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1.
Curr Pharm Biotechnol ; 20(3): 254-260, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30806307

RESUMO

BACKGROUND: Neuropilins (NRPs) participate in many processes related to cancer development such as angiogenesis, lymphangiogenesis and metastasis. Although endometrial cancer is one of the most common gynecological cancers, it has not been studied in terms of NRPs expression. OBJECTIVE: The aim of this study was to investigate the potential utility of NRPs as important factors in the diagnosis and treatment of endometrial cancer. METHODS: Our study consisted of 45 women diagnosed with endometrial cancer at the following degrees of histological differentiation: G1, 17; G2, 15; G3, 13 cases. The control group included 15 women without neoplastic changes. The immunohistochemical reactions were evaluated using light microscopy. RESULTS: We did not detect the expression of NRP-1 and NRP-2 in the control group. NRP-1 expression was found exclusively in cancer cells. It was higher in G2 and G3 and reached about 190% of G1. NRP-2 expression was observed in the endothelium and was similar across all three cancer grades. In cancer cells, NRP-2 expression increased with the degree of histological differentiation. CONCLUSION: NRP1 and NRP2 are candidates for complementary diagnostic molecular markers and promising new targets for molecular, personalized anticancer therapies.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias do Endométrio/metabolismo , Regulação Neoplásica da Expressão Gênica , Neuropilina-1/biossíntese , Neuropilina-2/biossíntese , Biomarcadores Tumorais/genética , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/cirurgia , Feminino , Humanos , Histerectomia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/cirurgia , Neuropilina-1/genética , Neuropilina-2/genética , Células Tumorais Cultivadas
2.
J Immunol Methods ; 475: 112429, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-29530508

RESUMO

BACKGROUND: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. METHODS: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. RESULTS: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. CONCLUSIONS: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.


Assuntos
5'-Nucleotidase/biossíntese , Antígeno B7-2/biossíntese , Biomarcadores Tumorais/análise , Neuropilina-1/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , 5'-Nucleotidase/análise , Antígeno B7-2/análise , Criança , Pré-Escolar , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/biossíntese , Humanos , Masculino , Neoplasia Residual , Neuropilina-1/análise , Células Precursoras de Linfócitos B/patologia
3.
Adv Clin Exp Med ; 27(6): 721-725, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29790686

RESUMO

BACKGROUND: Psoriasis is a chronic autoinflammatory disease whose underlying molecular mechanisms remain unclear. The disease is mediated by the cells and molecules of both the innate and adaptive immune systems. Some T cell surface molecules, including neuropilin-1 (NRP1), programmed death 1 (PD-1) and the human leukocyte antigen G (HLA-G), are known to play a role in the maintenance of immune tolerance. OBJECTIVES: The aim of this study was to investigate HLA-G, NRP1 and programmed cell death gene (PDCD1) mRNA expression in psoriatic patients. MATERIAL AND METHODS: The study included 72 psoriatic patients and 35 healthy individuals. Twentyone patients (29.17%) suffered from concomitant psoriatic arthritis. The mRNA expression of HLA-G, NRP1, and PDCD1 were determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The severity of skin lesions was assessed by means of the Psoriasis Area and Severity Index (PASI), Body Surface Area (BSA), the Patient Global Assessment (PGA), and the Dermatology Life Quality Index (DLQI). RESULTS: The median value of the PASI was 11.5, and of BSA was 15.8%. The expressions of NRP1 and PDCD1, but not HLA-G, were significantly lower in psoriatic patients in comparison with the control group. The expression of HLA-G, NRP1 and PDCD1 were not significantly different in the psoriatic arthritis and psoriasis vulgaris patients. CONCLUSIONS: The results of this study suggest that the molecular markers of immune tolerance, i.e., HLA-G, NRP1, and PD-1, may be involved in the immune response in psoriatic patients.


Assuntos
Antígenos HLA-G/biossíntese , Tolerância Imunológica/imunologia , Neuropilina-1/biossíntese , Receptor de Morte Celular Programada 1/biossíntese , Psoríase/imunologia , Adulto , Idoso , Biomarcadores/análise , Feminino , Antígenos HLA-G/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Neuropilina-1/imunologia , Receptor de Morte Celular Programada 1/imunologia , Adulto Jovem
4.
J Cancer Res Clin Oncol ; 144(7): 1329-1337, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29777301

RESUMO

PURPOSE: To determine if inhibiting neuropilin-1 (NRP-1) affects the radiosensitivity of NSCLC cells through a vascular endothelial growth factor receptor 2 (VEGFR2)-independent pathway, and to assess the underlying mechanisms. METHODS: The expression of VEGFR2, NRP-1, related signaling molecules, abelson murine leukemia viral oncogene homolog 1 (ABL-1), and RAD51 were determined by RT-PCR and Western blotting, respectively. Radiosensitivity was assessed using the colony-forming assay, and the cell apoptosis were analyzed by flow cytometry. RESULTS: We selected two cell lines with high expression levels of VEGFR2, including Calu-1 cells that have high NRP-1 expression, and H358 cells that have low NRP-1 expression. Upon inhibition of p-VEGFR2 by apatinib in Calu-1 cells, the expression of NRP-1 protein and other related proteins in the pathway was still high. Upon NRP-1 siRNA treatment, the expression of both NRP-1 and RAD51 decreased (p < 0.01; p < 0.05). Upon ABL-1 siRNA treatment, the expression of NRP-1 was increased and the expression of RAD51 was unchanged. Calu-1 cells treated with NRP-1 siRNA exhibited significantly higher apoptosis and radiation sensitivity in radiation therapy compared to Calu-1 cells treated with apatinib alone (p < 0.01; p < 0.01). The apoptosis and radiation sensitivity in H358 cells with NRP-1 overexpression was similar to the control group regardless of VEGFR2 inhibition. CONCLUSIONS: We demonstrated that when VEGFR2 was inhibited, NRP-1 appeared to regulate RAD51 expression through the VEGFR2-independent ABL-1 pathway, consequently regulating radiation sensitivity. In addition, the combined inhibition of VEGFR2 and NRP-1 appears to sensitize cancer cells to radiation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Neuropilina-1/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Apoptose/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/biossíntese , Piridinas/farmacologia , Rad51 Recombinase/biossíntese , Tolerância a Radiação/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
5.
J Neurosci ; 38(24): 5478-5494, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29773756

RESUMO

Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease's etiology is not fully understood, it is thought to involve a non-cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalized in vitro cocultures, we demonstrated that myocytes expressing diverse ALS-causing mutations promote axon degeneration and NMJ dysfunction, which were inhibited by applying Neuropilin1 blocking antibody. Finally, overexpressing miR126-5p is sufficient to transiently rescue axon degeneration and NMJ disruption both in vitro and in vivo Thus, we demonstrate a novel mechanism underlying ALS pathology, in which alterations in miR126-5p facilitate a non-cell-autonomous mechanism of motor neuron degeneration in ALS.SIGNIFICANCE STATEMENT Despite some progress, currently no effective treatment is available for amyotrophic lateral sclerosis (ALS). We suggest a novel regulatory role for miR126-5p in ALS and demonstrate, for the first time, a mechanism by which alterations in miR126-5p contribute to axon degeneration and NMJ disruption observed in ALS. We show that miR126-5p is altered in ALS models and that it can modulate Sema3 and NRP protein expression. Furthermore, NRP1 elevations in motor neurons and muscle secretion of Sema3A contribute to axon degeneration and NMJ disruption in ALS. Finally, overexpressing miR126-5p is sufficient to transiently rescue NMJ disruption and axon degeneration both in vitro and in vivo.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , MicroRNAs/metabolismo , Degeneração Neural/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Axônios/metabolismo , Axônios/patologia , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Degeneração Neural/genética , Degeneração Neural/patologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Neuropilina-1/biossíntese , Neuropilina-1/genética , Semaforina-3A/biossíntese , Semaforina-3A/genética
6.
J Cell Biochem ; 119(8): 6604-6613, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29693748

RESUMO

Acute lymphoblastic leukemia (ALL) is one of the most common and most malign childhood cancers. In this work, we investigated the expression and function of human mature microRNA-9 (miR-9) in ALL. In ALL in vitro cell lines and in situ clinical specimens, gene expression of miR-9 was tested by qRT-PCR. MiR-9 was overexpressed in CEM/C1 and Molt-3 cells to investigate its possible anti-cancer effects on ALL in vitro proliferation, cell-cycle progression, and in vivo explant growth. The possible downstream target of miR-9, neuropilin-1 (NRP1), was examined by dual-luciferase activity assay, qRT-PCR, and Western blot. NRP1was upregulated in miR-9-overexpressed CEM/C1 and Molt-3 cells to investigate the functional involvement of NRP1 in miR-9-mediated regulation on ALL in vitro proliferation and cell-cycle progression. MiR-9 was downregulated in ALL cell lines and leukemic T-cells of ALL patients. Lentivirus-mediated miR-9 overexpression inhibited ALL in vitro proliferation, cell-cycle progression, and in vivo explant growth. NRP1 was confirmed be the downstream target of miR-9, and inversely modulated by miR-9 in ALL. NRP1 upregulation reversed the anti-cancer regulations of miR-9 on ALL in vitro proliferation and cell-cycle progression. MiR-9 is downregulated in ALL. Overexpressing miR-9 may inhibit ALL development, possible through its downstream target of NRP1.


Assuntos
Ciclo Celular , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , Neuropilina-1/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Neoplásico/biossíntese , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , MicroRNAs/genética , Proteínas de Neoplasias/genética , Neuropilina-1/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Neoplásico/genética
7.
Cell Reprogram ; 19(5): 324-330, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28910136

RESUMO

Neuropilin-1 (NRP1) is one of the members of neuropilin family. It can combine with disparate ligands involved in regulating cell proliferation, apoptosis, and differentiation. The binding of NRP1 to Sema3A stimulates osteoblast differentiation through the classical Wnt/ß-catenin pathway. However, the functions of NRP1 in dental pulp stem cells (DPSCs) are not clear. The aim of our study was to investigate how NRP1 controlled odontoblast differentiation in DPSCs and clarified the underlying mechanisms. NRP1 expression was increased in time-dependent manner along with cell odontoblast differentiation. Overexpression of NRP1 upregulated dentin matrix protein-1, dentin sialophosphoprotein, alkaline phosphatase protein level, and mineralization in DPSCs, while knockdown of NRP1 induced the opposite effects. SiNRP1 similar to DKK1 availably blocked classical Wnt/ß-catenin signaling and odontoblast differentiation. In summary, NRP1, as a promoter of odontoblast differentiation, regulates DPSCs via the classical Wnt/ß-catenin pathway.


Assuntos
Diferenciação Celular , Polpa Dentária/metabolismo , Regulação da Expressão Gênica , Neuropilina-1/biossíntese , Odontoblastos/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt , Adolescente , Adulto , Polpa Dentária/citologia , Feminino , Humanos , Masculino , Neuropilina-1/genética , Odontoblastos/citologia , Células-Tronco/citologia
8.
Oncol Rep ; 36(5): 2444-2454, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27666723

RESUMO

Lymph node (LN) metastasis has been suggested as a major prognostic factor for oral cancer. Knockdown of the growth factors and receptors involved in these metastatic mechanisms could significantly reduce LN metastasis and improve the survival of oral cancer patients after treatment. The present study, therefore, aimed to evaluate the expression levels of the following growth factors and receptors in squamous cell carcinoma (SCC) of the tongue: the vascular endothelial growth factor (VEGF)­C and VEGF­D, which bind to the cell surface tyrosine kinase receptor VEGF receptor­3 (VEGFR­3); C­C motif chemokine receptor 7 (CCR7); neuropilin (NRP)1 and NRP2; and semaphorin 3E (SEMA3E). Furthermore, we assessed microvessel density (MVD) and lymphatic vessel density (LVD) to demonstrate the correlation between these factors and regional LN metastasis, with respect to the clinicopathological features. Finally, we analyzed the correlation between these proteins and overall or disease­free survival, in order to demonstrate their prognostic value. Univariate analysis revealed a significant association between LN metastasis and the expression levels of VEGF­C, VEGFR­3, CCR7, NRP1, and SEMA3E, as well as LVD, in SCC cells. In contrast, multivariate analysis identified associations between LN metastasis and NRP1 expression, as well as between LN metastasis and LVD; however, no correlation was found between LN metastasis and the expression levels of the other proteins. The expression levels of VEGF­C, VEGFR­3, NRP1, and SEMA3E, as well as LVD, were correlated with disease­free survival time. These results indicate that LN metastasis is associated with poor survival in SCC. This study suggests that NRP1 expression and LVD are independent factors that are likely to predict the risk of LN metastasis in SCC of the tongue, whereas the expression of VEGF­C, VEGFR­3, CCR7, and SEMA3E are non­independent predictive factors.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Neuropilina-1/genética , Receptores CCR7/genética , Semaforinas/genética , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/patologia , Metástase Linfática , Vasos Linfáticos/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Neuropilina-1/biossíntese , Prognóstico , Receptores CCR7/biossíntese , Semaforinas/biossíntese , Fator C de Crescimento do Endotélio Vascular/biossíntese , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/biossíntese
9.
PLoS One ; 11(9): e0161441, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27598321

RESUMO

The in vivo cutaneous nerve regeneration model using capsaicin is applied extensively to study the regenerative mechanisms and therapeutic efficacy of disease modifying molecules for small fiber neuropathy (SFN). Since mismatches between functional and morphological nerve fiber recovery are described for this model, we aimed at determining the capability of the capsaicin model to truly mimic the morphological manifestations of SFN in diabetes. As nerve and blood vessel growth and regenerative capacities are defective in diabetes, we focused on studying the key regulator of these processes, the neuropilin-1 (NRP-1)/semaphorin pathway. This led us to the evaluation of NRP-1 receptor expression in epidermis and dermis of subjects presenting experimentally induced small fiber neuropathy, diabetic polyneuropathy and of diabetic subjects without clinical signs of small fiber neuropathy. The NRP-1 receptor was co-stained with CD31 vessel-marker using immunofluorescence and analyzed with Definiens® technology. This study indicates that capsaicin application results in significant loss of epidermal NRP-1 receptor expression, whereas diabetic subjects presenting small fiber neuropathy show full epidermal NRP-1 expression in contrast to the basal expression pattern seen in healthy controls. Capsaicin induced a decrease in dermal non-vascular NRP-1 receptor expression which did not appear in diabetic polyneuropathy. We can conclude that the capsaicin model does not mimic diabetic neuropathy related changes for cutaneous NRP-1 receptor expression. In addition, our data suggest that NRP-1 might play an important role in epidermal nerve fiber loss and/or defective regeneration and that NRP-1 receptor could change the epidermal environment to a nerve fiber repellant bed possibly through Sem3A in diabetes.


Assuntos
Complicações do Diabetes/genética , Neuropatias Diabéticas/genética , Neuropilina-1/biossíntese , Pele/metabolismo , Neuropatia de Pequenas Fibras/genética , Adulto , Idoso , Biópsia , Capsaicina/metabolismo , Complicações do Diabetes/patologia , Neuropatias Diabéticas/patologia , Epiderme/metabolismo , Epiderme/patologia , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Regeneração Nervosa/genética , Neuropilina-1/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Pele/patologia , Neuropatia de Pequenas Fibras/patologia
10.
Acta Biomater ; 30: 311-318, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26602825

RESUMO

UNLABELLED: Peri-prosthetic osteolysis (PPO) occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. Semaphorin-3a (SEM3A), neuropilin-1 (NRP1) and plexin-A1 (PLEXA1) are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This study investigated SEM3A, NRP1 and PLEXA1 protein and mRNA expression in human PPO tissue and polyethylene (PE) particle-stimulated human peripheral blood mononuclear cell (PBMC)-derived osteoclasts in vitro. In addition, the effects of tumour necrosis factor alpha (TNFα) on cultured osteoclasts was assessed. In PPO tissues, a granular staining pattern of SEM3A and NRP1 was observed within large multi-nucleated cells that contained prosthetic wear particles. Immunofluorescent staining confirmed the expression of SEM3A, NRP1 and PLEXA1 in large multi-nucleated human osteoclasts in vitro. Furthermore, SEM3A, NRP1 and PLEXA1 mRNA levels progressively increased throughout osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL), and the presence of PE particles further increased mRNA expression of all three molecules. Soluble SEM3A was detected in human osteoclast culture supernatant at days 7 and 17 of culture, as assessed by ELISA. TNFα treatment for 72h markedly decreased the mRNA expression of SEM3A, NRP1 and PLEXA1 by human osteoclasts in vitro. Our findings suggest that SEM3A, NRP1 and PLEXA1 may have important roles in PPO, and their interactions, alone or as a complex, may have a role in pathological bone loss progression. STATEMENT OF SIGNIFICANCE: Peri-prosthetic osteolysis occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. The rate of hip and knee arthroplasty is increasing by at least 5% per year. However, these joint replacements have a finite lifespan, with data from the National Joint Replacement Registry (Australia) showing that the major cause of failure of total hip replacements is aseptic loosening. In aseptic loosening, wear particles liberated from prostheses are phagocytosed by macrophages, leading to release of inflammatory cytokines and up-regulation of osteoclast formation and activity. Semaphorin-3a, neuropilin-1 and plexin-A1 are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This is the first report to show that these molecules may be involved in the implant failure.


Assuntos
Prótese de Quadril/efeitos adversos , Prótese do Joelho/efeitos adversos , Proteínas do Tecido Nervoso/biossíntese , Neuropilina-1/biossíntese , Osteoclastos/metabolismo , Osteólise/metabolismo , Receptores de Superfície Celular/biossíntese , Semaforina-3A/biossíntese , Feminino , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Osteoclastos/patologia , Osteólise/patologia
11.
Biomed Res Int ; 2015: 862485, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26509169

RESUMO

Neonatal hypoxic-ischemic (HI) brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs) from adipose-derived stem cells (ASCs) and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1) and vascular endothelial growth factor receptor 2 (VEGFR2) was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.


Assuntos
Células Endoteliais/metabolismo , Hipóxia-Isquemia Encefálica/terapia , Infarto/terapia , Neuropilina-1/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Infarto/genética , Infarto/patologia , Neuropilina-1/biossíntese , Proteína Oncogênica v-akt/biossíntese , Proteína Oncogênica v-akt/genética , Ratos , Transdução de Sinais , Transplante de Células-Tronco , Células-Tronco/metabolismo , Estresse Mecânico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
12.
Cancer Biomark ; 15(5): 599-608, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26406949

RESUMO

OBJECTIVE: The role of miR-365 in cancer cells seemed controversial in previous studies. We thereby in this article aimed to define the role of miR-365 in malignant melanoma (MM) pathogenesis. METHODS: We detected miR-365 expression in malignant melanoma cell lines and then investigated the effects of miR-365 on the metastasis and malignancy of melanoma cells. The correlation between miR-365 level and NRP1 (neuropilin1) was further investigated in clinical malignant melanoma specimens. RESULTS: MiR-365 was strongly down-regulated in malignant melanoma (MM) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of MM. We also found that ectopic expression of miR-365 inhibited MM cell proliferation and MM metastasis in vitro and in vivo. We further identified a novel mechanism of miR-365 to suppress MM growth and metastasis. NRP1 was proved to be a direct target of miR-365, using luciferase assay and western blot. NRP1 over-expression in miR-365 expressing cells could rescue invasion and growth defects of miR-365. In addition, miR-365 expression inversely correlated with NRP1 protein levels in MM. CONCLUSION: Our data suggest that miR-365 functions as a tumor suppressor in MM development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for MM.


Assuntos
Biomarcadores Tumorais/genética , Melanoma/genética , MicroRNAs/biossíntese , Neuropilina-1/biossíntese , Animais , Biomarcadores Tumorais/biossíntese , Proliferação de Células/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Melanoma/patologia , Camundongos , MicroRNAs/genética , Invasividade Neoplásica/genética , Metástase Neoplásica , Neuropilina-1/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Immunology ; 146(1): 144-56, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26059660

RESUMO

The role of Foxp3(+) regulatory T (Treg) cells in the course of the early hyper-inflammatory and subsequent hypo-inflammatory phases of sepsis is ambiguous. Whereas Nrp1 expression has been reported to discriminate natural Treg cells from induced Treg cells, the Treg cell stability depends on the methylation status of foxp3-TSDR. To specifically evaluate the role of Foxp3(+) Treg cells in the early and late phases of sepsis, we induced sepsis by caecal ligation and puncture and subsequent Pseudomonas aeruginosa lung infection in a DEREG (DEpletion of REGulatory T cells) mouse model. We found an increase of Foxp3(+) Treg cells to all CD4(+) T cells during murine sepsis. Using a new methylation-sensitive quantitative RT-PCR method and deep amplicon sequencing, we demonstrated that natural (Nrp1(+) Foxp3(+) ) Treg cells and most induced (Nrp1(-) Foxp3(+) ) Treg cells are stable and exhibit unmethylated foxp3-TSDR, and that both Treg populations are functionally suppressive in healthy and septic mice. DEREG mice depleted of Foxp3(+) Treg cells exhibit higher disease scores, mortality rates and interleukin-6 expression levels than do non-depleted DEREG mice in early-phase sepsis, a finding indicating that Foxp3(+) Treg cells limit the hyper-inflammatory response and accelerate recovery. Treg cell depletion before secondary infection with P. aeruginosa 1 week after caecal ligation and puncture does not influence cytokine levels or the course of secondary infection. However, a moderate Treg cell recurrence, which we observed in DEREG mice during secondary infection, may interfere with these results. In summary, Treg cells contribute to a positive outcome after early-phase sepsis, but the data do not support a significant role of Treg cells in immune paralysis during late-phase sepsis.


Assuntos
Pulmão/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Sepse/imunologia , Linfócitos T Reguladores/imunologia , Animais , Ceco/cirurgia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-6/biossíntese , Pulmão/microbiologia , Depleção Linfocítica , Masculino , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neuropilina-1/biossíntese , Neuropilina-1/imunologia , Infecções por Pseudomonas/mortalidade , Sepse/microbiologia
14.
J Immunol ; 195(3): 944-52, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26109645

RESUMO

Regulatory T cells (Tregs), a subset of CD4(+) T cells, dramatically accumulate with age in humans and mice and contribute to age-related immune suppression. Recently, we showed that a majority of accumulating Tregs in aged mice expressed low levels of CD25, and their accrual is associated with declining levels of IL-2 in aged mice. In this study, we further investigated the origin of CD25(lo) Tregs in aged mice. First, aged Tregs had high expression of neuropilin-1 and Helios, and had a broad Vß repertoire. Next, we analyzed the gene expression profile of Tregs, naive T cells, and memory T cells in aged mice. We found that the gene expression profile of aged CD25(lo) Tregs were more related to young CD25(lo) Tregs than to either naive or memory T cells. Further, the gene expression profile of aged Tregs was consistent with recently described "effector" Tregs (eTregs). Additional analysis revealed that nearly all Tregs in aged mice were of an effector phenotype (CD44(hi)CD62L(lo)) and could be further characterized by high levels of ICOS and CD69. ICOS contributed to Treg maintenance in aged mice, because in vivo Ab blockade of ICOSL led to a loss of eTregs, and this loss was rescued in Bim-deficient mice. Further, serum levels of IL-6 increased with age and contributed to elevated expression of ICOS on aged Tregs. Finally, Treg accrual was significantly blunted in aged IL-6-deficient mice. Together, our data show a role for IL-6 in promoting eTreg accrual with age likely through maintenance of ICOS expression.


Assuntos
Envelhecimento/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Interleucina-6/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Proteínas Reguladoras de Apoptose/genética , Sequência de Bases , Proteína 11 Semelhante a Bcl-2 , Morte Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/biossíntese , Perfilação da Expressão Gênica , Receptores de Hialuronatos/biossíntese , Memória Imunológica/genética , Memória Imunológica/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis/antagonistas & inibidores , Proteína Coestimuladora de Linfócitos T Induzíveis/biossíntese , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Interleucina-6/sangue , Interleucina-6/genética , Selectina L/biossíntese , Lectinas Tipo C/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropilina-1/biossíntese , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de DNA , Fatores de Transcrição/biossíntese
15.
Mol Med Rep ; 12(1): 513-9, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25738638

RESUMO

Lentiviral expression vectors carrying human NRP-1 short hairpin RNA (shRNA) were constructed and selected to present highly efficient NRP-1/shRNA interference sequences, in order to investigate the effects of RNA interference (RNAi)-mediated NRP-1 silencing on the biological activities of breast cancer cells. Three pairs of human NRP-1 targeted specific interference sequences and one pair of non-specific control sequences were designed, synthesized and subcloned into pLB lentiviral vectors, which were further identified by polymerase chain reaction (PCR) and sequencing. Recombinant and lentiviral packaging plasmids were co-transfected into 293FT cell lines in order to produce lentiviral particles and to infect breast cancer cells with high NRP-1 expression. Flow cytometry was used to sort green fluorescent protein-positive cells. Fluorescence quantitative-reverse transcription-PCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. A cell counting kit-8 assay and an Annexin V-propidium iodide method in combination with flow cytometry were used to examine the effects of RNA interference-mediated NRP-1 gene silencing on cell proliferation, apoptosis and sensitivity to chemotherapy. The recombinant lentiviral plasmid pLB-NRP-1/shRNA was constructed successfully, as confirmed by PCR and sequencing. After the infection of recombinant lentiviral plasmids, the expression profiles of NRP-1 mRNA, and proteins of MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) were significantly lower than that of the control group (P<0.05). Compared with the control group, the MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) showed lower optical density values and higher apoptotic rates at 48, 72 and 96 h; these differences were statistically significant (P<0.05). EPI administration resulted in increased apoptosis in the MCF-7 and SK-BR-3 cell-specific interference groups compared with the control group (P<0.05). Lentiviral vectors encoding the human NRP-1 gene were constructed successfully and highly efficient NRP-1/shRNA interference sequences were selected. Furthermore, RNA interference (RNAi)-mediated NRP-1 silencing may induce proliferation suppression, apoptosis promotion, as well as enhanced sensitivity to chemotherapeutic agents.


Assuntos
Apoptose/genética , Neoplasias da Mama/genética , Proliferação de Células/genética , Neuropilina-1/genética , Sequência de Bases , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Lentivirus/genética , Células MCF-7 , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/biossíntese , Interferência de RNA , RNA Mensageiro/biossíntese
16.
Cancer Res ; 75(2): 330-43, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25414138

RESUMO

Beta human papillomaviruses (HPV) have been suspected to be carcinogenic in nonmelanoma skin cancers (NMSC), but the basis for potential viral contributions to these cancers is poorly understood. In particular, it is unresolved how HPV-infected keratinocytes escape cell-cycle control and whether their cross-talk with immune cells is critical for tumorigenesis. In nonviral preclinical models, the angiogenic cytokine VEGF-A has been identified as a critical regulator of NMSC. In this study, we dissected the contribution of epidermal versus myeloid cell-derived VEGF-A in HPV-mediated skin cancer by interbreeding an HPV8 transgenic mouse model with a conditional disruption of VEGF-A restricted to either epidermal or myeloid cells. Although only epidermal-derived VEGF-A was essential for initiation of skin tumor development, both spontaneously and UV-light triggered, both epidermal and myeloid cell-derived VEGF-A contributed to regeneration-induced tumorigenesis upon HPV8 overexpression, partly not only through a paracrine effect on endothelial cells, but also most probably through an additional autocrine effect on epidermal cells. Our findings offer new mechanistic insights into distinct functions of epidermal versus myeloid cell-derived VEGF-A during HPV-mediated tumorigenesis, with possible implications for preventing this disease.


Assuntos
Betapapillomavirus/fisiologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/virologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Epiderme/metabolismo , Epiderme/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/virologia , Neuropilina-1/biossíntese , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese
17.
Oncotarget ; 5(22): 11121-32, 2014 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-25333267

RESUMO

PURPOSE: To determined Neuropilin-1 (NRP-1) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression in the tumoral and peritumoral tissues of 214 treatment-naïve HCC patients and its correlation with overall survival (OS) and time to recurrence (TTR). EXPERIMENTAL DESIGN: NRP-1 and VEGFR-2 expression were examined by tissue microarray and peritumoral hypoxia by pimonidazole staining and angiogenesis by microvessel density (MVD). OS and TTR were evaluated by Kaplan-Meier analysis and log-rank test. RESULTS: Peritumoral NRP-1 and VEGFR-2 expression were significantly higher than that of the tumoral tissue (p < 0.001 for both), and high peritumoral expression of both factors was negatively associated with tumor size (p < 0.001 for both). Patients with high peritumoral expression of both proteins had the longest median OS (>94.0 months) and TTR (>84.0 months). The multivariate Cox proportional hazards analysis revealed that patients with high peritumoral expression of both NRP-1 and VEGFR-2 were more than 4 times less likely to have recurrence (p = 0.004) and more than 10 times likely to survive (p < 0.001). CONCLUSIONS: Peritumoral NRP-1 and VEGFR-2 expression is associated with prolonged TTR and extended OS of HCC patients and both may be useful as predictors of surgical outcome of HCC patients and explored as potential therapeutic targets.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Recidiva Local de Neoplasia/metabolismo , Neuropilina-1/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/cirurgia , Hipóxia Celular/fisiologia , Intervalo Livre de Doença , Feminino , Hepatectomia , Xenoenxertos , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neuropilina-1/genética , Prognóstico , Estudos Prospectivos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
18.
Pathology ; 46(5): 416-23, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24977735

RESUMO

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF). A soluble isoform of Nrp1 (sNrp1) has not been described in the mouse. Our goal was to examine the expression of mouse sNrp1 during liver development and regeneration.sNrp1 was cloned from mouse liver. The expression of sNrp1 and VEGF was examined in mouse liver during post-natal development and regeneration using northern blot, western blot, in situ hybridisation, and immunohistochemical analyses. HGF/NRP1 binding was examined in vitro.A novel 588-amino acid sNrp1 isoform was found to contain the ligand binding regions of Nrp1. The adult liver expressed more sNrp1 than full-length Nrp1. In vivo, hepatocytes constitutively expressed VEGF and sNrp1 in the quiescent state. sNrp1 was highly up-regulated at P20, a time point coinciding with a plateau in liver and body weights. Following hepatectomy, endogenous levels of sNrp1 decreased during the rapid growth phase, and VEGF levels were highest just prior to and during the angiogenic phase. sNrp1 levels again rose 5-10 days post-hepatectomy, presumably to control regeneration. HGF protein bound NRP1 and binding was competed with sNRP1.We cloned a novel mouse sNrp1 isoform from liver and provide evidence that this endogenous angiogenesis inhibitor may regulate VEGF or HGF bioavailability during normal physiological growth and development as well as during liver regeneration.


Assuntos
Regeneração Hepática/fisiologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Neovascularização Fisiológica/fisiologia , Neuropilina-1/biossíntese , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Fator A de Crescimento do Endotélio Vascular/biossíntese
19.
Dev Biol ; 391(2): 241-50, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24809797

RESUMO

Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1(Sema-) mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.


Assuntos
Córnea/irrigação sanguínea , Cristalino/irrigação sanguínea , Neuropilina-1/genética , Semaforina-3A/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Geneticamente Modificados , Movimento Celular , Células Cultivadas , Embrião de Galinha , Córnea/embriologia , Células Endoteliais , Cristalino/embriologia , Camundongos , Neovascularização Fisiológica , Neuropilina-1/biossíntese , Codorniz/embriologia , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/genética , Semaforina-3A/antagonistas & inibidores , Semaforina-3A/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/biossíntese
20.
Tumour Biol ; 35(7): 6919-24, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24737589

RESUMO

According to the previous studies, numerous biomarkers impact on the prognosis of acute myeloid leukemia (AML) and the prediction for AML had been improved tremendously in the past decades. However, accurate risk-stratification at diagnosis or prognosis remained difficult. In order to further investigate the prognosis evaluation biomarker, the transcription or expression of neuropilin-1 (NRP-1) in 87 AML patients and 32 non-malignant controls were examined. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect the NRP-1 expression. Clinical data were collected and analyzed for the 87 AML patients. The results indicated that high NRP-1 expression discriminated the complete remission (CR) rate of AML patients (22.12 % vs. 68.04 % for AML, P < 0.01). De novo AML patients tended to express higher NRP-1 proteins than relapsed AML patients. The overall survival (OS) and relapse-free survival (RFS) rate of the high NRP-1 expression patients decreased significantly compared with the low NRP-1 expression patients (P < 0.001). NRP-1 was revealed to be an independent risk factor for OS in AML (P = 0.003). In conclusion, NRP-1 could predict the shorter OS and RFS rate, and also related with the CR response in AML. Therefore, NRP-1 may act as a more aggressive and promising predictor for the poor prognosis of AML.


Assuntos
Biomarcadores Tumorais/biossíntese , Leucemia Mieloide Aguda/genética , Recidiva Local de Neoplasia/genética , Neuropilina-1/biossíntese , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Criança , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Neuropilina-1/genética , Prognóstico , Taxa de Sobrevida
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