Abstract
Purpose: :
B-crystallin is a chaperone belonging to the small heat shock protein family. The aim of this study was to examine a role of B-crystallin in choroidal angiogenesis in vivo and in vitro.
Methods: :
Choroidal neovascularization (CNV) was induced by laser in B-crystallin knockout (B(-/-)) and wild-type mice. Fluorescein angiography, histopathology, immunohistochemistry with anti-VEGF-A antibody, and TUNEL were analyzed in murine eyes 7 and 14 days after the treatment. VEGF concentration was determined by ELISA in serum obtained from treated mice. Cultured human retinal pigment epithelial cells (RPE) were transfected with B-crystallin siRNA, and VEGF concentration was measured in the culture supernatants of serum-starved cells after 48 hr. Ubiquitinated VEGF was detected by Western blot in cell lysates of RPE after conjugation with polyubiquitin. MG132, a proteasome inhibitor, was administrated to RPE cells isolated from B(-/-) and wild-type mice, and the supernatants were harvested 8 hr after the stimulation.
Results: :
CNV was prominently attenuated in B(-/-) mice compared to wild-type mice as evaluated by fluorescein angiography and histopathology. VEGF protein expression in CNV lesions, and VEGF concentration in the serum were induced during CNV formation in wild-type mice, but VEGF expression remained low in B(-/-) lesions and serum. Cultured RPE derived from B(-/-) mice showed low VEGF secretion under serum-starved condition compared to wild-type cells. VEGF protein was ubiquitinated in human RPE after B-crystallin siRNA transfection and VEGF increased after proteasomal inhibition. Endothelial cell apoptosis in newly formed vessels was significantly greater in B(-/-) than in wild-type mice.
Conclusions: :
Our studies point to the important role of B-crystallin in choroidal angiogenesis and its potential as a therapeutic target; attenuation of angiogenesis in B(-/-) mice is mediated by downregulation of VEGF through posttranslational mechanisms.
Keywords: choroid: neovascularization • chaperones • vascular endothelial growth factor