RESUMEN
ß2glycoprotein I (ß2GPI), also known as apolipoprotein H, is a phospholipidbinding plasma protein consisting of five homologous repeated units. ß2GPI downregulates vascular endothelial growth factor (VEGF) signaling pathways and inhibits angiogenesis in vitro. However, the in vivo roles and effectors of reduced ß2GPI and ß2GPI in retinal angiogenesis are still not fully understood. In this study, an oxygeninduced retinopathy model was used to investigate the effects of reduced ß2GPI and ß2GPI, and to monitor the expression of VEGF, VEGF receptor (VEGFR) 1, VEGFR2 and hypoxiainducible factor 1 (HIF1) mRNA and the phosphorylation of extracellular signalregulated kinase (ERK) and Akt. The data showed that both ß2GPI and reduced ß2GPI inhibited retinal angiogenesis and suppressed the expression of VEGF, VEGFR1, VEGFR2, HIF1, phosphorylated- (p) ERK and pAkt. The effects of reduced ß2GPI were significantly stronger than those of ß2GPI. In conclusion, this study showed that ß2GPI and reduced ß2GPI could inhibit retinal angiogenesis by downregulating the expression of VEGF and its downstream targets. This suggests that ß2GPI and reduced ß2GPI may have potential antiangiogenic activity in vivo.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/patología , Neovascularización Patológica/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , beta 2 Glicoproteína I/metabolismo , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos C57BL , Oxígeno/efectos adversos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/metabolismo , Retina/patología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Advanced glycation end products (AGEs) are a contributing factor in the angiogenesis that is characteristic of proliferative diabetic retinopathy. However, a previous study made a promising observation that domain IIV of ß2glycoprotein I (DIIV) inhibits angiogenesis in human umbilical vein cells. The present study aimed to confirm the inhibition of AGEinduced angiogenesis in retinal endothelial cells by DIIV and to investigate the potential underlying mechanisms. The RF/6A rhesus macaque choroidretinal vascular endothelial cell line was cultured in vitro and treated with AGEs in the presence or absence of different concentrations of DIIV. The proliferation, migration and tube formation of the RF/6A cells were evaluated using MTS assays, in vitro wound healing assays and in vitro Matrigel angiogenesis assays, respectively. The mRNA expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR) 2, VEGFR 1 and receptor for AGE (RAGE) were quantified by reverse transcription quantitative polymerase chain reaction. The expression of VEGFR1, VEGFR2 and the activation of protein kinase B (Akt) and extracellular signalregulated kinase (ERK) were also assessed by western blot analysis. The results indicated that AGEs promoted the migration, proliferation and tube formation of RF/6A cells in vitro (P<0.05), increased the expression of VEGF, VEGFR2 and RAGE (P<0.05) and increased the phosphorylation of Akt and ERK (P<0.05). DIIV inhibited the increase in VEGFR2 mRNA and protein, but did not inhibit the increase in VEGF or RAGE mRNAs. These results led to the conclusion that DIIV inhibited AGEinduced angiogenesis in the RF/6A cells, which was accompanied by a downregulation in the expression of VEGFR2 and its downstream phosphatidylinosol 3kinase/Akt and mitogenactivated protein kinase/ERK1/2 pathways. These findings provide further support towards the treatment of proliferative diabetic retinopathy by interventions that act via a mechanism similar to that of DIIV.
Asunto(s)
Productos Finales de Glicación Avanzada/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , beta 2 Glicoproteína I/farmacología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Expresión Génica , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , beta 2 Glicoproteína I/químicaRESUMEN
BACKGROUND: Reduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms. METHODS: The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK. RESULTS: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 µg/mg protein vs. 114.35 ± 10.38 µg/mg protein, p < 0.05;74.44 ± 5.27 µg/mg protein vs. 114.35 ± 10.38 µg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein. CONCLUSIONS: Both beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.