Department of Neuroscience and Experimental Therapeutics, Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Rosselló 161, Planta 6, 08036, Barcelona, Spain.
Cerebrovascular Research Group, Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain.
J Neuroinflammation. 2023 Sep 11;20(1):207. doi: 10.1186/s12974-023-02888-6.
Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31 endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.
血管内皮功能在脑缺血再灌注期间受到挑战。内皮细胞的反应涉及炎症性白细胞的吸引、黏附和浸润、血脑屏障的渗漏和血管生成。本研究使用转录组学和转译组学研究了急性缺血性脑卒中后脑内皮细胞的基因表达变化。我们通过以下两种方法分离脑内皮细胞的 mRNA：（i）翻译核糖体亲和纯化，使脑内皮核糖体附着的 mRNA 能够进行免疫沉淀，用于转译组测序；（ii）通过荧光激活细胞分选分离 CD31 内皮细胞，用于经典转录组分析。这两种技术都揭示了缺血调节的相似途径，但它们在一些来源于非内皮细胞的转录本中显示出特定的差异。我们通过选择两种技术共有的差异表达基因，定义了一个特征急性脑卒中（24 小时）内皮反应的基因集，从而对应转译组并最小化非内皮 mRNA 的污染。富集的途径与炎症和免疫调节、血管生成、细胞外基质、氧化应激以及脂质转运和储存有关。我们通过流式细胞术和免疫荧光验证了几个编码细胞表面蛋白的基因的蛋白质表达。炎症反应与内皮细胞中与脂质储存功能相关的基因上调有关，我们在缺血后发现了内皮细胞中脂质滴的生物发生。该研究报告了急性脑卒中后脑内皮细胞的稳健转译组学特征，并确定了参与膜信号和脂质储存的新途径的富集。综上所述，这些结果强调了内皮细胞在炎症反应中的贡献，并确定了可能成为改善缺血性脑卒中后血管功能的新靶点的新型分子。
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