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1.
Invest Ophthalmol Vis Sci ; 48(12): 5750-5, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18055828

RESUMO

PURPOSE: Originally identified as a lipopolysaccharide binding protein with Gram-negative bactericidal activity in the leukocytes, bactericidal/permeability-increasing protein (BPI) has been shown to induce various effects in retinal cells in vivo and in vitro. METHODS: The authors recently reported that BPI can induce ERK1/2 and Akt activity and that it increases DNA synthesis in the bovine retinal pigment epithelial (RPE) and pericyte cells. The authors have extended the characterization of BPI interaction with membrane proteins from bovine RPE. Crude membrane pools from RPE were isolated, solubilized, and bound to rBPI(21) affinity column. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue, which showed an intense band at 36 kDa consistently displaced by rBPI(21). RESULTS: Tandem mass spectrometry of the 36-kDa band suggested that cell surface protein glypican 4 (GPC4) serves as a putative BPI-binding protein. Heparitinase, phosphatidylinositol-specific phospholipase C, and anti-GPC4 antibody suppressed BPI-induced ERK and Akt phosphorylation in bovine RPE. Moreover, heparitinase also inhibited BPI actions on VEGF and PDGF-B mRNA expression induced by H(2)O(2). CONCLUSIONS: These new findings suggest that GPC4 is a specific binding protein for BPI on RPE to mediate the activation of ERK1/2, Akt, and the mRNA expressions of PDGF-B and VEGF.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Glipicanas/metabolismo , Proteínas de Membrana/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Transdução de Sinais/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/química , Bovinos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Glipicanas/química , Immunoblotting , Proteínas de Membrana/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Epitélio Pigmentado Ocular/efeitos dos fármacos , Polissacarídeo-Liases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética
2.
FASEB J ; 20(12): 2058-67, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17012258

RESUMO

Bactericidal/permeability-increasing protein (BPI) was originally identified as a lipopolysaccharide (LPS) binding protein with gram-negative bactericidal activity in the leukocytes. In this study, we characterized the previously unknown effects of BPI in the eye and the molecular mechanisms involved in its action. BPI mRNA was detected in bovine retina; retinal pigment epithelium; and primary cultures of bovine retinal pigment epithelial cells (RPE), pericytes (RPC), and endothelial cells (REC); while BPI protein was measured in human vitreous and plasma. BPI, but not control protein thaumatin, activated extracellular regulated kinase (ERK) and AKT, and increased DNA synthesis in RPE and RPC but not in REC. A human recombinant 21 kDa modified amino-terminal fragment of BPI (rBPI21) reduced H2O2-induced apoptosis in RPE and inhibited vascular endothelial growth factor (VEGF)-stimulated ERK phosphorylation in REC when preincubated with VEGF. Intraperitoneal (i.p.)-injected rBPI21 reduced ischemia-induced retinal neovascularization and diabetes-induced retinal permeability. Since BPI has unusual dual properties of promoting RPC and RPE growth while suppressing VEGF-induced REC growth and vascular permeability, the mechanistic understanding of BPI's action may provide novel therapeutic opportunities for diabetic retinopathy and age-related macular degeneration.


Assuntos
Inibidores da Angiogênese/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Sanguíneas/farmacologia , Proteínas de Membrana/farmacologia , Retina/citologia , Vasos Retinianos/efeitos dos fármacos , Transdução de Sinais , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/análise , Permeabilidade Capilar/efeitos dos fármacos , Bovinos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Proteínas de Membrana/análise , Neovascularização Patológica/tratamento farmacológico , Pericitos/citologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Plasma/química , Proteínas Recombinantes/farmacologia , Retina/efeitos dos fármacos , Retina/metabolismo , Vasos Retinianos/crescimento & desenvolvimento , Fator A de Crescimento do Endotélio Vascular/farmacologia , Corpo Vítreo/química
3.
Invest Ophthalmol Vis Sci ; 47(6): 2701-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16723489

RESUMO

PURPOSE: Although vascular endothelial growth factor (VEGF) is a key mediator of retinal vascular permeability (RVP), there may be additional humoral contributors. Hepatocyte growth factor (HGF) induces endothelial cell separation, regulates expression of cell adhesion molecules and is increased in the vitreous fluid of patients with proliferative diabetic retinopathy. The purpose of this study was to evaluate the in vivo effects of HGF on RVP and retinal hemodynamics and delineate the signaling pathways. METHODS: RVP was assessed by vitreous fluorescein fluorophotometry in rats. Time course and dose-response were determined after intravitreal HGF injection. MAP kinase (MAPK), phosphatidylinositol 3-kinase (PI-3 kinase), and protein kinase C (PKC) involvement were examined by using selective inhibitors. Retinal blood flow (RBF) and mean circulation time (MCT) were evaluated by video fluorescein angiography. RESULTS: HGF increased RVP in a time- and dose-dependent manner. HGF-induced RVP was evident 5 minutes after injection, and reached maximal levels after 25 minutes (+107% versus vehicle, P=0.002). This effect was comparable to that of maximum VEGF stimulation (134%+/-128% at 25 ng/mL). Selective inhibitors of MAPK (PD98059) and PI-3 kinase (LY294002) suppressed HGF-induced RVP by 86%+/-44% (P=0.015) and 97%+/-59% (P=0.021), respectively. Non-isoform-selective inhibition of PKC did not significantly decrease HGF-induced RVP. Although VEGF increases RBF and reduces MCT, HGF did not affect either. CONCLUSIONS: HGF increases RVP in a time- and dose-dependent manner at physiologically relevant concentrations with a magnitude and profile similar to that of VEGF, without affecting retinal hemodynamics. Thus, HGF may represent another clinically significant contributor to retinal edema distinct from the actions of VEGF.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Vasos Retinianos/fisiologia , Animais , Circulação Sanguínea , Western Blotting , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Angiofluoresceinografia , Fluorofotometria , Hemodinâmica/fisiologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Vasos Retinianos/enzimologia , Fatores de Tempo
4.
FASEB J ; 17(1): 76-8, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12475915

RESUMO

Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood-retinal barrier breakdown) is demonstrated to result from a leukocyte-mediated Fas-FasL-dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin-induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody-based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood-retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte-mediated Fas-FasL-dependent retinal endothelial cell apoptosis as a major cause of blood-retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas-FasL pathway may prove beneficial in the treatment of diabetic retinopathy.


Assuntos
Apoptose , Barreira Hematorretiniana , Diabetes Mellitus Experimental/imunologia , Retinopatia Diabética/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Receptor fas/fisiologia , Animais , Adesão Celular , Morte Celular , Técnicas de Cocultura , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Endotélio Vascular/citologia , Proteína Ligante Fas , Leucócitos/imunologia , Glicoproteínas de Membrana/fisiologia , Modelos Imunológicos , Neutrófilos/imunologia , Ratos , Retina/citologia
5.
Cancer Res ; 62(3): 789-95, 2002 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11830534

RESUMO

Antiangiogenic therapy, although effective in shrinking tumors, has not yet been established as a standalone treatment for cancer. This therapeutic limitation can be overcome by combining angiogenesis inhibitors with chemotherapeutic agents. NM-3, a small molecule isocoumarin, is a recently discovered angiogenesis inhibitor. Here we demonstrate that NM-3 inhibits the proliferation of human umbilical vein endothelial cells in vitro, at concentrations 10-fold less than those required to inhibit normal fibroblasts or tumor cells (HT29, MKN28, and MCF-7). NM-3 alone inhibits endothelial sprouting and tube formation in vitro. The results also show that synergistic antiproliferative activity is observed when human umbilical vein endothelial cells are treated with NM-3 in combination with 5-fluorouracil. The effects of treatment with NM-3 and various chemotherapeutic agents were also evaluated in tumor xenografts. The results demonstrate that combined treatment with NM-3 and chemotherapeutic agents significantly reduced mean tumor volume compared with either treatment alone, with no effects on body weight changes. Taken together, these findings demonstrate that NM-3 is a well-tolerated angiogenesis inhibitor that significantly increases the efficacy of existing antineoplastic agents.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Cumarínicos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/crescimento & desenvolvimento , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Ciclofosfamida/farmacologia , Sinergismo Farmacológico , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Fluoruracila/farmacologia , Inibidores do Crescimento/farmacologia , Humanos , Isocumarinas , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/patologia , Paclitaxel/farmacologia , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto
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