Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke

Abstract

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.

Microglia at the blood brain barrier in health and disease

“…Endothelial cell injury within the stroke site and surrounding area triggers a series of molecular signaling events and protein expression that stimulates microglia, which attempt to mitigate the damage caused by the stroke. Inflammation-associated transcripts may be enriched in endothelial cells following a stroke, as observed in a middle cerebral artery occlusion (MCAO) and reperfusion mouse models ( Arbaizar-Rovirosa et al, 2023 ). This enrichment corresponds with increased cytokine and chemokine activity and immunoglobulin Fc-gamma receptor I complex binding that may further inflammation ( Arbaizar-Rovirosa et al, 2023 ).…”

“…Inflammation-associated transcripts may be enriched in endothelial cells following a stroke, as observed in a middle cerebral artery occlusion (MCAO) and reperfusion mouse models ( Arbaizar-Rovirosa et al, 2023 ). This enrichment corresponds with increased cytokine and chemokine activity and immunoglobulin Fc-gamma receptor I complex binding that may further inflammation ( Arbaizar-Rovirosa et al, 2023 ). For example, signaling through the chemokine receptor, CCR2, or Fc receptor promote inflammation through upregulation of the IL-6 pathway, leading to activation of signal transducer and activator of transcription 3 (STAT3) by ischemic endothelium ( Arbaizar-Rovirosa et al, 2023 ).…”

“…This enrichment corresponds with increased cytokine and chemokine activity and immunoglobulin Fc-gamma receptor I complex binding that may further inflammation ( Arbaizar-Rovirosa et al, 2023 ). For example, signaling through the chemokine receptor, CCR2, or Fc receptor promote inflammation through upregulation of the IL-6 pathway, leading to activation of signal transducer and activator of transcription 3 (STAT3) by ischemic endothelium ( Arbaizar-Rovirosa et al, 2023 ). The proinflammatory transcriptional shift and BBB breach prompts microglia to migrate to the area of injury.…”

“…HIF-1 binds hypoxia response element (HRE) ( Rashid et al, 2019 ) in promoter regions of target genes, influencing angiogenesis, cell proliferation, erythropoiesis, and cell metabolism ( Hong et al, 2020 ), as well as a initiating inflammatory responses and compromising BBB integrity. HIF-1α stabilization/upregulation is associated with increased expression of vascular endothelial growth factor (VEGF) ( Mu et al, 2003 ; Shen et al, 2018 ; Zhang et al, 2018 ), glucose transporter 1 (GLUT1) ( Lum et al, 2007 ; Zhong et al, 2010 ), and multiple chemokines ( Arbaizar-Rovirosa et al, 2023 ) in brain endothelial cells ( Hong et al, 2020 ) along with other transcriptional changes. The temporal sequence and regulation of HIF-1-dependent downstream proteins play a crucial role in the pathogenesis and recovery of stroke, potentially affecting the balance between inflammatory and angiogenetic responses.…”

β-asarone induces viability and angiogenesis and suppresses apoptosis of human vascular endothelial cells after ischemic stroke by upregulating vascular endothelial growth factor A

“…Following the rupture of the BBB in IS, reactive oxygen species trigger morphological changes in astrocytes to form reactive astrocytes, which can alter the extracellular matrix (ECM), leading to complete remodeling of the ECM and the formation of ECM bundles ( Williamson et al, 2021 ). The endothelial cells then migrate through the ECM bundles to establish new capillary sprouts ( Arbaizar-Rovirosa et al, 2023 ). Therefore, induction of endothelial cell angiogenesis can effectively improve vascular remodeling and neurovascular function recovery after IS.…”