Phillips Chelsea, Stamatovic Svetlana, Keep Richard, Andjelkovic Anuska
University of Michigan.
University of Michigan-Ann Arbor.
Res Sq. 2023 Jan 13:rs.3.rs-2444060. doi: 10.21203/rs.3.rs-2444060/v1.
Incomplete recovery of blood-brain barrier (BBB) function contributes to stroke outcomes. How the BBB recovers after stroke remains largely unknown. Emerging evidence suggests that epigenetic factors play a significant role in regulating post-stroke BBB recovery. This study aimed to evaluate the epigenetic and transcriptional profile of cerebral microvessels after thromboembolic (TE) stroke to define potential causes of limited BBB recovery. RNA-sequencing and reduced representation bisulfite sequencing (RRBS) analyses were performed using microvessels isolated from young (6 months) and old (18 months) mice seven days poststroke compared to age-matched sham controls. DNA methylation profiling of poststroke brain microvessels revealed 11287 differentially methylated regions (DMR) in old and 9818 DMR in young mice, corresponding to annotated genes. These DMR were enriched in genes encoding cell structural proteins (e.g., cell junction, and cell polarity, actin cytoskeleton, extracellular matrix), transporters and channels (e.g., potassium transmembrane transporter, organic anion and inorganic cation transporters, calcium ion transport), and proteins involved in endothelial cell processes (e.g., angiogenesis/vasculogenesis, cell signaling and transcription regulation). Integrated analysis of methylation and RNA sequencing identified changes in cell junctions (occludin), actin remodeling (ezrin) as well as signaling pathways like Rho GTPase (RhoA and Cdc42ep4). Aging as a hub of aberrant methylation affected BBB recovery processes by profound alterations (hypermethylation and repression) in structural protein expression (e.g., claudin-5) as well as activation of a set of genes involved in endothelial to mesenchymal transformation (e.g., , ), repression of angiogenesis and epigenetic regulation. These findings revealed that DNA methylation plays an important role in regulating BBB repair after stroke, through regulating processes associated with BBB restoration and prevalently with processes enhancing BBB injury.
血脑屏障（BBB）功能的不完全恢复会影响中风的预后。中风后血脑屏障如何恢复在很大程度上仍不清楚。新出现的证据表明，表观遗传因素在调节中风后血脑屏障的恢复中起重要作用。本研究旨在评估血栓栓塞性（TE）中风后脑微血管的表观遗传和转录谱，以确定血脑屏障恢复受限的潜在原因。与年龄匹配的假手术对照组相比，在中风后7天，使用从小鼠（6个月）和老年小鼠（18个月）分离的微血管进行RNA测序和简化代表性亚硫酸氢盐测序（RRBS）分析。中风后脑微血管的DNA甲基化谱显示，老年小鼠中有11287个差异甲基化区域（DMR），年轻小鼠中有9818个DMR，这些区域对应于注释基因。这些DMR在编码细胞结构蛋白（如细胞连接、细胞极性、肌动蛋白细胞骨架、细胞外基质）、转运蛋白和通道（如钾跨膜转运蛋白、有机阴离子和无机阳离子转运蛋白、钙离子转运）以及参与内皮细胞过程（如血管生成/血管发生、细胞信号传导和转录调节）的蛋白质的基因中富集。甲基化和RNA测序的综合分析确定了细胞连接（闭合蛋白）、肌动蛋白重塑（埃兹蛋白）以及Rho GTPase（RhoA和Cdc42ep4）等信号通路的变化。衰老作为异常甲基化的中心，通过结构蛋白表达（如claudin-5）的深刻改变（高甲基化和抑制）以及一组参与内皮-间充质转化的基因（如 ， ）的激活、血管生成的抑制和表观遗传调节，影响血脑屏障的恢复过程。这些发现表明，DNA甲基化通过调节与血脑屏障恢复相关的过程，特别是与增强血脑屏障损伤的过程，在中风后血脑屏障修复中起重要作用。
