Cardiovascular Research Center (CSIC-ICCC), Hospital de Sant Pau (UAB), IIB-Sant Pau, Barcelona CiberOBN, Instituto de Salut Carlos III, Spain.
J Thromb Haemost. 2012 Sep;10(9):1895-905. doi: 10.1111/j.1538-7836.2012.04848.x.
Tissue factor (TF) and its signaling mediators play a crucial role in angiogenesis. We have previously shown that TF-induced endothelial cell (EC) CCL2 release contributes to neovessel formation.
In this study, we have investigated the signaling pathways involved in TF-induced EC tube formation.
The human microvascular endothelial cell line (HMEC-1) cultured onto basement membrane-like gel (Matrigel) was used to study TF signaling pathways during neovessels formation.
Inhibition of endogenous TF expression in ECs using siRNA resulted in inhibition of a stable tube-like structure formation in three-dimensional cultures, associated with a down-regulation of Akt activation, an increased phosphorylation of Raf at Ser(259) with a subsequent reduction of Raf kinase and a reduction of ERK1/2 phosphorylation ending up in Ets-1 transcription factor inhibition. Conversely, overexpression of TF resulted in an increase in tube formation and up-regulation of Akt protein. Moreover, immunoprecipitation of Akt and western blotting of the immunoprecipitates with anti-TF antibody revealed a direct interaction between TF and Akt. The effects of silencing TF were partially reversed by a PAR2 agonist that rescued tube formation, indicating that the TF-Akt pathway induces PAR2-independent effector signaling. Finally, enforced expression of Akt in TF-silenced ECs rescued tube formation in a Matrigel assay and induced Ets-1 phosphorylation.
In EC, TF forms a complex with Akt activating Raf/ERK and Ets-1 signaling induces microvessel formation.
组织因子（TF）及其信号介质在血管生成中起着至关重要的作用。我们之前已经表明，TF 诱导的内皮细胞（EC）CCL2 释放有助于新血管形成。
在这项研究中，我们研究了 TF 诱导的 EC 管形成涉及的信号通路。
用人微血管内皮细胞系（HMEC-1）在基底膜样凝胶（Matrigel）上培养，用于研究新血管形成过程中 TF 信号通路。
用 siRNA 抑制 EC 中的内源性 TF 表达导致三维培养中稳定的管状结构形成受到抑制，伴随着 Akt 激活的下调，Raf 丝氨酸（Ser259）磷酸化增加，随后 Raf 激酶减少，ERK1/2 磷酸化减少，导致 Ets-1 转录因子抑制。相反，TF 的过表达导致管形成增加和 Akt 蛋白的上调。此外，用抗 TF 抗体进行 Akt 的免疫沉淀和免疫沉淀物的 Western 印迹显示 TF 和 Akt 之间存在直接相互作用。PAR2 激动剂部分逆转了沉默 TF 的作用，挽救了管形成，表明 TF-Akt 通路诱导了 PAR2 非依赖性效应信号。最后，在 TF 沉默的 EC 中强制表达 Akt 挽救了 Matrigel 测定中的管形成，并诱导了 Ets-1 磷酸化。
在 EC 中，TF 与 Akt 形成复合物，激活 Raf/ERK，Ets-1 信号诱导微血管形成。
