The effect of fibroblast growth factor 2 on neovascular vessels depends on the stage of angiogenesis

Figures

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  Fig. 1. Schematic map of the rhodopsin/FGF2 fusion gene [9] and immunolabeled imaging of the retinal frozen section of FGF2-tg. A: Schematic map of the rhodopsin/FGF2 fusion gene. The bovine rhodopsin promoter is the previously described −2174 to +70 fragment. The FGF2 fragment extends from +84 to +1018 of the FGF2 gene with translation start sites at +280, +322, and +445 and the stop codon at +910 to +912. The oligonucleotide primers used to screen genomic DNA for the presence of the transgene are shown as P1 and P2. The primers used for RT-PCR are shown as P3 and P2. The transcription start site is indicated by +1 [9]. B: A frozen section of FGF2-tg mouse immunolabeled with anti-human FGF2 antibody (center). The left slide shows nuclei immunolabeled with DAPI from the same specimen. The right slide shows a merged view of the left two slides, showing that FGF2 is mainly localized in the outer nuclear layer of the retina. Scale bar = 200 μm.

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  Fig. 2. Schematic figure of Li-CNV models, Left figure: One-time PC group mice receive 3 intense PC shots altogether (1st PC, drawn as blue circles). After 1st PC, Li-CNV gradually developed around the 1st PC spots (drawn as red circular nest). Right figure: Two-times PC group mice receive 3 intense PC shots followed by 6 additional weak PC shots (2 nd PC, drawn as white circles) around existing Li-CNV on the 7th day of 1st PC. The laser conditions for 1st and 2 nd PC are shown below the two figures.

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  Fig. 3. One-time PC group of FGF2-tg and WT mice at 7 days post-1st PC. A: Left is OCTA imaging in one-time PC group of WT mouse at 7 days post-1st PC. Li-CNV was observed as a highly signaled lesion. Right is Li-CNV of the same mouse as the left figure and was immunolabeled with anti-rat CD31 antibody (green). Scale bar = 100 μm B: Left is OCTA imaging in one-time PC group of FGF2-tg mouse at 7 days post-1st PC. Compared to A figures, Li-CNV shows a more clearly defined highly signaled lesion and seems larger in size. Right is Li-CNV of the same mouse as shown in the left figure and immunolabeled with anti-rat CD31 antibody (green). Scale bar = 100 μm C: This figure represents the time-course change in Li-CNV size in FGF2-tg and WT mice. At day 3, 7, and 14 post-1st PC, the size of Li-CNV in FGF2-tg mice were significantly larger than that in WT mice on the same days. In contrast, there were no differences in the size of Li-CNV at day 21 and 28 post-1st PC between FGF2-tg and WT mice. Additionally, the size of Li-CNV in FGF2-tg mice at day 7 post-1st PC was significantly larger than that in FGF2-tg mice at day 3 post-1st PC. (FGF2-tg mice: n = 6, WT mice: n = 6, respectively).

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  Fig. 4. Immunolabeled imaging. A) a) OCTA imaging is Li-CNV in two-times PC group of FGF2-tg mouse at 7 days post-1st PC. The white circle indicated to 1st PC area. Scale bar = 100 μm b, c) Comparison of OCTA and immunolabeled imaging using anti-rat CD31 antibody (green) from same mouse at 28 days post-2nd PC. Li-CNV enlarged toward to 2 nd PC area (yellow circle). Scale bar = 100 μm d)A flat-mounted choroid preparation from of the same mouse as shown in the figure Bb) and Bc) immunolabeled with anti-mouse α-SMA antibody (red). New vessels branching toward to 2 nd PC area surrounded α-SMA positive smooth muscle cells were observed. A α-SMA detected smooth muscle cells and RPE cell activity. Scale bar = 100 μm. B) a) OCTA imaging is Li-CNV in two-times PC group of WT mouse at 7 days post-1st PC. The white circle indicated to 1st PC area. Scale bar = 100 μm b, c) Comparison of OCTA and immunolabeled imaging using anti-rat CD31 antibody (green) from same mouse at 7 days post-2nd PC. Li-CNV enlarged toward to 2 nd PC area (yellow circle). Scale bar = 100 μm d) A flat-mounted choroid preparation from of the same mouse as shown in the figure Cb) and Cc) immunolabeled with anti-mouse α-SMA…

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  Fig. 5. The time-course changes of Li-CNV in FGF2-tg and WT mice observed by OCTA. We observed Li-CNV in FGF2-tg and WT mice over time in the same individual until 28 days post-1st PC in the one-time PC group and post-2nd PC in the two-times PC group using OCTA.A. In both the one-time PC group of FGF2-tg and WT mice, Li-CNV size reached its maximum at 7 days post-1st PC and highly signaled lesion of Li-CNV gradually became indistinct by 28 days post-1st PC. Scale bar = 100 μm B. The one-time PC group of FGF2-tg mice clearly showed a decrease in Li-CNV size by 28 days post-1st PC compared with those of the Li-CNV size at 7 days post-1st PC. In contrast, in two-times PC group of FGF2-tg, highly signaled lesion of Li-CNV was illustrated clearly and Li-CNV size maintained until 28 days post-2nd PC. Scale bar = 100 μm, C. The highly signaled lesion of Li-CNV gradually became indistinct by 28 days post-1st PC in the one-time PC group of WT mice. In the two-times PC group of WT mice, Li-CNV enlarged at 7 days post-2nd PC compared with those of the Li-CNV at 7 days post-1st PC and CNV networks of both groups gradually became indistinct by 28 days post-2nd PC. Scale bar = 100 μm.

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  Fig. 6. The real-time qPCR in FGF2-tg and WT mice. Gene expression levels were calculated using the 2−ΔΔCt method, GAPDH was used as a reference gene, and the results were presented as relative expression to the control. Untreated WT mice were used as controls. A: FGF2 mRNA expression level in the one-time PC group of FGF2-tg mice at 7 days post-1st PC was significantly higher than that in WT mice on the same day. Additionally, FGF2 mRNA expression level in the two-times PC group of FGF2-tg mice at 7 days post-2nd PC was significantly higher than that in WT mice on the same day. On the other hand, there was no significant increase in FGF2 mRNA expression level at 7 days post-2nd PC in WT mice compared with the untreated WT control mice on the same day. (FGF2-tg mice: n = 6, WT mice: n = 6, respectively), B: FGFR1 mRNA expression level in the two-times PC group of FGF2-tg mice at 7 days post-2nd PC was significantly higher than that in WT mice on the same day. On the other hand, there was no significant increase in FGFR1 mRNA expression level at 7 days post-2nd PC in WT mice compared with the untreated WT control mice on the same day. (FGF2-tg mice: n = 5 or 6, WT mice: n = 5 or 6,…

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  Fig. 7. The size ratio of Li-CNV compared to Li-CNV size at day7 post PC. To confirm the change in Li-CNV size, we examined the size ratio at 14 and 28 days post-PC by setting the Li-CNV size at 7 days post-PC to 100 % using OCTA imaging. There was no difference in the size ratio of Li-CNV at day 14 post-PC in any of the groups. On the other hand, the size ratio of Li-CNV at day 28 post-2nd PC in two-times PC group of FGF2-tg mice was statistically higher than that in the one-time PC group of FGF2-tg mice. Furthermore, the size ratio of Li-CNV at day 28 post-2nd PC in the two-times PC group of FGF2-tg mice was statistically higher than that in WT mice. In other words, the Li-CNV size in two-times PC group of FGF2-tg mice was maintained until 28 days post-2nd PC. (FGF2-tg mice: n = 6, WT mice: n = 6, respectively).