Effect of integrin α5β1 inhibition on SDF-l/CXCR4-mediated choroidal neovascularization

Abstract

Aim: To investigate the roles of integrins in choroidal neovascularization (CNV) and their associations with the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis.

Methods: CNV lesions were induced in mice using laser photocoagulation. After CNV induction, all animals were randomly assigned to: control, SDF-1, SDF-1+age-related macular degeneration (AMD) 3100 (CXCR4 inhibitor), and SDF-1+ATN161 (integrin α5β1 inhibitor) groups; their effects on CNV progression were observed using hematoxylin eosin (HE) staining, fundus fluorescein angiography (FFA) grading and optical coherence tomography (OCT), and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining. Hypoxia-exposed endothelial cells (ECs) were used to simulate CNV in vitro, they were treated with SDF-1, combined with CXCR4 siRNA/AMD3100 or ATN161, and expression of integrin α5, cell migration and tube formation were analyzed.

Results: Integrin subunit α5 increased at 3^rd and 7^th day and decreased at 14^th day in CNV mice, with no significant change of β1-integrin. CXCR4 expression in CNV mice had persistent increase within 14d after induction. SDF-1 treatment significantly promoted the CNV progression during 3-14d. The mean CNV length in AMD3100 and ATN161 group at day 7 was 270.13 and 264.23 µm in HE images, significantly lower than the mean length in SDF-1 (345.70 µm) group. AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1. Mean integrin α5 positive area in SDF-1 group reached 2.31×104 µm², significantly higher than control (1.25×104 µm²), which decreased to 1.78×104 µm² after AMD3100 treatment. About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group, which significantly decreased to 43.12% after AMD3100 treatment. In vitro, integrin α5 peaked by 6 folds after 6h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24h of hypoxia. Approximately 25.12% of ECs expressed integrin α5 after SDF-1 stimulation, which decreased to 7.2%-9.5% after si-CXCR4 or AMD3100 treatment. ATN161 exerted an inhibitory effect comparable to that of si-CXCR4 on EC migration and tube formation in the presence of SDF-1.

Conclusion: SDF-1/CXCR4 signaling induces integrin α5β1 expression in ECs to promote CNV.

Keywords: CXCR4; choroidal neovascularization; endothelial cells; hypoxia; integrin α5β1; stromal cell-derived factor-1.

Figure 1. Profiles of SDF-1/CXCR4 and integrin expression in hypoxia-exposed RPE or RF/6A cells

A: ELISA and qRT-PCR analysis of SDF-1 levels in hypoxia-stimulated RPE cells at the indicated time points; B: Western blot and qRT-PCR analyses of CXCR4 expression in hypoxia-exposed RF/6A cells; C: Western blot and quantitative analyses (D) of the levels of integrin subunits and CXCR4 in RF/6A cells cultured under hypoxic conditions for the indicated times. ^aP<0.01; ^bP<0.05; NS: P>0.05 compared with the control group.

Figure 2. Profiles of SDF-1/CXCR4 and integrin expression in mice with laser-induced CNV

Western blot and quantitative analyses of the levels of integrin subunits and CXCR4 in mice with laser-induced CNV at the indicated times. n=3, ^aP<0.01; ^bP<0.05; NS: P>0.05 compared with the control group.

Figure 3. SDF-1/CXCR4 signaling promotes laser-induced CNV in mice

CNV was induced in animals using laser photocoagulation, followed by SDF-1 and SDF-1+AMD3100 injections on the indicated days. A-C: HE staining of CNV lesions and quantification of the CNV thickness and length in cross sections from the three groups. The margins of the CNV lesion are outlined by solid red lines. Scale bar, 50 µm; D-E: Representative OCT images and results of the analysis of CNV thickness using OCT in laser-treated mice; FFA imaging (F) and CNV grading (G) of the three groups on day 7. CNV lesions are circled by dashed black lines (the line represents the median of each group); H: Representative CNV areas in choroidal flatmounts from the indicated groups. Blood vessels in choroidal flatmounts were stained with rhodamine-conjugated agglutinin. A CNV lesion (dashed line) formed around the optic disc (solid line). Scale bar, 100 µm. Magnified image of a single CNV lesion. Scale bar, 50 µm; I-J: CNV areas and volumes were quantified in laser-induced mice. n=6; ^aP<0.01, ^bP<0.05.

Figure 4. Expression of integrin α5 and CXCR4 in CNV mice

CNV was induced in animals using laser photocoagulation, followed by SDF-1 and SDF-1+AMD3100 injections on day 7. Representative images of choroidal flatmounts (A, C) and quantification of the α5- and CXCR4-expressing areas and ratios in CNV lesions (B, D) after laser induction. CNV lesions (dashed line) formed around the optic disc (solid line). Scale bar, 100 µm. n=6; ^aP<0.01, ^bP<0.05.

Figure 5. Expression of integrin α5 and CXCR4 in ECs from CNV mice

CNV was induced in animals using laser photocoagulation, followed by SDF-1 and SDF-1+AMD3100 injections on day 7. Representative confocal images of the CNV lesion areas in different groups on day 7 (A, C) and quantification of α5 and CXCR4 expression relative to CD31 (B, D). Scale bar, 100 µm. n=6; ^aP<0.01, ^bP<0.05.

Figure 6. Effects of SDF-1/CXCR4 signaling on integrin α5 expression on ECs

A: Inhibitory effect of the CXCR4 siRNA on CXCR4 expression in RF/6A cells. ^aP<0.01 compared with the si-NC group; ^bP<0.01, ^cP<0.05 compared with the group transfected with the three siRNA sequences. Integrin α5 expression (B) and flow cytometry analysis of the percentage of α5-positive RF/6A cells (C) after CXCR4 inhibition in the presence of SDF-1, ^aP<0.05 compared with the control, ^bP<0.05 compared with the SDF-1 group, ^cP<0.05 compared with the SDF-1+si-NC group.

Figure 7. Effects of SDF-1/CXCR4 and integrin α5 on EC migration and tube formation

A: Tube formation of RF/6A cells in response to SDF-1 induction and CXCR4 inhibition or integrin α5β1 inhibition using ATN161 (2 mg/mL), scale bar, 500 µm; B: Transwell assay of RF/6A cell migration, scale bar, 500 µm; C: HE staining of CNV lesions and quantification of the CNV length in cross sections from the two groups on day 7; D: Fundus fluorescence angiography imaging of the two groups on day 7. n=6; ^aP<0.05 compared with the control, ^bP<0.05 compared with the SDF-1 group.