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Abstract
Hypoxic injury to cerebrovascular endothelial cells (ECs) after stroke leads to blood-brain barrier (BBB) dysfunction, which is commonly associated with disruptions of endothelial tight junctions (TJs) and increased permeability. Therefore, maintaining the structural integrity and proper function of the BBB is essential for the homeostasis and physiological function of the central nervous system (CNS). Our previous study revealed that autophagy functions on protecting the BBB by regulating the dynamics of Claudin-5, the essential TJ protein, under short-term starvation or hypoxia conditions. Here, we show that in zebrafish and in vitro cells, loss of membranous Claudin-5 conversely determine the occurrence of hypoxia-induced autophagy in cerebrovascular ECs. Absence of endothelial Claudin-5 could partly attenuate endothelial cell apoptosis caused by short-term hypoxic injury. Mechanism studies revealed that under hypoxic conditions, the existence of membranous Claudin-5 affects the stimulation of hypoxia inducible factor 1 subunit alpha (HIF-1a) and the inducible nitric oxide synthase (iNOS), which are responsible for the translocation of and endocytosis of caveole-packaged Claudin-5 into cytosol. Meanwhile, loss of Claudin-5 affects the generation of reactive oxygen species (ROS) and the downstream expression of BCL2/adenovirus E1B 19kDa protein interacting protein 3 (Bnip3). These together suppress the endothelial autophagy under hypoxia. This finding provides a theoretical basis for clarifying the mechanism of hypoxia-induced BBB injury and its potential protection mechanisms.
Highlights
As a main part of the neurovascular units, the blood-brain barrier (BBB) is a physical barrier for the central nervous system (CNS) and is mainly composed of endothelial cells (ECs), tight junction (TJ) proteins, capillary basement membranes, pericytes, and astrocytes (Sweeney et al, 2019; Lochhead et al, 2020)
The Claudin-5112Δ5-mutated bEnd.3 cell line was generated by the Abbreviations: BBB, Blood-brain barrier; CNS, Central nervous system; bEnd.3 cells, Brain microvascular endothelial cells; hypoxia inducible factor 1 subunit alpha (HIF-1a), Hypoxia inducible factor 1 subunit alpha; inducible nitric oxide synthase (iNOS), Inducible nitric oxide synthase; Cav-1, Caveolin-1; reactive oxygen species (ROS), Reactive oxygen species; ECs, Endothelial cells; TJ, Tight junction; Zonula Occludens-1 (ZO-1), Zonula occludens-1; MO, Morpholino; CRISPR/Cas9, Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; DCFH-DA, 2',7'-dichlorofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, Fetal bovine serum; Bnip3, BCL2/adenovirus E1B 19 kDa protein interacting protein 3; MFI, Mean fluorescence intensity; trans-endothelial electrical resistance (TEER), Trans-endothelial electrical resistance
The expression of Claudin-5 was examined by immunofluorescence staining and immunoblotting respectively, and the results indicated a total loss of Claudin-5 from cell membrane in these lines (Supplementary Figures S2B,C)
Summary
As a main part of the neurovascular units, the blood-brain barrier (BBB) is a physical barrier for the central nervous system (CNS) and is mainly composed of endothelial cells (ECs), tight junction (TJ) proteins, capillary basement membranes, pericytes, and astrocytes (Sweeney et al, 2019; Lochhead et al, 2020). TJs are mainly composed of the proteins Claudin, Occludin, and Zonula Occludens-1 (ZO-1), which form the TJ structural skeleton that limits and regulates the permeability between cells and maintains barrier function (Wilhelm et al, 2016; Kadry et al, 2020). The clinicopathological characteristics of the BBB after ischemic stroke are evacuation of the TJ structure between ECs, a decrease in Claudin-5 protein expression, and an increase in permeability (Lv et al, 2018; Gholami et al, 2020). To explore the mechanism by which Claudin-5 participates in the damage of BBB during cerebral ischemia has important clinical importance for the diagnosis and treatment of acute stroke, thrombolytic reperfusion, prognosis, and the prevention and treatment of hemorrhagic transformation after cerebral infarction
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