The Monocyte/Macrophage Role in Experimental Deep Vein Thrombosis Resolution and Vein Wall Injury

摘要 血栓后综合征(PTS)是深静脉血栓形成(DVT)最常见的后遗症， 以静脉壁纤维化、瓣膜破坏、常闭塞为特征。据估计，它发生在~40年- 50%的患者患有深静脉血栓。目前还没有治疗PTS的直接药物疗法，最近的失败凸显了这一点 分级压缩长袜，以防止PTS(SOX试验)。吸引力试验表明，即使是那些 接受溶栓治疗的患者在2年内仍有~40%的PTS发生率，而侵入性药物- 机械性血栓清除并不能改善结果。即使在新的情况下，重大出血风险仍然存在 非维生素K拮抗剂。单核/巨噬细胞(Mo/MΦ)是主要的白细胞导向两个关键 病理生物学过程：静脉血栓消退和相关的静脉壁纤维化损伤。MO/MΦ为 根据其炎症或抗炎功能进行分类，这在体内是一个动态过程。为 例如，IL-1、IL-12分泌和细胞表面Ly6chi、CCR2+、CX3CR1+抗原表达 以典型的活化或促炎的Mo/MΦ为特征。相反，IL-10的分泌、转录 因子Nr4a1依赖，细胞表面Ly6Clo、CCR2-、CX3CR1++抗原表达 交替激活具有促进愈合和消炎活性的Mo/MΦ。从已发布的和 初步数据显示，促炎症的Ly6chi Mo/MΦ与早期室速有关，随后转变为 Ly6Clo。我们证明，在室性心动过速的停滞小鼠模型中，存在促炎细胞因子环境，早期 LyC6hi Mo/MΦ转变为LyC6lo含量，并且Ly6Clo Mo/MΦ也可能驱动两种VT分辨率 静脉壁纤维性损伤。然而，Mo/MΦ在VT消退和静脉壁损伤中的作用机制 至于这是否是血栓形成模型，性别和年龄相关尚不清楚。我们的总体假设是 VT分辨率和静脉壁纤维化损伤依赖于Mo/MΦ作用，依赖于模型、年龄、 和性别，并可通过增加血栓静脉中Ly6Clo Mo/MΦ而改善。我们将解决这一问题 假设有三个具体的目的。具体目标1：确定当地环境和细胞因素， 血栓静脉中Ly6CH1和Ly6Clo Mo/MΦ表型与性别、年龄和血栓形成模型的关系 变种。特异性目标2：直接确定Mo/MΦ介导的室性心动过速消退和静脉机制 墙壁受伤。具体目标3：确定血栓环境中靶向的Mo/MΦ极化是否可以 促进室性心动过速消退和静脉壁愈合。不同年龄、性别、Mo/MΦ的室性心动过速小鼠模型 条件删除和转基因小鼠以及分子、免疫学和活体成像将用于 实现这些目标。这里提出的实验将显著地推动该领域的发展，通过定义 Mo/MΦ介导的室性心动过速消退和静脉壁损伤的机制将试验新的 评估和药物以增加促进愈合的Mo/MΦ活性，并有可能转化为PTS疗法。

ABSTRACT Post-thrombotic syndrome (PTS) is the most common sequelae from deep vein thrombosis (DVT), characterized by vein wall fibrosis, valve destruction, and often occlusion. It is estimated to occur in ~40 – 50% of those suffering a DVT. No direct medical therapy exists to treat PTS, highlighted by the recent failure of graded compression stockings to prevent PTS (SOX trial). The ATTRACT trial suggested that even those patients treated with thrombolysis still have a ~40% incident PTS at 2 years, and invasive pharmaco- mechanical thrombus removal did not improve outcomes. Significant bleeding risks remain even with the new non-vitamin-K antagonists. Monocyte/macrophages (Mo/MΦ) are the primary leukocyte directing two key pathobiologic processes: venous thrombosis resolution and the associated vein wall fibrotic injury. Mo/MΦ are classified by their inflammatory or anti-inflammatory functions, which is a dynamic process in vivo. For example, interleukin-1 (IL-1), IL-12 secreting and cell surface Ly6Chi, CCR2++, CX3CR1+ antigen expression characterizes classically activated, or pro-inflammatory Mo/MΦ. Conversely, IL-10 secreting, transcription factor Nr4a1 dependent, and cell surface Ly6Clo, CCR2-, CX3CR1++ antigen expression characterizes alternatively activated Mo/MΦ with pro-healing and inflammation resolving activities. From published and preliminary data, pro-inflammatory Ly6Chi Mo/MΦ are involved with early VT, followed by a later transition to Ly6Clo. We show that in a stasis murine model of VT that a pro-inflammatory cytokine milieu exists, that early LyC6hi Mo/MΦ changes over to a LyC6lo content, and that Ly6Clo Mo/MΦ may drive both VT resolution as well as vein wall fibrotic injury. However, the mechanism of Mo/MΦ actions in VT resolution and vein wall injury, as well as whether this is thrombogenic model, sex and age dependent is not known. Our overall hypothesis is that VT resolution and vein wall fibrotic injury is dependent on Mo/MΦ actions, is dependent on model, age, and sex, and can be ameliorated with increasing Ly6Clo Mo/MΦ in the thrombosed vein. We will address this hypothesis by three specific aims. Specific Aim 1: To define the local environmental and cellular factors that drive Ly6Chi and Ly6Clo Mo/MΦ phenotypes in the thrombosed vein, with sex, age, and thrombogenic model variation. Specific Aim 2: To directly determine the Mo/MΦ mediated mechanisms of VT resolution and vein wall injury. Specific Aim 3: To determine if targeted Mo/MΦ polarization within the thrombus environment can promote VT resolution and vein wall healing. Murine models of VT, with variations of age and sex, Mo/MΦ conditional deleted and transgenic mice and molecular, immunological, and in vivo imaging will be used to accomplish these aims. The proposed experiments herein will significantly move the field forward by defining the mechanisms of Mo/MΦ mediated actions in VT resolution and vein wall injury, and will test novel assessments and agents to increase pro-healing Mo/MΦ activity with potentially translation to PTS therapies.