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VWF-mediated thromboinflammation in stroke
ed by VWF, we performed flow cytometric analysis of single-cell suspensions prepared from brain tissue isolated from WT and VWF KO mice subjected to stroke. After stroke, mice were perfused, their brains harvested and subsequently divided into ipsilateral (affected by stroke) and contralateral (unaffected) hemispheres.
In agreement with previous reports,13,14 we observed significantly reduced cerebral infarcts in VWF KO mice, compared to WT mice (P<0.01) (Figure 1A). Twenty-four hours after stroke, the ipsilateral hemisphere showed increased numbers of infiltrated white blood cells (WBC) compared to the contralateral hemisphere in both the WT (P<0.005) and VWF KO (P<0.05) mice. However, average recruitment of WBC in the ipsilateral hemisphere of VWF KO mice was two-fold lower than in WT mice (5596 ± 1644 vs. 12435 ± 2083, respectively; P<0.05) (Figure 1B). The numbers of both myeloid and lymphoid WBC were significantly reduced in the brains of VWF KO mice com- pared to those in WT mice (P<0.05) (Figure 1C and D).
von Willebrand factor deficiency leads to reduced recruitment of inflammatory monocytes, neutrophils and T cells
To better determine which inflammatory cells were potentially recruited by VWF to the affected brain tissue during stroke, we used two antibody cocktails that allowed discrimination and quantification of recruited inflammatory monocytes, neutrophils, T cells and CD3neg lymphocytes (B cells and natural killer cells) (Table 1).
As shown in Figure 1E-H, all four subsets of immune cells were significantly increased in the ipsilateral brain of WT mice 24 h after stroke, compared to their numbers in the unaffected contralateral hemisphere. However, despite the presence of an infarct core in the affected ipsi- lateral hemisphere of VWF KO mice, the numbers of neu- trophils and T cells did not increase and remained similar to the baseline values of the contralateral hemisphere (Figure 1E and F). Hence, the absolute numbers of recruit- ed neutrophils and T cells was significantly higher in the ischemic brain of WT mice than in VWF KO mice (2017 ±733vs.512±203,P<0.05and974±184vs.244±44, P<0.01, respectively). These results suggest an important role for VWF in recruiting both neutrophils and T cells to the affected brain tissue during ischemic stroke.
A similar, but less marked, trend was observed for inflammatory monocytes (Figure 1G). In VWF KO mice, the ischemic hemisphere contained significantly more inflammatory monocytes than did the unaffected hemi- sphere, but still significantly fewer than the number of inflammatory monocytes that were recruited to the ischemic brain of WT mice (2466 ± 955 vs. 6760 ± 1414; P< 0.05).
No differences regarding CD3neg lymphocytes were observed between WT and VWF KO mice as similar numbers were recruited in the ischemic hemispheres of both groups (1162 ± 245 vs. 585 ± 188 respectively, P>0.05) (Figure 1H). Leukocyte blood counts and circulat- ing platelet-leukocyte complexes were similar between VWF KO and VWF WT mice 24 h after stroke (data not shown).
Visualization of von Willebrand factor-mediated thromboinflammation in the ischemic stroke brain
For visualization of VWF-mediated thromboinflamma- tion, immunofluorescent staining of platelets and leuko-
cytes was performed on brains obtained from VWF WT and KO mice 24 h after stroke (Figures 2 and 3). In VWF KO mice, very few platelet accumulations were found within the ipsilateral, stroke-affected brain (Figure 2A). In contrast, platelet/VWF-rich microthrombi were found frequently throughout the ipsilateral brain of VWF WT mice, underscoring the importance of VWF-mediated platelet adhesion in the ischemic stroke brain (Figure 2B- D). Platelet/VWF-rich microthrombi were absent in the contralateral hemisphere of both WT and VWF KO mice (data not shown).
Next, we visualized immune cell recruitment in VWF WT and KO mice. Since previous studies found no major role for monocytes,27 but an important detrimental role for both T cells28 and neutrophils29 in the acute phase of ischemic stroke, we focused on visualizing neutrophils and T cells in the stroke brain. To stain the vasculature in both VWF WT and KO mice, a sensitive lectin staining of the endothelium was performed (Figure 3). Using a spe- cific histological marker for neutrophils (Ly6G), we observed that neutrophils were more frequently present within the ipsilateral side of the brain of VWF WT mice than in that of VWF KO mice (Figure 3A and B). Since the smaller infarct sizes observed in VWF KO mice might bias neutrophil quantification by flow cytometry, we also quantified neutrophil recruitment in the ischemic infarct core in both VWF KO and WT mice by histology. Importantly, analysis of fixed areas of 1 mm2 in the infarct core corroborated our flow cytometric data, arguing against a nonspecific effect related to smaller infarct sizes. Quantification of neutrophil recruitment to the infarct core revealed a two-fold reduction of neutrophil density in VWF KO brains compared to that in WT mice (Figure 3C). Intriguingly, neutrophils were frequently observed within the microcirculation (Figure 3A and B). To investi- gate this further, both intravascular and extravascular neutrophils were counted in brain sections of VWF WT mice. On average, 66 ± 4% of neutrophils were found within the vasculature of the ischemic hemisphere, while the remaining neutrophils were already extravasated (Figure 3D). A comparable observation was made when we stained for T cells, which were also occasionally found within the microvasculature of VWF WT mice (Figure 3E). Due to the low number of T cells within the ischemic brain, quantification of T cells was not feasible. Lastly, T cells were virtually absent in the ipsilateral brain hemisphere of VWF KO mice (Figure 3F), confirming our flow cytometric data.
Inhibition of the von Willebrand factor A1 domain reduces the recruitment of neutrophils, monocytes and T cells and limits ischemic stroke brain injury
Given the central role of the VWF A1 domain in cere- bral ischemia/reperfusion injury,30 we next wanted to unravel its potential inflammatory contribution in ischemic stroke. To this end, we used a nanobody (KB- VWF-006bv) that specifically binds the VWF A1 domain, inhibiting its interaction with the platelet receptor GPIba.22 Intravenous treatment with 10 mg/kg of the nanobody was started immediately after establishment of reperfusion. The mean residence time of the nanobody is 3.5 h which allows blocking of the VWF-A1 domain dur- ing the acute reperfusion phase. As a control, a nonspecif- ic nanobody (KB-VWF-004bv) was administered. Twenty-four hours after stroke, mice treated with the
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