Boras Emhamed, Slevin Mark, Alexander M Yvonne, Aljohi Ali, Gilmore William, Ashworth Jason, Krupinski Jerzy, Potempa Lawrence A, Al Abdulkareem Ibrahim, Elobeid Adila, Matou-Nasri Sabine
Healthcare Science Research Institute, Manchester Metropolitan University, Manchester M1 5GD, UK.
Healthcare Science Research Institute, Manchester Metropolitan University, Manchester M1 5GD, UK; Institute of Cardiovascular Sciences, The University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9NT, UK.
Cytokine. 2014 Oct;69(2):165-79. doi: 10.1016/j.cyto.2014.05.027. Epub 2014 Jun 25.
C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel(TM) with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.
C反应蛋白（CRP）是炎症最急性期反应血清蛋白，也是心血管疾病的有力预测指标。其表达与动脉粥样硬化斑块不稳定性及未成熟微血管形成相关。我们之前已表明，CRP上调内皮细胞来源的Notch-3，这是一种参与血管发育、重塑和成熟的关键受体。在本研究中，我们调查了生物活性单体CRP（mCRP）与血管生成中Notch-3信号传导之间的联系。我们使用体外（细胞计数、伤口愈合和管腔形成试验）和体内（绒毛尿囊膜）血管生成试验以及蛋白质印迹法，研究由mCRP和Notch-3激活嵌合蛋白（Notch-3/Fc）诱导的血管生成信号通路。我们的结果显示，mCRP刺激作用与Notch-3/Fc促进牛主动脉内皮细胞（BAEC）增殖、迁移、在基质胶（Matrigel™）中形成管腔并上调磷酸化Akt表达的作用相结合，对血管生成具有累加效应。LY294002对PI3K/Akt存活通路的药理学阻断完全抑制了mCRP/Notch-3/Fc组合诱导的体外和体内血管生成，而γ-分泌酶抑制剂（DAPT）阻断Notch信号传导则部分抑制了mCRP/Notch-3/Fc诱导的血管生成。使用BAEC与血管平滑肌细胞共培养发芽血管生成试验和透射电子显微镜，我们表明mCRP和Notch-3信号传导的激活均诱导形成更粗的芽，蛋白质印迹法后来显示这与N-钙黏蛋白表达上调和VE-钙黏蛋白表达下调相关。因此，mCRP与Notch-3激活剂联合通过PI3K/Akt途径促进血管生成，它们的治疗性联合有潜力促进和稳定血管形成，同时降低不稳定斑块出血的风险。
