EDA-Containing Fibronectin Aggravates Ischemic Brain Injury In Mice

Fibronectin (FN) is a dimeric glycoprotein that plays an important role in several cellular processes, such as embryogenesis, malignancy, hemostasis, wound healing and maintenance of tissue integrity. FN is a ligand for many members of the integrin family (e.g. αIIbβ3, α5β1, α4β1, α9β1, αvβ3 and αvβ5) and also binds to thrombosis-related proteins including heparin, collagen and fibrin. FN generates protein diversity as a consequence of alternative processing of a single primary transcript. Two forms of FN exist; soluble plasma FN (pFN), which lacks the alternatively-spliced Extra Domain A (EDA); and insoluble cellular FN (cFN), which contains EDA. FN containing EDA (EDA+FN) is normally absent in plasma of human and mouse but EDA+FN has been found in patients with vascular injury secondary to vasculitis, sepsis, acute major trauma or ischemic stroke. We tested the hypothesis that elevated levels of plasma EDA+FN increase brain injury in an experimental model of ischemic stroke in mice.

We used two genetically modified mouse strains: EDA^+/+ mice contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+FN, whereas EDA^-/- mice contain an EDA-null allele of the EDA exon and express only FN lacking EDA. Control EDA^wt/wt mice contain the wild-type FN allele. Transient focal cerebral ischemia was induced by 60 minutes of occlusion of the right middle cerebral artery with a 7.0 siliconized filament in male mice (8-10 weeks in age). Mice were anesthetized with 1–1.5% isoflurane mixed with medical air. Body temperature was maintained at 37°C ± 1.0 using a heating pad. Laser Doppler flowmetry was used to confirm induction of ischemia and reperfusion. At 23 hours after MCAO, mice were evaluated for neurological deficits as a functional outcome and were sacrificed for quantification of infarct volume. For morphometric measurement eight 1 mm coronal sections were stained with 2% triphenyl-2, 3, 4-tetrazolium-chloride (TTC). Sections were digitalized and infarct areas were measured blindly using NIS elements.

In EDA^+/+ mice the percentage of infarct volume (mean ± SEM: 37.25 ± 4.11, n= 12,) in the ipsilateral (ischemic) hemisphere was increased by approximately two-fold compared to EDA ^wt/wt mice (mean ± SEM: 22.33 ± 3.39, n=11; P< 0.05, ANOVA) or EDA^-/- mice (mean ± SEM: 21.72 ± 2.94, n=9). Regional cerebral blood flow during ischemia was not different among groups as assessed by laser Doppler flowmetry. The percentage increase in infarct volume in the EDA^+/+ mice correlated well with severe neurological deficits (motor-deficit assessed by a four-point neurological score scale) compared to EDA ^wt/wt or EDA^-/- mice. Because both thrombosis and inflammation contributes to brain injury during ischemic stroke, we investigated the time to form an occlusive thrombus in ferric-chloride carotid artery injury model by intravital microscopy. EDA^+/+ mice demonstrated significantly faster time to occlusion (mean ± SEM: 12.35 ± 1.51 n=12,) compared to EDA^wt/wt (Mean ± SEM: 17.27 ± 1.72 min, n=13, P<0.05, ANOVA) or EDA^-/- (Mean ± SEM: 15.61 ± 1.76, n=11) mice. Additionally, the inflammatory response in the ischemic region was increased by two fold in EDA^+/+ mice compared to EDA ^wt/wt and EDA^-/- mice as sensed by myeloperoxidase activity and immunohistochemical analysis of neutrophils.

EDA-containing FN is pro-thrombotic and pro-inflammatory, and aggravates ischemic brain injury in an experimental model of stroke in mice. The presence of EDA⁺FN in plasma may be a risk factor for vascular injury secondary to ischemic stroke.

No relevant conflicts of interest to declare.