Bone Marrow Endothelial VEGFR3 Regulation of Leukocyte Trafficking in Myocardial Infarction

在美国，心血管疾病（CVD）每年的死亡人数超过癌症和肺部疾病 结合强调了在这种情况下迫切需要新的治疗干预。最近的成功 CANTOS试验支持这样的观点，即通过干预来减轻心血管疾病诱导的炎症可能会抑制心血管疾病的发生， 未来治疗的关键我们和其他人的大量证据表明，CVD诱导 骨髓（BM）中的紧急造血刺激白细胞释放，白细胞前往梗死或 动脉粥样硬化组织恶化病理，这是一种在人类中描述的现象。我们 未发表的工作发现CVD模型在BM脉管系统中诱导促血管生成表型， 在一个不完全理解的过程中驱动白细胞释放到血液中。我们观察到 循环血管内皮生长因子（VEGF）水平与CVD小鼠。有趣的是， 内皮生长因子受体3（VEGFR3）在骨髓的窦状隙中高度表达， 造血细胞和白细胞所在的血管区室。VEGF-C/VEGFR3信号转导已被 据报道，在癌症诱导的淋巴管内皮细胞转移过程中， 炎症，一个让人想起BM窦状隙的守门功能的过程。我的初步数据 在骨髓脓毒症样模型中通过延时活体显微镜检查中性粒细胞内流 炎症应激发现，中性粒细胞进入特定部位增加，我们称之为"EC门"， 门数量的变化表明白细胞内渗可能发生在特定的部位， 内皮细胞因此，我们假设内皮细胞VEGFR3信号通过以下途径调节白细胞运输： 炎症应激期间BM窦内皮内的"门"。我们将通过以下方式检验这一假设： 使用时间推移活体内研究炎症应激和EC门活性之间的关系， 显微镜下追踪中性粒细胞内渗频率、不同进入"门"的频率和 在LPS和对照组中，将内渗事件设为门控。我们将在分选的BM中测量VEGFR3信号传导， 内皮细胞，并测试VEGFR3的抑制是否抑制炎性应激后的EC门活性。 这些实验为验证心肌梗死（MI）诱导 类似的变化，以EC门活动。我们将对成年小鼠进行假手术或MI手术，并检查EC门控。 通过延时活体显微镜观察的活性，分选的BM内皮细胞中的VEGFR3信号传导和相关性 这些发现与循环白细胞水平的流式细胞术分析。最后，我们建议测试 通过研究缺乏VEGFR3的小鼠的EC门活性， 内皮VEGFR3或用VEGFR3抑制剂预处理的小鼠。这项工作旨在研究新的 VEGF 3依赖性机制调节CVD诱导后白细胞从BM释放 炎症应激，并测试该途径的调节是否可以为未来的治疗干预提供信息。

Cardiovascular disease (CVD) accounts for more annual deaths in the US than cancer and lung disease combined highlighting the pressing need for novel therapeutic interventions in this setting. Recent success of the CANTOS trial supports the notion that interventions to attenuate CVD-induced inflammation may hold the key to future therapeutics. A body of evidence from us and others have demonstrated that CVD induces emergency hematopoiesis in the bone marrow (BM) inciting the release of leukocytes which travel to infarct or atherosclerotic tissue worsening pathology, a phenomenon that has been described in humans. Our unpublished work found that models of CVD induce a pro-angiogenic phenotype in the BM vasculature which drives leukocyte release into the blood in an incompletely understood process. We observed elevated circulating vascular endothelial growth factor (VEGF) levels in mice with CVD. Interestingly, vascular endothelial growth factor receptor 3 (VEGFR3) is highly expressed in the sinusoidal niche of the marrow, the vascular compartment where hematopoietic cells and leukocytes reside. VEGF-C/VEGFR3 signaling has been reported to regulate transendothelial migration of cells across lymphatic endothelium during cancer-induced inflammation, a process reminiscent of the gate-keeping function of BM sinusoids. My preliminary data examining neutrophil intravasation by time-lapse intravital microscopy in a sepsis-like model of BM inflammatory stress found that neutrophil entry increases at specific sites which we term “EC gates” with no change in the number of gates suggesting that leukocyte intravasation likely occurs at specialized sites of the endothelium. Thus, we hypothesize that endothelial VEGFR3 signaling regulates leukocyte trafficking through “gates” within the BM sinusoidal endothelium during inflammatory stress. We will test this hypothesis by investigating the relationship between inflammatory stress and EC gate activity using time-lapse intravital microscopy to track neutrophil intravasation frequency, the frequency of distinct “gates” of entry and the ratio of intravasation events to gates across LPS and control groups. We will measure VEGFR3 signaling in sorted BM endothelial cells and test whether inhibition of VEGFR3 inhibits EC gate activity following inflammatory stress. These experiments build the foundation for testing the hypothesis that myocardial infarction (MI) induces similar changes to EC gate activity. We will perform sham or MI-surgery on adult mice and examine EC gate activity by time-lapse intravital microscopy, VEGFR3 signaling in sorted BM endothelial cells and correlate these findings with flow cytometric analysis of circulating leukocyte levels. We finally propose to test the contributions of VEGFR3 signaling in the context of MI by investigating EC gate activity in mice lacking endothelial VEGFR3 or mice pre-treated with VEGFR3 inhibitor. This work proposes to investigate novel VEGFR3-dependent mechanisms that regulate leukocyte release from the BM following CVD-induced inflammatory stress and to test whether modulation of this pathway may inform future therapeutic interventions.