Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension.

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH

In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1

Adult epicardial cells are required for endogenous cardiac repair. After myocardial injury, they are reactivated, undergo epithelial-to-mesenchymal transformation (EMT) and migrate into the injured myocardium where they generate various cell types, including coronary smooth muscle cells and cardiac interstitial fibroblasts, which contribute to cardiac repair. To understand what drives epicardial EMT, we used an in vitro model for human adult epicardial cells. These cells have an epithelium-like morphology and markedly express the cell surface marker vascular cell adhesion marker (VCAM-1). In culture, epicardial cells spontaneously undergo EMT after which the spindle-shaped cells now express endoglin. Both epicardial cells before and after EMT express the epicardial marker, Wilms tumor 1 (WT1). Adding transforming growth factor beta (TGFβ) induces loss of epithelial character and initiates the onset of mesenchymal differentiation in human adult epicardial cells. In this study, we show that TGFβ-induced EMT is dependent on type-1 TGFβ receptor activity and can be inhibited by soluble VCAM-1. We also show that epicardial-specific knockdown of Wilms tumor-1 (WT1) induces the process of EMT in human adult epicardial cells, through transcriptional regulation of platelet-derived growth factor receptor alpha (Pdgfrα), Snai1 and VCAM-1. These data provide new insights into the process of EMT in human adult epicardial cells, which might provide opportunities to develop new strategies for endogenous cell-based cardiac repair

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast

Sub-Lethal Irradiation of Human Colorectal Tumor Cells Imparts Enhanced and Sustained Susceptibility to Multiple Death Receptor Signaling Pathways

Background: Death receptors (DR) of the TNF family function as anti-tumor immune effector molecules. Tumor cells, however, often exhibit DR-signaling resistance. Previous studies indicate that radiation can modify gene expression within tumor cells and increase tumor cell sensitivity to immune attack. The aim of this study is to investigate the synergistic effect of sub-lethal doses of ionizing radiation in sensitizing colorectal carcinoma cells to death receptor-mediated apoptosis. Methodology/Principal Findings: The ability of radiation to modulate the expression of multiple death receptors (Fas/ CD95, TRAILR1/DR4, TRAILR2/DR5, TNF-R1 and LTbR) was examined in colorectal tumor cells. The functional significance of sub-lethal doses of radiation in enhancing tumor cell susceptibility to DR-induced apoptosis was determined by in vitro functional sensitivity assays. The longevity of these changes and the underlying molecular mechanism of irradiation in sensitizing diverse colorectal carcinoma cells to death receptor-mediated apoptosis were also examined. We found that radiation increased surface expression of Fas, DR4 and DR5 but not LTbR or TNF-R1 in these cells. Increased expression of DRs was observed 2 days post-irradiation and remained elevated 7-days post irradiation. Sub-lethal tumor cell irradiation alone exhibited minimal cell death, but effectively sensitized three of three colorectal carcinoma cells to both TRAIL and Fasinduced apoptosis, but not LTbR-induced death. Furthermore, radiation-enhanced Fas and TRAIL-induced cell death lasted as long as 5-days post-irradiation. Specific analysis of intracellular sensitizers to apoptosis indicated that while radiation di