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. Stable overexpression of proNGF induced acellular capillaries in rat retina. Recent studies showed that modulating p75NTR expression mitigates proNGF-mediated release of inflammatory mediators in Muller cells and diabetic retina. Although the link between retinal inflammation and vascular injury has been well-established, the role of p75NTR remains unexplored. The aim of this study is to elucidate the role and mechanism by which knocking down expression of p75NTR can prevent development of acellular capillaries, a hall mark of diabetic retinopathy, in vivo.
Methods:
Overexpression of proNGF in SD rats was achieved by intravitreal injection of the GFP-proNGF123 plasmid (20 μg). Expression of p75NTR was silenced using co-intravitreal injection of shRNA for 6-weeks. Expression of NGF, proNGF, p75NTR and apoptotic markers was quantified by Western-Blot. Acellular capillary formation was detected in retinal trypsine digest. Human retinal endothelial cells were cultured in high glucose and stimulated with mutant proNGF and the interaction between p75NTR intracellular domain (ICDp75) and neurotrophin receptor interacting factor (NRIF) was examined using immunoprecipitation.
Results:
Knocking down expression of p75NTR using shRNA significantly reduced proNGF- induced acellular capillary formation and the number of TUNEL-positive cells in retinal tryptic digest. Overexpression of proNGF induced significant increases of ICDp75 and decreased NGF expression compared with GFP or GFP-shRNA. In high glucose maintained endothelial cultures, proNGF accelerated endothelial apoptosis and stimulated expression of ICDp75 and its association with NRIF protein as indicated by immunoprecipitation.
Conclusions:
Silencing p75NTR expression restored NGF levels and exerted vascular protective action in retina that stably overexpresses cleavage-resistant proNGF. P75-mediated apoptosis in retinal vascular endothelial cells involve recruitment of NRIF. Therefore, targeting p75NTR may be an effective strategy in treatment of diabetic retinopathy.
Keywords: 688 retina •
499 diabetic retinopathy •
449 cell survival