探討動脈粥狀硬化中發育程序化影響巨噬細胞極化之分子機制

Abstract

動脈粥狀硬化形成的過程，巨噬細胞會吞噬血液中過多的氧化態低密度脂蛋白（Oxidized low-density lipoproteins, ox-LDL），形成泡沫細胞堆積在血管內層，進而形成纖維性斑塊。已知母鼠高膽固醇血症能透過表觀遺傳的方式使胎兒罹患動脈粥狀硬化的機會增加。初步研究已發現降低親代母鼠DNA甲基化程度可以改善子代雄鼠動脈粥狀硬化的程度，可能和促發炎M1巨噬細胞數量的減少、抑制發炎M2巨噬細胞數量的增加有關，但是對於表觀遺傳和巨噬細胞之間的關係仍不清楚。因此本實驗欲探討親代營養狀況如何透過表觀遺傳的調控影響子代巨噬細胞極化的分子機制。以ApoE－/－小鼠作為動物模型，在孕前、哺乳期間給予DNA甲基轉移酶抑制劑5-Aza-2‘-deoxycytidine（5-aza-dC），子代離乳後皆餵食西方飲食。首先，主動脈RNA定序結果顯示親代母鼠經過5-aza-dC處理後雄性子代主動脈差異表現基因（differentially expressed genes, DEG）與巨噬細胞極化相關路徑高度相關，接著利用從骨髓衍生的巨噬細胞（bone marrow-derived macrophage, BMDM），我們確認了親代母鼠經過5-aza-dC處理可以降低雄性子代BMDM誘導成M1巨噬細胞數量，功能性分析上發現M1巨噬細胞糖解能力下降，M2巨噬細胞氧化磷酸化能力增加，之後，為了進一步探討分子機制，我們利用BMDM進行RNA定序，透過Gene Ontology（GO）分析得知DEG與細胞週期、轉錄因子結合及轉錄調控等生物路徑高度相關，並且我們發現親代母鼠經過5-aza-dC處理後雄性子代巨噬細胞中轉錄因子HIF2α表現量增加，最後，在Raw264.7細胞過度表現Hif2a，證明部分M2巨噬細胞相關基因表現量上升，表示高表現HIF2α可能促進巨噬細胞傾向往M2極化，然而，我們在BMDM中Hif2a啟動子的特定序列上並未觀察到DNA甲基化有明顯改變的情形。綜合以上結果，我們發現親代母鼠孕前及哺乳期間給予5-aza-dC使雄性子代巨噬細胞Hif2a表現量增加，可能將巨噬細胞趨勢M2極化，進而改善子代雄鼠動脈粥狀硬化。

During the development of atherosclerosis, macrophages uptake excessive oxidized low-density lipoproteins (ox-LDL) and transform into foam cells. The accumulation of foam cells in the arterial intima leads to the formation of fibrous plaques. Our previous study shows that maternal hypercholesterolemia increases the risk of developing atherosclerosis in adult offspring, potentially through epigenetic mechanism. Furthermore, our preliminary results indicate that reducing DNA methylation levels in dams attenuates atherosclerosis development in male offspring, which may be associated with a reduction in M1 macrophages and an increase in M2 macrophages. However, the relationship between epigenetic inheritance and macrophages remains unclear. Here, we aim to investigate the molecular mechanism of maternal effect in macrophages polarization through epigenetic regulation. In this study, DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-aza-dC), was given to ApoE－/－ female mice before pregnancy and during lactation. First, RNA-sequence data obtained from the aorta in male offspring derived from 5-aza-dC-treated dams exhibited a high correlation with pathways related to macrophage polarization. Subsequently, we used bone marrow-derived macrophage (BMDM) and confirmed that maternal 5-aza-dC treatment reduced the cell populations of M1 macrophages in male offspring. For functional analysis, the glycolytic capacity of M1 macrophages decreased and the oxidative phosphorylation capacity of M2 macrophages increased in male offspring derived from 5-aza-dC-treated dams. Moreover, we analyzed RNA-sequence data obtained from BMDM using Gene Ontology (GO) enrichment analysis. We identified differentially expressed genes were highly enriched in pathways associated with cell cycle, transcription factor binding and positive regulation of transcription. We specifically focused on transcription factor HIF2α, which is implicated in macrophage polarization and was upregulated in the macrophages isolated from male offspring derived from 5-aza-dC-treated dams. In particular, we overexpressed HIF2α in Raw264.7 cells and observed an increase in the expression of genes related to M2 macrophages. However, the DNA methylation level of the specific region within HIF2α promoter was unaltered in the macrophages isolated from male offspring derived from 5-aza-dC-treated dams compared to those isolated from male offspring derived from PBS-treated dams. In summary, our findings suggest that maternal 5-aza-dC treatment before pregnancy and during lactation can increase the expression of HIF2α in the macrophages from male offspring and potentially drive macrophage polarization toward M2 macrophages. This, in turn, partially ameliorates atherosclerosis development in male offspring mice.