Genetic Ablation of Extra Domain A of Fibronectin in Hypercholesterolemic Mice Improves Stroke Outcome by Reducing Thrombo-Inflammation

Abstract

Background The fibronectin splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with comorbid conditions including diabetes and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe−/−) mouse. Methods and Results In a transient cerebral ischemia/reperfusion injury model, Apoe−/− mice expressing Fn deficient in EDA (Fn-EDA−/−Apoe−/− mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 vs. Apoe−/− mice). Concomitantly, intracerebral thrombosis (assessed by fibrin (ogen) deposition) and postischemic inflammation (phospho-NF-κB p65, phospho IKKα/β, IL1-β and TNFα) within lesions of Fn-EDA−/−Apoe−/− mice were markedly decreased (P<0.05 vs. Apoe−/− mice). In a FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA−/−Apoe−/− mice (P<0.05 vs. Apoe−/− mice). Genetic ablation of TLR4 improved stroke outcome in Apoe−/− mice (P<0.05) but had no effect on stroke outcome in Fn-EDA−/−Apoe−/− mice. Bone marrow transplantation experiments revealed that non-hematopoietic cell-derived Fn-EDA exacerbates stroke through TLR4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe−/− mouse 15 minutes after reperfusion significantly improved stroke outcome. Conclusions Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites

“…In contrast, FN circulates in the soluble form in plasma or accumulates in tissue as insoluble ECM components (32). There is a marked upregulation of FN in the plasma during inflammatory conditions (33,34), and there is extravasation of this plasmatic protein in the basement membrane of the inflamed tissue (35). Moreover, FN has the ability to act as an endogenous ligand for TLR4 and to activate its signaling pathway, which leads FN-PMN).…”

Protein methionine oxidation augments reperfusion injury in acute ischemic stroke

“…Sections were scanned and infarct area was analyzed using NIH ImageJ software. To correct for brain swelling due to edema after ischemia the corrected total infarct volume (%) was calculated as described (68): corrected infarct volume (%) = (volume of contralateral hemisphere − [volume of ipsilateral hemisphere − volume of infarct])/volume of contralateral hemisphere × 100.…”

“…BMT was performed in MsrA +/+ and MsrA −/− mice as described previously (68). Bone marrow cells were collected from the femurs and tibias of 6- to 7-week-old donor mice using Ficoll gradient centrifugation.…”

Toward a Biological Therapy to Improve Stroke Outcomes After Thrombolytic Therapy

“…Over the past few decades, substantial work has been undertaken to develop treatments to reduce the ischemia-reperfusion injury of thrombolytic therapy and thereby reduce further the disability and death after acute ischemic cerebral infarctions. It is in this context that the work of Dhanesha et al 4 raises hope that a biological therapy can be developed to reduce the clinical consequences of ischemia-reperfusion injury after thrombolytic therapy for acute stroke.…”

“…In the plasma under normal conditions, Fn lacks both domains, whereas cellular Fn contains either domain A or B. 6 In their study in this issue of Circulation, Dhanesha et al 4 used multiple single knockouts and double knockouts to characterize the role of Fn-EDA in the ischemia-reperfusion injury in a hypercholesterolemic mouse model of transient severe cerebral ischemia. They show conclusively in a compelling preclinical model that Fn-EDA plays a central role in the pathogenesis of cerebral ischemia-reperfusion injury and neurological outcomes.…”