Antiphospholipid antibodies exacerbate damage following oxygen deprivation-reperfusion injury in an in vivo model for stroke and in ex vivo blood 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% O2). 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement C.P is supported by Versus Arthritis (21223) and the Imperial College-Wellcome Trust Institutional Strategic Support Fund. D.J.S. was supported by British Heart Foundation Intermediate and Senior Basic Science Research Fellowships (FS/15/33/31608, FS/SBSRF/21/31020), the BHF Centre for Regenerative Medicine (RM/17/1/33377), the MRC (MR/R026416/1) and the Wellcome Trust (212937/Z/18/Z). D.J.A is funded by the MRC (MR/V037633/1). This work was partly funded by the Rosetrees Trust (C.P, I.P.G). Infrastructure support for this research was provided by the NIHR Imperial Biomedical Research Centre (BRC). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Patient & controls consent - ethical approval: approved by the appropriate local, regional and/or national Ethics board (University College London, Imperial College London, University of Texas Medical Branch). Animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Guidance for the Operation of Animals (Scientific Procedures) Act 1986 (Home Office, London, United Kingdom). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data can be made available following request to the corresponding author.