Figure 3.

NGF gene therapy increases NGF expression in aging GECs, reverses impaired in vitro angiogenesis, and reduced cell proliferation. Aging GECs were transduced with human LV-NGF in medium containing polybrene linker. Mock transductions in aging GECs performed using polybrene linker without LV-NGF served as negative controls (-, control) for LV-NGF gene therapy. The efficiency of transfection in aging GECs was determined using LV-GFP. (A) The percentage of LV-GFP–transfected aging GECs showing GFP-derived fluorescence was 82% ± 6%. (B) Western blot for NGF showed that after NGF gene therapy (+), aging GECs express both endogenous (rat) NGF and human NGF from gene therapy vs negative controls (-, control), which express only endogenous (rat) NGF. (C) NGF gene therapy with LV-NGF resulted in a significant 3.7-fold increase in in vitro angiogenesis at 6 hours in aging GECs vs negative controls (-, control). Panels are representative images of capillary-like tube formation. Original magnification, ×200. Data are means ± SD (N = 6). (D) Immunofluorescence staining for NGF (red fluorescence) showed that NGF gene therapy in aging GECs strongly increased the expression of NGF by 3.3-fold and induced long filopodia (arrows) vs aging GECs without gene therapy (negative controls). (E) Immunoperoxidase staining for NGF showed that NGF gene therapy of aging GECs induced a significant increase in NGF expression (brown staining) and extensive long filopodia (arrows) reflecting a change in these cells to an angiogenic phenotype; aging GECs without gene therapy (negative controls) have minimal NGF expression and lack filopodia. Data are means ± SD (N = 6). (F) Cell proliferation assessment after LV-NGF gene therapy of aging GECs. LV-NGF gene therapy of aging ECs significantly increased cell proliferation. The Student t test and 1-way analysis of variance with the Tukey multiple comparison test were used to determine statistical significance between 2 or multiple groups, respectively.