Proangiogenic M2-type macrophages and choroidal neovascularization

摘要 巨噬细胞渗入炎症部位并极化为抗血管生成的M1型巨噬细胞或 促血管生成的M2型巨噬细胞，取决于各种细胞因子和其他外部因素的存在 各种因素。促血管生成M2型巨噬细胞是许多炎性血管生成的主要贡献者 常见疾病，部分是通过分泌促血管生成因子，如血管内皮生长因子-A或白介素1b。值得注意的是，M2类型 新生血管性AMD患者脉络膜新生血管(CNV)区聚集巨噬细胞。这个 新生血管性AMD患者眼内M2型巨噬细胞衍生细胞因子的增加可能与此有关 细胞群是CNV发病的主要驱动力。这一假设进一步得到了观察结果的支持 M2型巨噬细胞在激光诱导的新生血管或遗传的血管内皮生长因子-A诱导的小鼠中的聚集 新生血管性AMD模型。激光诱导的CNV中以M2型为主的巨噬细胞的消融 巨噬细胞阻断CNV，而眼内注射M2型巨噬细胞促进CNV。基座 根据这些发现，我们提出了防止巨噬细胞M2型极化的药物疗法 可以作为一种新的方法来有效地抑制新生血管性AMD患者的CNV进展。有限的 巨噬细胞M1型与M2型极化调节的信号机制 阻碍了选择性阻断M2型的分子靶点和药物抑制剂的确定 巨噬细胞极化。为了解决这一未得到满足的需求，我们进行了全球量化时间进程 蛋白质组学和磷酸蛋白质组学以及已确定的与M1-VS相关的激酶激活事件 M2型巨噬细胞极化。此外，我们在化学筛选中发现了药物抑制物 M2型偏振，选择性地阻挡M2型偏振，而不是M1型偏振。因此，这两者的结合 方法现在为我们提供了一种独特的资源来建立选择性阻断的新疗法 CNV中促血管生成M2-而不是抗血管生成M1型巨噬细胞。在原则证明实验中，我们 可以证明两种已确认的抑制剂，MEK抑制剂曲美替尼和HDAC抑制剂Panobinostat， 阻断CNV损伤的M2型巨噬细胞极化并有效地抑制CNV损伤的形成 诱导CNV实验。我们提议的实验将在一个 在详细的体外实验中，系统地研究了它们选择性阻断M2型极化的能力 在三种已建立的新生血管性AMD小鼠模型中也是如此。我们的建议是基于广泛的 提供了强有力的科学前提的初步数据。拟议中的实验具有高度的严谨性和 重要的临床相关性，并可能导致新的治疗方法的新生血管性AMD。

SUMMARY Macrophages infiltrate the site of inflammation and polarize to either antiangiogenic M1-type macrophages or proangiogenic M2-type macrophages, depending on the presence of various cytokines and other external factors. Proangiogenic M2-type macrophages are major contributors to inflammatory angiogenesis in many common diseases, in part by secreting proangiogenic factors, such as VEGF-A or IL-1b. Notably, M2-type macrophages accumulate in areas of choroidal neovascularization (CNV) in patients with neovascular AMD. The increase in M2-type macrophage-derived cytokines in eyes of patients with neovascular AMD has implicated this cell population as a major driver of CNV pathogenesis. This hypothesis is further supported by the observation of an accumulation of M2-type macrophages in laser-induced CNV or in a genetic VEGF-A-induced mouse model of neovascular AMD. Ablation of macrophages in laser-induced CNV, which are predominantly M2-type macrophages, blocked CNV, whereas administration of M2-type macrophages in the eye promoted CNV. Based on these findings we propose that pharmacologic therapies that prevent M2-type polarization of macrophages can serve as a novel approach to potently inhibit CNV progression in patients with neovascular AMD. The limited understanding of the signaling mechanisms that are regulating M1- versus M2-type polarization of macrophages has hindered the identification of molecular targets and pharmacologic inhibitors to selectively block M2-type macrophage polarization. To address this unmet need, we have performed global quantitative time-course proteomics and phosphoproteomics and identified kinase activation events that are associated with M1- versus M2-type macrophage polarization. Furthermore, we identified in chemical screens pharmacologic inhibitors of M2-type polarization that selectively block M2- but not M1-type polarization. Thus, the combination of these two approaches provides us now with a unique resource to establish novel therapies that selectively block proangiogenic M2- but not antiangiogenic M1-type macrophages in CNV. In proof-of-principle experiments we can show that two of the identified inhibitors, the MEK inhibitor trametinib and the HDAC inhibitor panobinostat, blocked M2-type macrophage polarization in CNV lesions and potently inhibited CNV lesion formation in laser- induced CNV experiments. Our proposed experiments will test identified kinase and non-kinase inhibitors in a systematic manner for their ability to selectively block M2-type polarization in detailed in vitro experiments as well as in three well-established mouse models of neovascular AMD. Our proposal is based on extensive preliminary data that provide a strong scientific premise. The proposed experiments have high rigor and important clinical relevance and will likely lead to novel therapeutic approaches for neovascular AMD.