by an IL-17A-neutralizing antibody, and small molecules that were identified as inhibitors by binding to the IL-17A-binding region of IL-17RA. IL-17A signalling also altered sulphated glycosaminoglycan deposition and spheroid colony formation, while treatment with small-molecule inhibitors of IL-17A attenuated this response. Furthermore, mitogen-activated protein kinase pathways were activated by IL-17A stimulation and induced IL-6 and COX-2 expression, while small-molecule inhibitors of IL-17A suppressed their expression. Taken together, these results show that IL-17A is a valid target for IVD disease therapy and that small-molecule inhibitors that inhibit the IL-17A-IL-17RA interaction may be useful for pharmacotherapy of IVD disease.

The migration of epidermal stem cells (EpSCs) is critical for wound re-epithelization and wound healing. Recently, growth/differentiation factor-5 (GDF-5) was discovered to have multiple biological effects on wound healing; however, its role in EpSCs remains unclear. In this work, recombinant mouse GDF-5 (rmGDF-5) was found via live imaging in vitro to facilitate the migration of mouse EpSCs in a wound-scratch model. Western blot and real-time PCR assays demonstrated that the expression levels of RhoA and matrix metalloproteinase-9 (MMP9) were correlated with rmGDF-5 concentration. Furthermore, we found that rmGDF-5 stimulated mouse EpSC migration in vitro by regulating MMP9 expression at the mRNA and protein levels through the RhoA signalling pathway. Moreover, in a deep partial-thickness scald mouse model in vivo, GDF-5 was confirmed to promote EpSC migration and MMP9 expression via RhoA, as evidenced by the tracking of cells labelled with 5-bromo-2-deoxyuridine (BrdU). The current study showed that rmGDF-5 can promote mouse EpSC migration in vitro and in vivo and that GDF-5 can trigger the migration of EpSCs via RhoA-MMP9 signalling.

Inflammation promotes colorectal carcinogenesis. Tumour growth often generates a hypoxic environment in the inner tumour mass. We here report that, in colon cancer cells, the expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) associates with that of the hypoxia response gene carbonic anhydrase-IX (CA-IX). The COX-2 knockdown, achieved by the stable infection of a COX-2 specific short harpin RNA interference (shCOX-2), down-regulates CA-IX gene expression. In colorectal cancer (CRC) cells, PGE(2), the main COX-2 gene products, promotes CA-IX gene expression by ERK1/2 activation. In normoxic environment, shCOX-2 infected/CA-IX siRNA transfected CRC cells show a reduced level of active metalloproteinase-2 (MMP-2) that associates with a decreased extracellular matrix invasion capacity. In presence of hypoxia, COX-2 gene expression and PGE(2) production increase. The knockdown of COX-2/CA-IX blunts the survival capability of CRC cells in hypoxia. At a high cell density, a culture condition that creates a mild pericellular hypoxic environment, the expression of COX-2/CA-IX genes is increased and triggers the invasive potential of colon cancer cells. In human colon cancer tissues, COX-2/CA-IX protein expression levels, assessed by Western blot and immunohistochemistry, correlate each other and increase with tumour stage. In conclusion, these data indicate that COX-2/CA-IX interplay promotes the aggressive behaviour of CRC cells.

Tendon injuries cause considerable morbidity in the general adult population. The tenocytes within the tendon have the full capacity to heal the tendon intrinsically. Activated protein C (APC) plays an important role in coagulation and inflammation and more recently has been shown to promote cutaneous wound healing. In this study we examined whether APC can induce a wound healing phenotype in tenocytes. Sheep tenocytes were treated with APC, endothelial protein C receptor (EPCR) blocking antibody (RCR252) and/or EPCR small interfering (si)RNA. Cell proliferation and migration were measured by crystal violet assay and a scratch wounding assay, respectively. The expression of EPCR, matrix metalloproteinase (MMP)-2, type I collagen and MAP kinase activity were detected by real time PCR, zymography, immunofluorescence, immunohistochemistry and Western blotting. APC stimulated proliferation, MMP-2 activity and type I collagen deposition in a dose-dependent manner and promoted migration of cultured tenocytes. APC dose-dependently stimulated phosphorylated (P)-ERK2 and inhibited P-p38. Interestingly, tenocytes expressed EPCR protein, which was up-regulated by APC. When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked. APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes. Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases. These data implicate APC as a potential healing agent for injured tendons.