von Willebrand factor contributes to poor outcome in a mouse model of intracerebral haemorrhage

Abstract

Spontaneous intracerebral haemorrhage (ICH) is the most devastating stroke subtype and has no proven treatment. von Willebrand factor (VWF) has recently been demonstrated to promote inflammation processes. The present study investigated the pathophysiological role of VWF after experimental ICH. Functional outcomes, brain edema, blood-brain barrier (BBB) permeability, cerebral inflammation and levels of intercellular adhesion molecule-1 (ICAM-1) and matrix metalloproteinase-9 (MMP-9) were measured in a mouse model of ICH induced by autologous blood injection. We show that VWF were increased in the plasma and was accumulated in the perihematomal regions of mice subjected to ICH. Injection of VWF resulted in incerased expression of proinflammatory mediators and activation of ICAM-1 and MMP-9, associated with elevated myeloperoxidase, recruitment of neutrophils and microglia. Moreover, mice treated with VWF showed dramatically decreased pericyte coverage, more severe BBB damage and edema formation, and neuronal injury was increased compared with controls. In contrast, blocking antibodies against VWF reduced BBB damage and edema formation and improved neurological function. Together, these data identify a critical role for VWF in cerebral inflammation and BBB damage after ICH. The therapeutic interventions targeting VWF may be a novel strategy to reduce ICH-related injury.

Introduction

von Willebrand factor (VWF) is an ultra-large multimeric glycoprotein, which is present in Weibel-Palade bodies of endothelial cells and alpha granules of platelets and is released in circulation upon activation¹. VWF plays a crucial role in platelet adhesion and aggregation after vascular injury and under conditions of high shear stress^2,3. Recently, VWF was also shown to mediate leukocyte extravasation and inflammatory response. Animal studies have shown that VWF deficiency reduced inflammatory cell recruitment, atherosclerotic lesion and ischemic cerebral injury^4,5,6, and blocking antibody against VWF inhibited neutrophil extravasation in peritonitis and was protected from myocardial ischemic injury^5,7.

Spontaneous intracerebral haemorrhage (ICH), defined as spontaneous bleeding from intraparenchymal blood vessels in the absence of trauma, represents roughly 10–15% of all stroke subtypes^{Gelatin zymography}

Zymography was performed as described47. In brief, 40 μg protein samples were mixed with SDS sample buffer and loaded onto 10% Tris-Glycine gels containing 0.1% gelatin. After electrophoresis, gels were washed with 2.5% Triton X-100 for 30 minutes and incubated in develo** buffer (50 mM Tris pH 7.6, 0.2 mM NaCl, 5 mM CaCl₂, 0.02% Brij-35) at 37 °C for 48 hours. Gels were stained with 0.5% Coomassie blue R-250 (Sigma-Aldrich) for 3 hour and destained appropriately. Gels were scanned and analyzed using Quantity One software (Bio-Rad).

Immunofluorescence

Twenty four hours after ICH induction, mice were euthanized by overdose of chloral hydrate, perfused transcardially with ice-cold PBS and 4% paraformaldehyde (pH 7.4; Sigma-Aldrich). The brains were removed and fixed in 4% paraformaldehyde overnight and then cryoprotected in 30% sucrose overnight at 4 °C. Frozen sections were cut in the coronal plane at 20 μm on a cryostat (Leica Microsystems Inc., Buffalo Grove, IL, USA). Immunohistochemistry was performed as described^49,50. Images were obtained using an Olympus BX 51 microscope and an Olympus FV 1,000 confocal microscope. Colocalization was verified and reconstructed using Olympus FV 10-ASW software. Primary antibodies used were: rabbit anti-VVW (ab154193, Abcam), rabbit anti-human VWF (Dako), rat anti-CD31 (PECAM-1, BD Pharmingen, San Diego, CA, USA), rat anti-mouse LY-6B.2 (AbD Serotec, Raleigh, NC, USA), goat anti-Iba1 (Ionized calcium binding adaptor molecule 1, Abcam, Cambridge, MA, USA) and FITC-conjugated rat anti-mouse aminopeptidase N (CD13) (BD Pharmingen). Secondary antibodies used were: Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG), Alexa Fluor 594 donkey anti-rat IgG, Alexa Fluor 594 donkey anti-goat IgG, and Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen). Nuclei were stained with DAPI. For quantification of LY6B and Iba1 labeled cells in the perihematoma area, four fields from each section were captured under 40X objective. Each image was traced using ImageJ software. The total numbers of positive cells in the traced area were counted and expressed as per mm². Pericyte coverage was determined as a percentage of CD13-positive area covering CD31-positive area in 0.42 mm² regions. For each animal, 3 sections (400 μm apart) from the ipsilateral hemispheres were analyzed.

Western blotting

Twenty four hours after ICH induction, mice were transcardially perfused and the brains were removed and separated into hemispheres ipsilateral and contralateral to haemorrhage. Ipsilateral hemispheres were cut into 4 mm thickness block around the needle track. The brain tissues were homogenized in RIPA lysis buffer (Millipore) containing protease inhibitor cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Equal amounts of protein samples were loaded on 10% SDS-PAGE gel, electrophoresed, and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 and incubated with goat anti-mouse ICAM-1 (R&D systems), and rabbit anti-β-actin (Cell Signaling Technology) antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected with an enhanced chemoluminescence solution (Millipore) and quantified by scanning densitometry using a Bio-Image Analysis System (Bio-Rad).

BBB permeability

Mice were injected with 2% Evans blue in PBS (4 ml/kg; Sigma-Aldrich) at 21 hours after ICH induction, followed 3 hours later by transcardiac perfusion. The hemorrhagic brain hemispheres were removed and placed in formamide (Sigma-Aldrich) for 72 hours. The amount of extravasated Evans blue dye was evaluated at 620 nm⁴⁹.

Neurobehavioral scores

Neurological deficits were assessed by an investigator blinded to the treatment of the animals at 1 and 3 days after ICH. For the quantification of neurological deficits, a 5 point neurological score was employed: 0, no neurological deficit; 1, forelimb weakness; 2, spontaneous circling; 3, partial paralysis on one side; 4, absence of spontaneous movement or unconsciousness; 5, death.

Statistics

Data are represented as means ± standard errors of the means. Statistical analysis were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. Differences were determined by using the Student two-tailed t test when two groups were compared. Behavior data were compared using Mann-Whitney U test. P values less than 0.05 were considered as statistically significant.