Beneficial Effects of LOX‐1 Inhibition on Brain Endothelial Proinflammatory Mediators and Barrier Proteins Following OxLDL Plus Acute Ischemic Injury
Authors: Wendt Trevor S. , Gonzales Rayna J. 
Published: May 2022

Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular injury is elevated levels of oxidized low‐density lipoprotein (OxLDL). Via the lectin‐like OxLDL receptor 1 (LOX‐1), OxLDL contributes to mechanisms associated with vascular dysfunction and inflammation. The impact of OxLDL/LOX‐1 on cerebrovascular endothelial dysfunction and inflammation in the setting of AIS remains to be elucidated. The aim of this study is to investigate the effect of OxLDL via LOX‐1 on endothelial proinflammatory mediator and integral barrier protein levels during in vitro ischemic‐like conditions. We hypothesized that acute ischemic injury would increase endothelial barrier dysfunction as well as inflammation and that OxLDL would exacerbate these responses in a LOX‐1 dependent manner. Primary male human brain microvascular endothelial cells (HBMEC) were preconditioned with human OxLDL (50μg/dL; 12h) or vehicle using a serum dose reported in AIS patients. Next cells were treated with BI‐0115 (selective LOX‐1 inhibitor; 10μM) or vehicle (<0.1% DMSO) for 0.5h prior to being exposed to normoxia (21% O2) or hypoxia plus glucose deprivation (HGD; 1% O2) for 6h in the continued presence of OxLDL or vehicle. Levels of HBMEC barrier protein (ZO‐1), adhesion molecule (VCAM‐1), and inflammatory proteins (iNOS, IL‐1β) were examined using FIJI mediated immunocytochemical analysis. In terms of barrier protein levels, we observed that ZO‐1 levels were decreased following HGD. Contrary to our hypothesis, OxLDL plus HGD resulted in increased levels of ZO‐1; and moreover, we observed a greater increase in ZO‐1 levels when treated with BI‐0115 alone or BI‐0115 plus OxLDL. During normoxic conditions OxLDL increased levels of ZO‐1 and this response was LOX‐1 independent. Concomitantly, HGD increased levels of the adhesion molecule, VCAM‐1. The presence of HGD plus OxLDL or OxLDL plus BI‐0115 had no effect on VCAM‐1 expression. During normoxic conditions, OxLDL increased VCAM‐1 levels in a LOX‐1 dependent manner. We next observed that HGD increased the levels of proinflammatory mediators, iNOS and IL‐1β. OxLDL under normoxic conditions significantly increased the levels of iNOS in a LOX‐1 dependent manner but had no effect on the levels of IL‐1β. HGD plus OxLDL had no effect on iNOS levels; however, treatment with BI‐0115 alone or BI‐0115 plus OxLDL significantly attenuated HGD‐mediated increases in iNOS levels. Moreover during HGD, OxLDL increased levels of IL‐1β in a LOX‐1 dependent manner suggesting that OxLDL acting through LOX‐1 leads to an increase in a central mediator of vascular inflammation and permeability. In conclusion, the beneficial effect of LOX‐1 inhibition on cerebrovascular endothelial permeability and inflammation suggests that this receptor may be a novel, viable therapeutic target in the treatment of AIS.