The TEAD4216 Isoform Inhibits VEGF Expression Figure 9. TEAD4 is upregulated and alternatively spliced within the eye in an animal model of ocular ischemic disease. RT-PCR for TEAD4 from choroidal, retinal and iris tissue isolated from a non-human primate eye (Rhesus macaque) 24 hrs after occlusion of the central retinal artery (CRAO), indicates that the full length TEAD4434 transcript is increased and the TEAD4148 enhancer isoform is produced in the lasered eye. L = Lasered CRAO eye; C = control eye. doi:10.1371/journal.pone.0031260.g009 some normal vessels in the choroid and pars plana. Tissue sections stained with rabbit IgG revealed no positive staining. Discussion We report the discovery of a novel TEAD4 isoform, isolated from vascular endothelial cells derived from human eyes, that represses gene expression from the VEGF promoter. We note that through alternative splicing of TEAD4 transcripts, different proteins are synthesized; some of these isoforms act as strong enhancers of transcription from the VEGF promoter, while others act as strong repressors. Vascular endothelial growth factor has been implicated as a key component responsible for the onset and promotion of neovascu- larization. VEGF-associated neovascularization is involved in the pathology of blinding eye diseases such as proliferative diabetic retinopathy, retinopathy of prematurity (ROP) and AMD. In addition to ocular disease, solid tumor growth requires abundant neovascularization to supply nutrients for survival and growth. Thus, it is not surprising that drugs that inhibit the actions of VEGF proteins are useful in the treatment of ocular neovascular disease and neoplasia. [22,23] On the other hand, the protein factors that control VEGF transcription in health or disease is less understood; these proteins may also prove to be potential therapeutic targets. Under hypoxic conditions, expression of VEGF is upregulated in a variety of tissues. This hypoxia-mediated stimulation of VEGF is attributed mainly to the stabilization of the transcription factor HIF-1 alpha. [13,24] Targeting HIF-1 alpha may be useful in inhibiting neovascular disease, but as with most biological systems, alternative mechanisms and pathways provide functional redun- dancy. Recently, the full length TEAD4 protein (TEAD4434) was shown to stimulate VEGF gene transcription under hypoxic Figure 10. Human TEAD4 protein is present on new vessels in neovascular AMD lesion. Photomicrograph showing a subretinal neovascular membrane in a human ocular tissue section (Fast Red, TEAD4; hematoxylin counterstain; original magnification x 100). Insert (location indicated by rectangle; original magnification x 1000) shows positive staining for TEAD4 by vascular endothelium of a choroidal new vessel that has bridged the elastic lamina of Bruch’s membrane. doi:10.1371/journal.pone.0031260.g010 PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e31260