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S.H. Chavala, L. Vincent, F. Zhang, D. Cobrinik, A. Hooper, S. Rafii, T.C. Lee; The MDM2 Inhibitor, Nutlin–3A, is Capable of Inhibiting Endothelial Cell Proliferation via p53 Mediated Apoptosis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5344.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize and develop a novel approach to the treatment of pathologic ocular neovascularization. The protein p53 is a transcription factor that acts to prevent cellular proliferation of stressed cells by promoting cell cycle arrest or apoptosis. Nutlin–3A is a small molecule antagonist of MDM2, a key negative regulator of p53. By inhibiting p53–MDM2 interaction, Nutlin–3A prevents proteosomal degradation of p53. Nutlin–3A is actively being studied in tumor biology; however, we set forth to study a novel use and mechanism of this compound in treating ocular neovascularization.
Human umbilical vein endothelial cells (HUVEC) cultures were used for endothelial cell proliferation studies and Matrigel capillary tube formation assays. We performed a dose–response experiment with Nultin–3A, Nutlin–3B (an enantiomer of Nutlin–3A with 200 times less activity), and DMSO/control. Endothelial cells were stimulated with basic fibroblast growth factor (bFGF) at 5ng/ml or with human vascular endothelial growth factor (VEGF) at 20 ng/mL. Immunohistochemistry, Western blot, and flow cytometry for annexin V and propidium iodide (PI) were performed. In anticipation for an oxygen–induced retinopathy (OIR) mouse model, postnatal day 17 mice were treated with one injection of subconjunctival Nutlin–3A (50 µM to 10mM) in one eye and with DMSO in the other eye. Mice were sacrificed 5 days after injection and retinal wholemounts were labeled with a lectin to study the vasculature.
Nutlin–3A prevents endothelial cell proliferation in a dose–dependent fashion (95.8% (VEGF) and 82.8% (bFGF) cell death compared to respective controls) as well as formation of Matrigel capillary tubes at 48 hours. Immunohistochemistry and Western blot confirms an increased expression of p53 in the presence of Nutlin–3A compared to control. Flow cytometry for apoptosis by annexin V and PI reveals increased co–staining for Nutlin–3A treated cells. Ocular toxicity experiments did not reveal any scleral thinning or other abnormalities at the site of injection. Nutlin–3A treated mice showed no evidence of retinal toxicity with normal appearing retinal vasculature and architecture.
Nutlin–3A inhibits in vitro endothelial cell proliferation and may have potential benefit in treating ocular neovascular disorders.
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