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1.
Invest Ophthalmol Vis Sci ; 56(12): 7209-13, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26540659

RESUMO

PURPOSE: MicroRNAs are a class of small noncoding RNAs that negatively regulate gene expression by binding to complimentary sequences of target messenger RNA. Their roles in corneal lymphangiogenesis are largely unknown. This study was to investigate the specific role of microRNA-184 (mir-184) in corneal lymphangiogenesis (LG) in vivo and lymphatic endothelial cells (LECs) in vitro. METHODS: Standard murine suture placement model was used to study the expressional change of mir-184 in corneal inflammatory LG and the effect of synthetic mir-184 mimic on this process. Additionally, a human LEC culture system was used to assess the effect of mir-184 overexpression on cell functions in vitro. RESULTS: Expression of mir-184 was significantly downregulated in corneal LG and, accordingly, its synthetic mimic suppressed corneal lymphatic growth in vivo. Furthermore, mir-184 overexpression in LECs inhibited their functions of adhesion, migration, and tube formation in vitro. CONCLUSIONS: These novel findings indicate that mir-184 is involved critically in LG and potentially could be used as an inhibitor of the process. Further investigation holds the promise for divulging new therapies for LG disorders, which occur inside and outside the eye.


Assuntos
Neovascularização da Córnea/genética , Regulação da Expressão Gênica , Linfangiogênese/genética , Vasos Linfáticos/patologia , MicroRNAs/genética , RNA Mensageiro/genética , Animais , Contagem de Células , Movimento Celular , Células Cultivadas , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Vasos Linfáticos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/biossíntese , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real
2.
Invest Ophthalmol Vis Sci ; 56(11): 6313-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26431485

RESUMO

PURPOSE: We recently reported that corneal lymphatic vessels develop integrin alpha-9 (Itga-9)-positive valves during inflammatory lymphangiogenesis. The purpose of this study was to further investigate the role of Itga-9 in corneal lymphatic valve formation in vivo and lymphatic endothelial cell (LEC) functions in vitro. METHODS: Standard murine suture placement model was used to study the effect of Itga-9 blockade on lymphatic valve formation in vivo using Itga-9 neutralizing antibody. Whole-mount corneas were harvested for immunofluorescent microscopic analysis. Additionally, human LEC culture system was used to examine the effect of Itga-9 gene knockdown on cell functions using small interfering RNAs (siRNAs). RESULTS: Itga-9 blockade in vivo significantly reduced the number of lymphatic valves formed in the inflamed cornea. Moreover, Itga-9 gene knockdown in human LECs suppresses cell functions of proliferation, adhesion, migration, and tube formation. CONCLUSIONS: Itga-9 is critically involved in corneal lymphatic valve formation. Further investigation of the Itga-9 pathway may provide novel strategies to treat lymphatic-related diseases occurring both inside and outside the eye.


Assuntos
Neovascularização da Córnea/tratamento farmacológico , Cadeias alfa de Integrinas/uso terapêutico , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/patologia , Animais , Células Cultivadas , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Vasos Linfáticos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C
3.
Int J Biochem Cell Biol ; 55: 109-18, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25161149

RESUMO

Tumor necrosis factor superfamily 15 (TNFSF15) suppresses angiogenesis by specifically inducing apoptosis in proliferating endothelial cells. Death receptor 3 (DR3), a member of the TNF receptor superfamily (TNFRSF25), has been identified as a receptor for TNFSF15 to activate T cells. It is unclear, however, whether DR3 mediates TNFSF15 activity on endothelial cells. Here we show that siRNA-mediated knockdown of DR3 in an in vivo Matrigel angiogenesis assay, or in adult bovine aortic endothelial (ABAE) cell cultures, leads to resistance of endothelial cells to TNFSF15-induced apoptosis. Interestingly, DR3-depleted cells also exhibited markedly diminished responsiveness to TNFα cytotoxicity, even though DR3 is not a receptor for TNFα. Treatment of the cells with either TNFSF15 siRNA or a TNFSF15-neutralizing antibody, 4-3H, also results in a significant inhibition of TNFα-induced apoptosis. Mechanistically, DR3 siRNA treatment gives rise to an increase of ERK1/2 MAPK activity, and up-regulation of the anti-apoptotic proteins c-FLIP and Bcl-2, thus strengthening apoptosis-resisting potential in the cells. These findings indicate that DR3 mediates TNFSF15-induced endothelial cell apoptosis, and that up-regulation of TNFSF15 expression stimulated by TNFα is partly but significantly responsible for TNFα-induced apoptosis in endothelial cells.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspases/metabolismo , Bovinos , Linhagem Celular , Doxorrubicina/farmacologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Expressão Gênica , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética
4.
Invest Ophthalmol Vis Sci ; 55(5): 3320-7, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24781940

RESUMO

PURPOSE: Lymphatic research has progressed rapidly in recent years. Lymphatic dysfunction has been found in myriad disorders from cancer metastasis to transplant rejection; however, effective treatment for lymphatic disorders is still limited. This study investigates the role of angiopoietin-2 (Ang-2) in corneal inflammatory lymphangiogenesis (LG) in vivo and in lymphatic endothelial cell (LEC) functions in vitro. METHODS: Standard suture placement model was used to study Ang-2 expression in inflamed cornea, and corneal LG and hemangiogenesis (HG) responses in Ang-2 knockout mice. Moreover, human LEC culture system was used to examine the effect of Ang-2 gene knockdown on LEC functions using small interfering RNAs (siRNAs). The effect of siRNA treatment on corneal LG was also assessed in vivo. RESULTS: Angiopoietin-2 was expressed on lymphatic vessels and macrophages in inflamed cornea. While corneal LG response was abolished in Ang-2 knockout mice, the HG response was also significantly suppressed with disorganized patterning. Moreover, anti-Ang-2 treatment inhibited LEC proliferation and capillary tube formation in vitro and corneal LG in vivo. CONCLUSIONS: Angiopoietin-2 is critically involved in lymphatic processes in vivo and in vitro. Further investigation of the Ang-2 pathway may provide novel insights and therapeutic strategies for lymphatic-related disorders, which occur both inside and outside the eye.


Assuntos
Angiopoietina-2/genética , Córnea/irrigação sanguínea , Neovascularização da Córnea/genética , Regulação da Expressão Gênica , Linfangiogênese/genética , Vasos Linfáticos/patologia , RNA/genética , Angiopoietina-2/biossíntese , Animais , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Vasos Linfáticos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Invest Ophthalmol Vis Sci ; 52(9): 6529-35, 2011 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-21715348

RESUMO

PURPOSE. High-risk corneal transplantation refers to grafting performed on inflamed and highly vascularized host beds. It represents a clinical dilemma because the rejection rate can be as high as 90%, irrespective of current treatment modalities. This study was conducted to investigate whether combined blockade of VEGFR-3 (vascular endothelial growth factor receptor-3) and VLA-1 (very late antigen-1) promotes high-risk transplant survival and how it correlates with corneal lymphangiogenesis and hemangiogenesis before and after transplantation. METHODS. High-risk corneal transplantation was performed between normal C57BL/6 (donor) and inflamed BALB/c (recipient) mice. The recipients were randomized to receive intraperitoneal injections of VEGFR-3 and VLA-1-neutralizing antibodies or their controls twice a week for up to 8 weeks after transplantation. Corneal grafts were evaluated by ophthalmic slit-lamp biomicroscopy and analyzed by Kaplan-Meier survival curve. Additionally, whole-mount corneas before and after transplantation were examined by immunofluorescent microscopic assays, and the correlation between lymphatic or blood vessel distribution and transplant outcome was analyzed. RESULTS. The combined blockade markedly promotes 90% survival of high-risk transplants. This strategy specifically modified host beds by selective inhibition of lymphangiogenesis but not hemangiogenesis. A strong correlation was also identified between high-risk transplant rejection and severe lymphatic invasion reaching the donor-graft border. CONCLUSIONS. These novel findings not only provide a new and potentially powerful strategy to promote high-risk transplant survival, they also confirm a critical role of high-degree lymphangiogenesis in mediating high-risk transplant rejection. Results from this study may also shed new light on our understanding and management of other lymphatic- and immune-related diseases in general.


Assuntos
Anticorpos Neutralizantes/farmacologia , Neovascularização da Córnea/cirurgia , Transplante de Córnea , Sobrevivência de Enxerto/fisiologia , Integrina alfa1beta1/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Córnea/irrigação sanguínea , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/fisiopatologia , Injeções Intraperitoneais , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fatores de Risco
6.
Invest Ophthalmol Vis Sci ; 52(7): 4808-12, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21372020

RESUMO

PURPOSE: To investigate the specific role of very late antigen-1 (VLA-1; also known as integrin α1ß1) in corneal inflammatory lymphangiogenesis in vivo and lymphatic endothelial cell functions in vitro. METHODS: A standard suture-induced corneal inflammatory lymphangiogenesis model was used in normal adult BALB/c mice to test the effect of systemic administration of VLA-1-neutralizing antibody on lymphatic formation and macrophage infiltration in vivo. Additionally, a human lymphatic endothelial cell culture system was used to examine the effect of VLA-1 gene depletion on lymphatic endothelial cell functions in vitro using small interfering RNAs. RESULTS: These data demonstrated, for the first time, that VLA-1 blockade significantly suppressed corneal lymphangiogenesis and macrophage infiltration during inflammation. Moreover, VLA-1 gene depletion led to a marked inhibition of lymphatic endothelial cell processes of adhesion, proliferation, and capillary tube formation. CONCLUSIONS: These novel findings together indicate that VLA-1 is critically involved in the processes of lymphangiogenesis. Further investigation on this factor may provide novel therapies for corneal inflammation, transplant rejection, and other lymphatic-related disorders in the body.


Assuntos
Neovascularização da Córnea/imunologia , Endotélio Linfático/metabolismo , Integrina alfa1beta1/metabolismo , Linfangiogênese/imunologia , Vasos Linfáticos/patologia , Animais , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Endotélio Linfático/patologia , Seguimentos , Regulação da Expressão Gênica , Humanos , Integrina alfa1beta1/genética , Integrina alfa1beta1/imunologia , Vasos Linfáticos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , RNA/genética
7.
Apoptosis ; 14(6): 788-95, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19418226

RESUMO

Vascular endothelial growth inhibitor (VEGI) is an endogenous inhibitor of endothelial cell growth and a promising candidate for cancer therapy. VEGI is able to inhibit tumor growth by specifically targeting the tumor neovasculature. Increasing the anti-angiogenic potential of this cytokine is of great interest for its therapeutic potential. NF-kappaB is known to have an integral role in TNF superfamily signaling, acting as a pro-survival factor. A role of VEGI-induced NF-kappaB activation in endothelial cells has yet to be described. Here we show that suppression of the NF-kappaB pathway can increase the apoptotic potential of VEGI. We used siRNA to deplete NF-kappaB or its activator IKK2 from adult bovine aortic endothelial cells. The siRNA treatments diminished VEGI-induced NF-kappaB activation, evidenced from a reduced extent of NF-kappaB nuclear translocation and diminished expression of NF-kappaB-target genes such as interleukins-6 and -1beta. The siRNA-treated endothelial cells when exposed to VEGI exhibited a marked decrease in cell viability and a significant increase in apoptosis. These results confirm that VEGI utilizes NF-kappaB as a pro-survival role factor in endothelial cells. We then examined whether a combination of VEGI with NF-kappaB inhibitors would constitute a more potential therapeutic regiment. We found that in the presence of the NF-kappaB inhibitors curcumin or BMS-345541 there was a marked increase in the apoptotic potential of VEGI on endothelial cells. These findings indicate that a combination therapy using VEGI and NF-kappaB inhibitors could be a potent approach for cancer treatment.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , NF-kappa B/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/farmacologia , Animais , Bovinos , Células Endoteliais/enzimologia , Quinase I-kappa B/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
8.
J Immunol ; 179(6): 3742-51, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17785811

RESUMO

Angiogenesis is an essential component of chronic inflammation that is linked to carcinogenesis. In this study, we report that human vascular endothelial growth inhibitor (VEGI, TNF superfamily 15), an endothelial cell-produced antiangiogenic cytokine, induces mouse dendritic cell (DC) maturation, a critical event in inflammation-initiated immunity. VEGI-stimulated bone marrow-derived immature DCs display early activation of maturation signaling molecules NF-kappaB, STAT3, p38, and JNK, and cytoskeleton reorganization and dendrite formation. The activation signals are partially inhibited by using a neutralizing Ab against death domain-containing receptor-3 (DR3) or a truncated form of DR3 consisting of the extracellular domain, indicating an involvement of DR3 in the transmission of VEGI activity. A VEGI isoform, TL1A, does not induce similar activities under otherwise identical experimental conditions. Additionally, the cells reveal significantly enhanced expression of mature DC-specific marker CD83, secondary lymphoid tissue-directing chemokine receptor CCR7, the MHC class-II protein (MHC-II), and costimulatory molecules CD40, CD80, and CD86. Functionally, the cells exhibit decreased Ag endocytosis, increased cell surface distribution of MHC-II, and increased secretion of IL-12 and TNF. Moreover, VEGI-stimulated DCs are able to facilitate the differentiation of CD4+ naive T cells in cocultures. These findings suggest that the anticancer activity of VEGI arises from coupling the inhibition of endothelial cell growth with the promotion of the adaptive immune mechanisms through the stimulation of DC maturation.


Assuntos
Inibidores da Angiogênese/fisiologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologia , Inibidores da Angiogênese/biossíntese , Animais , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Bovinos , Diferenciação Celular/genética , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/citologia , Endotélio Vascular/citologia , Feminino , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/biossíntese
9.
Biomed Pharmacother ; 57(1): 4-14, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12642031

RESUMO

The HIV-1 regulatory protein Tat and the accessory protein Vpr are thought to stimulate viral replication and contribute to viral pathogenesis as extracellular proteins. Humoral immune responses to these early viral proteins may therefore be beneficial. We examined serum anti-Tat and anti-Vpr IgG by ELISA in the GRIV cohort of HIV-1 seropositive slow/non-progressors (NP) and fast-progressors (FP), and in seronegative controls. Based on information obtained during a brief follow-up period (median = 20 months), NPs were sub-grouped as those maintaining non-progression status and therefore stable (NP-S), and those showing signs of disease progression (NP-P). As the primary comparison, initial serum anti-Tat and anti-Vpr IgG (prior to follow-up) were analyzed in the NP sub-groups and in FPs. Anti-Tat IgG was significantly higher in stable NP-S compared to unstable NP-P (P = 0.047) and FPs (P < 0.0005); the predictive value of higher anti-Tat IgG for maintenance of non-progression status was 92% (P = 0.029). In contrast, no-difference was observed in anti-Vpr IgG between NP-S and NP-P, although both were significantly higher than FPs (P

Assuntos
Produtos do Gene tat/imunologia , Produtos do Gene vpr/imunologia , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Estudos de Coortes , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Seguimentos , Sobreviventes de Longo Prazo ao HIV , Soropositividade para HIV/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Mapeamento de Peptídeos/métodos , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência Humana
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