Abstract
Purpose::
Juxafoveal Telangiectasia (JFT) is a progressive bilateral disorder that leads to blindness. During the disease, retinal blood vessels become tortuous, dilated and leaky and at later stages subretinal neovascularisation occurs. We aim to identify the genes that are involved in driving these vascular abnormalities.
Methods::
In the absence of an animal model of JFT, several models of retinal degeneration have been chosen which exhibit similar vascular changes to those in JFT: The Royal College of Surgeons (RCS) rat, very low density lipoprotein receptor (VLDLR) knock out mouse, curlytail mouse and retinal degeneration 1 and 10 (RD1 and RD10) mice. We have optimised a novel method for obtaining whole vessel fragments from mouse and rat retinae. Magnetic beads coated with an anti-PECAM antibody are incubated with enzymatically-digested retinae and bind specifically to endothelial cells within blood vessels. This enables vessel-enriched and vessel-depleted fractions to be obtained. RNA from these and control animals is used in microarray analyses, to identify genes involved in the development of the abnormal vessels. Protein and RNA from these fractions is also used for validation of the microarray data.
Results::
Extracted vessels appear as bead-bound fragments by light microscopy, and are positive for collagen IV. Good yields of endothelial cells in the vessel-enriched fraction have been confirmed by western blotting for VE-cadherin. Some purified vessel fragments are also positive for the pericyte marker, alpha-smooth muscle actin (ASMA), by immunofluorescent staining. Semi-quantitative PCR confirmed higher levels of VEGFR1 and ASMA in the vessel-enriched fraction, than in the vessel depleted fraction, indicating good purity and yields of microvessel fragments. Microarray experiments using microvessels from RCS rat has yielded several interesting genes enriched in the abnormal vessel fraction, such as the tachykinin receptor, membrane frizzled-related protein and a sprouty-related protein. We are confirming these changes by real-time PCR and immunofluorescent staining.
Conclusions::
Microvessels can be rapidly purified from mice and rat retinae using magnetic beads. These fragments are positive for endothelial and pericyte markers, and can be used to extract RNA for microarray analysis. Several interesting genes that could be involved in driving vascular abnormalities in the RCS rat have been identified. Microarray analysis of the remaining mice models may reveal genes that are altered commonly across all the models, and which may therefore be important regulators of abnormal vascular growth in retinal disease.
Keywords: retinal neovascularization • gene microarray • vascular cells