Laminins in an in�vitro anterior lens capsule model established using HLE B-3 cells

Abstract

Cataracts are the most common eye disease to cause blindness in patients. The abnormal deposition of laminins (LMs) in the lens capsule and the disruption of capsular epithelium contribute to cataract development, although the mechanism by which this occurs is currently unclear. The present study aimed to reproduce HLE B-3 basement membranes (BMs) using HLE B-3 cells and to analyze the similarities of LM expression between HLE B-3 BMs and human anterior lens capsule (ALC). Immunohistochemistry (IHC), ELISA, western blot analysis and immunoprecipitation (IP)-western blot analysis were used to detect total LMs, LM trimers and 11 LM subunits in HLE B-3 cells, HLE B-3 BMs and human ALCs. In IHC staining, HLE B-3 cells and human ALCs were positive for LMs. In LM ELISA, all samples analyzed were positive for LMs. Western blot analysis detected all LM subunits except for LMγ3 in HLE B-3 cell lysate, 4 subunits (LMα4, LMα2, LMα1 and LMγ1) in HLE B-3 cell culture supernatant, 5 subunits (LMα4, LMα2, LMα1, LMβ3 and LMγ1) in HLE B-3 BMs, and 3 subunits (LMα4, LMγ2 and LMγ1) in human ALCs. The results of IP-western blot analysis revealed that the LM411 trimer was detected in HLE B-3 cell culture supernatant. These results indicated that HLE B-3 BMs were similar to human ALCs in terms of LM expression. Therefore, HLE B-3 BMs could be used as an in vitro ALC model to determine the role of LMs in ALC in the pathogenesis of cataracts and to select potential anti-cataract drugs.

A novel silent mutation E83E of αA-crystallin in age-related cataract with impaired chaperone function

“…Protein levels of CRYAA, histone H3, GAPDH, α-SMA,lamininγ2, bronectin (FN), bax, bcl-2 in HLE B-3 celllysates,supernatants,BMs and human ALCs were analyzed by IB. Afterplasmidtransfectionfor 48 h, HLE B-3 cell lysates,supernatants andBMs wereharvested as described previously [25]. Human ALCs lysates werecollectedasdescribed below.…”

“…The 42℃ treatment assay was performed asdescribedpreviously [25]. Brie y,HLE B-3cellstransfected with plasmids were cultured in 6-wellplates andputinto humidi ed atmosphere with 5% CO 2 for24 h.Cells were cultured at 42℃ in humidi edatmospherewithoutCO 2 for 4 h. With those culturedat 37℃inhumidi ed atmosphere without CO 2 for4 hasnegative control.…”

“…Fornegativecontrol, cells werekept at room temperature in PBS withoutUVirradiation for 1 h.HLE B-3 cells transfected withplasmidswere cultured in low-glucoseDMEM containing 10% FBS at 37℃inhumidi ed atmosphere with 5%CO 2 for 24 h.Cellswere then treated with UV for1 h as described above. HLEB-3cell lysates, supernatants andbasement membranes were harvestedasdescribed previously [25].…”

“…Onedish wastreated by UV(253.7 nm, 100 µW/cm 2 )for30 min. Aftertreated, changed PBS with 1.5 mllow-glucoseDMED containing10% FBS, 100 units/ml penicillin and100 µg/mlstreptomycin.Human ALCs were cultured at 37℃ with 5%CO 2 for24 h. The UV group was again treated with UVfor30 min,turned-over each pieces of Coomassie brilliant blue (CBB) staining HLE B-3 basement membranes were separated by 10%SDS-PAGE,thenstained by CBB staining R250 as describedpreviously [25].…”

“…Recently,humananterior lenscapsules (ALCs) models in vitro wasconstructedsuccessfully usingthe human lens epithelial B3 (HLE B-3)cell linebasement membrane(BM) [22]. Cellstransfected with CRYAA indicateditsability to prevent UVA inducedapoptosis in human lensepithelialcells [23][24][25].…”