OxLDL Mediates Brain Vascular Smooth Muscle Cell LOX‐1 Splicing, Oxidative Stress, and Inflammation Following Ischemic‐like Injury

Abstract

A critical risk factor known to mediate vascular injury and found to be clinically correlated with acute ischemic stroke is elevated oxidized low‐density lipoprotein (OxLDL) levels. OxLDL, via the lectin‐like OxLDL receptor 1 (LOX‐1), has been shown to alter vascular wall function in part by mediating mechanisms associated with oxidative stress and inflammation. LOX‐1 is characterized by three alternative splice variants. Variant (V)1 corresponds to the full‐length protein that is able to bind OxLDL; whereas, this binding activity is disrupted in V2 and V3. LOX‐1 splice variant characterization as well as mediators of inflammation and oxidative stress in the cerebrovasculature has yet to be addressed following OxLDL exposure alone or in the context of acute ischemic injury. Therefore, we investigated the expression profile of LOX‐1 (V1, 2, 3), proinflammatory mediators (PTGS2, IL‐1β), and oxidative stress mediator (SOD1) in primary male human brain vascular smooth muscle cells (VSMCs) following an in vitro acute ischemic injury (HGD; hypoxia plus glucose depravation) ± OxLDL. We hypothesized that HGD would alter LOX‐1 variant expression as well as induce a proinflammatory and oxidative stress response that would be exacerbated by OxLDL via LOX‐1 receptor. VSMCs were preconditioned with human OxLDL (50 and 25μg/dL; 12h) or vehicle. Next VSMCs were treated with BI‐0115 (selective LOX‐1 inhibitor; 10 μM) or vehicle (<0.1% DMSO) for 0.5h prior to being exposed to normoxia (21% O2) or HGD (1% O2) for 6h in the continued presence of OxLDL or vehicle. LOX‐1 variants were visualized using semi‐quantitative RT‐PCR analysis. PTGS2, IL‐1β, and SOD1 gene expression were assessed using quantitative real time‐PCR. COX‐2 glycosylation (active form) was measured via western blotting and used as a functional proinflammatory outcome. VSMCs exhibited a differential expression profile of the LOX‐1 variants under normoxia: V1 > V3 > V2. OxLDL alone increased V1 expression, had no effect on V2 levels, and increased V3 expression in a dose dependent manner. Following HGD, we observed a decrease in V1 expression with no change in V2 or V3 in comparison to normoxia. HGD plus OxLDL increased V1 and V2 expression; however, V2 expression was OxLDL dose dependent. Similarly, V3 expression was increased, however only at OxLDL 50 μg/dL in comparison to HGD alone. We next observed that HGD increased PTGS2, IL‐1β, and SOD1 mRNA expression, while OxLDL alone increased IL‐1β and SOD1 in a dose and LOX‐1 receptor dependent manner. Paradoxically HGD plus OxLDL decreased levels of IL‐1β and SOD1 in a LOX‐1 dependent manner. Moreover, LOX‐1 inhibition as well as OxLDL alone decreased levels of PTGS2 compared to HGD plus vehicles. Finally, we observed that HGD and OxLDL individually increased COX‐2 glycosylation, suggesting enzyme activation. In conclusion, the beneficial down‐regulation of LOX‐1 V1 by HGD was attenuated by the presence of OxLDL, suggesting that the combinatory effect predisposes the cerebrovasculature to an increased OxLDL/LOX‐1 response during acute ischemic stroke. Furthermore, LOX‐1 inhibition and the resulting attenuation of OxLDL‐mediated increases in VSMC IL‐1β and SOD1 transcription suggests that LOX‐1 may be a potential therapeutic target for patients with a higher risk of stroke.