Fortunato Tiago M, Vara Dina S, Wheeler-Jones Caroline P, Pula Giordano
Department of Pharmacy and Pharmacology, University of Bath, Bath Spa, United Kingdom.
Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, United Kingdom.
PLoS One. 2014 Oct 7;9(10):e109375. doi: 10.1371/journal.pone.0109375. eCollection 2014.
Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.
内皮祖细胞（ECFCs）是从人外周血单个核细胞（hPBMNC）组分培养物中获得的，其特点是具有高增殖和促血管生成潜力，这使其在细胞治疗中备受关注。在此，我们描述了在ECFCs中表达的表面蛋白中蛋白酶激活受体（PAR）1和2的检测。这两种受体在功能上均与细胞外信号调节激酶（ERK）1和2偶联，ERK1和2在响应选择性PAR1或PAR2激活肽时会被激活并磷酸化。PAR1而非PAR2的特异性刺激显著抑制了ECFCs在体外形成毛细血管样管，这表明血管生成受到能够刺激PAR1的蛋白酶（如凝血酶）的负调控。如使用MEK抑制剂PD98059以及PAR2刺激激活ERK但不影响毛细血管管形成这一事实所表明的，ERK的激活不参与体外血管生成的调节。定量聚合酶链反应（qPCR）和免疫印迹均显示，响应PAR1刺激，血管内皮生长因子2（VEGFR2）显著下调。此外，添加血管内皮生长因子（VEGF，50 - 100 ng/ml）而非碱性成纤维细胞生长因子（FGF，25 - 100 ng/ml）可挽救经PAR1激活肽处理的ECFCs的管形成。因此，我们提出，VEGFR2下调导致的VEGF反应性降低是PAR1激活的抗血管生成作用的基础。尽管PAR2的作用仍不明确，但本研究为ECFCs血管生成活性的调节提供了新的线索，并提示了成人血管生成与凝血级联反应之间的潜在联系。
