PTEN对血管瘤内皮细胞VEGF-A自分泌环的调控作用与机制

Infantile hemangioma, which is the most common tumor in infants, is a benign vascular neoplasm resulting from the abnormal proliferation of hemangioma-derived endothelial cells. Recently, we and others have shown that hemangioma-derived endothelial cells demonstrate the phenotype of a constitutively active autocrine vascular endothelial growth factor-A (VEGF-A) loop, which results in increased clonality and enhanced survival ability. However, the mechanisms accounting for the upregulated autocrine VEGF-A loop in hemangioma-derived endothelial cells have not yet been elucidated. We recently indicated tumor blood suppressor phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene was downregulated in proliferating infantile hemangioma blood vessels compared with that of normal fetal skin. Relative to normal vascular endothelial cells, lower levels of PTEN protein were also observed in the hemangioma-derived endothelial cells. We propose that autocrine VEGF-A loop in hemangioma-derived endothelial cells may be modulated by PTEN. To determine whether PTEN expression modulate autocrine VEGF-A loop, we decided to over-express and knockdown PTEN in hemangioma-derived endothelial cells using recombinant DNA technology. We measured the effect of PTEN on hemangioma-derived endothelial cell characteristics and functions. We explored the potential mechanisms of PTEN-regulated autocrine VEGF-A loop using protein microarray. Our study will elucidate mechanisms by which PTEN regulates autocrine VEGF-A loop in hemangioma-derived endothelial cells and signaling pathways involves in the development of IH. These issues will highlight the important role that the increased knowledge of the autocrine VEGF-A loop and PTEN involved in the pathogenesis of infantile hemangioma will have in guiding the development of effective, rationally designed therapeutic strategies.

婴幼儿血管瘤是婴幼儿期最常见的良性肿瘤，血管瘤内皮细胞过度增殖所导致的病理性血管生成是血管瘤的主要生物学行为。近期我们及国外学者证实，血管瘤内皮细胞中存在着异常活化的VEGF-A自分泌环，从而维持血管瘤内皮细胞异常的克隆形成与耐受凋亡能力，但其形成机制不明。本课题组预实验发现与VEGF-A调控相关的肿瘤抑制因子PTEN在增殖期血管瘤微血管与内皮细胞中表达下调，我们推测PTEN可能参与对血管瘤内皮细胞中VEGF-A自分泌的调控。故本实验拟运用基因重组、蛋白质芯片及激光共聚焦等检测PTEN不同表达水平对血管瘤内皮细胞VEGF-A自分泌环活性、细胞生物学特征及功能的影响，分析PTEN调控VEGF-A自分泌环的信号转导通路网络调控机制。本课题将阐明血管瘤内皮细胞中VEGF-A自分泌环异常活化的始动机制，揭示PTEN与血管瘤发生发展的关系及其信号传导途径，为血管瘤的治疗提供新思路和新靶点。

婴幼儿血管瘤是婴幼儿期最常见的良性肿瘤，血管瘤内皮细胞与血管瘤周细胞过度增殖所导致的病理性血管生成是血管瘤的主要生物学行为。近期我们及国外学者证实，血管瘤内皮细胞（HemEC）中存在着异常活化的VEGF-A自分泌环，但其形成机制不明；此外，HemEC与周细胞之间的存在相互作用，血管瘤周细胞（HemPC）表现出不同的增殖与分化能力，但HemPC的增殖与分化是受什么机制调控的呢？.针对第一个疑问，我们选取增殖期IH组织与正常皮肤组织，以及HemEC与正常血管内皮细胞进行对比研究。结果发现，PTEN基因在IH微血管组织中的表达较正常皮肤血管组织出现显著的下调。与异常活化的VEGF-A/VEGFR-2信号通路相反，HemEC中PTEN蛋白表达较正常血管内皮细胞低。PTEN高表达可显著抑制VEGF的表达，并抑制VEGF-A对VEGFR-2磷酸化的激活，但对VEGFR-2蛋白表达水平无显著作用。上调PTEN后HemEC增殖能力显著降低、凋亡率显著升高。.针对第二个疑问，我们选取增殖期与消退前期IH瘤体组织进行研究。我们发现，Jagged1、Notch3及Hes1在增殖期与消退前期血管瘤中均有表达，其中Jagged1主要分布于HemEC，Notch3与Hes1则主要表达于HemPC。与增殖期HemPC相比，消退前期HemPC中Notch3与Hes1基因表达水平显著升高，与周细胞分化/收缩力相关的基因：smMHC与αSMA的表达水平在消退前期HemPC中也显著升高。外源性Jagged1能通过HenPC上的Notch3受体，激活通路下游靶点Hes1与HEYL活性；活化后的HemPC细胞增殖受到显著抑制使细胞停留在有丝分裂的G0/G1期，同时也提示Jagged1/Notc3信号通路激活后能作用于与细胞分裂相关的调控因子如细胞周期蛋白或细胞周期蛋白依赖激酶。进一步研究证实，激活Jagged1/Notc3能通过p21Cip1对cyclin D1、CDK-4及磷酸化Rb参与HemPC增殖的调控。此外， Jagged1/Notc3激活后，HemPC中smMHC与αSMA表达，细胞趋于成熟。因此，HemPC中存在活化的Jagged1/Notc3信号通路；Jagged1/Notc3参与了对体外培养HemPC增殖与分化的调控。