Richardson Matthew R, Robbins Emilie P, Vemula Sasidhar, Critser Paul J, Whittington Catherine, Voytik-Harbin Sherry L, Yoder Mervin C
Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut Street, R4-W125, Indianapolis, IN, 46202, USA.
Angiogenesis. 2014 Jul;17(3):675-83. doi: 10.1007/s10456-014-9423-8. Epub 2014 Feb 23.
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.
血管生成素样蛋白2（ANGPTL2）已被报道可诱导发芽血管生成；然而，其在血管发生（内皮细胞（EC）的新生管腔形成）中的作用仍未被探索。我们试图通过siRNA介导的ANGPTL2基因表达抑制来研究ANGPTL2在调节人脐血来源的内皮集落形成细胞（ECFC）血管发生中的潜在作用。我们发现，ANGPTL2减少的ECFC在体外管腔面积减少了三倍，而向ECFC中添加外源性ANGPTL2蛋白结构域导致在三维（3D）血管生成胶原试验中管腔形成增加。尽管增殖和凋亡不受影响，但通过ANGPTL2敲低（KD），ECFC迁移减弱了36%。我们随后发现，ANGPTL2 KD后，c-Jun NH2末端激酶（JNK）而非ERK1/2的磷酸化降低，膜型1基质金属蛋白酶（MT1-MMP）的表达在体外由ANGPTL2沉默的ECFC衍生的管腔化结构中降低，MT1-MMP已知受JNK调节且是EC迁移和3D管腔形成的关键调节因子。用JNK抑制剂或外源性rhTIMP-3（MT1-MMP活性抑制剂）处理3D胶原基质中的ECFC，导致与ANGPTL2 KD观察到的相似的血管管腔形成减少表型，而JNK活性的刺激增加了血管生成。基于基因沉默、药理学、细胞和生化方法，我们得出结论，ANGPTL2可能通过其对迁移的影响以及部分通过激活JNK和增加MT1-MMP表达来正向调节ECFC血管管腔形成。
