The promoting effects of insulin-like growth factor 1 on the biological behaviour of human retinal vascular endothelial cells

Background

The suppression of retinal angiogenesis is one of primary treatment targets for retinal vascular diseases, so seeking the intervention targets of retinal neovascularization is a hot research.Studies showed that insulin-like growth factor 1 (IGF-1) can promote the growth and restrain the apoptosis of vascular endothelial cells.However, whether IGF-1 is an intervention target for the treatment of retinal vascular diseases is unelucidated.

Objective

This study was to address the effects of IGF-1 on the migration, apoptosis and capillary tube formation of human retinal vascular endothelial cells (HRECs) and mechanism.

Methods

HRECs were cultured in vitro, and the cells in the exponential phase were prepared for subsequent experiments.The expression of IGF-1R mRNA in the cells was examined using reverse transcriptase PCR assay.Different concentrations of IGF-1 were added in the medium based on the difference of tests.The relative free-cell area difference (ΔS) after test was measured by Photoshop CS4 software and compared among 0, 10 and 200 ng/ml IGF-1 groups 12 and 24 hours after cell scratching, respectively.The cell apoptotic rate was assayed by flow cytometry and compared between 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group, and the number of capillary tubes was examined by Matrigel test and assessed among 0, 10, 100 and 200 ng/ml IGF-1 groups 24 hours after addition of IGF-1.The expressions of platelet derived growth factor (PDGF)-BB mRNA and caspase-3 mRNA in the cells of the 0, 500 and 1 000 ng/ml IGF-1 groups were detected by real-time fluorescence quantitative PCR after adding IGF-1 for 6 hours.

Results

Cultured cells grew well and attached 90% confluence 2-3 days after incubation, and IGF-1R mRNA was positively expressed in the cells.In 12 and 24 hours after scratching, the relative migrating area of the cells was gradually reduced with the increase of IGF-1 contents.The ΔS was (4.83±0.61)×10⁵ μm² in the 200 ng/ml IGF-1 group, which was significantly larger than (3.28±0.64) ×10⁵ μm² in the 0 ng/ml IGF-1 group 24 hours after stretching (t=-3.707, P=0.021). The apoptotic rate in the 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group was (18.77±2.37)% and (12.05±0.88)%, with a significant difference between them (t=2.869, P=0.046). The number of intact tubes was significantly increased in the 200 ng/ml IGF-1 group compared with the 0 ng/ml IGF-1 group ([20.33±2.83]/well vs.[17.94±1.96]/well; t=-2.940, P=0.042). Compared with 0 ng/ml IGF-1 group, the relative expression level of PDGF-BB mRNA was elevated and that caspase-3 mRNA was evidently reduced in the 1 000 ng/ml IGF-1 group (t=-3.489, P=0.025; t=7.287, P=0.002).

Conclusions

IGF-1 can promote the migration and angiogenesis of HRECs and inhibit the apoptosis of HRECs.These effects of IGF-1 probably are associated with the up-regulation of PDGF-BB and down-regulation of caspase-3 in the cells.

Insulin-like growth factor 1/metabolism; Retinal vascular endothelial cells; Angiogenesis; RNA, messenger/analysis; Migration; Apoptosis; Human