title: Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis.
abstract: Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression.
authors.affiliation: Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut Street, R4-W125, Indianapolis, IN, 46202, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Richardson
authors.forename: Emilie P
authors.initials: EP
authors.lastname: Robbins
authors.forename: Sasidhar
authors.initials: S
authors.lastname: Vemula
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Critser
authors.forename: Catherine
authors.initials: C
authors.lastname: Whittington
authors.forename: Sherry L
authors.initials: SL
authors.lastname: Voytik-Harbin
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
mesh_terms: D000091302:Angiopoietin-Like Protein 2; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D054443:Cell Migration Assays; D002465:Cell Movement / Q000187:drug effects; D003114:Colony-Forming Units Assay; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C485792:ANGPTL2 protein, human; D000091302:Angiopoietin-Like Protein 2; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14