Ang-2调控妊娠早期螺旋动脉血管平滑肌细胞去分化和侵袭的机制研究

Spiral artery remodeling is one of the key processes in the establishment of a complication free pregnancy; failure in this process being associated with pre-eclampsia and fetal growth restriction. During remodeling the spiral arteries of the decidua become dilated. We have previously demonstrated that the early stages of spiral artery remodeling involve separation and disorganization of the vascular smooth muscle cells (VSMCs), VSMC dedifferentiation and their eventual migration away from the vessel wall. However, their biological activity has only previously been described on endothelial cells with no direct effects on VSMCs being demonstrated. Ang-2 is correlated with poor prognosis in a number of different tumours and recent data demonstrates that Ang-2 can bind to and activate cancer cell expressed integrin subunits inducing increased invasion. Our pilot data demonstrates that Ang-2 can bind to VSMCs and stimulates phosphorylation of a number of signaling pathways in VSMCs, as well as induce morphological changes and VSMC invasion. We therefore hypothesise that Ang-2 induces VSMC de-differentiation and invasion via a mechanism involving Tie-2 and/or integrins signaling pathways. To test this hypothesis we aim to determine which VSMC cell surface molecules bind to Ang-2, which signaling pathways are subsequently activated and finally what is the functional consequence of Ang-2 activation of VSMCs? We will test this hypothesis using immunoprecipitation, Real-time monitoring technique of cell invasion and migration, RNA-Seq techniques, to explore the VSMC dedifferentiation, alterations in VSMC cytoskeleton and invasion/migration, then to find out the Ang-2 receptors and involved signaling pathways. These studies will define a role for Ang-2 in VSMC plasticity that was wide reaching consequences for a number of pathologies involving vascular remodeling.

子宫螺旋动脉重塑是妊娠成功的关键步骤之一，重塑失败可致子痫前期、胎儿生长受限等。重塑时蜕膜膨胀，失去肌肉弹性壁，这主要与血管平滑肌细胞（VSMCs)相关。我们前期研究发现重塑早期VSMCs发生分离、重排、迁移、去分化并最终从血管壁迁出；VSMCs去分化主要由血管生成素2（Ang-2）介导。但Ang-2对VSMCs的作用鲜有报道。我们的预实验显示Ang-2结合VSMC并激活多个信号通路，改变VSMCs形态和侵袭力。据此我们提出假说：Ang-2通过Tie-2和/或整合素信号通路诱导VSMCs去分化和侵袭，从而促进螺旋动脉重塑。本项目拟通过免疫共沉淀、细胞侵袭迁移实时监测技术、RNA-Seq等技术，探讨Ang-2对VSMCs去分化、细胞骨架重排和侵袭/迁移的作用，挖掘VSMCs中Ang-2的受体及其信号转导机制。本研究将阐明Ang-2在VSMCs中的作用，为涉及血管重塑疾病的治疗提供新思路。

子痫前期（Pre-eclampsia，PE）和胎儿生长受限（Fetal Growth Restriction，FGR）是产科常见的并发症，增加母胎病死率。这两种疾病具有共同的病理特征：绒毛外滋养层细胞（Extravillous Trophoblast，EVT）侵袭不足和子宫螺旋动脉（Uterine Spiral Arteries，SpA）重塑不足。其中血管平滑肌细胞（Vascular Smooth Muscle Cells，VSMCs）去分化，从血管壁中分离并迁移至周围的间质是SpA重塑过程中的重要一步。VSMCs去分化定义为高分化的收缩型VSMCs重新转换表型成为合成型的VSMCs。我们的前期数据表明血管生成素-2（Angiopoietin-2, Ang-2）对VSMCs的去分化具有直接作用，但其机制尚未清楚。所以，本项目我们主要探索Ang-2的生物学作用及其分子机制。我们的主要发现包括：Ang-2与VSMCs的整合素β6结合，并引起FAK磷酸化（S910和Y397），AKT磷酸化（S473）。Ang-2可以诱导VSMCs去分化，表现为VSMCs的收缩性蛋白如钙调蛋白结合蛋白h-caldesmon和钙调蛋白calponin的表达降低，同时增加VSMCs的增殖和迁移能力。Ang-2可以通过FAK信号通路增加组蛋白甲基转移酶EZH2的含量从而下调ATG-7的表达，抑制细胞自噬，促进VSMCs增殖能力。在PE患者蜕膜的SpA中，我们发现EZH2的蛋白含量下调，而ATG-7则升高。以上的结果阐明了Ang-2 在VSMCs 去分化过程中的重要作用。因为EVT侵袭不足也是PE和FGR的重要病理特征，我们还研究了Ang-2对滋养层细胞侵袭的作用。我们发现Ang-2通过结合滋养层细胞的整合素受体和Tie2受体，激活JNK通路，引起细胞骨架的改变和细胞侵袭；Ang-2还可以使降解细胞外基质的MMP2和MMP9表达上调，从而促进滋养层细胞的迁移和侵袭。综上所述，Ang-2可以诱导VSMCs去分化和增加EVT的侵袭能力，在孕早期SpA重塑起了重要的作用。本研究加深了我们对于Ang-2在妊娠过程中的作用的理解，为早期干预和治疗PE和FGR提供新的靶点和理论依据。