Proangiogenic M2-type macrophages and choroidal neovascularization

总结 巨噬细胞浸润炎症部位，并与抗血管生成的M1型巨噬细胞或 促血管生成的M2型巨噬细胞，这取决于各种细胞因子和其他外部 因素促血管生成M2型巨噬细胞是许多肿瘤中炎性血管生成的主要贡献者。 常见疾病，部分通过分泌促血管生成因子，如VEGF-A或IL-1b。特别是M2型 巨噬细胞在患有新生血管性AMD的患者的脉络膜新生血管形成（CNV）区域中积聚。的 新生血管性AMD患者眼内M2型巨噬细胞源性细胞因子的增加与此有关， 细胞群体作为CNV发病机制的主要驱动因素。这一假设得到以下观察结果的进一步支持： 在激光诱导的CNV或遗传VEGF-A诱导的小鼠中M2型巨噬细胞的积累 新生血管性AMD模型。激光诱导CNV中巨噬细胞的消融，主要是M2型 巨噬细胞，阻断CNV，而在眼睛中给予M2型巨噬细胞促进CNV。基于 基于这些发现，我们提出预防巨噬细胞M2型极化的药物疗法， 可以作为一种新的方法，有效地抑制新生血管性AMD患者的CNV进展。有限 了解调节巨噬细胞M1型与M2型极化的信号机制 阻碍了分子靶点和药理学抑制剂的鉴定，以选择性地阻断M2型 巨噬细胞极化。为了解决这一未满足的需求，我们进行了全球定量时间过程 蛋白质组学和磷酸蛋白质组学，并确定了与M1-vs M2型巨噬细胞极化。此外，我们在化学筛选中鉴定了 选择性地阻挡M2型偏振而不阻挡M1型偏振M2型偏振。因此，这两者的结合 方法为我们提供了一个独特的资源，以建立新的治疗方法，选择性地阻止 CNV中的促血管生成M2型巨噬细胞，而非抗血管生成M1型巨噬细胞。在原理验证实验中， 可以显示两种鉴定的抑制剂，MEK抑制剂曲美替尼和HDAC抑制剂帕比司他， 阻断CNV损伤中的M2型巨噬细胞极化，并有效抑制激光照射下CNV损伤的形成。 诱导CNV实验。我们提出的实验将测试确定的激酶和非激酶抑制剂， 在详细的体外实验中， 以及在三种成熟的新生血管性AMD小鼠模型中。我们的建议是基于广泛的 初步数据提供了强有力的科学前提。所提出的实验具有高度的严谨性， 重要的临床相关性，并可能导致新的治疗方法新生血管性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.