Abstract
Purpose: :
Genetic deletion of very low-density lipoprotein receptor (VLDLR) has been linked to subretinal neovascularization in the knockout (vldlr-/-) mice. To determine the effects of VLDLR on the function of retinal vascular endothelial cells (RVEC), we tested the hypothesis that lack of VLDLR promotes RVEC proliferation and angiogenic functions.
Methods: :
Primary RVEC culture isolated from wildtype and vldlr-/- mice carrying a SV40 gene was used to assess angiogenic properties by various assays for MTS proliferation, tube formation, and migration in the presence or absence of angiogenic factors. Immunofluorescent staining was used to characterize the isolated cells with various markers (CD31, αSMA) and to determine the expression and activation of NFkB.
Results: :
Characteristic endothelial morphology and immuno marker staining confirmed that over 95% of isolated cells were endothelial cells. MTS assay revealed that the proliferation rate of RVECs from vldlr-/- retina was about 130% higher than that of wildtype RVECs under the same culture condition. Growth curve analysis showed that vldlr-/- RVECs grew at a much faster rate (20-100% increase) than wildtype RVECs from day 1 to day 5 with either basal or growth media. Tube formation assay revealed a striking upsurge of total tubule length by vldlr-/- RVECs in all three conditions of basal, 10 ng/ml bFGF, and growth media. The difference ranges 3-11 folds increase compared to those formed by wildtype RVECs. Similarly, migration assay in these conditions also showed that the number of vldlr-/- RVECs migrated through the 8-µm pore membrane was 1.3-3.7 times of those of wildtype RVECs. In addition, NFΚB activation in vldlr-/- RVECs was noticeable with increased immunofluorescent staining intensity and nucleus staining especially after 10 ng/ml bFGF stimulation.
Conclusions: :
Lack of VLDLR significantly augmented a spectrum of angiogenic functions of RVECs. It suggests that VLDLR is a critical molecule regulating multiple intrinsic angiogenic properties of RVEC.
Keywords: retinal neovascularization • age-related macular degeneration • retinal culture