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. Although the link between retinal inflammation and vascular injury has been well-established, the role of proNGF/p75NTR remains unexplored. The aim of this study is to examine whether proNGF can directly induce apoptosis of retinal endothelial cells in vitro and 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-proNGF plasmid (20 μg) or mutant protein expressing the cleavage-resistant proNGF. Expression of p75NTR was silenced using co-intravitreal injection of shRNA for 4-weeks. Expression of proNGF, p75NTR and apoptotic markers was quantified by Western-Blot. Acellular capillary formation was detected in retinal trypsin digest. Human retinal endothelial cells were cultured in high glucose and stimulated with proNGF in the presence or absence of p75NTR antagonist (A) or the γ-secretase inhibitor (E).
Results: :
Overexpression of proNGF in rat retina induced expression of p75NTR and activated glial Muller cells as indicated by GFAP staining. After 4-weeks, proNGF induced acellular capillary formation that was blocked by p75NTR shRNA. In high glucose maintained endothelial cultures, proNGF induced expression of p75NTR, activation of JNK, cleaved-PARP and caspase-3. Treatment of cells with p75NTR antagonist (A) or preventing proteolysis of p75NTR intracellular domain by inhibiting γ-secretase (E) prevented proNGF-induced caspase-3 activation in HG-treated cells.
Conclusions: :
Overexpression of proNGF induced up-regulation of p75NTR and development of acellular capillary formation. These effects were abrogated by silencing p75NTR expression in vivo or its pharmacological antagonists in vitro. Therefore, targeting p75NTR or its proteolysis may be an effective strategy in treatment of diabetic retinopathy.
Keywords: diabetic retinopathy • vascular occlusion/vascular occlusive disease • ischemia