FoxO1调控Fb/TF介导的CTEPH内皮重塑和功能障碍中自噬的作用机制

Chronic thromboembolic pulmonary hypertension (CTEPH) is a disease with high disability and mortality. Endothelial dysfunction can be caused by fibrin (Fb) and tissue factor (TF), as well as autophagy changes of pulmonary artery endothelial cell (PAEC). Forkhead box transcription factor O-1 (FoxO1) plays a key role in regulating cellular autophagy, however, its effect and mechanisms on PAEC autophagy remain unclear during CTEPH vascular endothelium remodeling induced by Fb and TF. Our preliminary studies have showed that the expression of FoxO1 can be significantly down-regulated along with the duration of embolization in the rat model of CTEPH. To explore the effect and mechanisms of FoxO1 on regulating cell autophagy and dysfunction of PAEC in CTEPH, the clinical specimens of patients and animal models with CTEPH will be combined to be used in this study. The role of FoxO1 will be explored by specifically blocking the upstream Fb/TF-MAPK pathway, knocking out FoxO1 gene,using the FoxO1 inhibitors as well as agonists in vitro and in vivo experiments. The autophagy of PAEC will be also analyzed by the methods of PCR array and RFP-GFP-LC3B kits and the morphological and functional changes of the PAECs will be observed. Furthermore, the constitutively active mutant of FoxO1 Ad-Foxo1: AAA will be selectively delivered into the lungs of rats to investigate if it can improve the pulmonary artery vascular endothelium remodeling. It can be expected that the constitutively active mutant of FoxO1 may offer a potential therapeutic option for CTEPH vascular remodeling.

慢性血栓栓塞性肺动脉高压（CTEPH）致残率和病死率高。纤维蛋白（Fb）、组织因子（TF）及肺动脉内皮细胞（PAEC）自噬改变可致内皮功能障碍。转录因子FoxO1是调控细胞自噬的关键点，但其对Fb/TF介导的CTEPH内皮重塑和功能障碍中自噬的作用及机制未明。我们前期研究表明FoxO1在CTEPH鼠模型中表达随着栓塞时间延长下调更明显。本研究拟通过CTEPH患者临床标本与CTEPH动物模型结合，离体及在体实验特异性阻断FoxO1上游Fb/TF-MAPK通路、FoxO1基因敲除、FoxO1抑制剂及激动剂等方面探讨FoxO1调控CTEPH中PAEC自噬和功能的作用；通过PCR阵列技术及RFP-GFP-LC3B等方法分析PAEC自噬情况，观察PAEC形态及功能改变；再通过应用FoxO1基因的组成型活化突变体进行干预，期望能改善肺血管重塑，为早期干预、防治CTEPH的发生发展提供新的思路和方法。

慢性血栓栓塞性肺动脉高压（CTEPH）致残率和病死率高。纤维蛋白（Fb）、组织因子（TF）及肺动脉内皮细胞（PAEC）自噬改变可致内皮功能障碍。转录因子FoxO1是调控细胞自噬的关键点，但其对Fb/TF介导的CTEPH内皮重塑和功能障碍中自噬的作用及机制未明。.本研究主要研究CTEPH动物模型的建立、肺栓塞慢性化至CTEPH内皮功能障碍、肺血管重塑机制，分析组织因子（TF）、FoxO1、炎症反应、细胞自噬和凋亡在CTEPH发生、发展过程中的作用。研究FoxO1与凋亡和自噬相互影响在肺栓塞慢性化、肺动脉内皮重塑中的关键作用。.上述实验，我们成功建立了CTEPH犬与大鼠模型，研究结果显示实验组大鼠平均肺动脉高压（mPAP）、肺动脉管壁面积/管总面积（WA/TA）明显高于假手术组（P<0.05），且mPAP与肺动脉WA/TA 成正相关；实验组大鼠血浆TF抗原、TF活性含量、TFmRNA 和蛋白均明显高于假手术组（P<0.05），且血浆TF 活性与肺动脉WA% 呈正相关；实验组大鼠肺动脉自噬水平指标（Beclin-1和LC3B）mRNA 和蛋白的表达水平明显低于假手术组（P<0.05），二者与 WA/TA 、肺动脉TF蛋白表达均成负相关；实验组促凋亡蛋白Bad 抗原、mRNA、和蛋白(1周、2周、4周组）表达与同周数的假手术组比较明显上升（P<0.05）；实验组FoxO1和抗凋亡蛋白Bcl-2 的抗原、mRNA表达和蛋白(1周、2周、4周组）表达与同周数的假手术组比较下降明显（P<0.05）；实验组大鼠肺动脉Bad 蛋白表达与 WA/TA和mPAP成正相关；实验组大鼠肺动脉Bcl-2蛋白表达与WA/TA和mPAP成负相关。.上述实验，我们发现CTEPH模型大鼠的mPAP、WA/TA、FoxO1、TF、自噬水平和凋亡水平之间具有良好的相关性；提示TF、FoxO1、自噬和凋亡在CTEPH大鼠模型，特别是肺血管重塑过程中起着重要作用，为临床CTEPH的防治提供新的靶点。 .另外，我们还收集CTEPH患者血标本，检测血中TF、易栓状态危险因素如炎症指标的表达水平，研究发现TF活性、抗原、mRNA和炎症指标表达水平较对照组明显升高。表明TF、炎症在CTEPH可能通过炎症反应-凝血-血栓循环在发生、发展过程中起重要作用，为早期干预、防治CTEPH的发生发展提供新思路