ABSTRACT
The generation of functional arterial endothelial cells (aECs) from embryonic stem cells (ESCs) holds great promise for vascular tissue engineering. However, the mechanisms underlying their generation and the potential of aECs in revascularizing ischemic tissue are not fully understood. Here, we observed that hypoxia exposure of mouse ESCs induced an initial phase of HIF1α-mediated upregulation of the transcription factor Etv2, which in turn induced the commitment to the EC fate. However, sustained activation of HIF1α in these EC progenitors thereafter induced NOTCH1 signaling that promoted the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the mouse hindlimb ischemia model. Furthermore, transplantation of aECs in mice showed engraftment in ischemic myocardium and restored cardiac function in contrast to ECs derived under normoxia. Thus, HIF1α activation of Etv2 in ESCs followed by NOTCH1 signaling is required for the generation aECs that are capable of arteriogenesis and revascularization of ischemic tissue.
Subject(s)
Arteries/cytology , Cell Differentiation , Endothelial Cells/cytology , Endothelial Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mouse Embryonic Stem Cells/cytology , Receptors, Notch/metabolism , Transcription Factors/metabolism , Animals , Cell Hypoxia , Cell Lineage , Endothelial Cells/transplantation , Hindlimb/pathology , Ischemia/pathology , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/metabolism , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Organogenesis , Signal Transduction , Transcription Factors/genetics , Up-Regulation/geneticsABSTRACT
Tumor endothelial marker 8 (TEM8; ANTXR1) is one of two anthrax toxin receptors; the other is capillary morphogenesis gene 2 protein (CMG2; ANTXR2). TEM8 shows enhanced expression in certain tumor endothelia, and is thought to be a player in tumor vasculature formation. However, a comprehensive expression profile of individual TEM8 variants in normal or cancerous tissues is lacking. In this work we carried out an extensive analysis of all splice variants of human TEM8 in 12 digestive tissues, and 8 each fetal and adult tissues, 6 of them cognate pairs. Using variant-specific primers, we first ascertained the status of full-length transcripts by nested PCR. We then carried out quantitative analysis of each transcript by real-time PCR. Three splice variants of TEM8 were reported before, two single-pass integral membrane forms (V1 and V2) and one secreted (V3). Our analysis has revealed two new variants, one encoding a membrane-bound form of the receptor and the other secreted, which we have designated V4 and V5, respectively. All tissues had V1, V2, V3, and V4, but only prostate had V5. Real-time PCR revealed that all variants are present at different levels in various tissues. V3 appeared the most abundant of all. To ascertain its functionality for anthrax toxin, we expressed the newly identified form V4 in a receptor-negative host cell, and included V1 and V2 for comparison. Cytotoxicity, toxin binding, and internalization assays showed V4 to be as efficient a receptor as V1 and V2.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Neoplasm Proteins/metabolism , Protein Isoforms/metabolism , Receptors, Cell Surface/metabolism , Transcriptome/genetics , Adult , Base Sequence , Brain/metabolism , Computational Biology , DNA Primers/genetics , Electrophoresis , Fetus/metabolism , Gene Expression Regulation, Developmental/genetics , Humans , Male , Microfilament Proteins , Molecular Sequence Data , Prostate/metabolism , Protein Isoforms/genetics , Real-Time Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNAABSTRACT
BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) facilitates intercellular communication between the epithelial carcinoma and its surrounding stromal tissue during metastatic invasion through interaction with its proto-oncogenic receptor, Met, found on carcinoma cells. This study utilizes the C31/Tag transgenic mouse prostate cancer cell line model in an attempt to characterize the interaction between HGF/SF and Met on the metastatic potential of prostate cancer. METHODS: Exogenous HGF was supplied to the prostate adenocarcinoma cell line (Pr-14) and metastatic cell line (Pr-14c) to evaluate mitogenicity by proliferation assays, morphological characteristics on an extracellular matrix substrate, and motogenic properties using the scatter assay, invasion chambers, and zymogram studies to analyze secretory enzymes produced by the cell lines. RESULTS: RNA and protein analyses show that the cell lines express similar amounts of Met. Pr-14 cells have an increased growth rate following HGF/SF treatment, whereas the metastatic Pr-14c cells show little change. Morphological studies of Pr-14c cells on extracellular matrix demonstrate negligible changes when compared to the tubular formation of Pr-14 cells after HGF/SF stimulation. Motility studies of the metastatic cells following HGF/SF treatment reveal a potentially faulty signaling pathway downstream of Met activation in the metastatic prostate cells. CONCLUSIONS: Our studies suggest that proliferation, invasion, and cell morphological characteristics may be induced independently from the HGF/SF-Met pathway in C31/Tag metastatic prostate cancer cells.
