Abstract 127: Genetic Ablation of Alternatively-spliced Extra Domain A of Fibronectin Protects Hypercholesterolemic Mice from Acute Stroke Injury
Background and Hypothesis: Fibronectin containing the alternatively-spliced extra domain A (EDA+-FN) is absent in the arteries or circulation of healthy humans and mice; but is specifically expressed in the endothelium of atherosclerotic arteries, and elevated in circulation during pathological settings, including stroke. We tested the hypothesis that deletion of EDA in FN protects hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice from acute stroke injury.
Methods: We generated EDAhi/hi/ApoE-/- mice (constitutively overexpress EDA+-FN in plasma) and EDA-/-/ApoE-/- mice (EDA-deleted allele of FN). ApoE-/-mice (express EDA+-FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings) were used as control. Susceptibility to thrombosis was assessed using FeCl3-induced carotid thrombosis model. Stroke outcome was evaluated in male mice following 60 min of ischemia/ 23 h of reperfusion injury by measuring infarct size, neurological deficit and markers of inflammation within the infarct and surrounding regions (immunohistochemistry).
Results: Using intravital microscopy, we found that the time to stable occlusion of the carotid artery was accelerated in EDAhi/hi/ApoE-/- mice (P<0.05 vs ApoE-/- mice), whereas EDA-/-/ApoE-/- mice showed opposite effect. EDA-/-/ApoE-/- mice had smaller infarcts and improved neurological outcome (P<0.01 vs. ApoE-/- mice), concordant with decreased vascular inflammation (neutrophils, macrophages, and NF-κB p65 expression). Conversely, EDAhi/hi/ApoE-/- mice had larger infarcts and more severe neurological deficits, concordant with increased vascular inflammation (P<0.01 vs. ApoE-/- mice). Because EDA+-FN activates toll-like receptor 4 (TLR4) in vitro, we investigated the role of TLR4 in the EDA+-FN-mediated aggravated stroke injury. Genetic ablation of TLR4 in EDAhi/hi/ApoE-/- mice completely reversed aggravated stroke injury (P<0.05 vs. EDAhi/hi/ApoE-/- mice), whereas TLR4 deficiency in EDA-/-/ApoE-/- had no effect.
Conclusions: EDA+-FN is pro-thrombotic and pro-inflammatory, and promotes acute stroke injury via a TLR4 -dependent mechanism. We suggest that monitoring plasma EDA+-FN levels may have prognostic value for patients at high risk for stroke.
Author Disclosures: A. Ahmad: None. C. Gandhi: None. A. Chauhan: Research Grant; Significant; American Society of Hematology.
- © 2014 by American Heart Association, Inc.