Antiphospholipid antibodies exacerbate damage following oxygen deprivation-reperfusion injury in anin vivomodel for stroke and inex vivoblood derived endothelial cells

Background Prothrombotic antiphospholipid antibodies (aPL) found in patients with antiphospholipid syndrome (APS) are a recognised risk factor for ischemic stroke. However, it is unclear if aPL cause injury post thrombolysis leading to worse outcomes. We investigated whether aPL exacerbate reperfusion injury and sought to translate our findings in endothelial colony forming cells (ECFC) isolated from patients with APS. Methods Transient ischemic stroke was induced in adult rats injected with serum-derived IgG from patients with APS (APS-IgG, containing aPL) or healthy controls (HC-IgG). Infarct size and intracellular signalling processes involved in ischemia-reperfusion injury were determined post reperfusion. In vitro , human umbilical vein endothelial cells (HUVEC) treated with IgG, as well as APS and HC ECFC, were exposed to hypoxia (0.1% O 2 ). Cell death and relevant signalling mechanisms were assessed following reperfusion and compared to matched normoxic cultures. Results In vivo , APS-IgG induced >2-fold larger infarcts and lower levels of active phosphorylated Akt, a key pro-survival kinase, compared to HC-IgG. In vitro , aPL-mediated cell death and suppression of Akt phosphorylation was confirmed in HUVEC exposed to IgG and hypoxia-reperfusion. Consistent with these findings, higher rates of cell death and reduced Akt phosphorylation following reperfusion were observed in ex vivo APS ECFC compared to HC ECFC. Treatment with the immunomodulating agent hydroxychloroquine ameliorated ECFC death and this effect was more pronounced in APS-derived cells. Conclusion Patient-derived IgG aPL exacerbate cell death following reperfusion in a novel in vivo stroke model for APS, as well as in vitro HUVEC cultures. These observations are mimicked in ex vivo APS ECFC. Our findings describe a novel pathogenic role for aPL in mediating tissue injury in addition to their known thrombogenic properties and indicate potential for pharmacological intervention.