Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis. Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-β for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo. Approximately 20~30% of F4/80+ (or Iba-1+) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-β and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4+ lesions. The treatment did not significantly alter collagen-1+ fibrotic lesions. MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-β and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-β1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch’s membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor β. a-SMA = alpha smooth muscle actin. C3a = complement C3a.

中文翻译：

---

巨噬细胞向肌成纤维细胞的转变导致新生血管性年龄相关性黄斑变性继发的视网膜下纤维化


即使采用抗血管内皮生长因子（VEGF）治疗，黄斑纤维化也会导致新生血管性年龄相关性黄斑变性（nAMD）造成不可挽回的视力丧失。众所周知，炎症在黄斑纤维化中发挥着重要作用，尽管其潜在机制仍不清楚。本研究的目的是了解浸润巨噬细胞和补体蛋白如何导致黄斑纤维化。使用我们小组开发的两阶段激光方案在 C57BL/6J 小鼠中诱导视网膜下纤维化。在第二次激光后 10、20、30 和 40 天收集眼睛，并进行免疫组织化学处理，以检测浸润巨噬细胞（F4/80 和 Iba-1）、补体成分（C3a 和 C3aR）和纤维血管病变（胶原蛋白-1、异凝集素） B4 和 α-SMA）。研究中还使用了患有黄斑纤维化的人类视网膜切片。用重组 C3a、C5a 或 TGF-β 处理来自 C57BL/6J 小鼠的骨髓源性巨噬细胞 (BMDM) 48 和 96 小时。 qPCR、Western blot 和免疫组织化学用于检测肌成纤维细胞标志物的表达。使用 C3aR 拮抗剂 (C3aRA) 和 C3a 阻断抗体在体外和体内进一步研究 C3a-C3aR 通路在巨噬细胞向肌成纤维细胞转变 (MMT) 和视网膜下纤维化中的参与。在人类 nAMD 眼和小鼠模型的视网膜下纤维化病变中，大约 20~30% 的 F4/80+（或 Iba-1+）浸润巨噬细胞共表达 α-SMA。 TGF-β和C3a（而非C5a）处理显着上调BMDM中α-SMA、纤连蛋白和胶原蛋白1的表达。 C3aRA 治疗可防止 BMDM 中 C3a 诱导的 α-SMA、纤连蛋白和胶原蛋白 1 上调。 在诱导视网膜下纤维化的两阶段激光模型中，使用 C3a 阻断抗体而非 C3aRA 治疗可显着减少血管渗漏和异凝集素 B4+ 病变。该治疗没有显着改变胶原蛋白-1+纤维化病变。 MMT 在继发于 nAMD 的黄斑纤维化中发挥作用。 MMT 可以被 TGF-β 和 C3a 诱导，但不能被 C5a 诱导。需要进一步的研究来充分了解 MMT 在黄斑纤维化中的作用。巨噬细胞向肌成纤维细胞转变（MMT）导致视网膜下纤维化。视网膜下纤维化病变含有多种细胞类型，包括巨噬细胞和肌成纤维细胞，并且是纤维血管。肌成纤维细胞是驱动致病性纤维化的关键细胞，它们通过产生过量的细胞外基质蛋白来实现这一点。我们发现浸润性巨噬细胞可以转分化为肌成纤维细胞，这种现象在黄斑纤维化中被称为巨噬细胞向肌成纤维细胞转变（MMT）。除了 TGF-β1 之外，CNV 中补体激活过程中产生的 C3a 也可以诱导导致黄斑纤维化的 MMT。 RPE = 视网膜色素上皮。 BM = 布鲁赫膜。 MMT = 巨噬细胞向肌成纤维细胞的转变。 TGFB = 转化生长因子β。 a-SMA = α 平滑肌肌动蛋白。 C3a = 补充 C3a。