Figure 5

DCZ19931 inhibits vascular permeability via downregulation of ICAM-1 expression. (A) HUVECs were cultured with VEGF (Ctrl, 10 ng/mL), VEGF (10 ng/mL) plus DCZ19931 (500 nM), VEGF (10 ng/mL) plus Ranibizumab (250 μg/mL), VEGF (10 ng/mL) plus DCZ19931 (500 nM) and Ranibizumab (250 μg/mL) for 24 h. qRT-PCR assays were performed to detect the expression of ICAM-1 mRNA in HUVECs (n = 4). (B) HUVECs were cultured with VEGF (Ctrl, 10 ng/mL), VEGF (10 ng/mL) plus DCZ19931 (500 nM), VEGF (10 ng/mL) plus Ranibizumab (250 μg/mL), VEGF (10 ng/mL) plus DCZ19931 (500 nM) and Ranibizumab (250 μg/mL), or left untreated (WT) for 24 h. WT indicated normal HUVECs without VEGF induction. EB-transwell assays were conducted to detect cell permeability (n = 4). (C,D) Laser-induced CNV mice received intravitreal injections of 10% DMSO (2 μL, Ctrl), DCZ19931 (2 μL, 1 μg/μL), Ranibizumab (2 μL, 10 mg/mL), Ranibizumab (1 μL, 10 mg/mL) plus DCZ19931 (1 μL, 1 μg/μL) after laser injury. WT indicated normal mice without CNV induction. qRT-PCR assays were performed to detect the expression of ICAM-1 mRNA in choroidal tissues at day 7 after treatment (C, n = 5). Western blots were performed to determine ICAM-1 expression in choroidal tissues. GAPDH was used as the internal control. Representative immunoblots along with the quantitative results were shown (D, n = 5). (E) The pups of OIR mice at P12 received intravitreal injections of 10% DMSO (1 μL, Ctrl), DCZ19931 (1 μL, 1 μg/μL), Ranibizumab (1 μL, 10 mg/mL), 1 μL of Ranibizumab (10 mg/mL) plus DCZ19931 (1 μg/μL) mixture. ICAM-1 immunofluorescence assays were conducted to determine ICAM-1 expression in OIR model. The representative images with the quantitative results were shown (n = 5, scale bar, 50 μm; nuclei, blue; ICAM-1-positive cells, green). Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc test. *P < 0.05 indicated significant difference between the marked groups.