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Ram H Nagaraj, Scott Howell, Rooban B Nahomi; Exogenous αB-crystallin promotes tubulogenesis in human retinal endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5817.
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© ARVO (1962-2015); The Authors (2016-present)
We have investigated the role of exogenous αB-crystallin in tubulogenesis of human retinal capillary endothelial cells.
Human retinal endothelial cells (HREC) were seeded on plates coated with a basement membrane extract that had low levels of growth factors (from Trevigen). Cells were cultured for 16 to 24 hrs with either serum-free medium or serum-free medium containing endothelial cell growth factors. To verify the role of αB-crystallin on tubulogenesis, the medium was depleted of αB-crystallin by immunoprecipitation. To test the role of exogenous αB-crystallin, 0-50 μg of recombinant human αB-crystallin (wild type or R120G mutant) was added to the culture media. HREC overexpressing human αB-crystallin or treated with an siRNA for αB-crystallin were used to determine effects of the endogenous αB-crystallin on tubulogenesis. Tubulogenesis was assessed by staining endothelial cells with Calcein AM followed by measuring total tube length. To determine if a functional domain within αB-crystallin was responsible, cells were cultured with chaperone or chaperone-null (scrambled) peptides of αB-crystallin.
Cells cultured in the αB-crystallin depleted medium showed significantly lower tubulogenesis than those in the complete medium. A concentration-dependent increase in tubulogenesis was observed in cells treated with exogenous recombinant αB-crystallin and such an effect was absent in cells treated with either chaperone-compromised R120G mutant protein or ovalbumin. HREC overexpressing αB-crystallin displayed higher levels and cells treated with an siRNA for αB-crystallin showed lower levels of tubulogenesis relative to control cells. The negative effect of αB-crystallin siRNA was partially reversed by the addition of recombinant αB-crystallin. The functional domain peptide of αB-crystallin mirrored the effects of the whole protein whereas the scrambled peptide showed no such effects. Addition of αB-crystallin to the medium enhanced cell survival pathway as determined by the enhanced Akt phosphorylation.
Both exogenous and endogenous αB-crystallin promote tubulogenesis of HREC. The effect appears to be related to the chaperone function of αB-crystallin. The observed higher expression of αB-crystallin in the diabetic retina could promote retinal angiogenesis during proliferative diabetic retinopathy.
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