Many diseases of the eye such as retinoblastoma, diabetic retinopathy, and retinopathy of prematurity are associated with blood-retinal barrier (BRB) dysfunction. Identifying the factors that contribute to BRB formation during human eye development and maintenance could provide insights into such diseases. Here we show that A-kinase anchor protein 12 (AKAP12) induces BRB formation by increasing angiopoietin-1 and decreasing vascular endothelial growth factor (VEGF) levels in astrocytes. We reveal that AKAP12 downregulates the level of hypoxia-inducible factor-1␣ (HIF-1␣) protein by enhancing the interaction of HIF-1␣ with pVHL (von Hippel-Lindau tumor suppressor protein) and PHD2 (prolyl hydroxylase 2). Conditioned media from AKAP12-overexpressing astrocytes induced barriergenesis by upregulating the expression of tight junction proteins in human retina microvascular endothelial cells (HRMECs). Compared with the retina during BRB maturation, AKAP12 expression in retinoblastoma patient tissue was markedly reduced whereas that of VEGF was increased. These findings suggest that AKAP12 may induce BRB formation through antiangiogenesis and barriergenesis in the developing human eye and that defects in this mechanism can lead to a loss of tight junction proteins and contribute to the development of retinal pathologies such as retinoblastoma.

During mammalian embryogenesis, the early embryo grows in a relatively hypoxic environment due to a restricted supply of oxygen. The molecular mechanisms underlying modulation of self-renewal and differentiation of mouse embryonic stem cells (mESCs) under such hypoxic conditions remain to be established. Here, we show that hypoxia inhibits mESC self-renewal and induces early differentiation in vitro, even in the presence of leukemia inhibitory factor (LIF). These effects are mediated by down-regulation of the LIF-STAT3 signaling pathway. Under conditions of hypoxia, hypoxia-inducible factor-1␣ (HIF-1␣) suppresses transcription of LIF-specific receptor (LIFR) by directly binding to the reverse hypoxia-responsive element located in the LIFR promoter. Ectopic expression and small interference RNA knockdown of HIF-1␣ verified the inhibitory effect on LIFR transcription. Our findings collectively suggest that hypoxia-induced in vitro differentiation of mESCs is triggered, at least in part, by the HIF-1␣-mediated suppression of LIF-STAT3 signaling. Analysis of mESCs2 isolated from the inner cell mass of preimplantation embryos has aided in elucidating the early molecular events and mechanisms responsible for maintenance of ESC pluripotency (1). In contrast to human ESCs (hESCs), selfrenewal of mESCs can be sustained indefinitely in feeder-free conditions if supplemented with LIF (2). mESC lines can be derived directly from embryos in the presence of LIF, indicating that this factor is specifically required for the maintenance of pluripotency. Intracellular signaling cascades initiated by LIF are mediated through the heterodimerization of LIFR and glycoprotein 130 (gp130). This complex activates Janus-associated tyrosine kinase and the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Activation of STAT3 by LIF is crucial in mediating self-renewal signals in mESCs (3, 4).During mammalian development, oxygen needed for cellular metabolism in the early embryo is supplied by simple diffusion from fluids within the oviduct and uterus. Thus, the early embryo develops in a relatively hypoxic environment until the onset of vascularization after implantation (5). Actually, oxygen tension in the mammalian reproductive tract is reported to be less than half the atmospheric oxygen tension (6). Low oxygen tension triggers a wide range of cellular events centered on the regulation of hypoxia-inducible factor-1 (HIF-1) (7), which consists of a common ␤ subunit (HIF-1␤) and an oxygen-sensitive ␣ subunit (HIF-1␣). Under normoxia, HIF-1␣ is rapidly degraded via the von Hippel-Lindau protein-mediated ubiquitin-proteasome pathway (8). Although the role and biological relevance of HIF-1 during murine embryonic development have been established (9 -11), our understanding of how HIF-1 affects the early differentiation of mESCs at the molecular level is beginning to emerge.Our previous study demonstrated the presence of hypoxic regions during normal mouse development (12), which prompted us to study the role of hy...

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.