BACKGROUND:Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis.METHODS:Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies.RESULTS:Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT² PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V⁺ procoagulant endothelial CD62E⁺ (E-selectin) and neutrophil (Ly6G⁺) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082.CONCLUSIONS:Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

中文翻译：

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内皮 PTP1B 缺失促进 VWF 胞吐作用和静脉血栓炎症

背景：内皮激活促进促凝血细胞外囊泡和专门储存颗粒中炎症介质的释放。内皮膜胞吐作用受磷酸化控制。我们假设内皮细胞中PTP1B（蛋白酪氨酸磷酸酶1B）的缺失通过触发内皮膜融合和胞吐作用来促进静脉血栓炎症。方法：对诱导性内皮缺失PTP1B（End.PTP1B-KO）的小鼠进行下腔静脉结扎以诱导狭窄和静脉血栓形成。使用转基因小鼠的原代内皮细胞和人脐静脉内皮细胞进行机制研究。结果：血管超声和组织学显示，内皮 PTP1B 小鼠中静脉血栓明显变大，含有更多 Ly6G（淋巴细胞抗原 6 家族成员 G）阳性中性粒细胞。缺失和活体显微镜证实下腔静脉结扎后中性粒细胞的募集更加明显。 RT ² PCR 分析仪阵列和免疫细胞化学分析显示，原代 End.PTP1B-KO 内皮细胞中内皮活化和粘附分子表达增加，包括 CD62P（P-选择素）和 VWF（冯维勒布兰德因子）。用 NF-κB（核因子 kappa B）激酶抑制剂 BAY11-7082、中和 CD162（P-选择素糖蛋白配体-1）或 VWF 的抗体或精氨酰甘氨酰天冬氨酸整合素阻断肽进行预处理，可消除中性粒细胞对 End.PTP1B-KO 的粘附体外的内皮细胞。下腔静脉结扎后，End.PTP1B-KO 小鼠中膜联蛋白 V ⁺促凝血内皮 CD62E ⁺（E-选择素）和中性粒细胞 (Ly6G ⁺ ) 细胞外囊泡的循环水平也有所升高。较高的血浆 MPO（髓过氧化物酶）和 Cit-H3（瓜氨酸组蛋白 3）水平以及中性粒细胞弹性蛋白酶活性表明中性粒细胞活化和细胞外陷阱形成。将 End.PTP1B-KO 细胞外囊泡输注到 C57BL/6J 野生型小鼠中最显着地增强了内源性中性粒细胞的募集，而这种反应在 VWF 缺陷小鼠或 VWF 阻断抗体中减弱。 PTP1B 结合减少和 SNAP23（突触体相关蛋白 23）酪氨酸去磷酸化导致 VWF 胞吐作用和中性粒细胞粘附增加被确定为机制，所有这些都可以通过使用 BAY11-7082 抑制 NF-κB 激酶来恢复。结论：我们的研究结果显示内皮 PTP1B 缺失通过增强 SNAP23 磷酸化、内皮 VWF 胞吐作用和中性粒细胞募集来促进静脉血栓炎症。