Abstract
Abstract: :
Purpose: To determine the differences in angiogenic potential of primary cultures of retinal endothelial cells (REC) derived from normal (NL), diabetic (DM) and diabetic retinopathy (DR) patients. Methods: We used various in vitro angiogenic assays as described by Castellon et al., Exp Eye Res. 74(4):523–35. These include angiogenic growth factor–induced cell proliferation and survival, capillary–like tube formation on Matrigel ®, and the novel secondary sprouting assay. mRNA derived from confluent REC monolayers treated with single or combined growth factors (GF) was analyzed by RT–PCR. Results: DM and DR cells formed more mature tubular networks than NL cells; however, they collapsed sooner. Addition of GF had minimal effects on tube formation/collapse. NL REC had lower basal proliferation and survival rates compared to DM and DR cells. They also had lower proliferative and survival responses to single or combined angiogenic GF than DM and DR REC. NL cells had a very limited capacity to form secondary sprouts in basal or growth–factor containing medium. However, DM and especially DR, REC were capable of forming larger secondary sprouting colonies when grown in basal or growth–factor containing medium. Because secondary sprouting involves spontaneous cell survival and invasion, we analyzed the expression of genes known to regulate these cellular behaviors, including angiogenic GF and their receptors. IGF–1 was only produced by NL REC whereas EGF was mainly present in DR cells. Members of the plasminogen activator cascade (uPA, uPAR, PAI–1, PAI–2, tPA) were also analyzed. uPA was greatly upregulated in DR cells as compared to NL, whereas the endogenous inhibitor PAI–2 was decreased. Other differentially–expressed genes included survivin, tie–1, tie–2, ang–1, ang–2, sprouty–4, and jagged, all of which have been implicated in angiogenesis. Conclusions: Using a variety of in vitro assays for angiogenesis, we demonstrate that DM, and especially DR, cells are inherently different from NL REC, even though culture conditions are the same. These differences imply that "diabetic memory" may involve increased cellular responses to angiogenic growth factors resulting in enhanced cell survival/invasion, eventually leading to neovascular conditions.
Keywords: neovascularization • gene/expression • growth factors/growth factor receptors