factors from IRIS ciliary body (I/CB) cell cultures upregulate bcl-2 which prevents apoptosis of corneal endothelial cells

Purpose. To determine the effect of soluble factors secreted by iris ciliary body (I/CB) cells on apoptosis of corneal endothelial cells. Methods. Human and rabbit corneal endothelial cells (HCN and RCN, respectively) were cultured in the presence of either 50% I/CB cell supernatant, aqueous humor (AH), or TGF-β (2ng/ml). Fifty percent human epithelium (HCE), rabbit cornea epithelium (RCE) or RCN conditioned culture supematants were added to HCN cultures as controls. HCN were also treated with anti-Fas mAb. Different fractions of I/CB factor were separated by exclusion membrane filtration and co-cultured with HCN. Apoptosis of HCN was assessed by flow cytometry and Giemsa staining. Expression of Bcl-2 protein was assessed by flow cytometry. Fractions of I/CB were treated with proteinase K at 5 u.g/ml. Results. I/CB supematants and AH reduced spontaneous apoptosis of HCN approximately 50% after 3 days in culture. I/CB supematants stimulated upregulatkm of intracytoplasmic and cell membrane-associated Bcl-2 protein expression by 58% and 270% respectively. By contrast, supematants from HCE, RCN, and RCE did not affect Bcl-2 protein expression or apoptosis. Proteinase K treatment and fractionation of I/CB supematants indicated that the Bcl-2inducing fraction was a 30-50 KD peptide. Conclusions. A 30-50 KD peptide secreted by I/CB cells and present in the AH upregulates Bcl-2 protein expression and protects comeal endothelial cells from apoptosis. This AH-borne factor may play a key role in protecting the corneal endothelium from cellular attrition.