Figure 4
Figure 4. Attenuation of laser-induced CNV in αB-crystallin knockout mice. In panels A-D, CNV was induced by the use of laser photocoagulation and relative FA score and CNV volume measured 14 days after laser. (A) FA score and CNV volume were significantly reduced in αB crystallin−/− (n = 16) compared with wild-type mice (n = 17; P < .02; P < .05; respectively). (B) Representative FA in laser-induced CNV in wild-type and αB crystallin−/− mice at day 14 after laser. (C-D) Representative confocal image of FITC-labeled isolectin-B4–stained flat mounts on day 14. In panel E, VEGF-A concentrations (ELISA in pg/mL) in peripheral blood of wild-type and αB crystallin−/− mice on day 0, 3, 7, and 14 after laser treatment. In panel F-K, histology (hematoxylin and eosin stain), and VEGF-A (arrows) and CD31 (arrows) expression in wild-type (F,H,J) and αB crystallin−/− mice (G,I,K) in CNV lesions 14 days after laser treatment. Bar indicates 50 μm. Original magnification, ×200. In panels L through O, detection of apoptotic cells in CNV lesions by TUNEL assay (red; arrows) and relationship with nuclei (DAPI+ nuclei; blue) and endothelial cells (isolectin B4; green; L-O). Increased numbers of TUNEL+ endothelial cells (arrows) are seen in merged image in αB crystallin−/− mice (L-M; arrows). Bar indicates 50 μm. Original magnification, ×200. In panel P, VEGF concentrations (ELISA in pg/mL) in supernatants of cultured RPE cells exposed to serum-free medium for 48 hours. VEGF concentrations are significantly lower in αB crystallin−/− mice than that in wild-type mice (*P < .01). In panel Q, mono-tetra ubiquitinated proteins are prominent in RPE cells transfected with αB-crystallin siRNA compared with controls; however, polyubiquitinated proteins are similarly prominent after αB-crystallin siRNA treatment and in controls. In panel R, mono-tetra ubiquitinated VEGF-A and polyubiquitinated VEGF-A were detected by Western blot of ubiquitinated proteins by the use of anti-VEGF-A antibody. Images were taken with a SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA (A-D,F-I) and 20×/0.5NA lens (J-U).

Attenuation of laser-induced CNV in αB-crystallin knockout mice. In panels A-D, CNV was induced by the use of laser photocoagulation and relative FA score and CNV volume measured 14 days after laser. (A) FA score and CNV volume were significantly reduced in αB crystallin−/− (n = 16) compared with wild-type mice (n = 17; P < .02; P < .05; respectively). (B) Representative FA in laser-induced CNV in wild-type and αB crystallin−/− mice at day 14 after laser. (C-D) Representative confocal image of FITC-labeled isolectin-B4–stained flat mounts on day 14. In panel E, VEGF-A concentrations (ELISA in pg/mL) in peripheral blood of wild-type and αB crystallin−/− mice on day 0, 3, 7, and 14 after laser treatment. In panel F-K, histology (hematoxylin and eosin stain), and VEGF-A (arrows) and CD31 (arrows) expression in wild-type (F,H,J) and αB crystallin−/− mice (G,I,K) in CNV lesions 14 days after laser treatment. Bar indicates 50 μm. Original magnification, ×200. In panels L through O, detection of apoptotic cells in CNV lesions by TUNEL assay (red; arrows) and relationship with nuclei (DAPI+ nuclei; blue) and endothelial cells (isolectin B4; green; L-O). Increased numbers of TUNEL+ endothelial cells (arrows) are seen in merged image in αB crystallin−/− mice (L-M; arrows). Bar indicates 50 μm. Original magnification, ×200. In panel P, VEGF concentrations (ELISA in pg/mL) in supernatants of cultured RPE cells exposed to serum-free medium for 48 hours. VEGF concentrations are significantly lower in αB crystallin−/− mice than that in wild-type mice (*P < .01). In panel Q, mono-tetra ubiquitinated proteins are prominent in RPE cells transfected with αB-crystallin siRNA compared with controls; however, polyubiquitinated proteins are similarly prominent after αB-crystallin siRNA treatment and in controls. In panel R, mono-tetra ubiquitinated VEGF-A and polyubiquitinated VEGF-A were detected by Western blot of ubiquitinated proteins by the use of anti-VEGF-A antibody. Images were taken with a SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA (A-D,F-I) and 20×/0.5NA lens (J-U).

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