Abstract
Purpose: :
We have previously shown significant accumulation of the pro-form of nerve growth factor (proNGF) and its receptor p75NTR in diabetic rat retinas and Muller cells. The purpose of this study is to elucidate the role of proNGF/p75NTR axis in sustaining glial activation, and expression of inflammatory mediators in vitro and in vivo.
Methods: :
Overexpression of proNGF in SD rats was achieved by intravitreal injection of the GFP-proNGF plasmid (20 µg) expressing the protease cleavage resistant proNGF construct. Expression of p75NTR was silenced using co-intravitreal injection of siRNA for 1-week or shRNA for 4-weeks. Expression of proNGF, TNF-alpha, IL-1-beta and NFkB were quantified by immunohistochemistry and Western-Blot. As an ex vivo correlate, Muller rMC-1 cells were cultured in high glucose and stimulated with proNGF in the presence or absence of p75NTR inhibitor.
Results: :
Overexpression of proNGF in rat retina induced expression of p75NTR, activated glial Muller cells as indicated by GFAP staining, and upregulated the pro-inflammatory transcription factor NFkB. After 1-week, proNGF induced expression of TNF-alpha and IL-1-beta, and this was blocked by p75NTR siRNA. After 4-weeks, proNGF sustained the expression of itself, TNF-alpha and IL-1-beta, and this was blocked by p75NTR shRNA. In high glucose maintained rMC-1 cultures, proNGF induced expression of p75NTR, release of its ICD, and upregulation of of NFkB, which resulted in sustained expression proNGF as well as TNF-alpha. These effects were blunted by the selective p75NTR antagonist.
Conclusions: :
Similar to diabetes, overexpression of proNGF in normal rat retina induced up-regulation of p75NTR and NFkB expression leading to inflammatory cascades. These effects were associated with induction and sustaining expression of proNGF, TNF-alpha and IL-1B that were blocked by inhibiting p75NTR expression. Thus, inhibiting p75NTR may be an effective strategy in diabetic retinopathy.
Keywords: diabetic retinopathy • inflammation • Muller cells