Abstract
Abstract: :
Purpose: To determine the expression of several angiogenesis–regulatory proteins on retinal endothelial cells (REC) from secondary sprouting colonies (SSC). Methods:We used the SSC model, described by Castellon et al., Exp Eye Res. 74(4):523–35. REC were plated on a thick layer of Matrigel R , later organizing into tubular networks which collapse within 48 hrs. However, some REC survive, proliferate and invade the matrix, sometimes forming thicker cords that resemble capillaries with lumens. After 14 to 17 days the SSC were embedded in OCT freezing medium, cryosectioned and stained with antibodies against von Willebrand factor (vWf), smooth muscle actin (SMA), survivin, tie–1 and tie–2, angiopoietin–1 and –2 (Ang–1 and Ang–2). As control, we used parallel REC cultures plated on LabTekII chamber slides. mRNA derived from confluent REC monolayers as well as SSC was analyzed by RT–PCR. Results: As expected, cells from monolayers and SSC were positive for vWf, a specific endothelial cell marker. However, some cells in both cultures were positive for SMA, a marker for smooth muscle cells and pericytes. There were SMA+ cells in REC from passages 1–12, but there were far fewer in the latter passages. Survivin, tie–2 and ang–2 were also positive by immunofluorescence. Interestingly, most survivin + cells also expressed vWf, suggesting most were of endothelial origin. However, some survivin+ cells were also SMA+, indicating that some pericytes were also upregulating survivin in culture. RT–PCR confirmed that both monolayers and SSC cells expressed ang–2, tie–2 and survivin and these molecules were upregulated by PDGF treatment. Conclusions: We identified some SMA+ cells on both monolayers and SSC. Pericytes were able to establish cell–cell interactions with REC in these in vitro systems, mimicking the contact between SMA+ cells and vWf+ cells in vivo. Markers related to pathological angiogenesis like survivin, tie–2 and ang–2 were also present, which demonstrates that the SSC model may be suitable for studies on retinal diseases like diabetic retinopathy and age related macular degeneration.
Keywords: vascular cells • neovascularization • immunohistochemistry