Bone Marrow Endothelial VEGFR3 Regulation of Leukocyte Trafficking in Myocardial Infarction

骨髓内皮 VEGFR3 对心肌梗死白细胞贩运的调节

• 批准号: 10226604
• 负责人: Fadi Emad Pulous
• 金额: $ 6.6万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226604
• 关键词: Bone; Marrow; Endothelial; VEGFR3; Regulation

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 Our unpublished work found that models of CVD induce a pro-angiogenic phenotype in the BM vasculature which drives leukocyte release into the blood in an incompletely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

在美国，心血管疾病 (CVD) 每年造成的死亡人数比癌症和肺部疾病还要多 结合起来强调了在这种情况下迫切需要新的治疗干预措施。 CANTOS 试验支持这样的观点，即减轻 CVD 引起的炎症的干预措施可能会控制病情 我们和其他人的大量证据表明，CVD 是未来治疗的关键。 骨髓（BM）中的紧急造血作用刺激白细胞的释放，这些白细胞会进入梗塞或 动脉粥样硬化组织使病理恶化，这种现象在人类中已有描述。 未发表的研究发现，CVD 模型在 BM 脉管系统中诱导促血管生成表型，这 我们观察到以一个尚不完全清楚的过程驱动白细胞释放到血液中。 CVD 小鼠循环血管内皮生长因子 (VEGF) 水平。 内皮生长因子受体 3 (VEGFR3) 在骨髓的窦状微环境中高度表达， 造血细胞和白细胞所在的血管室已被 VEGF-C/VEGFR3 信号传导。 据报道在癌症诱导期间调节细胞跨淋巴内皮的跨内皮迁移 炎症，这个过程让人想起 BM 正弦波的看门功能。我的初步数据。 通过延时活体显微镜检查败血症样 BM 模型中的中性粒细胞内渗 炎症应激发现，中性粒细胞进入的特定位点增加，我们称之为“EC 门”，而没有 门数量的变化表明白细胞内渗可能发生在特定的部位 因此，我们认为内皮 VEGFR3 信号传导通过调节白细胞运输。 炎症应激期间骨髓窦内皮内的“门”我们将通过以下方式检验这一假设。 使用延时活体内研究炎症应激与 EC 门活性之间的关系 显微镜追踪中性粒细胞内渗频率、不同进入“门”的频率以及 我们将测量分选的 BM 中的 VEGFR3 信号传导。 内皮细胞并测试 VEGFR3 的抑制是否会抑制炎症应激后 EC 门的活性。 这些实验为检验心肌梗死 (MI) 诱发的假设奠定了基础 我们将对成年小鼠进行假手术或 MI 手术并检查 EC 门活动。 通过延时活体显微镜观察分选的 BM 内皮细胞中 VEGFR3 信号传导的活性并关联 我们最终建议通过流式细胞术分析循环白细胞水平来测试这些发现。 通过研究缺乏 EC 门的小鼠的 VEGFR3 信号传导在 MI 背景下的贡献 内皮 VEGFR3 或用 VEGFR3 抑制剂预处理的小鼠本工作旨在研究新的。 CVD 诱导后调节 BM 中白细胞释放的 VEGFR3 依赖性机制 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。