Bone Marrow Endothelial VEGFR3 Regulation of Leukocyte Trafficking in Myocardial Infarction

在美国，心血管疾病(CVD)每年导致的死亡人数超过癌症和肺部疾病 联合强调了在这种情况下对新的治疗干预的迫切需要。最近取得的成功 CANTOS试验支持这样一种观点，即减轻CVD诱导的炎症的干预措施可能会 未来治疗学的关键。来自我们和其他人的大量证据表明，心血管疾病诱导 骨髓(BM)中的紧急造血刺激白细胞的释放，这些白细胞游走到脑梗塞或 动脉粥样硬化组织恶化病理，这一现象已经在人类中被描述过。我们的 未发表的工作发现，CVD模型在BM血管系统中诱导出促血管生成的表型， 促使白细胞在一个尚未完全了解的过程中释放到血液中。我们观察到高空 脑血管病小鼠循环血管内皮生长因子水平的变化有趣的是，血管 血管内皮细胞生长因子受体3(VEGFR3)在骨髓窦壁龛中高表达。 造血细胞和白细胞所在的血管隔间。血管内皮生长因子C/血管内皮生长因子受体3信号转导通路 据报道，在致癌过程中调节细胞通过淋巴管内皮细胞的跨内皮迁移 炎症，这一过程使人联想到BM血窦的把关功能。我的初步数据 应用时间推移活体显微镜检查脓毒症样骨髓模型中中性粒细胞的渗入 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”，没有NO。 门的数量变化表明白细胞可能发生在特定的部位 内皮细胞。因此，我们假设血管内皮细胞VEGFR3信号通过 炎症应激时骨髓血窦内皮细胞内的“门”。我们将通过以下方式验证这一假设 应用延时活体技术研究炎症应激与EC门活性的关系 显微镜跟踪中性粒细胞进入血管内的频率，不同的“门”进入的频率和比率 脂多糖组和对照组的血管内事件。我们将在分类的BM中测量VEGFR3信号 并检测抑制VEGFR3是否抑制炎症应激后EC门的活性。 这些实验为检验心肌梗死(MI)引起的假说奠定了基础 与EC GATE活动类似的变化。我们将在成年小鼠上进行假手术或MI手术，并检查EC GATE 分选骨髓内皮细胞的时间推移活体显微镜活性、VEGFR3信号转导及其相关性 这些发现与循环白细胞水平的流式细胞仪分析相结合。我们最终建议测试一下 VEGFR3信号在心肌梗死中的作用--通过研究缺乏EC门的小鼠的活性 血管内皮细胞VEGFR3或用VEGFR3抑制剂预先处理的小鼠。这项工作提出要研究小说 血管内皮生长因子受体3依赖的调节血管内皮生长因子受体诱导的骨髓白细胞释放的机制 并测试这一通路的调节是否可以为未来的治疗干预提供参考。

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 Our unpublished work found that models of CVD induce a pro-angiogenic phenotype in the BM vasculature which drives leukocyte release into the blood in an incompletely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.