Session Title: Antiphospholipid Syndrome
Session Type: Abstract Submissions (ACR)
Circulating pathogenic antiphospholipid antibodies (aPL) are the hallmark of antiphospholipid syndrome (APS) and a major risk factor for ischemic stroke, with up to one third of stroke cases in patients under 50 years of age due to APS. Additionally, stroke is the most common recurrent thrombotic manifestation in APS. While aPL are convincingly prothrombotic in both venous and arterial in vivo vessel thrombosis models, their effect upon stroke size post ischemia remains unexplored. We therefore employed an established rat model of ischemic / reperfusion (I/R) stroke injury utilizing transient endovascular filament middle cerebral artery (MCA) occlusion to assess the pathogenicity of APS-derived IgG.
Polyclonal IgG was purified from sera of two female patients with APS (triple positive for lupus anticoagulant, IgG anti-cardiolipin (aCL) and anti-beta-2-glycoprotein I (aβ2GPI)) and four female healthy controls (HC). Two pooled IgG populations, APS-IgG and HC-IgG, were prepared at a final concentration of 1mg/ml and confirmed to be endotoxin-free (<0.1EU/mg).
Male Sprague-Dawley rats (200-215g) were injected once intravenously with 1mg APS-IgG or HC-IgG (5 rats per group). After 15min, a filament inserted through the common carotid artery was positioned so as to occlude the right MCA for 30min. The filament was then removed to allow reperfusion. After 24hr reperfusion, rats were sacrificed and histology was performed to determine brain infarct size, quantified by analyzing digital images scanned from 1mm brain slices stained with triphenyl tetrazolium chloride (TTC) which identifies metabolically active tissue. A small portion of the brain was kept for analysis of intracellular pathways by Western blot. Blood was collected prior to occlusion and at sacrifice. Researchers administering the IgG and performing TTC analysis were blinded to the groups.
APS-IgG induced significantly larger infarcts compared to HC-IgG (mean infarct area as a percentage of total brain area ± SD: APS-IgG 32.2±4.6% versus HC-IgG 14.3±4.5%, p<0.01).
Reduced phosphorylation of the pro-survival kinase Akt was seen in brain tissue lysates of rats injected with APS-IgG compared to HC-IgG (ratio of phosphorylated : total Akt protein ± SD: APS-IgG 0.4±0.2 versus HC-IgG 0.9±0.2, p=0.03). Interestingly, there was a strong negative correlation between infarct size and the level of Akt activation (r=-0.9, p<0.01). Brain tissue lysates showed no difference in phosphorylation of ERK, JNK or p38-MAPK between APS-IgG and HC-IgG groups. Human IgG aCL/aβ2GPI activity was present in the sera of rats treated with APS-IgG at sacrifice; human IgG was detected in brain tissue lysates from animals treated with APS-IgG and HC-IgG with no difference between the groups.
This study is the first to directly demonstrate the ability of APS-IgG to exacerbate stroke severity post I/R injury, causing larger infarcts compared to rats treated with HC-IgG. Furthermore, this finding correlated with inhibition of the pro-survival kinase Akt and warrants further validation studies to confirm Akt dependency and dissect mechanisms through which this inhibition may occur.
S. S. Pierangeli,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/igg-antiphospholipid-antibodies-enhance-stroke-damage-an-in-vivo-ischemiareperfusion-study/