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C. S. Jadhao, A. D. Bhatwadekar, M. E. Boulton, J. J. Steinle, M. B. Grant; Nerve Growth Factor Promotes Endothelial Progenitor Cell-Mediated Angiogenic Responses. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2668.
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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.
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.
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.
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.
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