Analysis of VEGF-A Regulated Gene Expression in Endothelial Cells to Identify Genes Linked to Angiogenesis

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America.
PLoS ONE (Impact Factor: 3.23). 09/2011; 6(9):e24887. DOI: 10.1371/journal.pone.0024887
Source: PubMed


Angiogenesis is important for many physiological processes, diseases, and also regenerative medicine. Therapies that inhibit the vascular endothelial growth factor (VEGF) pathway have been used in the clinic for cancer and macular degeneration. In cancer applications, these treatments suffer from a "tumor escape phenomenon" where alternative pathways are upregulated and angiogenesis continues. The redundancy of angiogenesis regulation indicates the need for additional studies and new drug targets. We aimed to (i) identify novel and missing angiogenesis annotations and (ii) verify their significance to angiogenesis. To achieve these goals, we integrated the human interactome with known angiogenesis-annotated proteins to identify a set of 202 angiogenesis-associated proteins. Across endothelial cell lines, we found that a significant fraction of these proteins had highly perturbed gene expression during angiogenesis. After treatment with VEGF-A, we found increasing expression of HIF-1α, APP, HIV-1 tat interactive protein 2, and MEF2C, while endoglin, liprin β1 and HIF-2α had decreasing expression across three endothelial cell lines. The analysis showed differential regulation of HIF-1α and HIF-2α. The data also provided additional evidence for the role of endothelial cells in Alzheimer's disease.

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    • "Moreover, our data had shown that MEF2C is another factor specifically induced by VEGF-A and also bFGF, implicating MEF2C also in the control of angiogenesis [6], [11]. An important angiogenesis-related function of MEF2C was further corroborated by its demonstrated expression in tip cells [12], however its mechanism of action in endothelial cells during angiogenesis has not yet been fully determined. "
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    ABSTRACT: The MADS box transcription factor MEF2C has been detected by us to be upregulated by the angiogenic factors VEGF-A and bFGF in endothelial cells. We have here investigated its potential role for angiogenesis. MEF2C was surprisingly found to strongly inhibit angiogenic sprouting, whereas a dominant negative mutant rather induced sprouting. The factor mainly affected migratory processes of endothelial cells, but not proliferation. In gene profiling experiments we delineated the alpha-2-macroglobulin gene to be highly upregulated by MEF2C. Further data confirmed that MEF2C in endothelial cells indeed induces alpha-2-macroglobulin mRNA as well as the secretion of alpha-2-macroglobulin and that conditioned supernatants of cells overexpressing MEF2C inhibit sprouting. Alpha-2-macroglobulin mediates, at least to a large extent, the inhibitory effects of MEF2C as is shown by knockdown of alpha-2-macroglobulin mRNA by lentiviral shRNA expression which reduces the inhibitory effect. However, under hypoxic conditions the VEGF-A/bFGF-mediated upregulation of MEF2C is reduced and the production of alpha-2-macroglobulin largely abolished. Taken together, this suggests that the MEF2C/alpha-2-macroglobulin axis functions in endothelial cells as a negative feed-back mechanism that adapts sprouting activity to the oxygen concentration thus diminishing inappropriate and excess angiogenesis.
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    ABSTRACT: The process of sprouting angiogenesis is extremely complex involving hundreds of proteins that regulate transcription and participate in signaling pathways controlling cellular movement, proliferation and phenotype alteration. Modeling has been attempted to understand all these mechanisms, and hence, in this chapter, we will focus on models that deal individually with each one of these mechanisms relevant to angiogenesis, as well as with platforms that integrate various models into multiscale models of the whole process.
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    ABSTRACT: Hypoxia inducible factor (HIF) is a master heterodimeric transcriptional regulator of oxygen (O(2)) homeostasis critical to proper angiogenic responses. Due to the distinctive coexpression of HIF-1α and HIF-2α subunits in endothelial cells, our goal was to examine the genetic elimination of HIF transcriptional activity in response to physiological hypoxic conditions by using a genetic model in which the required HIF-β subunit (ARNT, Aryl hydrocarbon Receptor Nuclear Translocator) to HIF transcriptional responses was depleted. Endothelial cells (ECs) and aortic explants were isolated from Arnt ( loxP/loxP ) mice and infected with Adenovirus-Cre/GFP or control-GFP. We observed that moderate levels of 2.5 % O(2) promoted vessel sprouting, growth, and branching in control aortic ring assays while growth from Adenovirus-Cre infected explants was compromised. Primary Adenovirus-Cre infected EC cultures featured adverse migration and tube formation phenotypes. Primary pulmonary or cardiac ARNT-deleted ECs also failed to proliferate and survive in response to 8 or 2.5 % O(2) and hydrogen peroxide treatment. Our data demonstrates that ARNT promotes EC migration and vessel outgrowth and is indispensible for the proliferation and preservation of ECs in response to the physiological environmental cue of hypoxia. Thus, these results demonstrate that ARNT plays a critical intrinsic role in ECs and support an important collaboration between HIF-1 and HIF-2 transcriptional activity in these cells.
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