Defects in vein valve PROX1/FOXC2 antithrombotic pathway in endothelial cells drive the hypercoagulable state induced by trauma and critical illness.
Defects in vein valve PROX1/FOXC2 antithrombotic pathway in endothelial cells drive the hypercoagulable state induced by trauma and critical illness.
复制标题
内皮细胞中静脉瓣膜 PROX1/FOXC2 抗血栓通路的缺陷导致创伤和危重疾病引起的高凝状态。
DOI：
10.1097/ta.0000000000003945
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Turnbull,IsaiahR
中科院分区：
文献类型：
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作者：
Hoofnagle,MarkH;Hess,Annie;Nalugo,Margaret;Ghosh,Sarbani;Hughes,Shin-Wen;Fuchs,Anja;Welsh,JohnD;Kahn,MarkL;Bochicchio,GrantV;Randolph,GwendalynJ;Leonard,JenniferM;Turnbull,IsaiahR
目的：深静脉血栓形成（DVT）是创伤后发病率和死亡率最高的疾病。最近，我们发现静脉瓣膜处的血流模式诱导振荡应激基因，这些基因维持一种抗凝内皮表型，抑制静脉瓣膜和鼻窦处的自发凝血，在人类病理样本中存在DVT时丢失，并且依赖于转录因子FOXC2的表达。我们描述了一个实验，修改了我们的小鼠多重损伤系统，它显示了临床相关的微血栓形成和高凝性的证据，适用于创伤自发性DVT的研究，而不需要直接的血管损伤或结扎。最后，我们通过定量聚合酶链反应和免疫荧光检测重症患者静脉中基因表达的变化，研究了这些模型的发现是否与人类危重疾病模型相关。方法建立sc57 /Bl6小鼠肝挤压损伤、单侧下肢挤压假性骨折、总血容量15%出血的改良小鼠多重损伤模型。在损伤后2、6、24和48小时用酶联免疫吸附法检测血清中d-二聚体的含量。凝血酶凝血实验：暴露腿部静脉，经眶后注射1 mM罗丹明（6 g） 100 μL，静脉表面涂凝血酶450 μg/mL，体内免疫荧光显微镜实时观察血栓形成情况。然后检查图像中可见小鼠隐静脉和股总静脉的血栓覆盖面积百分比。如前所述，他莫昔芬处理PROX1 Ert2Cre FOXC2 fl/fl小鼠，诱导FOXC2静脉瓣膜特异性敲除。然后，动物接受改良的小鼠多重损伤模型，肝挤压损伤，单下肢挤压和假性骨折，总血容量出血15%。损伤24小时后，我们通过凝血酶测定检测了静脉瓣膜中FOXC2基因缺失和未缺失的情况下，单纯损伤动物和多次损伤动物的瓣膜表型。然后检查图像是否靠近小鼠隐静脉、胫静脉和股浅静脉交界处的瓣膜，以及在接触凝血酶之前静脉中是否存在自发性微血栓。人类静脉样本来自于选择性心脏手术后保存的多余组织和器官获取后的器官捐献者。切片石蜡包埋，免疫荧光法检测PROX1、FOXC2、血栓调节蛋白、内皮蛋白C受体和血管性血癌因子。所有动物研究均由机构动物护理和使用委员会审查和批准，所有人体研究均由机构审查委员会审查和批准。结果小鼠多次损伤后，d-二聚体酶联免疫吸附试验显示纤维蛋白分解产物与损伤、纤维蛋白溶解和/或微血栓形成一致。凝血酶凝血试验显示，与未受伤的动物相比，暴露于凝血酶的多重损伤动物的静脉覆盖面积百分比更高（45%对27% p= 0.0002），这与我们的模型系统中创伤后高凝状态的表型一致。与未处理的野生型小鼠相比，未处理的FoxC2基因敲除小鼠在静脉瓣膜处的凝血明显增加。多次损伤后，野生型小鼠经凝血酶治疗后，静脉凝血明显增加。
OBJECTIVESDeep venous thrombosis (DVT) causes significant morbidity and mortality after trauma. Recently, we have shown that blood flow patterns at vein valves induce oscillatory stress genes, which maintain an anticoagulant endothelial phenotype that inhibits spontaneous clotting at vein valves and sinuses, is lost in the presence of DVT in human pathological samples, and is dependent on expression of the transcription factor FOXC2. We describe an assay, modifying our mouse multiple injury system, which shows evidence of clinically relevant microthrombosis and hypercoagulability applicable to the study of spontaneous DVT in trauma without requiring direct vascular injury or ligation. Finally, we investigated whether these model findings are relevant to a human model of critical illness by examining gene expression changes by quantitative polymerase chain reaction and immunofluorescence in veins collected from critically ill.METHODSC57/Bl6 mice were subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Serum was assayed for d-dimer at 2, 6, 24, and 48 hours after injury by enzyme-linked immunosorbent assay. For the thrombin clotting assay, veins of the leg were exposed, 100 μL of 1 mM rhodamine (6 g) was injected retro-orbitally, and 450 μg/mL thrombin was then applied to the surface of the vein with examination of real-time clot formation via in vivo immunofluorescence microscopy. Images were then examined for percentage area of clot coverage of visible mouse saphenous and common femoral vein. Vein valve specific knockout of FOXC2 was induced with tamoxifen treatment in PROX1 Ert2Cre FOXC2 fl/fl mice as previously described. Animals were then subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Twenty-four hours after injury, we examined the valve phenotype in naive versus multiple injury animals, with and without loss of the FOXC2 gene from the vein valve (FOXC2 del) via the thrombin assay. Images were then examined for proximity of clot formation to the valve present at the junction of the mouse saphenous, tibial, and superficial femoral vein and presence of spontaneous microthrombi present in the veins before exposure to thrombin. Human vein samples were obtained from excess tissue preserved after harvest for elective cardiac surgery and from organ donors after organ procurement. Sections were submitted for paraffin embedding and then assayed by immunofluorescence for PROX1, FOXC2, thrombomodulin, endothelial protein C receptor, and von Willebrand's factor. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee, and all human studies reviewed and approved by the institutional review board.RESULTSAfter mouse multiple injuries, enzyme-linked immunosorbent assay for d-dimer showed evidence of products of fibrin breakdown consistent with formation of clot related to injury, fibrinolysis, and/or microthrombosis. The thrombin clotting assay demonstrated higher percentage area of vein covered with clot when exposed to thrombin in the multiple injury animals compared with uninjured (45% vs. 27% p= 0.0002) consistent with a phenotype of hypercoagulable state after trauma in our model system. Unmanipulated FoxC2 knockout mice manifest increased clotting at the vein valve as compared with unmanipulated wild type animals. After multiple injuries, wild type mice manifest increase clotting at the vein after thrombin …