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1.
Mol Med ; 27(1): 158, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34906074

RESUMO

BACKGROUND: The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells. METHODS: Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays. RESULTS: Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression. CONCLUSIONS: Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells.


Assuntos
Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal/genética , Fator de Crescimento Transformador beta1/genética , Proteínas rab de Ligação ao GTP/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Fator de Crescimento Transformador beta1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
2.
Res Pract Thromb Haemost ; 7(4): 100177, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37333992

RESUMO

Background: Tissue factor (TF) is essential for hemostasis. TF-expressing extracellular vesicles (TF+ EVs) are released in pathological conditions, such as trauma and cancer, and are linked to thrombosis. Detection of TF+ EV antigenically in plasma is challenging due to their low concentration but may be of clinical utility. Objectives: We hypthesised that ExoView can allow for direct measurement of TF+ EV in plasma, antigenically. Methods: We utilized the anti-TF monoclonal antibody 5G9 to capture TF EV onto specialized ExoView chips. This was combined with fluorescent TF+ EV detection using anti-TF monoclonal antibody IIID8-AF647. We measured tumor cell-derived (BxPC-3) TF+ EV and TF+ EVs from plasma derived from whole blood with or without lipopolysaccharide (LPS) stimulation. We used this system to analyze TF+ EVs in 2 relevant clinical cohorts: trauma and ovarian cancer. We compared ExoView results with an EV TF activity assay. Results: BxPC-3-derived TF+ EVs were identified with ExoView using 5G9 capture with IIID8-AF647 detection. 5G9 capture with IIID8-AF647 detection was significantly higher in LPS+ samples than in LPS samples and correlated with EV TF activity (R2 = 0.28). Trauma patient samples had higher levels of EV TF activity than healthy controls, but activity did not correlate with TF measurements made by ExoView (R2 = 0.15). Samples from patients with ovarian cancer have higher levels of EV TF activity than those from healthy controls, but activity did not correlate with TF measurement by ExoView (R2 = 0.0063). Conclusion: TF+ EV measurement is possible in plasma, but the threshold and potential clinical applicability of ExoView R100, in this context, remain to be established.

3.
Mol Cancer ; 11: 62, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22920728

RESUMO

BACKGROUND: Rab proteins constitute a large family of monomeric GTP-binding proteins that regulate intracellular vesicle transport. Several Rab proteins, including rab31, have been shown to affect cancer progression and are related with prognosis in various types of cancer including breast cancer. Recently, the gene encoding rab31 was found to be overexpressed in estrogen receptor-positive breast cancer tissue. In a previous study we found a significant association of high rab31 mRNA expression with poor prognosis in node-negative breast cancer patients. In the present study, we aimed to investigate the impact of rab31 (over)-expression on important aspects of tumor progression in vitro and in vivo. METHODS: Breast cancer cells displaying low (MDA-MB-231) or no (CAMA-1) endogenous rab31 expression were stably transfected with a rab31 expression plasmid. Batch-transfected cells as well as selected cell clones, expressing different levels of rab31 protein, were analyzed with regard to proliferation, cell adhesion, the invasive capacity of tumor cells, and in vivo in a xenograft tumor model. Polyclonal antibodies directed to recombinantly expressed rab31 were generated and protein expression analyzed by immunohistochemistry, Western blot analysis, and a newly developed sensitive ELISA. RESULTS: Elevated rab31 protein levels were associated with enhanced proliferation of breast cancer cells. Interestingly, weak to moderate overexpression of rab31 in cell lines with no detectable endogenous rab31 expression was already sufficient to elicit distinct effects on cell proliferation. By contrast, increased expression of rab31 in breast cancer cells led to reduced adhesion towards several extracellular matrix proteins and decreased invasive capacity through Matrigel(TM). Again, the rab31-mediated effects on cell adhesion and invasion were dose-dependent. Finally, in a xenograft mouse model, we observed a significantly impaired metastatic dissemination of rab31 overexpressing MDA-MB-231 breast cancer cells to the lung. CONCLUSIONS: Overexpression of rab31 in breast cancer cells leads to a switch from an invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced adhesion and invasion in vitro, and a reduced capacity to form lung metastases in vivo.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Proteínas rab de Ligação ao GTP/genética , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Transplante Heterólogo , Proteínas rab de Ligação ao GTP/imunologia , Proteínas rab de Ligação ao GTP/metabolismo
4.
Biol Chem ; 393(12): 1449-55, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23667902

RESUMO

mRNA levels of the urokinase receptor splice variant uPAR-del4/5 are associated with prognosis in breast cancer. Its overexpression in cancer cells affects tumor biologically relevant processes. In the present study, individual breast cancer cell clones displaying low vs. high uPAR-del4/5 expression were analyzed demonstrating that uPAR-del4/5 leads to reduced cell adhesion and invasion in a dose-dependent manner. Additionally,matrix metalloproteinase-9 (MMP-9) was found to be strongly upregulated in uPAR-del4/5 overexpressing compared to vector control cells. uPAR-del4/5 may thus play an important role in the regulation of the extracellular proteolytic network and, by this, influence the metastatic potential of breast cancer cells.


Assuntos
Neoplasias da Mama/genética , Mama/metabolismo , Splicing de RNA , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Mama/citologia , Mama/patologia , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/genética , Invasividade Neoplásica/genética , Regulação para Cima
5.
Blood ; 116(5): 806-14, 2010 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-20410508

RESUMO

Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TF(flox/flox),LysM(Cre) mice) or in both endothelial cells (ECs) and hematopoietic cells (TF(flox/flox),Tie-2(Cre) mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).


Assuntos
Coagulação Sanguínea/fisiologia , Endotoxemia/fisiopatologia , Células Mieloides/metabolismo , Tromboplastina/fisiologia , Animais , Antitrombina III/análise , Plaquetas/metabolismo , Células Cultivadas , Coagulação Intravascular Disseminada/etiologia , Coagulação Intravascular Disseminada/fisiopatologia , Células Endoteliais/metabolismo , Endotoxemia/sangue , Deleção de Genes , Leucócitos/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/análise , Precursores de RNA/biossíntese , Precursores de RNA/sangue , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Quimera por Radiação , Especificidade da Espécie , Tromboplastina/antagonistas & inibidores , Tromboplastina/deficiência , Tromboplastina/genética
6.
Breast Cancer Res Treat ; 127(3): 649-57, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20635136

RESUMO

uPAR, the three-domain membrane receptor of the serine protease urokinase, plays a crucial role in tumor growth and metastasis. Several mRNA splice variants of this receptor have been reported. One of these, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, is specifically overexpressed in breast cancer and represents a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. The aim of the present study was to examine the molecular and cellular properties of the encoded uPAR-del4/5 protein. To investigate the impact of the uPAR-del4/5 overexpression on in vitro and in vivo aspects of tumor progression (e.g., proliferation, adhesion, invasion, metastatic seeding, and/or metastatic growth), we combined the analysis of transfected cancer cell lines with a murine xenograft tumor model. Increased expression of uPAR-del4/5 in human cancer cells led to reduced adhesion to several extracellular matrix proteins and decreased invasion through Matrigel, while cell proliferation was not affected in vitro. Moreover, invasion of uPAR-del4/5 overexpressing cells was not altered by addition of urokinase, while that of uPAR-wild-type overexpressing cells was drastically increased. Accordingly, we observed that, in contrast to uPAR-wild-type, uPAR-del4/5 does not interact with urokinase. On the other hand, when overexpressed in human breast cancer cells, uPAR-del4/5 distinctly impaired metastatic dissemination and growth in vivo. We demonstrate that the uPAR-del4/5 mRNA splice variant mediates tumor-relevant biological processes in vitro and in vivo. Our results thus illustrate how tumor-specific alternative splicing can distinctly impact the biology of the tumor.


Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , RNA Mensageiro/genética , Deleção de Sequência
7.
BMC Cancer ; 11: 273, 2011 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-21702998

RESUMO

BACKGROUND: Members of the urokinase-type plasminogen activator (uPA) system are up-regulated in various solid malignant tumors. High antigen levels of uPA, its inhibitor PAI-1 and its receptor uPAR have recently been shown to be associated with poor prognosis in soft-tissue sarcoma (STS) patients. However, the mRNA expression of uPA system components has not yet been comprehensively investigated in STS patients. METHODS: The mRNA expression level of uPA, PAI-1, uPAR and an uPAR splice variant, uPAR-del4/5, was analyzed in tumor tissue from 78 STS patients by quantitative PCR. RESULTS: Elevated mRNA expression levels of PAI-1 and uPAR-del4/5 were significantly associated with clinical parameters such as histological subtype (P = 0.037 and P < 0.001, respectively) and higher tumor grade (P = 0.017 and P = 0.003, respectively). In addition, high uPAR-del4/5 mRNA values were significantly related to higher tumor stage of STS patients (P = 0.031). On the other hand, mRNA expression of uPA system components was not significantly associated with patients' survival. However, in STS patients with complete tumor resection (R0), high PAI-1 and uPAR-del4/5 mRNA levels were associated with a distinctly increased risk of tumor-related death (RR = 6.55, P = 0.054 and RR = 6.00, P = 0.088, respectively). Strikingly, R0 patients with both high PAI-1 and uPAR-del4/5 mRNA expression levels showed a significant, 19-fold increased risk of tumor-related death (P = 0.044) compared to the low expression group. CONCLUSION: Our results suggest that PAI-1 and uPAR-del4/5 mRNA levels may add prognostic information in STS patients with R0 status and distinguish a subgroup of R0 patients with low PAI-1 and/or low uPAR-del4/5 values who have a better outcome compared to patients with high marker levels.


Assuntos
Regulação Neoplásica da Expressão Gênica , Inibidor 1 de Ativador de Plasminogênio/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Sarcoma/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sarcoma/patologia , Sarcoma/cirurgia , Análise de Sobrevida , Adulto Jovem
8.
Histopathology ; 57(3): 461-71, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20840675

RESUMO

AIMS: The urokinase-type plasminogen activator receptor (uPAR) is a key molecule for pericellular proteolysis in tumour cell invasion and metastasis. The aim was to evaluate the prognostic impact of uPAR in invasive breast cancer dependent on which cell types within the tumour express uPAR. METHODS AND RESULTS: uPAR expression was analysed by immunohistochemistry in 270 tumour tissue specimens of invasive ductal breast carcinomas using tissue microarrays. For evaluation of uPAR immunoexpression we used the epitope-mapped, uPAR domain II-specific monoclonal antibody IID7. High uPAR score values in both tumour cells (uPAR-Tc) and stromal cells were significantly related to high tumour grade (G3), and inversely correlated with oestrogen receptor status. On multivariate analysis, high uPAR-Tc values contributed independent prognostic information for disease-free survival (hazard ratio 1.93, P = 0.007) when adjusted for prognostically relevant clinicopathological parameters, whereas uPAR expression in stromal cells was not related to prognosis. In addition, elevated uPAR-Tc values were found to be prognostic indicators in clinically relevant subgroups of patients with invasive breast cancer. CONCLUSIONS: In invasive breast cancer uPAR expression in invasive carcinoma cells, but not in stromal cells, has a significant impact on patients' prognosis, and contributes to a more aggressive tumour phenotype.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/genética , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Análise em Microsséries , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética
9.
Circulation ; 116(20): 2298-306, 2007 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17967980

RESUMO

BACKGROUND: Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. METHODS AND RESULTS: We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. CONCLUSIONS: Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.


Assuntos
Cardiomegalia/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/diagnóstico por imagem , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/fisiopatologia , Ecocardiografia , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Infarto do Miocárdio/diagnóstico por imagem , Miócitos Cardíacos/fisiologia , Fenótipo , Traumatismo por Reperfusão/fisiopatologia , Tromboplastina/genética , Miosinas Ventriculares/genética
10.
Int J Mol Med ; 21(2): 251-9, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18204793

RESUMO

Urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) play a key role in tumor-associated processes such as the degradation of extracellular matrix proteins, tissue remodeling, cell adhesion, migration, and invasion. High antigen levels of uPA and PAI-1 in tumor tissue of various solid malignant tumors, including breast cancer, are associated with poor patient prognosis. In the present study, we examined whether analysis of uPA and PAI-1 mRNA expression represents an alternative to the measurement of the respective antigen levels in breast cancer. Highly sensitive quantitative real-time PCR (QPCR) assays, based on the LightCycler technology, were established to quantify uPA and PAI-1 mRNA expression in breast cancer cell lines as well as in tumor tissue of breast cancer patients. mRNA concentrations were normalized to the expression level of the housekeeping gene h-G6PDH. The respective uPA and PAI-1 antigen concentrations were determined by established ELISA formats. QPCR mean interassay variation coefficients were 11% (uPA) and 8% (PAI-1). In breast cancer cell lines, mRNA and antigen values were highly correlated for both uPA and PAI-1 (each: rs=0.95; p<0.001). In contrast, correlations between uPA/PAI-1 mRNA and protein in the breast cancer samples were found to be distinctly weaker or not significant. Thus, quantitative determination of mRNA expression for both factors does not mirror antigen levels in breast cancer tissue, possibly due to posttranscriptional regulation. Except for nodal status being inversely correlated with uPA mRNA levels, no significant interrelations were observed between uPA or PAI-1 mRNA expression and clinicopathological parameters. On the protein level, elevated uPA and PAI-1 values were associated with a negative steroid hormone receptor status. In conclusion, the implementation of mRNA quantification of uPA and PAI-1 in breast tumors is unable to serve as a one-to-one substitution for antigen determination by ELISA.


Assuntos
Antígenos de Neoplasias/análise , Neoplasias da Mama/genética , Ensaio de Imunoadsorção Enzimática/métodos , Regulação Neoplásica da Expressão Gênica , Inibidor 1 de Ativador de Plasminogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ativador de Plasminogênio Tipo Uroquinase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/análise
11.
Am J Cancer Res ; 7(9): 1959-1970, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28979817

RESUMO

Dysregulated expression of rab31, a member of the large Rab protein family of the Ras superfamily of small GTPases, has been observed in several types of cancer, including breast cancer. Rab31, depending on its expression level, may regulate the switch between an invasive versus proliferative phenotype of breast cancer cells in vitro. Moreover, gene expression of rab31 is induced by the C-terminal subunit of mucin-1 (MUC1-C) and estrogen receptors (ER). To gain further insights into the clinical relevance of rab31 and mucin-1 expression in breast cancer, we analyzed the relation between rab31 and mucin-1 (CA15-3) antigen levels in detergent tissue extracts of ER-positive (ER+) tumors and clinicopathological parameters as well as patients' prognosis. No significant correlation was observed between rab31 and CA15-3 antigen levels. Elevated rab31 antigen levels in tumor tissue extracts were significantly associated with higher tumor grade (P = 0.021). Strikingly, an inverse significant association was observed for CA15-3 with tumor grade (P = 0.032). Furthermore, high rab31 antigen levels were significantly associated with a high S-phase fraction (SPF, P = 0.047), whereas a trend for lower CA15-3 antigen levels in tumor tissue displaying higher SPF was observed. High rab31 antigen levels were significantly associated with poor 5-year disease-free survival (DFS) of ER+ breast cancer patients in univariate Cox regression analysis (HR = 1.91, 95% CI = 1.14-3.17, P = 0.013). In contrast, high levels of CA15-3 antigen levels were associated with better patients' prognosis (HR = 0.56, 95% CI = 0.33-0.95, P = 0.031). In multivariable analysis, rab31 antigen levels contributed independent prognostic information for DFS when adjusted for prognostically relevant clinicopathological parameters with a HR for high versus low values of 1.97 (95% CI = 1.09-3.54, P = 0.024), whereas CA15-3 antigen levels were not significant. Our results strongly suggest that rab31 antigen levels in tumor tissue are associated with the proliferative status, and rab31 represents an independent biomarker for prognosis in ER+ breast cancer patients. Total mucin-1 (CA 15-3) levels are rather inversely associated with tumor grade and SPF, and elevated levels even indicate prolonged DFS in ER+ breast cancer patients.

12.
Diabetes ; 54(7): 2143-54, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15983216

RESUMO

The initial phase of wound repair involves inflammation, induction of tissue factor (TF), formation of a fibrin matrix, and growth of new smooth muscle actin (alpha-SMA)-positive vessels. In diabetes, TF induction in response to cutaneous wounding, which ordinarily precedes increased expression of vascular endothelial growth factor (VEGF) and alpha-SMA transcription, is diminished, though not to a degree causing excessive local bleeding. Enhanced TF expression in wounds of diabetic mice caused by somatic TF gene transfer increased VEGF transcription and translation and, subsequently, enhanced formation of new blood vessels and elevated blood flow. Furthermore, increased levels of TF in wounds of diabetic mice enhanced wound healing; the time to achieve 50% wound closure was reduced from 5.5 days in untreated diabetic mice to 4.1 days in animals undergoing TF gene transfer (this was not statistically different from wound closure in nondiabetic mice). Thus, cutaneous wounds in diabetic mice display a relative deficiency of TF compared with nondiabetic controls, and this contributes to delayed wound repair. These data establish TF expression as an important link between the early inflammatory response to cutaneous wounding and reparative processes.


Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Tromboplastina/genética , Cicatrização/fisiologia , Ferimentos e Lesões/fisiopatologia , Animais , Primers do DNA , Diabetes Mellitus Tipo 1/genética , Inflamação , Camundongos , Camundongos Endogâmicos NOD , Reação em Cadeia da Polimerase , Pele/lesões , Tromboplastina/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização/genética , Ferimentos e Lesões/genética
13.
Eur J Cancer ; 41(17): 2760-8, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16256342

RESUMO

Recently, two components of important protease systems in cancer, i.e., the urokinase plasminogen activator receptor (uPAR) mRNA splice variant uPAR-del4/5 and the tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), were independently reported to be of prognostic value in breast cancer. In the present study, we have evaluated the impact of both these factors on disease-free survival (DFS) in 205 breast cancer patients by assessing mRNA expression in tumour tissue by quantitative PCR. High uPAR-del4/5 mRNA expression was associated with shorter DFS in breast cancer patients (P=0.0363), whereas high TIMP-3 mRNA levels were associated with a good prognosis (P=0.0049). Furthermore, by combining uPAR-del4/5 with TIMP-3 values, we demonstrate that breast cancer patients with high uPAR-del4/5 and low TIMP-3 mRNA levels had a highly significantly shorter DFS in comparison to those patients with low uPAR-del4/5 and high TIMP-3 mRNA expression (P<0.0001). These patients had a more than 6-fold higher risk for disease recurrence or death in multivariate analysis. Therefore, considering the prognostic impact of two proteolytic factors stemming from complementary protease systems may improve the prediction of disease recurrence in breast cancer.


Assuntos
Neoplasias da Mama/enzimologia , Receptores de Superfície Celular/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/cirurgia , DNA Complementar/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática , Mastectomia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Prognóstico , RNA Mensageiro/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase
14.
FASEB J ; 17(3): 476-8, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12514112

RESUMO

Although tissue factor (TF), the principial initiator of physiological coagulation and pathological thrombosis, has recently been proposed to be present in human blood, the functional significance and location of the intravascular TF is unknown. In the plasma portion of blood, we found TF to be mainly associated with circulating microvesicles. By cell sorting with the specific marker CD42b, platelet-derived microvesicles were identified as a major location of the plasma TF. This was confirmed by the presence of full-length TF in microvesicles acutely shedded from the activated platelets. TF was observed to be stored in the alpha-granules and the open canalicular system of resting platelets and to be exposed on the cell surface after platelet activation. Functional competence of the blood-based TF was enabled when the microvesicles and platelets adhered to neutrophils, as mediated by P-selectin and neutrophil counterreceptor (PSGL-1, CD18 integrins) interactions. Moreover, neutrophil-secreted oxygen radical species supported the intravascular TF activity. The pools of platelet and microvesicle TF contributed additively and to a comparable extent to the overall blood TF activity, indicating a substantial participation of the microvesicle TF. Our results introduce a new concept of TF-mediated coagulation crucially dependent on TF associated with microvesicles and activated platelets, which principally enables the entire coagulation system to proceed on a restricted cell surface.


Assuntos
Coagulação Sanguínea , Plaquetas/química , Vesículas Citoplasmáticas/química , Tromboplastina/análise , Tromboplastina/fisiologia , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Vasos Sanguíneos/química , Adesão Celular , Vesículas Citoplasmáticas/ultraestrutura , Fibrina/biossíntese , Humanos , Modelos Biológicos , Neutrófilos/fisiologia , Ativação Plaquetária , Espécies Reativas de Oxigênio/metabolismo
15.
Sarcoidosis Vasc Diffuse Lung Dis ; 22(1): 66-74, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15881283

RESUMO

BACKGROUND AND AIM: For sarcoidosis it is generally hypothesized that inherited factors and environmental antigens may contribute to pathogenesis. Since M. tuberculosis DNA was found in a significant percentage of sarcoidosis patients, we analyzed the relationship between HLA-DRB1 alleles, inflammatory markers and the presence of M. tuberculosis DNA in sarcoidosis and its influence on clinical course. METHODS: From 144 patients with sarcoidosis lung tissue, BAL and/or blood were investigated by means of PCR assays to detect an 123 bp multicopy element of M. tuberculosis DNA and HLA-DRB1 alleles, respectively. ACE was measured spectrophotometrically, sIL-2R by ELISA. Clinical data describing the disease course were available from 63 patients. RESULTS: The percentage of M. tuberculosis positive sarcoidosis patients was significantly increased in the chronic patients group compared to acute disease. The percentage of HLA-DRB 1*03 positive patients was significantly higher in acute sarcoidosis, whereas in chronic disease the HLA-DRB1*11 positive patients were clearly over-represented. In addition, we found a highly significant correlation of HLA-DRB1*11 or -DRB1*15 alleles and/or the presence of M. tuberculosis DNA to a chronic disease course, whereas HLA-DRB1*03 or -DRB1*04 alleles combined with the absence of M. tuberculosis DNA were associated with an acute sarcoidosis (p = 0.009). ACE and sIL2-R serum levels were significantly higher in M. tuberculosis positive sarcoidosis independent of the HLA-DRB1 specificity, but did not differ between acute and chronic disease course alone. CONCLUSIONS: The association between certain HLA-DR antigens, the presence of M. tuberculosis DNA and disease course indicates that specific antigens of M. tuberculosis may play a pathogenetic role in chronic sarcoidosis.


Assuntos
Antígenos HLA-DR/genética , Inflamação , Mycobacterium tuberculosis/genética , Sarcoidose/genética , Sarcoidose/patologia , Adulto , Antígenos de Bactérias , Biomarcadores , Líquido da Lavagem Broncoalveolar , DNA Bacteriano , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Frequência do Gene , Cadeias HLA-DRB1 , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , Reação em Cadeia da Polimerase , Prognóstico
16.
Cardiovasc Res ; 60(1): 131-5, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-14522414

RESUMO

BACKGROUND: Tissue factor (TF), the main trigger of coagulation is important in the propagation of cardiovascular diseases. Based on an in vitro study, we hypothesised that enalapril may blunt the endotoxin-induced, TF-triggered coagulation in humans. METHODS: In a randomised, controlled trial, 30 healthy male volunteers received 2 ng/kg of lipopolysaccharide (LPS) after pre-treatment with placebo or enalapril for 5 days or with enalapril 2 h before LPS infusion. RESULTS: Infusion of LPS increased interleukin-6 levels 400 fold, and induced a 10-fold increase in prothrombin fragment, a fourfold increase in D-dimer, and a fivefold increase in plasmin-antiplasmin complexes. However, pre-treatment with enalapril did not blunt LPS-induced coagulation. CONCLUSIONS: Our trial provides evidence against a modulatory role of angiotensin converting enzyme in LPS-induced, TF-triggered coagulation.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Coagulação Intravascular Disseminada/prevenção & controle , Enalapril/uso terapêutico , Adulto , Análise de Variância , Coagulação Intravascular Disseminada/imunologia , Coagulação Intravascular Disseminada/microbiologia , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinolisina/análise , Humanos , Infusões Intravenosas , Interleucina-6/sangue , Leucócitos Mononucleares/química , Lipopolissacarídeos/administração & dosagem , Masculino , Protrombina/análise , Estatísticas não Paramétricas , Tromboplastina/análise
17.
Invest Ophthalmol Vis Sci ; 43(6): 1965-70, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12037006

RESUMO

PURPOSE: To analyze duration of gene expression in the retinal pigment epithelium (RPE) in immunocompetent animals after gene transfer with a high-capacity adenoviral (HC-Ad) vector. METHODS: An HC-Ad vector was constructed to express the enhanced green fluorescence protein (EGFP) from the human CMV promoter. This vector (HC-AdFK7) was used to transduce rat RPE cells in cell culture and after subretinal injection in vivo in adult immunocompetent Wistar rats. In cell culture, expression of EGFP was analyzed by fluorescence microscopy. In vivo expression was monitored by scanning laser ophthalmoscopy and stereo fluorescence microscopy. After enucleation of the eyes, immunohistochemical and morphologic analyses by fluorescence light microscopy and electron microscopy were performed. RESULTS: In vitro, RPE cells were efficiently transduced with HC-AdFK7. Expression persisted for the observation time of 8 weeks. In vivo, the RPE was efficiently transduced with low doses of HC-AdFK7. EGFP synthesis was confirmed by antibody staining and found to be stable for the complete study period of 6 months. The neuroretina was well preserved over areas of subretinal vector administration, and significant morphologic changes were not detected. There was no accumulation of inflammatory T cells or macrophages. CONCLUSIONS: In contrast to previous results with earlier generation adenoviral vectors, subretinal injection of an HC-Ad vector in immunocompetent rats resulted in long-term transgene expression without evidence of adverse immune reactions or significant toxicity, probably because of the absence of expression of the viral gene from this vector. Thus, HC-Ad vectors are suitable for the treatment of eye disorders that require durable gene expression in the RPE.


Assuntos
Adenoviridae/genética , Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas Luminescentes/genética , Epitélio Pigmentado Ocular/metabolismo , Animais , Células Cultivadas , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Macrófagos/imunologia , Microscopia de Fluorescência , Monócitos/imunologia , Oftalmoscopia , Epitélio Pigmentado Ocular/citologia , Ratos , Ratos Long-Evans , Ratos Wistar , Linfócitos T/imunologia , Fatores de Tempo , Transgenes
18.
Invest Ophthalmol Vis Sci ; 43(7): 2474-80, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12091453

RESUMO

PURPOSE: To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous. METHODS: Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR. RESULTS: Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells. CONCLUSIONS: Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein-protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.


Assuntos
Proteínas do Olho/genética , Epitélio Pigmentado Ocular/metabolismo , Corpo Vítreo/fisiologia , Células Cultivadas , Primers do DNA/química , Proteínas do Olho/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Thromb Haemost ; 89(1): 3-8, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12540946

RESUMO

The loss of blood through vessel wall ruptures is initially prevented by the rapid adhesion of platelets to the subendothelium, and the formation of a thrombus consisting of platelets and different types of leukocytes. Concomitantly, the coagulation process is thought to be activated by vascular wall tissue factor (TF). Here, a new model for the initiation of coagulation is presented, based on unexpected findings on the presence and functional activation of TF within the blood itself. TF was recently found to be stored in the alpha-granules of resting platelets under physiological conditions. Activation by collagen exposes TF on the platelet cell membrane and on platelet derived microvesicles. Adhesive interactions of the TF bearing platelets and microvesicles to neutrophils and monocytes support the functional activation of the blood based TF. The intravascular TF pathway is proposed to play a significant role during hemostasis by enabling the generation of fibrin at the site of the developing thrombus.


Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Tromboplastina/metabolismo , Plaquetas/química , Plaquetas/ultraestrutura , Comunicação Celular , Humanos , Tromboplastina/fisiologia
20.
Thromb Haemost ; 88(6): 1054-9, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12529759

RESUMO

Experimental and clinical data suggest that tissue factor (TF), the major initiator of blood coagulation cascade, as well as proteases and components of the fibrinolytic system are involved in tumor growth at least in some solid tumors via effects on angiogenesis. Whereas the pro- and anti-angiogenic effects of the plasminogen/plasmin system and plasminogen kringle domains, respectively, are well characterized, the pathways responsible for the pro-angiogenic properties of TF remain poorly understood. To learn more about the biological significance of the recently described binding of plasminogen to the extracellular domain of TF, we examined the effects of soluble TF (sTF) on angiostatin-inhibited proliferation of endothelial cells. In solid phase binding assays, we found that sTF binds specifically to plasminogen, to the plasminogen kringle domains K1-3, K1-5, K4, as well as to mini-plasminogen. Inhibition of binding of plasminogen and its kringle domains to sTF by the lysine analog 6-aminohexanoic acid (AHA) suggests that lysine-binding sites are involved in plasminogen interaction with TF. Moreover, in the presence of sTF, the inhibitory effect of K1-5 on bFGF-mediated HUVEC proliferation was dose-dependently and saturably abolished. This suggests that TF can interfere with the antagonistic effect of K1-5 on endothelial cell proliferation. In contrast, sTF by itself had no effect on the endothelial cell proliferation. Whereas the interference of TF with K1-5-mediated effect was prevented by AHA, this lysine analog did not abolish the proliferation inhibition of K1-5. In conclusion, the binding of sTF to the plasminogen fragment K1-5 seems to antagonize the anti-angiogenic effects of this plasminogen fragment.


Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Plasminogênio/metabolismo , Plasminogênio/farmacologia , Tromboplastina/metabolismo , Angiostatinas , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/metabolismo , Humanos , Técnicas In Vitro , Kringles , Lisina/química , Fragmentos de Peptídeos/química , Plasminogênio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Solubilidade
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