巨噬细胞弹性蛋白酶（MMP12）对视网膜下纤维化的发展有重要贡献。
Macrophage elastase (MMP12) critically contributes to the development of subretinal fibrosis.
机构信息
Aier School of Ophthalmology, Central South University, Changsha, 410000, China.
Aier Institute of Optometry and Vision Science, Changsha, 410000, China.
出版信息
J Neuroinflammation. 2022 Apr 5;19(1):78. doi: 10.1186/s12974-022-02433-x.
BACKGROUND
Macular subretinal fibrosis is the end-stage complication of neovascular age-related macular degeneration (nAMD). We previously developed a mouse model of two-stage laser-induced subretinal fibrosis that mimics closely the dynamic course of macular fibrosis in nAMD patients. This study was aimed to understand the molecular mechanism of subretinal fibrosis.
METHODS
Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser-induced protocol. Twenty days later, eyes were collected and processed for RNA sequencing (RNA-seq) analysis. DESeq2 was used to determine the differentially expressed genes (DEGs). Gene Ontology (GO) and KEGG were used to analyze the enriched pathways. The expression of the selected DEGs including Mmp12 was verified by qPCR. The expression of MMP12 in subretinal fibrosis of mouse and nAMD donor eyes was examined by immunofluorescence and confocal microscopy. The expression of collagen 1, αSMA and fibronectin and cytokines in bone marrow-derived macrophages from control and subretinal fibrosis mice were examined by qPCR, immunocytochemistry and Luminex multiplex cytokine assay. The MMP12 specific inhibitor MMP408 was used to evaluate the effect of MMP12 on TGFβ-induced macrophage-to-myofibroblast transition (MMT) in vitro and its role in subretinal fibrosis in vivo.
RESULTS
RNA-seq analysis of RPE-choroid from subretinal fibrosis eyes uncovered 139 DEGs (fold change log2(fc) ≥ 0.5, FDR < 0.05), including 104 up-regulated and 35 were down-regulated genes. The top 25 enrichment GO terms were related to inflammation, blood vessels/cardiovascular development and angiogenesis. One of the most significantly upregulated genes, Mmp12, contributed to 12 of the top 25 GO terms. Higher levels of MMP12 were detected in subretinal fibrotic lesions in nAMD patients and the mouse model, including in F4/80 or Iba1 macrophages. BMDMs from subretinal fibrosis mice expressed higher levels of MMP12, collagen-1, αSMA and fibronectin. MMP408 dose-dependently suppressed TGFβ-induced MMT in BMDMs. In vivo treatment with MMP408 (5 mg/kg) significantly reduced subretinal fibrosis accompanied by reduced F4/80 macrophage infiltration.
CONCLUSIONS
MMP12 critically contributes to the development of subretinal fibrosis, partially through promoting MMT.
背景
黄斑部视网膜下纤维化是新生血管性年龄相关性黄斑变性（nAMD）的终末期并发症。我们之前开发了一种两阶段激光诱导的视网膜下纤维化小鼠模型，该模型非常类似于 nAMD 患者黄斑部纤维化的动态过程。本研究旨在了解视网膜下纤维化的分子机制。
方法
使用两阶段激光诱导方案在 C57BL/6J 小鼠中诱导视网膜下纤维化。 20 天后，收集眼睛并进行 RNA 测序（RNA-seq）分析。DESeq2 用于确定差异表达基因（DEGs）。基因本体论（GO）和 KEGG 用于分析富集途径。通过 qPCR 验证所选 DEGs 包括 Mmp12 的表达。通过免疫荧光和共聚焦显微镜检查小鼠和 nAMD 供体眼的视网膜下纤维化中 MMP12 的表达。通过 qPCR、免疫细胞化学和 Luminex 多重细胞因子测定检查来自对照和视网膜下纤维化小鼠的骨髓来源巨噬细胞中胶原 1、αSMA 和纤维连接蛋白和细胞因子的表达。使用 MMP12 特异性抑制剂 MMP408 评估 MMP12 在 TGFβ 诱导的巨噬细胞向肌成纤维细胞转化（MMT）中的作用及其在体内视网膜下纤维化中的作用。
结果
视网膜下纤维化眼睛的 RPE-脉络膜的 RNA-seq 分析揭示了 139 个 DEGs（倍数变化 log2(fc)≥0.5， FDR<0.05），包括 104 个上调和 35 个下调基因。前 25 个富集 GO 术语与炎症，血管/心血管发育和血管生成有关。上调最明显的基因之一，Mmp12，与前 25 个 GO 术语中的 12 个有关。在 nAMD 患者和小鼠模型的视网膜下纤维病变中检测到更高水平的 MMP12，包括 F4/80 或 Iba1 巨噬细胞。来自视网膜下纤维化小鼠的 BMDMs 表达更高水平的 MMP12、胶原蛋白 1、αSMA 和纤维连接蛋白。MMP408 剂量依赖性地抑制 BMDMs 中 TGFβ 诱导的 MMT。体内用 MMP408（5mg/kg）治疗可显著减少视网膜下纤维化，同时减少 F4/80 巨噬细胞浸润。
结论
MMP12 对视网膜下纤维化的发展至关重要，部分通过促进 MMT。
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