Attenuation of retinal neovascularization in an OIR model with the use of αB-crystallin knockout mice. In (A-H), FITC-Isolectin-B4 angiography in wild-type (A-D) and αB crystallin^−/− (E-H) mice at P12 (A,E) and P17 (B-D,F-H) with OIR. At P12, avascular areas identified with arrows. In panel H, the αB^−/− flat mount is seen at greater magnification with additional DAPI nuclear staining. Bars indicate 1 mm in panels A, B, E, and F; 200 μm in panels C, D, and G; and 50 μm in panel H. Original magnification, ×50. Images obtained by the use of a confocal microscope (Zeiss) with a 2.5×/0.12 NA (A,B,E,F) and a 40×/1.3 oil immersion NA (C,D,G,H). In panels I-L, hematoxylin and eosin staining in wild-type (I,J) and αB crystallin^−/− (K,L) mice at P12 (I,K) and P17 (J,L) with OIR. At P12, occluded existing retinal vessels identified with white arrows. At P17, neovascular tufts identified with black arrows. Bars indicate 100 μm in panels I-L. Original magnification, ×100). Images were obtained by the use of a Spot II digital camera and fluorescence microscope (Leica) with a 20×/0.5 NA. In panels M and N, immunofluorescent staining with isolectin-B4 (green) demonstrates prominent neovascularization (white arrows) in wild-type nice (M) and infrequent neovascularization in αB crystallin^−/− mice (N). Bar indicates 50 μm. Original magnification, ×100. Images were obtained by the use of a SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA lens.

Neovascular endothelial cell apoptosis in αB-crystallin knockout^−/− mice in OIR. In panels A-D, DAPI nuclear staining (A; blue), TUNEL staining (B,D; red), and isolectin-B4 endothelial cell staining (C-D; green) in OIR in αB^−/− mice at P17. Several TUNEL⁺ apoptotic cells are detected in neovascular tufts (A-B; arrows), some of which colocalize with isolectin-B4–positive endothelial cells (C,D) on the retinal surface. Bar indicates 50 μm. In panel E, the number of apoptotic cells is significantly greater in αB crystallin^−/− mice than that in wild-type mice (P < .01). In panels F through I, DAPI nuclear staining (F), and expression of cleaved caspase-3 (G,I; red), and isolectin-B4 endothelial cells (H-I; green) in αB crystallin^−/− mice at P17. Immunoreactivity for cleaved caspase-3 is observed in neovacular endothelial cells (F-I; arrow). Bar indicates 50 μm. Original magnification, ×200. In panels J through U, expression of VEGFR2 (J-M; red), NG2 (N-Q; red), and CD105 (R-U; red) and colocalization with DAPI (blue) and isolectin-B4 (green) in wild-type (J,K,N,O,R,S) and αB crystallin^−/− (L,M,P,Q,T,U) retinas in OIR model at P17. Bar indicates 50 μm. Original magnification, ×100. Images obtained with a confocal microscope (Zeiss) with a 40×/1.3 oil immersion NA (C,D,H,I) with SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA (F,G,J-O).

VEGF-A protein expression is reduced in αB-crystallin^−/− mice during OIR. In panels A through F, expression of VEGF-A (A,C,D,F; red) and relationship with isolectin-B4⁺ endothelial cells (B,C,E,F; green) in wild-type (A-C) and αB crystallin^−/− mice (D-F) with OIR at P17. Bar indicates 50 μm. Original magnification, ×100. Images were obtained by the use of a SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA lens. In panel G, VEGF-A protein concentration (pg/mg protein) in posterior eyecup homogenates, determined by ELISA in αB crystallin^−/− and wild-type mice with OIR. αB crystallin^−/− mice at P17 showed significantly lower concentrations of VEGF-A than wild-type (P < .001) mice. In panel H, VEGF-A mRNA expression determined by real-time PCR analysis and normalized to beta-actin mRNA in posterior eye cups of αB crystallin^−/− and wild-type mice with OIR (n = 6). VEGF-A mRNA is up-regulated at P17 in wild-type mice (P < .005) and αB crystallin^−/− mice (P < .02). In panel I, Hif-1α protein expression in posterior eyecups with or without hyperoxia at P12 and P17 as measured by Western blot. Hif-1α expression is up-regulated at P17 in wild-type and αB^−/− mice. In panel J, αB-crystallin and its phosphorylated serine 59 form in wild-type murine retinas at P12 with or without hyperoxia, as measured by Western blot. Expression of phosphorylated serine 59 form of αB-crystallin is significantly increased in the presence of hyperoxia (n = 3; P < .01). In panel K, Western blot for αB-crystallin, VEGF, and VEGF-R2 in anti–VEGF-A immunoprecipitates from wild-type and αB crystallin^−/− mice at P12 and P17 in OIR. αB-crystallin binds to VEGF-A at P12 and P17 in wild-type mice but not in αB crystallin^−/− mice. Knockout of αB-crystallin does not affect VEGF-R2 binding to VEGF-A. In panel L, Western blot for αB-crystallin and TGF-β in anti–TGF-β immunoprecipitates of wild-type and αB crystallin^−/− mice at P12 and P17 in OIR. There is no apparent binding of αB-crystallin to TGF-β at P12 and P17 in αB crystallin^−/− and wild-type mice.

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).