The TEAD4216 Isoform Inhibits VEGF Expression Figure 8. The TEAD4148 and TEAD4434 isoforms that enhance VEGF expression are localized to the nucleus whereas the TEAD4216 repressor isoform is maintained in the cytoplasm. TEAD4 isoforms were expressed as fusion proteins within 293T cells with various fluorescent proteins (FP) (TEAD4434 -Green-FP, and TEAD4148-Yellow-FP (pseudo-colored green) and TEAD4216-Green-FP) to visualize cellular localization. doi:10.1371/journal.pone.0031260.g008 portion of TEAD4216, lacking the NLS, and the non-DNA binding part of TEAD4148 (59- TEAD4216+39- TEAD4148), as well as the mutant isoform that consisted of the TEA DNA binding domain only (DNA BD, containing exons 2 to 4 of TEAD4434) also failed to enhance. Although, the combination of alternatively spliced sequences downstream of the complete TEA DNA binding domain may influence potency of enhancement, as evidenced by the TEAD4434 and TEAD4216+NLS isoforms, the same sequence loses enhancer function without a functional NLS (Figure S6). TEAD4 is Present in Ocular Tissue in Primate Ischemic or Neovascular Eye Disease Occlusion of the central retinal artery or vein leads to ocular hypoxia and often results in a rapid increase of intra-ocular VEGF. [18,19] Analysis of retinal, choroidal and iris tissue for TEAD4 RNA in a non-human primate (NHP) model of central retinal artery occlusion (CRAO) indicated that the TEAD4434 form of TEAD4 was upregulated in the ischemic eye and undetectable in the contralateral control eye (Figure 9). In addition, the NHP orthologue of the human TEAD4148 isoform was found to be present in iris tissue from the ischemic eye and not in the normal control eye (Figure 9). Sequence comparison of the human TEAD4148 and NHP TEAD4148 isoforms indicated 98.4% identity at the nucleotide and 96.6% identity at the protein level. Choroidal neovascularization is a common sequelae of age- related macular degeneration and is a VEGF-mediated process. [20,21] To test whether TEAD4 is present in neovascular complexes associated with AMD, antibodies raised against human TEAD4 were used in immunohistopathologic analysis of sections from eyes with neovascular AMD. Hematoxylin and eosin staining demonstrated subretinal neovascular membranes in the three globes obtained post-mortem from three patients with age-related macular degeneration. Endothelium of choroidal new blood vessels within the membrane stained positively for TEAD4 in two eyes (Figure 10); the third eye showed relatively weak staining overall. In addition and for all eyes, positive extracellular staining was observed within the membrane. This finding was attributed to leakage of serum from the abnormal vasculature into the tissue, since similar staining of serum was also present within vessels. Subretinal blood stained positively in one eye. In addition to staining within the neovascular membrane, anti-TEAD4 antibody recognized some other ocular cells. Positive staining was identified in retinal pigment epithelium and corneal epithelium, and for one globe that showed most staining overall, there was also convincing staining of ciliary epithelium, retinal neurons and endothelium of PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e31260