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LncRNA-Profile-Based Screening of Extracellular Vesicles Released from Brain Endothelial Cells after Oxygen-Glucose Deprivation.

ABSTRACT: Brain microvascular endothelial cells (BMECs) linked by tight junctions play important roles in cerebral ischemia. Intercellular signaling via extracellular vesicles (EVs) is an underappreciated mode of cell-cell crosstalk. This study aims to explore the potential function of long noncoding RNAs (lncRNAs) in BMECs' secreted EVs. We subjected primary human and rat BMECs to oxygen and glucose deprivation (OGD). EVs were enriched for RNA sequencing. A comparison of the sequencing results revealed 146 upregulated lncRNAs and 331 downregulated lncRNAs in human cells and 1215 upregulated lncRNAs and 1200 downregulated lncRNAs in rat cells. Next, we analyzed the genes that were coexpressed with the differentially expressed (DE) lncRNAs on chromosomes and performed Gene Ontology (GO) and signaling pathway enrichment analyses. The results showed that the lncRNAs may play roles in apoptosis, the TNF signaling pathway, and leukocyte transendothelial migration. Next, three conserved lncRNAs between humans and rats were analyzed and confirmed using PCR. The binding proteins of these three lncRNAs in human astrocytes were identified via RNA pulldown and mass spectrometry. These proteins could regulate mRNA stability and translation. Additionally, the lentivirus was used to upregulate them in human microglial HMC3 cells. The results showed NR_002323.2 induced microglial M1 activation. Therefore, these results suggest that BMECs' EVs carry the lncRNAs, which may regulate gliocyte function after cerebral ischemia.

SUBMITTER: He X

PROVIDER: S-EPMC9405926 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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LncRNA-Profile-Based Screening of Extracellular Vesicles Released from Brain Endothelial Cells after Oxygen-Glucose Deprivation.

He Xiang X Zou Hecun H Lyu Qiang Q Tang Yujing Y Xiong Wenkui W Shang Fei-Fei FF

Brain sciences 20220803 8

Brain microvascular endothelial cells (BMECs) linked by tight junctions play important roles in cerebral ischemia. Intercellular signaling via extracellular vesicles (EVs) is an underappreciated mode of cell-cell crosstalk. This study aims to explore the potential function of long noncoding RNAs (lncRNAs) in BMECs' secreted EVs. We subjected primary human and rat BMECs to oxygen and glucose deprivation (OGD). EVs were enriched for RNA sequencing. A comparison of the sequencing results revealed 1 ...[more]

PMID: 36009090

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Project description:IntroductionAs with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells.MethodsWe conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells.ResultsSequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway.DiscussionThe results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.

Project description:Shuxuetong injection composed of leech (Hirudo nipponica Whitman) and earthworm (Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells (bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N2/5% CO2 for 6 hours, followed by high-glucose medium containing 95% O2 and 5% CO2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations (diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.

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Project description:As any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye, and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells. We conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture, and used bioinformatic tools to evaluate their potential role in the neuroprotective signalling attributed to these cells. Sequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b and the let-7 family. A total of 51 miRNA were found to be differentially enriched in EV compared to whole cells from which EV originated. Bioinformatic analyses also indicated preferential enrichment of species demonstrated to regulate genes involved in cell proliferation and survival, including PTEN the master inhibitor of the PI3K/AKT pathway. The results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks, is likely to represent a significant proportion of the neuroprotective effect observed after transplantation of these cells into animal models of retinal ganglion cell (RGC)-depletion. Future work will seek to evaluate the modulation of putative genes, as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.

Project description:BackgroundBrain microvascular endothelial cells form a highly selective blood brain barrier regulated by the endothelial tight junctions. Cerebral ischemia selectively targets tight junction protein complexes, which leads to significant damage to cerebral microvasculature. Short noncoding molecules called microRNAs are implicated in the regulation of various pathological states, including endothelial barrier dysfunction. In the present study, we investigated the influence of microRNA-155 (miR-155) on the barrier characteristics of human primary brain microvascular endothelial cells (HBMECs).Methods and resultsOxygen-glucose deprivation was used as an in vitro model of ischemic stroke. HBMECs were subjected to 3 hours of oxygen-glucose deprivation, followed by transfections with miR-155 inhibitor, mimic, or appropriate control oligonucleotides. Intact normoxia control HBMECs and 4 oxygen-glucose deprivation-treated groups of cells transfected with appropriate nucleotide were subjected to endothelial monolayer electrical resistance and permeability assays, cell viability assay, assessment of NO and human cytokine/chemokine release, immunofluorescence microscopy, Western blot, and polymerase chain reaction analyses. Assessment of endothelial resistance and permeability demonstrated that miR-155 inhibition improved HBMECs monolayer integrity. In addition, miR-155 inhibition significantly increased the levels of major tight junction proteins claudin-1 and zonula occludens protein-1, while its overexpression reduced these levels. Immunoprecipitation and colocalization analyses detected that miR-155 inhibition supported the association between zonula occludens protein-1 and claudin-1 and their stabilization at the HBMEC membrane. Luciferase reporter assay verified that claudin-1 is directly targeted by miR-155.ConclusionsBased on these results, we conclude that miR-155 inhibition-induced strengthening of endothelial tight junctions after oxygen-glucose deprivation is mediated via its direct target protein claudin-1.

Dataset Information

LncRNA-Profile-Based Screening of Extracellular Vesicles Released from Brain Endothelial Cells after Oxygen-Glucose Deprivation.

Publications

LncRNA-Profile-Based Screening of Extracellular Vesicles Released from Brain Endothelial Cells after Oxygen-Glucose Deprivation.

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