Inhibitory effect of recombinant tyrosine­sulfated madanin­1, a thrombin inhibitor, on the behavior of MDA­MB­231 and SKOV3 cells in vitro.

Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombin­targeting recombinant tyrosine­sulfated madanin­1 on cancer cell behavior and signaling pathways compared with madanin­1 wild­type (WT) were investigated. Recombinant madanin­1 2 sulfation (madanin­1 2S) and madanin­1 WT proteins were generated using Escherichia coli. SKOV3 and MDA­MB­231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA­MB­231 cells, which were significantly suppressed by madanin­1 2S (P<0.05). Madanin­1 2S also significantly suppressed thrombin­induced expression of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase in both cell lines (P<0.05), whereas madanin­1 WT had no effect on the expression levels of these proteins in MDA­MB­231 cells. Furthermore, madanin­1 2S significantly reversed the effects of thrombin on E­cadherin, N­cadherin and vimentin expression in MDA­MB­231 cells (P<0.05), whereas madanin­1 WT did not show any effect. In conclusion, madanin­1 2S suppressed the migration and invasion of cancer cells more effectively than madanin­1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin­1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.

Sulfated CXCR3 Peptide Trap Use as a Promising Therapeutic Approach for Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.

Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice.

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

Transforming Growth Factor-ß1-induced Human Subconjunctival Fibrosis is Mediated by MicroRNA 143/145 Expression.

Purpose: To investigate the roles and pathways of microRNAs 143 and 145 in transforming growth factor (TGF)-ß1-induced human subconjunctival fibrosis. Methods: Human tenon's capsule fibroblasts (HTFs) were obtained from a healthy eye. After treating cultured HTFs with TGF-ß1, the expression of microRNAs 143 and 145 was evaluated using polymerase chain reaction. To identify the pathways of TGF-ß1-induced microRNA 143/145 expression, HTFs were treated with specific inhibitors of p38MAPK, PI3K/Akt, JNK, ERK, and with siRNAs for SMAD2 and SMAD4. Mutagenesis studies were performed to evaluate the role of the CArG box and SMAD-binding element (SBE). To investigate the role of microRNA 143/145 in TGF-ß1-induced myofibroblast transdifferentiation, microRNA 143/145 mimics and microRNA 143/145 inhibitors were applied to the HTFs. Results: Array analysis revealed that TGF-ß1 induced the expression of microRNA 143/145 in a dose- and time-dependent manner. When inhibitors and siRNAs for p38MAPK, PI3K/Akt, ERK, and JNK were applied, the TGF-ß1-induced expression of microRNA 143/145 was inhibited; however, SMAD2 and SMAD4 inhibition did not affect the TGF-ß1-induced expression of these microRNAs. In the mutagenesis studies, both the CArG box and SBE were associated with TGF-ß1-induced expression of microRNA 143/145. Mimics of microRNA 143/145 induced increased myofibroblast formation, whereas their inhibitors had the opposite effect. Conclusions: TGF-ß1-induced human subconjunctival fibrosis was mediated by the expression of microRNA 143/145, mainly via SMAD-independent pathways. Inhibition of TGF-ß1-induced microRNA 143/145 expression in HTFs might represent a novel strategy to prevent subconjunctival fibrosis.

Salmonella­induced miR­155 enhances necroptotic death in macrophage cells via targeting RIP1/3.

Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), a virulent pathogen, is known to induce host­cell death. Using reverse transcription­quantitative polymerase chain reaction, a 28­fold increase of microRNA (miR)­155 expression in RAW 264.7 macrophages was observed following infection with Salmonella for 24 h. This miR­155 upregulation increased macrophage cell death by up to 40% in 48 h following infection. Western blot analysis revealed that receptor interacting protein 1 (RIP1) and 3 (RIP3) were increased at 18 h following miR­155 transfection to macrophages, similar to Salmonella infection. In addition, inhibition of RIP1 by pre­incubating macrophages with necrostatin­1, a RIP1 specific inhibitor, increased the viability of Salmonella­infected cells and miR­155­transfected cells by up to 20%. The cleavage of poly (adenosine diphosphate­ribose) polymerase­1 (PARP­1) was also enhanced by miR­155 induction upon Salmonella infection. Therefore, it was suggested that RIP1/3­induced necroptosis and PARP­1­mediated necrosis caused by miR­155 induction may represent distinct routes of programmed necrotic cell death of Salmonella­infected macrophages.

Expression of microRNAs in fibroblast of pterygium.

AIM: To screen microRNAs (miRNAs) and set up target miRNAs in pterygium. METHODS: Primary fibroblasts were isolated from pterygium and Tenon's capsule and cultured. Immunocytochemical analysis and Western blotting were performed to confirm the culture of fibroblasts. In all, 1733 miRNAs were screened in the first step by using GeneChip(®) miRNA3.0 Array. Specific miRNAs involved in the pathogenesis of pterygium were subsequently determined using the following criteria: 1) high reproducibility in a repetitive test; 2) base log value of >7.0 for both control and pterygial fibroblasts; and 3) log ratio of >1.0 between pterygial fibroblasts and control fibroblasts. RESULTS: Primary screening showed that 887/1733 miRNAs were up-regulated and 846/1733 miRNAs were down-regulated in pterygial fibroblasts compared with those in control fibroblasts. Of the 1733 miRNAs screened, 4 miRNAs, namely, miRNA-143a-3p, miRNA-181a-2-3p, miRNA-377-5p and miRNA-411a-5p, met the above-mentioned criteria. Primary screening showed that these 4 miRNAs were up-regulated in pterygial fibroblasts compared with control fibroblasts and that miRNA-143a-3p had the highest mean ratio compared with the miRNAs in control fibroblasts. CONCLUSION: miRNA-143a-3p, miRNA-181a-2-3p, miRNA-377-5p and miRNA-411a-5p are up-regulated in pterygial fibroblasts compared with control fibroblasts, suggesting their involvement in the pathogenesis of pterygium.

Lithium chloride inhibits TGF-ß1-induced myofibroblast transdifferentiation via PI3K/Akt pathway in cultured fibroblasts from Tenon's capsule of the human eye.

Excess scarring of the conjunctiva after glaucoma filtration surgery is a major cause of failure. Transforming growth factor (TGF)-ß is critically involved in post-operative scarring. Lithium inhibits TGF-ß-induced gene protein expression in corneal fibroblasts and inhibits TGF-ß-induced epithelial mesenchymal transition. Here, we investigated the effects of LiCl on TGF-ß1-mediated signaling pathways and on myofibroblast transdifferentiation of human Tenon's capsule fibroblasts (HTFs). LiCl treatment reduced expression of TGF-ß1-induced α-SMA expression in HTFs. LiCl also decreased Akt phosphorylation induced by TGF-ß1. TGF-ß1-induced α-SMA expression was significantly decreased by LY294002 and Akt siRNA indicating that these changes are mediated by the PI3K/Akt pathway. Thus, LiCl induces the suppression of transdifferentiation stimulated by TGF-ß1 by the regulation of PI3K/Akt signaling in HTFs.

Transduction of PTEN proteins using the tat domain modulates TGF-ß1-mediated signaling pathways and transdifferentiation in subconjunctival fibroblasts.

PURPOSE: This study investigated the effects of the tumor suppressor protein PTEN (phosphatase and tensin homolog) on transforming growth factor (TGF)-ß1-mediated signaling pathways and the transdifferentiation of human subconjunctival fibroblasts (SCFs) after the transduction of this protein containing a transactivator of transcription (Tat) domain. METHODS: The Tat-PTEN expression vector was constructed to express the Tat domain of HIV-1 fused to PTEN. After transduction of the fusion protein and TGF-ß1 stimulation, the dose-dependent effect of the transduced Tat-PTEN fusion protein on Akt phosphorylation and the stability of the Tat-PTEN fusion protein in SCF cells were evaluated by Western blot analysis. The effect of the Tat-PTEN fusion protein on the TGF-ß1-stimulated expression of α-SMA and fibronectin was also evaluated by Western blot analysis and immunocytochemistry. RESULTS: To increase the efficiency of enzyme activity and to successfully deliver this protein to cells, the authors used a PTEN fusion protein that contained the transduction domain of the Tat protein from HIV-1. By Western blot analysis, the transduced Tat-PTEN fusion protein was found to modulate TGF-ß1 signaling in SCF cells and result in the suppression of Akt phosphorylation. Furthermore, the transduction of the Tat-PTEN fusion protein was found to suppress the TGF-ß1-stimulated expression of α-SMA and fibronectin by Western blot analysis and immunocytochemical staining, and the effects of the transduced fusion protein could be controlled in a dose-dependent manner. CONCLUSIONS: The Tat-PTEN fusion proteins were successfully transduced into the SCF cells and induced the suppression of transdifferentiation and fibrosis through the regulation of TGF-ß-mediated signaling. The ability of the Tat-PTEN fusion protein to regulate cell survival could potentially be applied to protein therapy to counteract postoperative scarring in glaucoma surgery.

TGF-ß-stimulated aberrant expression of class III ß-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells.

The class III ß-tubulin isotype (ß(III)) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III ß-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-ß (TGF-ß) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-ß on the aberrant expression of class III ß-tubulin and the intracellular signaling pathway mediating these changes. TGF-ß-induced aberrant expression and O-linked-ß-N-acetylglucosamine (O-GlcNac) modification of class III ß-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-ß also stimulated phosphorylation of ERK. TGF-ß-induced aberrant expression of class III ß-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-ß stimulated aberrant expression of class III ß-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-ß stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.

Potentiation of bortezomib-induced apoptosis by TGF-beta in cultured human Tenon's fibroblasts: contribution of the PI3K/Akt signaling pathway.

PURPOSE: To investigate the effects of bortezomib on cell apoptosis and proliferation in human Tenon's capsule fibroblasts (HTFs) after cotreatment with TGF-ß. METHODS: The effect of bortezomib on the apoptosis and cell proliferation of cultured HTFs was determined with FACS analysis and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assay with or without cotreatment of TGF-ß. The apoptotic effect of cotreatment of bortezomib and TGF-ß through the PI3K/Akt pathway was determined by Western blot analysis. The mRNA level of Bcl-2 and Bax was determined by RT-PCR. p53 expression, DNA-PKcs cleavage, and c-Jun phosphorylation were determined. RESULTS: Cotreatment with bortezomib (5 µM) and TGF-ß (10 µM) increased the proportion of apoptotic cells in HTFs on FACS analysis, whereas either bortezomib or TGF-ß treatment alone did not. The MTT assay also showed that when bortezomib was cotreated with TGF-ß, the cell proliferation of HTFs induced by TGF-ß treatment was significantly decreased at 72-hour incubation. The cotreatment of bortezomib and TGF-ß specifically decreased the Akt phosphorylation induced by TGF-ß, indicating on Western blot analysis that these changes are mediated by the PI3K/Akt pathway. The mRNA level of an apoptosis-related factor, Bcl-2, was significantly reduced, and p53 expression, DNA-PKcs cleavage, and c-Jun phosphorylation were increased after cotreatment. CONCLUSIONS: Bortezomib-induced apoptosis is potentiated by TGF-ß cotreatment in cultured HTFs by inhibition of the PI3K/Akt pathway, indicating that the effect of bortezomib may be potentiated when the level of TGF-ß is elevated, as is observed in the postoperative period.

The Drosophila homolog of methionine sulfoxide reductase A extends lifespan and increases nuclear localization of FOXO.

Methionine sulfoxide reductase A (msrA) was previously found to increase resistance to oxidative stress and longevity in animals. We identified Drosophila msrA (dmsrA), a Drosophila homolog of human msrA, as a downstream effector of forkhead box O (FOXO) signaling in Drosophila, which enhances resistance to oxidative stress and increases survival under stressed conditions. Additionally, overexpression of dmsrA in neurons extended the lifespan of flies. Moreover, overexpression of dmsrA in fat body cells caused FOXO to translocate to the nucleus, implying that this possible positive feedback loop between dmsrA and FOXO could potentiate the antioxidant activity of dmsrA and increase the lifespan in Drosophila.

Effects of AG490 on lens epithelial cell death induced by H(2)O(2).

PURPOSE: To evaluate the influence of H(2)O(2) on lens epithelial cells (LECs) and to determine the effect of the Janus kinase (JAK) inhibitor AG490 and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 on LEC death after H(2)O(2) exposure. METHODS: Human lens epithelial (HLE) B-3 cells were cultured. Cells were treated for 45 min with H2O2 and signal transducers and activators transcription (STAT) 3, JAK2, and ERK1/2 phosphorylation were surveyed by Western blot analysis. After pretreatment with either 40 microM AG490 or 25 microM U0126, LECs were exposed to H(2)O(2). LEC death was evaluated by microscopy and flow cytometry. RESULTS: H(2)O(2) induced phosphorylation of Tyr-705 STAT3, JAK2, and ERK1/2 in LECs. In cells pretreated with both AG490 and U0126, phosphorylation of Tyr-705 STAT3, JAK2, and ERK1/2 was suppressed. Microscopic findings, however, showed that only AG490 noticeably enhanced cell survival, and flow cytometry showed that cell necrosis decreased to 4.05% after pretreatment with 40 microM AG490. CONCLUSIONS: AG490 may prevent H(2)O(2)-induced LEC death by blocking an unknown necrosis pathway. Further investigation is required to characterize that pathway.

TGF-beta-induced interleukin-6 participates in transdifferentiation of human Tenon's fibroblasts to myofibroblasts.

PURPOSE: To gain a better understanding of the roles of interleukins (ILs) in subconjunctival fibrosis, we investigated their expression in transforming growth factor-beta1 (TGF-beta1)-stimulated Tenon's fibroblasts and examined their association with the transdifferentiation of fibroblasts to myofibroblasts. METHODS: After primary culture, fibroblasts derived from human Tenon's capsule were exposed to TGF-beta1. The expression of alpha-smooth muscle actin (alpha-SMA) protein was assessed by western immunoblots and immunofluorescence. The mRNA levels of various ILs were also evaluated by multiplex reverse transcription (RT)-PCR. Using the small interfering RNAs (siRNAs) specific for IL-6 and IL-11 and the promoter deletion assay, the contributions of IL-6 and IL-11 to TGF-beta1-induced induction of alpha-SMA were determined. RESULTS: In human Tenon's fibroblasts, TGF-beta1 stimulated the expression of alpha-SMA protein determined by western blot analysis and also increased the mRNA levels of IL-6 and IL-11 determined by multiplex RT-PCR. On the western immunoblots and immunofluorescence, the increased expression of alpha-SMA was attenuated only by the siRNAs specific for IL-6 but not by the siRNAs specific for IL-11. When the activator protein-1 binding sites of the IL-6 promoter region were deleted, the stimulation effects of TGF-beta1 decreased. CONCLUSIONS: Our data show that autocrine IL-6 may participate in the TGF-beta1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is known to be an essential step for subconjunctival fibrosis.

PDGF stimulation of Müller cell proliferation: Contributions of c-JNK and the PI3K/Akt pathway.

Platelet-derived growth factor (PDGF) has a critical role in proliferative vitreoretinopathy (PVR) as a chemoattractant and mitogen for retinal pigment epithelial cells and retinal glial cells. Here, we investigated the potential effects of PDGF on the proliferation of Müller cells and the intracellular signaling pathway mediating these changes. PDGF induced Müller cell proliferation and increased phosphorylation of the PDGF receptor (PDGFR), as shown by an MTT assay and immunoprecipitation analyses. Both effects were blocked by JNJ, a PDGFR-selective tyrosine kinase inhibitor. PDGF also stimulated phosphorylation of c-JNK and Akt. PDGF-induced Müller cell proliferation was significantly reduced by pre-treatment with SP600125 and LY294002, inhibitors of c-JNK and Akt phosphorylation, respectively. Our findings collectively indicate that PDGF-stimulated Müller cell proliferation occurs via activation of the c-JNK and PI3K/Akt signaling pathways. These data provide useful information in establishing the role of Müller cells in the development of proliferative vitreoretinopathy.

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling.

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.

IGF-1 counteracts TGF-beta-mediated enhancement of fibronectin for in vitro human lens epithelial cells.

PURPOSE: To determine whether insulin-like growth factor (IGF-1) affects transforming growth factor (TGF-beta)- mediated fibronectin accumulation in human lens epithelial cell line (HLE B-3) cells. MATERIALS AND METHODS: HLE B-3 cells were incubated for 24 hours with TGF-beta (10 ng/ mL), IGF-1 (10 ng/mL), or both. Expression of the fibronectin gene was determined using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Fibronectin levels were examined using western blot analysis and immunofluorescence staining. RESULTS: Expression of the fibronectin gene was not different between the TGF-beta/IGF-1 treated group and the TGF-beta treated group (p= 0.116). However, western blot analysis demonstrated decreased fibronectin levels in human lens epithelial cells treated with TGF-beta and IGF-1 compared to those treated with TGF-beta only (p < 0.01). Immunofluorescence staining disclosed inhibition of TGF-beta-induced fibronectin in the presence of IGF-1. CONCLUSION: This study suggests that IGF-1 counteracts TGF-beta-mediated fibronectin accumulation in human lens epithelial cells.

Upregulation of TGF-beta-induced tissue transglutaminase expression by PI3K-Akt pathway activation in human subconjunctival fibroblasts.

PURPOSE: Excessive scarring in subconjunctival tissues after filtering surgery seems to be characterized by aberrant extracellular matrix (ECM) production, and tissue transglutaminase (tTgase) plays an important role in this process. In the present study, the effects of transforming growth factor (TGF)-beta2 on the expression of tTgase, its activity in subconjunctival fibroblasts and whether the effects of TGF-beta are mediated by prosurvival signaling pathways were examined. METHODS: Primary subconjunctival fibroblasts treated with TGF-beta2 were examined for the expression of tTgase with Western blot analysis. The modulation of extracellular tTgase activity by TGF-beta2 was measured by both the formation of fibronectin polymers and the ECM protein incorporation of fluorescein cadaverine. The expression of tTgase was analyzed by immunofluorescence staining and Western blot analysis of subconjunctival fibroblasts that were transiently transfected with an Akt dominant negative mutant gene or were treated with an Akt inhibitor or tTgase siRNA. RESULTS: Treatment of subconjunctival fibroblasts with TGF-beta2 caused an increase in activation and expression of tTgase. The effects of TGF-beta stimulation of subconjunctival fibroblasts were twofold, causing both rapid activation of the ERK pathway within minutes of treatment and a more delayed activation of the phosphatidylinositol3-kinase-protein kinase B (PKB)/Akt pathway; however, only Akt activation was necessary for TGF-beta-induced tTgase expression. Transient transfection of subconjunctival fibroblasts with an Akt dominant negative mutant gene, or treatment with an Akt inhibitor (but not with an ERK inhibitor) or tTgase siRNA led to decreased activation and expression of tTgase. CONCLUSIONS: TGF-beta2 activated the PI3K-Akt pathway, and this activation was essential for the expression and activity of tTgase in subconjunctival fibroblasts. The results indicate a novel biological function of the PI3K-Akt pathway in subconjunctival fibroblasts. Elevated expression and activity of tTgase may play an important role in the pathogenesis of diseases related to wound healing and fibrogenic reactions in subconjunctival fibroblasts.

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases.

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-alpha. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced.

N-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide, a novel hydroxamic acid-based inhibitor of aminopeptidase N and its anti-angiogenic activity.

In the course of our screening, N-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide (1), which contains a metal-chelating hydroxamate group, has been identified as a potent inhibitor of aminopeptidase N (APN, EC 3.4.11.2). Compound 1 potently inhibited APN activity with a K(i) value of 3.5 microM. It also inhibited the basic fibroblast growth-factor-induced invasion of bovine aortic endothelial cells at low micromolar concentrations.

Extra-articular soft tissue ganglion cyst around the knee: focus on the associated findings.

The aim of this study was to evaluate MR imaging findings of the associated findings in surrounding tissues of the extra-articular soft tissue ganglion cysts around the knee. We retrospectively reviewed MR images of 30 patients who had surgically confirmed extra-articular soft tissue ganglion cysts around the knee with focus on the associated findings in surrounding tissues, such as muscle, subcutaneous fat, bone, and nerve. The most common associated finding was the visualization of channel between ganglion cyst and the joint, which was demonstrated in 20 cases (continuous type in 12 cases and discontinuous type in 8 cases). Other associated findings were seen in 15 cases; pericystic edema ( n=9), bony remodelling ( n=3), and nerve involvement ( n=3). The bony remodelling involved the proximal metaphysis of tibia in all 3 cases. Two patients with nerve involvement had deep peroneal nerve in subacute phase and one involved common peroneal nerve in chronic phase. The MR imaging is a useful imaging modality to evaluate the associated findings in extra-articular soft tissue ganglion cysts around the knee. The evaluation of these associated findings is helpful for the differentiation of ganglion cysts from other cystic lesions around the knee.