BMP2 downregulates urokinase-type plasminogen activator via p38 MAPK: Implications in C2C12 cells myogenic differentiation.

Bone morphogenetic protein (BMP)2 strongly affects the differentiation program of myoblast cells by inhibiting myogenesis and inducing osteogenic differentiation. In turn, extracellular matrix (ECM) proteinases, such as urokinase-type plasminogen activator (uPA), can influence the fate of muscle stem cells by participating in ECM reorganization. Although both BMP2 and uPA have antagonistic roles in muscles cells differentiation, no connection between them has been elucidated so far. This study aims to determine whether BMP2 regulates uPA expression in the myogenic C2C12 cell line and its impact on muscle cell fate differentiation. Our results showed that BMP2 did not modify C2C12 cell proliferation in a growth medium or myogenic differentiation medium. Although BMP2 inhibited myogenesis and induced osteogenesis, these effects were achieved with different doses of BMP2. Low concentrations of BMP2 blocked myogenesis, while a higher concentration was needed to induce osteogenesis. Reduced uPA expression was noticed alongside myogenic inhibition at low concentrations of BMP2. BMP2 activated p38 MAPK signaling to inhibit uPA activity. Furthermore, ectopic human uPA expression reduced BMP2's ability to inhibit the myogenic differentiation of C2C12 cells. In conclusion, BMP2 inhibits uPA expression through p38 MAPK and in vitro myogenesis at non-osteogenic concentrations, while uPA ectopic expression prevents BMP2 from inhibiting myogenesis in C2C12 cells.

The expression of urokinase-type plasminogen activator system in upper tract urothelial carcinoma and its prognostic value after radical nephroureterectomy.

BACKGROUND: To evaluate the expression pattern and prognostic role of the urokinase-type plasminogen activator (uPA) system in patients who underwent radical nephroureterectomy (RNU) for nonmetastatic upper tract urothelial carcinoma (UTUC). METHODS: A total of 732 patients who were treated with RNU for clinically nonmetastatic UTUC comprised our analytical cohort. Immunohistochemical staining of uPA, uPA receptor (uPAR) and uPA inhibitor (PAI-1) was performed using Murine IgG1 monoclonal antibodies. Outcomes of interest were recurrence-free survival, cancer-specific survival, and overall survival. RESULTS: The median age of the patients was 69.8 years and 56.6% of them were males. Overall, overexpression of uPA, uPAR, and PAI-1 was observed in 292 (39.9%), 346 (47.3%), and 345 (47.1%) patients, respectively. The uPA system components showed a statistically significant association with adverse clinicopathologic features such as lymphovascular invasion, multifocality, sessile tumors, and advanced pathologic stage (P < 0.01). On multivariable models, higher pathologic tumor stage, multifocality, and lymph node involvement were associated with RFS, OS, and CSS, but not the overexpression of uPA, uPAR, or PAR-1. In patients with organ-confined disease (≤pT2N0), however, uPA was significantly associated with RFS (hazard ratio [HR]: 2.04, 95% confidence interval [CI]: 1.21-3.43), OS (HR: 1.59, 95% CI:1.08-2.24) and CSS (HR: 2.55, 95% CI:1.44-4.52). uPA improved the predictive accuracy of a standard post-RNU model for all 3 endpoints, in organ-confined disease, by a prognostically significant margin. CONCLUSIONS: Overexpression of uPA system components was associated with adverse clinicopathologic characteristics and survival outcomes on the univariable, but not multivariable analyses. uPA expression was an independent predictor of survival outcomes in patients with organ-confined disease. While the clinical value of the uPA system remains limited in this cohort, further studies are needed to identify a marker or constellation of markers of high predictive value to help in counseling and treatment planning of UTUC patients.

Expression of urokinase-type plasminogen activator system in non-metastatic prostate cancer.

PURPOSE: To investigate the prognostic role of expression of urokinase-type plasminogen activator system members, such as urokinase-type activator (uPA), uPA-receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in patients treated with radical prostatectomy (RP) for prostate cancer (PCa). METHODS: Immunohistochemical staining for uPA system was performed on a tissue microarray of specimens from 3121 patients who underwent RP. Cox regression analyses were performed to investigate the association of overexpression of these markers alone or in combination with biochemical recurrence (BCR). Decision curve analysis was used to assess the clinical impact of these markers. RESULTS: uPA, uPAR, and PAI-1 were overexpressed in 1012 (32.4%), 1271 (40.7%), and 1311 (42%) patients, respectively. uPA overexpression was associated with all clinicopathologic characteristics of biologically aggressive PCa. On multivariable analysis, uPA, uPAR, and PAI-1 overexpression were all three associated with BCR (HR: 1.75, p < 0.01, HR: 1.22, p = 0.01 and HR: 1.20, p = 0.03, respectively). Moreover, the probability of BCR increased incrementally with increasing cumulative number of overexpressed markers. Decision curve analysis showed that addition of uPA, uPAR, and PAI-1 resulted in a net benefit compared to a base model comparing standard clinicopathologic features across the entire threshold probability range. In subgroup analyses, overexpression of all three markers remained associated with BCR in patients with favorable pathologic characteristics. CONCLUSION: Overexpression of uPA, uPAR, and PAI-1 in PCa tissue were each associated with worse BCR. Additionally, overexpression of all three markers is informative even in patients with favorable pathologic characteristics potentially helping clinical decision-making regarding adjuvant therapy and/or intensified follow-up.

Circulating uPA as a potential prognostic biomarker for resectable esophageal squamous cell carcinoma.

Previous research showed that the 4 genes of matrix metallopeptidase 9 (MMP9), cyto-keratin 20 (CK20), cyto-keratin 19 (CK19) and urokinase type plasminogen activator (uPA) are detectable in the peripheral blood. All the 4 genes are related to tumor invasion and metastasis. However, whether their expression is associated with clinicopathologic factors and the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still confused. Expression levels of MMP9, CK20, CK19, and uPA were evaluated by quantificational real-time polymerase chain reaction (qRT-PCR) in peripheral blood of 205 ESCC patients who received radical resection. The cut-off value was 1000 copy numbers. Their impacts on clinicopathologic factors and survival were investigated. The uPA expression positively correlated with gender (P = .046) and tumor size (P = .046). Meanwhile, CK19 expression positively correlated with tumor size (P = .029), vascular invasion (P = .024), and CK20 expression positively correlated with tumor size (P = .035) and degrees of differentiation (P = .032). Moreover, the overexpression of MMP9 has a correlation with postoperative radiotherapy (P = .041) and chemotherapy (P = .012). Among the 4 genes, only uPA is a prognostic indicator for disease-free survival and overall survival both in univariate analysis and multivariate analysis (P = .015). This study suggests that circulating uPA mRNA in peripheral blood can serve as a potential unfavorable prognosis biomarker in ESCC. Further perspective, multi-center and large-scale study is still needed.

Influence of miR-34a on preeclampsia through the Notch signaling pathway.

OBJECTIVE: The aim of this study was to investigate the influence of micro-ribonucleic acid-34a (miR-34a) on preeclampsia through the Notch signaling pathway. PATIENTS AND METHODS: The expressions of miR-34a, Notch-1, Notch-2, and Notch-3 in the placenta of 39 preeclampsia patients and 42 normal patients were detected by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The correlations between miR-34a expression with the expressions of Notch-1, Notch-2 and Notch-3 were analyzed, respectively. Besides, placental trophoblasts were isolated from preeclampsia patients and cultured in vitro. The expressions of miR-34a, Notch-1, Notch-2 and Notch-3 in placental trophoblasts were analyzed. Furthermore, the influences of miR-34a on the protein expressions of Notch-1, Notch-2, Notch-3, and hairy and enhancer of split-1 (Hes-1) in the Notch signaling pathway were analyzed by Luciferase reporter gene assay and Western blotting. The role of Notch in trophoblast invasion was investigated through the Notch inhibitors. In addition, its influence on the expression of urokinase-type plasminogen activator (uPA) was studied by miR-34a overexpression. RESULTS: The expressions of miR-34a and Notch-1 were correlated with preeclampsia in the placentas of preeclampsia patients and normal patients to a certain degree. The expression of miR-34a in preeclamptic placenta was significantly higher than that of the normal placenta (p<0.05). However, Notch-1 expression was markedly lower in preeclamptic placenta (p<0.05). No significant differences were found in the expressions of Notch-2 and Notch-3 between the two types of placentas (p>0.05). MiR-34a had a remarkable negative correlation with Notch-1 expression in the Notch family (p<0.001, r=-0.5775). RT-PCR results revealed that the mRNA expression of miR-34a in placental trophoblasts of patients with preeclampsia was notably higher than that of normal people (p<0.01). However, Western blotting demonstrated that the protein expressions of Notch-1, Notch-2 and Notch-3 exhibited the opposite results. Additionally, the protein expression of Notch-1, Notch-2, Notch-3 and Hes-1 in trophoblasts transfected with pre-miR-34a was significantly decreased. The treatment with Notch inhibitors markedly reduced the trophoblast invasion. Furthermore, miR-34a overexpression or intracellular domain of Notch (ICN) overexpression regulated uPA expression. CONCLUSIONS: MiR-34a regulates uPA system through the Notch signal transduction, thereby regulating the invasion of placental trophoblasts in patients with preeclampsia.

Identification of the key genes associated with neuropathic pain.

Neuropathic pain is a chronic pain state associated with multiple etiologies that results in considerable social and economic burden. The identification of key genes associated with neuropathic pain is important for the development of novel therapies. Therefore, the present study downloaded the gene expression profile GSE15041 from the Gene Expression Omnibus database. The unverified gene chip was removed and the microarray data was normalized following quality control. The limma package in R was used to screen the differentially expressed genes (DEGs), followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Furthermore, a protein­protein interaction (PPI) network based on the identified DEGs was constructed to select hub proteins, and reverse transcription­quantitative polymerase chain reaction was performed to detect the expression of these proteins in a mouse model of neuropathic pain. In total, 86 common DEGs were identified. DEGs were significantly enriched in ̔extracellular space̓ and KEGG pathway enrichment analysis demonstrated that the DEGs were significantly enriched in inflammatory diseases and the mitogen­activated protein kinase signaling pathway. The PPI network consisted of 27 nodes (proteins) and 47 PPI edges (interactions). Interleukin (IL)­6, transcription factor AP­1 (c­Jun) and urikinase­type plasminogen activator (Plau) were identified as hub proteins and key genes in neuropathic pain. The mRNA expression of these hub proteins was significantly increased in the neuropathic pain model, compared with the sham group. IL­6, c­Jun, and Plau may be involved in development of neuropathic pain and further research investigating the exact role of these key genes is required.

Extracellular Collagen Promotes Interleukin-1ß-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

Purpose: Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1ß (IL-1ß). Methods: Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Results: Collagen and IL-1ß synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1ß, were all abolished by a selective uPA inhibitor. Conclusions: Collagen and IL-1ß cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1ß-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

Multiparity upregulates placental plasminogen and urokinase-type plasminogen activator.

PROBLEM: Multiparity increased the number of trophoblast cells in decidua of both low and high fetal loss mouse models. However, they differ in fetal survival rate and maternal thymocyte subpopulations, suggesting that trophoblast invasiveness is not equivalent. Our aim was to explore the involved mechanism. METHOD OF STUDY: We studied placentae from primiparous and multiparous females of low and high fetal loss models. We investigated invasiveness in vitro, expression of plasminogen, and its activators: tissue type (tPA)-urokinase type (uPA), and activity and expression of matrix metalloproteinases (MMP)-2 and MMP-9. RESULTS: Placental invasiveness is upregulated by multiparity, but lesser in the high fetal loss model. Multiparous animals showed elevated expression of plasminogen and uPA. However, the high fetal loss combination showed higher expression of a short and less active fragment of uPA (LMW-uPA). MMP-2, MMP-9, and tPA were unaffected. CONCLUSION: uPA would participate in the increased multiparity-associated placental invasiveness.

Aeroallergen Der p 2 promotes motility of human non-small cell lung cancer cells via toll-like receptor-mediated up-regulation of urokinase-type plasminogen activator and integrin/focal adhesion kinase signaling.

House dust mite (HDM) allergens are one of the major causes leading to respiratory hypersensitiveness and airway remodeling. Here we hypothesized that a major HDM allergen Der p 2 could increase cell motility and invasiveness of non-small cell lung cancer (NSCLC) cells. Our results showed that low dose (1 and 3 µg/mL) recombinant Der p 2 protein (DP2) enhanced the migration and invasiveness of human NSCLC cell A549, H1299 and CL1-5, but nonsignificantly altered their growth. Further investigation revealed that integrin αV level was increased and its downstream signaling including focal adhesion kinase (FAK) and paxillin were activated in A549 cells exposed to DP2. In parallel, DP2 also activated the FAK-associated signaling effectors such as Src, phosphatidyl inositol 3-kinase (PI3K), AKT, p38 mitogen-activated protein kinase (P38), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Our findings also revealed that DP2 increased expression level of urokinase type plasminogen-activated kinase (uPA) and uPA receptor (uPAR), and subsequently enhanced the binding of uPAR to integrin αV. Moreover, the involvement of toll-like receptor 2/4 (TLR2/4)-triggered ERK1/2 activation in the increased expression of uPA and uPAR was also demonstrated. Collectively, these findings indicate that DP2 can enhance cell motility and invasiveness of NSCLC cells, attributing to TLR2/4-ERK1/2 activation, increased uPA and uPAR expression, enhanced binding of uPAR to integrin αV, and the consequent FAK signaling cascades. Thus, we suggest that DP2 may exacerbate NSCLC via promoting metastatic ability of carcinoma cell.

Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera.

BACKGROUND: The big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types. RESULTS: To selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity. CONCLUSION: Our results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.

The Urokinase Receptor-Derived Peptide UPARANT Mitigates Angiogenesis in a Mouse Model of Laser-Induced Choroidal Neovascularization.

PURPOSE: A mouse model of age-related macular degeneration (AMD) was used to investigate the anti-angiogenic and anti-inflammatory role of UPARANT in laser-induced choroidal neovascularization (CNV). METHODS: Choroidal neovascularization was induced by laser photocoagulation, and UPARANT was intravitreally injected. Some experiments were also performed after either intravitreal injection of anti-VEGF drugs or systemic administration of UPARANT. Immunohistochemistry using CD31 antibodies was used to evaluate the area of CNV. Evans blue dye extravasation was quantitatively assessed. Transcripts of markers of outer blood retinal barrier were measured by quantitative RT-PCR, also used to evaluate angiogenesis and inflammation markers. Western blot was used to determine levels of transcription factors encoding genes involved in angiogenesis and inflammation. Levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and formyl peptide receptors (FPRs) were determined at the transcript and the protein level. RESULTS: Intravitreal UPARANT reduced the CNV area and the leakage from the choroid. The uPA/uPAR/FPR system was upregulated in CNV, but was not influenced by UPARANT. UPARANT recovered laser-induced upregulation of transcription factors encoding angiogenic and inflammatory markers. Accordingly, angiogenic and inflammatory factors were also reduced. UPARANT as compared to anti-VEGF drugs displayed similar effects on CNV area. CONCLUSIONS: UPARANT mitigates laser-induced CNV by inhibiting angiogenesis and inflammation through an action on transcription factors encoding angiogenesis and inflammatory genes. The finding that UPARANT is effective against CNV may help to establish uPAR and its membrane partners as putative targets in the treatment of AMD.

Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model.

A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis.

High glucose and insulin enhance uPA expression, ROS formation and invasiveness in breast cancer-derived cells.

BACKGROUND: Accumulating evidence indicates that type 2 diabetes is associated with an increased risk to develop breast cancer. This risk has been attributed to hyperglycemia, hyperinsulinemia and chronic inflammation. As yet, however, the mechanisms underlying this association are poorly understood. Here, we studied the effect of high glucose and insulin on breast cancer-derived cell proliferation, migration, epithelial-mesenchymal transition (EMT) and invasiveness, as well as its relationship to reactive oxygen species (ROS) production and the plasminogen activation system. METHODS: MDA-MB-231 cell proliferation, migration and invasion were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), scratch-wound and matrigel transwell assays, respectively. ROS production was determined using 2' 7'-dichlorodihydrofluorescein diacetate. The expression of E-cadherin, vimentin, fibronectin, urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) were assessed using qRT-PCR and/or Western blotting assays, respectively. uPA activity was determined using gel zymography. RESULTS: We found that high glucose stimulated MDA-MB-231 cell proliferation, migration and invasion, together with an increased expression of mesenchymal markers (i.e., vimentin and fibronectin). These effects were further enhanced by the simultaneous administration of insulin. In both cases, the invasion and growth responses were found to be associated with an increased expression of uPA, uPAR and PAI-1, as well as an increase in active uPA. An osmolality effect of high glucose was excluded by using mannitol at an equimolar concentration. We also found that all changes induced by high glucose and insulin were attenuated by the anti-oxidant N-acetylcysteine (NAC) and, thus, depended on ROS production. CONCLUSIONS: From our data we conclude that hyperglycemia and hyperinsulinemia can promote breast cancer cell proliferation, migration and invasion. We found that these features were associated with increased expression of the mesenchymal markers vimentin and fibronectin, as well as increased uPA expression and activation through a mechanism mediated by ROS.

Human urokinase-type plasminogen activator gene-modified bone marrow-derived mesenchymal stem cells attenuate liver fibrosis in rats by down-regulating the Wnt signaling pathway.

AIM: To evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) with human urokinase-type plasminogen activator (uPA) on liver fibrosis, and to investigate the mechanism of gene therapy. METHODS: BMSCs transfected with adenovirus-mediated human urokinase plasminogen activator (Ad-uPA) were transplanted into rats with CCl4-induced liver fibrosis. All rats were sacrificed after 8 wk, and their serum and liver tissue were collected for biochemical, histopathologic, and molecular analyzes. The degree of liver fibrosis was assessed by hematoxylin and eosin or Masson's staining. Western blot and quantitative reverse transcription-polymerase chain reaction were used to determine protein and mRNA expression levels. RESULTS: Serum levels of alanine aminotransferase, aminotransferase, total bilirubin, hyaluronic acid, laminin, and procollagen type III were markedly decreased, whereas the levels of serum albumin were increased by uPA gene modified BMSCs treatment. Histopathology revealed that chronic CCl4-treatment resulted in significant fibrosis while uPA gene modified BMSCs treatment significantly reversed fibrosis. By quantitatively analysing the fibrosis area of liver tissue using Masson staining in different groups of animals, we found that model animals with CCl4-induced liver fibrosis had the largest fibrotic area (16.69% ± 1.30%), while fibrotic area was significantly decreased by BMSCs treatment (12.38% ± 2.27%) and was further reduced by uPA-BMSCs treatment (8.31% ± 1.21%). Both protein and mRNA expression of ß-catenin, Wnt4 and Wnt5a was down-regulated in liver tissues following uPA gene modified BMSCs treatment when compared with the model animals. CONCLUSION: Transplantation of uPA gene modified BMSCs suppressed liver fibrosis and ameliorated liver function and may be a new approach to treating liver fibrosis. Furthermore, treatment with uPA gene modified BMSCs also resulted in a decrease in expression of molecules of the Wnt signaling pathway.

Resveratrol inhibits hypoxia-driven ROS-induced invasive and migratory ability of pancreatic cancer cells via suppression of the Hedgehog signaling pathway.

A hypoxic microenvironment is commonly found in the central region of solid tumors, including pancreatic cancer. Our previous study revealed that resveratrol plays an important role in suppressing the proliferation and EMT of pancreatic cancer cells. However, whether resveratrol could suppress hypoxia-induced cancer progression and the underlying mechanisms have not been fully elucidated. The aim of the present study was to evaluate whether resveratrol affects hypoxia-induced reactive oxygen species (ROS) production and the activation of the Hedgehog (Hh) signaling pathway as well as the invasion of pancreatic cancer. The human pancreatic cancer cell lines, BxPC-3 and Panc-1, were subjected to a hypoxic condition and three different concentrations of resveratrol. The intracellular ROS were determined using 2,7-dichlorodihydrofluorecein diacetate. Wound healing and Transwell invasion assays were used to detect the migratory and invasive potential of the cancer cells. Metastatic-related and Hh signaling-related factors were detected by qRT-PCR and western blot analysis. Immunofluorescence staining was used to test the nuclear translocation of GLI1. The results showed that the hypoxia-induced production of ROS was decreased by resveratrol in a concentration-dependent manner. Resveratrol significantly inhibited the hypoxia-stimulated invasion and migration of pancreatic cancer cells. Resveratrol inhibited hypoxia-induced HIF-1α protein expression. Resveratrol also suppressed hypoxia­induced expression of metastatic-related factors, uPA and MMP2. In addition, resveratrol markedly inhibited hypoxia-mediated activation of the Hh signaling pathway. Furthermore, the antioxidant N-acetylcysteine (NAC) significantly suppressed the invasive and migratory ability of pancreatic cancer cells during hypoxia. Taken together, these data indicate that resveratrol plays an important role in suppressing hypoxia-driven ROS-induced pancreatic cancer progression by inhibiting the Hh signaling pathway, providing evidence that resveratrol may be a potential candidate for the chemoprevention of cancer.

Downregulation of miR-193b in systemic sclerosis regulates the proliferative vasculopathy by urokinase-type plasminogen activator expression.

OBJECTIVES: To investigate the role of microRNA-193b-3p (miR-193b) in the vascular pathophysiology of systemic sclerosis (SSc). METHODS: Expression of miR-193b in skin biopsies and fibroblasts from patients with SSc and normal healthy (NH) controls were determined by real-time PCR. Transfection with miR-193b precursor and inhibitor were used to confirm targets of miR-193b. Proliferative effects of urokinase-type plasminogen activator (uPA) were determined by water-soluble tetrazolium salt-1 assay and by analysis of proliferating cell nuclear antigen expression. Fluorescence activated cell sorting analysis was performed to investigate the effect of uPA on apoptosis. For inhibition of the uPA-cellular receptor for uPA (uPAR) pathway, uPAR neutralising antibodies and low molecular weight uPA were used. RESULTS: We found that miR-193b was downregulated in SSc fibroblasts and skin sections as compared with NH controls. The expression of miR-193b was not affected by major profibrotic cytokines and hypoxia. Induction of miR-193b in SSc fibroblasts suppressed, and accordingly, knockdown of miR-193b increased the levels of messenger RNA and protein for uPA. uPA was found to be upregulated in SSc as compared with NH controls in a transforming growth factor-ß dependent manner, and uPA was strongly expressed in vascular smooth muscle cells in SSc skin section. Interestingly, uPA induced cell proliferation and inhibited apoptosis of human pulmonary artery smooth muscle cells, and these effects were independent of uPAR signalling. CONCLUSIONS: In SSc, the downregulation of miR-193b induces the expression of uPA, which increases the number of vascular smooth muscle cells in an uPAR-independent manner and thereby contributes to the proliferative vasculopathy with intimal hyperplasia characteristic for SSc.

Corneal Expression of SLURP-1 by Age, Sex, Genetic Strain, and Ocular Surface Health.

PURPOSE: Although secreted Ly6/urokinase-type plasminogen activator receptor-related protein-1 (Slurp1) transcript is highly abundant in the mouse cornea, corresponding protein expression remains uncharacterized. Also, SLURP1 was undetected in previous tear proteomics studies, resulting in ambiguity about its baseline levels. Here, we examine mouse corneal Slurp1 expression in different sexes, age groups, strains, and health conditions, and quantify SLURP1 in human tears from healthy or inflamed ocular surfaces. METHODS: Expression of Slurp1 in embryonic day-13 (E13), E16, postnatal day-1 (PN1), PN10, PN20, and PN70 Balb/C, FVBN, C57Bl/6, and DBA/2J mouse corneas, Klf4Δ/ΔCE corneas with corneal epithelial-specific ablation of Klf4, migrating cells in wild-type corneal epithelial wound edge, and in corneas exposed to pathogen-associated molecular patterns (PAMPs) poly(I:C), zymosan-A, or Pam3Csk4 was examined by QPCR, immunoblots, and immunofluorescent staining. Human SLURP1 levels were quantified by ELISA in tears from 34 men and women aged 18 to 80 years. RESULTS: Expression of Slurp1, comparable in different strains and sexes, was low in E13, E16, PN1, and PN10 mouse corneas, and increased rapidly after eyelid opening in a Klf4-dependent manner. We found Slurp1 was downregulated in corneas exposed to PAMPs, and in migrating cells at the wound edge. Human SLURP1 expression, comparable in different sexes and age groups, was significantly decreased in tears from inflamed ocular surfaces (0.34%) than those from healthy individuals (0.77%). CONCLUSIONS: These data describe the influence of age, sex, genetic background, and ocular surface health on mouse corneal expression of Slurp1, establish the baseline for human tear SLURP1 expression, and identify SLURP1 as a useful diagnostic and/or therapeutic target for inflammatory ocular surface disorders.

Immunohistochemical Detection of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor in Canine Vascular Endothelial Tumours.

Immunohistochemistry was used to assess the expression of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in 57 canine primary haemangiosarcomas (HSAs), 26 canine cutaneous haemangiomas (HAs) and in control sections of canine cutaneous granulation tissue. The correlation between uPA/uPAR expression and the Ki67 labelling index (LI) was estimated in the HSA and HA tissues. uPA was expressed by 73.2% and 75.0% of splenic HSAs and non-splenic HSAs, respectively. All HSA tissues tested expressed uPAR. Expression of both molecules was significantly higher in HSAs than in cutaneous HAs (3.8% for uPA and 30.7% for uPAR). The average Ki67 LI of the uPA(+)/uPAR(+) HSAs was significantly higher than that of uPA(-)/uPAR(+) HSAs and HA tissues (mean ± SDs 32.8 ± 15.3, 15.2 ± 7.2 and 2.1 ± 0.7, respectively; P <0.05). These results suggest that uPA and uPAR play a significant role in the malignant proliferation of canine HSA, regardless of the primary origin of the tumour.

Platelet-delivered therapeutics.

We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches.

Macrophages of M1 phenotype have properties that influence lung cancer cell progression.

Stromal macrophages of different phenotypes can contribute to the expression of proteins that affects metastasis such as urokinase-type plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), but knowledge of how essential their contribution is in comparison to the cancer cells in small cell lung cancer (SCLC) and lung squamous cell carcinoma (SCC) is lacking. The expression of uPA, uPAR, and PAI-1 and of the matrix metalloproteinases (MMP)-2 and MMP-9 were studied in human macrophages of M1 and M2 phenotype and compared to a lung SCC (NCI-H520) and a SCLC (NCI-H69) cell line. Effects of treatment with conditioned media (CM) from M1 and M2 macrophages on the expression of these genes in H520 and H69 cells as well as effects on the cell growth were investigated. In addition, data on the stromal macrophages immunoreactivity of uPAR, MMP-2, and MMP-9 in a few SCC and SCLC biopsies was included. uPAR, MMP-2, and MMP-9 were confirmed in stromal cells including macrophages in the SCC and SCLC biopsies. In vitro, both macrophage phenotypes expressed considerably higher mRNA levels of uPA, uPAR, PAI-1, and MMP-9 compared to the cancer cell lines, and regarding uPAR, the highest level was found in the M1 macrophage phenotype. Furthermore, M1 CM treatment not only induced an upregulation of PAI-1 in both H520 and H69 cells but also inhibited cell growth in both cell lines, giving M1 macrophages both tumor-promoting and tumor-killing potential.