Domain I-IV of ß2-glycoprotein I inhibits advanced glycation end product-induced angiogenesis by down-regulating vascular endothelial growth factor 2 signaling.

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.

Reduced beta2-glycoprotein I protects macrophages from ox-LDL-induced foam cell formation and cell apoptosis.

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.