conditions in bovine cells and to act independently of the HIF-1 pathway. From hypoxic ocular endothelial cells, we recently identified an alternatively spliced TEAD4 transcript able to encode a new isoform (TEAD4148) that is capable of enhancing VEGF promoter activity 3 to 5 times greater than that achieved by the full length TEAD4 protein and 10 times greater than the control. Whether specific alternatively spliced products of TEAD4 are biologically relevant to VEGF-dependent diseases is unknown. Nevertheless, disease specific splicing of TEAD4 within the mouse retina in a model of ROP has been noted. [11] Disease specific splicing within human tissue of other members of the TEAD family has also been observed. Whether repressing the transcrip- tion-enhancing isoforms or employing the transcription-inhibiting isoforms can help disrupt neovascularization remains to be determined. The initial discovery of VEGF as a secreted molecule was observed in rodent and human tumor cell lines, and the protein was believed to be a mitogen specific to endothelial cells.[25–28] Subsequently, VEGF was shown to act as a potent inducer of cell proliferation, migration or survival in a wide variety of cells not of endothelial origin, including smooth muscle cells, retinal cells and neurons, glial cells, pericytes and mammary tumor cells.[29–40] Inhibition of VEGF production in tumor cells may help in treatments for cancer. We demonstrate that lentiviral transduction of TEAD4216 into HEK 293T cells results in an inhibition of proliferation in both serum-containing and serum-free media as measured by a cell proliferation assay. The ability of the TEAD4216 isoform to act as a suppressor of cell division is intriguing and may prove to be of therapeutic benefit. We have previously demonstrated that TEAD4 transcripts are alternatively spliced within the retina of a mouse model of ROP. [11] We now show that the full length TEAD4434 is upregulated under ischemic conditions within the primate eye. Interestingly, in a primate CRAO model, expression of the TEAD4 isoform with highest stimulatory activity (i.e. TEAD4148) is seen at highest levels in iris tissue, a site of clinically relevant neovascularization in humans with vaso-occlusive disease. [41] Neovascularization of the iris is noted as a consequence of blockage of retinal blood vessels in primates and VEGF is known to play a significant role. [42,43] The VEGF receptors, VEGFR1 and VEGFR2, and VEGF are expressed within primate iris tissue. [44] It is possible that the TEAD4148 isoform may be produced within the iris under conditions of ocular hypoxia to initiate the VEGF cascade thereby stimulating neovascularization. Thus, it would appear that not only expression, but also alternative splicing of the TEAD4 gene, is regulated by hypoxia and this mechanism may be tissue and/or cell specific (Figure S1). Vascular endothelial growth factor is a key component to the etiology of AMD and treatments for blocking the action of VEGF have proven to be standard of care for this disease. [20] We have shown that human choroidal endothelial cells express TEAD4 transcripts in vitro and that expression is upregulated under conditions known to stimulate neovascularization. The potential role of hypoxia in the stimulation of VEGF expression in AMD is unclear. Here, we demonstrate the presence of TEAD4 protein in choroidal neovascular complexes in human eyes with neovascular AMD (Figure 10). Interestingly, HIF is absent from choroidal endothelial cells in AMD-related neovascular complexes, and thus a HIF-independent, TEAD4 pathway may regulate VEGF expression in these cells. [45]. Analysis of the TEAD4216 isoform suggests that it acts as an inhibitor of transcription of the VEGF gene even in the presence of stimulatory isoforms (Figure 4). Sequence analysis of the TEAD4216 isoform shows that it lacks a portion of the TEA DNA binding The TEAD4216 Isoform Inhibits VEGF Expression domain (the third alpha helix which includes a nuclear localization signal) that is retained by enhancer isoforms. Fusion protein experiments indicate that the TEAD4216 isoform remains in the cytoplasm, while the enhancing isoforms are found within the nucleus; suggesting that the repression effect may occur within the cytoplasm. It is possible that TEAD4216 mediated inhibition of VEGF occurs by some other indirect pathway, that involves protein degradation or regulation of other genes, that maybe cell- and cell- state specific. [46] Understanding the mechanism of action of this promoter repressor may lead to the discovery of essential cofactors that are required for the function of TEAD4. [47,48] How competitive inhibition is achieved when the enhancer isoforms are physically located within the nucleus is intriguing and warrants further investigation. Most recently, specific cofactors that are essential for TEAD4434 mediated induction of the VEGF gene in muscle cells and that TEAD4434 may also upregulate the HIF-1 alpha gene via MCAT sequences in endothelial cells have been noted. [49,50] Whether the other TEAD4 isoforms possess the ability to effect expression of the HIF-1 alpha gene or require the same specific cofactors for function and if this is a cell specific phenomenon needs further study. The full length TEAD4434 interacts with YAP65 to mediate transcription from muscle-specific genes. [51] Introduction of TEAD4 into cells lacking all TEAD proteins failed to elicit gene transcription until YAP was coin- troduced with a TEAD protein. [51] The carboxyl half of the TEAD4 protein, amino acids 224 to 434, is considered essential for binding to YAP and more specifically four specific amino acids, K297, W299, F337 and Y422 are critical for YAP interaction. [51,52] The amino acids K297 and W299 are within exon 9, F337 is contained in exon 10 while Y422 is in exon 12. Only TEAD4434 contains all 4 of these amino acids, whereas the repressor TEAD4216 possesses F337 and Y422 only, while the potent enhancer TEAD44148 contains Y422 only (Figure 1). Thus, it is unlikely that either TEAD4216 or TEAD4148 binds YAP with strong affinity and may use other cofactors to mediate their function. Other cofactors such as transcriptional coactivator with PDZ binding motif (TAZ), interferon response factor 2 binding protein 2 (IRF2BP2), a basic helix-loolp-helix leucine zipper protein (MAX), and serum response factor (SRF) a member of the MADS box superfamily of transcription factors have all been implicated as potential cofactors of TEAD4 and other members of the TEAD family of proteins. [47,48,50,53] Interestingly, both TAZ and YAP are involved in the Hippo signaling pathway, an organ growth control system that involves the regulation of genes responsible for cell proliferation and apoptosis.[52,54–56] The TAZ protein is a 14-3-3 binding protein and both YAP and TAZ activity are repressed by the Hippo pathway, by phosphorylation and sequestering of these proteins to the cytoplasm. Perturbation of the Hippo pathway has been implicated in cancer and uncon- trolled cell division. [57] Considering that TEAD is essential for YAP-mediated cell growth, it is not unreasonable to consider the possibility that inhibitor TEAD4216 isoform may play a role or be utilized to regulate the Hippo pathway. Characterizing the function of the new TEAD4216 isoform may lead to a better understanding of the control of VEGF gene regulation and may allow the development of therapeutics to inhibit VEGF gene transcription, pathologic neovascularization and cell proliferation through a HIF-independent mechanism. Materials and Methods Cultured Cells Primary human ocular vascular endothelial cells were isolated from human retinal, iris and choroidal tissue and cultured as PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e31260