Modulating p75NTR Prevents ProNGF- And Diabetes-Induced Acellular Capillaries Via Suppression of NRIF Nuclear Translocation

Abstract

Purpose: We have previously shown that knocking down the expression of the neurotrophin receptor p75NTR prevents development of acellular capillary formation in a model of proNGF overexpression. The aim of this study is to elucidate the mechanism by which p75NTR can stimulate retinal endothelial cell death and development of acellular capillaries, a hallmark of diabetic retinopathy.

Methods: Male wild-type (WT) and age-matched p75KO were rendered diabetic using injected streptozotocin for six-month. The number of acellular capillary formation and pericytes were counted in retinal trypsin-digest using PASH and or immunoflurescent staining. P75NTR was silenced using siRNA in human retinal endothelial (HRE) cells. Expressions of proNGF, p75NTR and apoptotic markers were quantified by Western-blot. Human mutant (hm-proNGF) was used to examine HRE cell viability with life and death kit and nuclear translocation of neurotrophin receptor interacting factor (NRIF) and p75NTR intra-cellular domain p75ICD (provided by Bruce Carter, Vanderbilt University) using immunocytochemistry.

Results: Deletion of p75NTR did not affect weight or blood glucose level in comparison to WT mice. Diabetes significantly induced acellular capillary formation 2.6-fold and significantly reduced pericyte count (63%) in WT mice but not in p75KO mice. In HRE cells, hm-proNGF induced a dose-dependent cell death that peaked at 50ng/ml. This effect was associated with nuclear translocation of NRIF. Overexpression of proNGF induced expression of p75NTR, its intracellular domain ICDp75 activation of JNK/p38 MAPK and cleaved-PARP. These effects were mitigated by knocking down expression of p75NTR using siRNA. NRIF nuclear translocation was prevented with inhibition of p75NTR in vitro.

Conclusions: Knocking-out of p75NTR expression exerted vascular protective action in the diabetic retina. P75NTR-mediated apoptosis in retinal vascular endothelial cells involve cleavage of p75NTR, release of ICDp75 and the nuclear translocation of NRIF. Therefore, targeting p75NTR may be an effective and novel strategy for the treatment of diabetic retinopathy.

Keywords: 499 diabetic retinopathy • 426 apoptosis/cell death • 715 signal transduction: pharmacology/physiology