Brain microvascular endothelial cell‐derived exosomes transmitting circ_0000495 promote microglial M1‐polarization and endothelial cell injury under hypoxia condition

Human brain microvascular endothelial cells (HBMVECs) and microglia play critical roles in regulating cerebral homeostasis during ischemic stroke. However, the role of HBMVECs‐derived exosomes in microglia polarization after stroke remains unknown. We isolated exosomes (Exos) from oxygen glucose deprivation (OGD)‐exposed HBMVECs, before added them into microglia. Microglia polarization markers were tested using RT‐qPCR or flow cytometry. Inflammatory cytokines were measured with ELISA. Endothelial cell damage was assessed by cell viability, apoptosis, apoptosis‐related proteins, oxidative stress, and angiogenic activity using CCK‐8, flow cytometry, western blot, ELISA, and endothelial tube formation assay, respectively. We also established middle cerebral artery occlusion (MCAO) mice model to examine the function of circ_0000495 on stroke in vivo. Our study found that HBMVECs‐Exos reduced M2 markers (IL‐10, CD163, and CD206), increased M1 markers (TNF‐α, IL‐1β, and IL‐12), CD86‐positive cells, and inflammatory cytokines (TNF‐α and IL‐1β), indicating the promotion of microglial M1‐polarization. Microglial M1‐polarization induced by HBMVECs‐Exos reduced viability and promoted apoptosis and oxidative stress, revealing the aggravation of endothelial cell damage. However, circ_0000495 silencing inhibited HBMVECs‐Exos‐induced alterations. Mechanistically, circ_0000495 adsorbed miR‐579‐3p to upregulate toll‐like receptor 4 (TLR4) in microglia; miR‐579‐3p suppressed HBMVECs‐Exos‐induced alterations via declining TLR4; furthermore, Yin Yang 1 (YY1) transcriptionally activated circ_0000495 in HBMVECs. Importantly, circ_0000495 aggravated ischemic brain injury in vivo via activating TLR4/nuclear factor‐κB (NF‐κB) pathway. Collectively, OGD‐treated HBMVECs‐Exos transmitted circ_0000495 to regulate miR‐579‐3p/TLR4/NF‐κB axis in microglia, thereby facilitating microglial M1‐polarization and endothelial cell damage. In OGD‐subjected BMVECs, the upregulated YY1 transcriptionally activated circ_0000495, then circ_0000495 was transported by exosomes to microglial cells. In microglial cells, circ_0000495 targeted miR‐579‐3p to upregulate TLR4, leading to M1 microglial polarization, which in turn promoted apoptosis and inhibited angiogenesis of BMVECs.