Nerve Growth Factor Promotes Endothelial Progenitor Cell-Mediated Angiogenic Responses

Abstract

Purpose: : Retinal neuronal cells are dysfunctional in diabetes and in response to injury express nerve growth factor (NGF) which, in some settings, can be pro-angiogenic. We postulated that the NGF that accompanies retinal injury/ischemia will a) contribute to an "angiogenic switch" in CD34⁺ cells taking them from beneficial reparative cells to cells that participate in pathological angiogenesis and b) have little effect on the resident endothelial cells of the retinal vasculature.

Methods: : CD34⁺ cells were isolated from healthy volunteers (n=10) using immunomagnetic separation. Human retinal endothelial cells (HREC) were obtained from retinas of healthy human donors (n=3). Cell proliferation in response to NGF (1, 2 and 4pM) was determined by cell counts for CD34⁺ cells and MTT assay for HREC. Migration of CD34⁺ cells and HREC was studied using a modified Boyden chamber assay. NGF stimulated CD34⁺ cell incorporation into HREC tubule formation was evaluated using 3-dimensional (3D) extracellular matrix. NGF receptor activation was assessed by Western blot analysis.

Results: : NGF induced a dose-dependent increase (1.5-3-fold; p<0.001) in proliferation of CD34⁺ cells at 24 hrs compared to controls but had no effect on HREC proliferation. However, NGF stimulated HREC migration in a dose-dependent manner. Pretreatment of CD34⁺ cells with NGF increased migration to SDF-1 (p<0.05). NGF-treated CD34⁺ cells enhanced in vitro tube formation by HRECs 2-fold (p<0.05) as assessed by increases in numbers of sprouts in a 3D model of angiogenesis Western blotting of cell lysates with phospho-specific antibodies revealed phosphorylation of TrKA (receptor for NGF) in CD34⁺ and HRECs. NGF stimulation resulted in activation of Akt in HRECs and ERK1 in CD34⁺ cells while pretreatment with LY294002 (PI3K inhibitor) and PD98059 (MAPK inhibitor) blocked this phosphorylation response by 1.2-fold and 5-fold respectively.

Conclusions: : NGF differentially regulates the angiogenic potential of CD34⁺ cells and resident endothelial cells and may contribute to the angiogenic switch in CD34⁺ cells which is associated with the pathological neovascularization in diabetic retinopathy.

Keywords: diabetic retinopathy • growth factors/growth factor receptors • neovascularization