Abstract
BackgroundThe blood–brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro.MethodsWe used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1β (IL-1β). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR.ResultsOGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1.ConclusionsOur data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.
Highlights
The blood–brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function
oxygen-glucose deprivation (OGD) and IL-1 induce loss of BBB integrity in vitro In order to investigate the effect of hypoxia and/or inflammation on BBB integrity, we first tested the effect of OGD and/or IL-1 on endothelial cell morphology, as well as expression and cellular localisation of specific tight junction proteins involved in maintaining BBB integrity, namely occludin and zona occludens-1 (ZO-1)
IL-1β treatment alone (4 h) induced a significant change in endothelial cell morphology similar to that seen after OGD and reoxygenation (Figs. 2a, b and 3a), whilst no change in cellular morphology was seen after OGD and reoxygenation in the presence of IL-1β (Figs. 2a, b and 3a)
Summary
The blood–brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. The blood–brain barrier (BBB) of the central nervous system (CNS) is a specialised vascular structure that is essential for normal brain function and homeostasis (see [1] for review). It is composed of brain endothelial cells (BEC), pericytes, and astrocyte endfeet that are embedded in a network of extracellular matrix (ECM) molecules that form the basement membrane of the cerebrovasculature (see [2, 3] for review). BEC interaction with the basement membrane occurs via integrins, the major cell surface adhesion molecules responsible for anchoring endothelial cells to the ECM [3, 6] that are critically involved in the interaction between the basement membrane and astrocyte endfeet, thereby further contributing to BBB integrity [3]
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