1. Blocking Von Willebrand Factor for Treatment of Cutaneous Inflammation
Carina Hillgruber, Annika K. Steingräber, Birgit Pöppelmann, Cécile V. Denis, Jerry Ware, Dietmar Vestweber, Bernhard Nieswandt, Stefan W. Schneider, Tobias Goerge 
Journal of Investigative Dermatology 
Volume 134, Issue 1, Pages (January 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Cutaneous inflammation leads to von Willebrand factor (VWF) accumulation in human and murine skin. (a) Representative photograph of a patient suffering from leukocytoclastic vasculitis (LcV), and hematoxylin and eosin (H&E) staining of inflamed skin presenting inflammatory infiltrates (black arrows) and red blood cell extravasation (red arrows). Bar=100 μm. (b) Representative murine back skin with Evans Blue leakage at the three sites of immune complex–mediated vasculitis (ICV) induction (dotted circles). H&E staining of an inflammatory spot reveals massive recruitment of inflammatory cells (black arrows). Bar=100 μm. Representative images of (c) human and (d, e) murine skin biopsies immunofluorescently stained for VWF (green) and CD31 (red). Ctrl, control; ICD, irritative contact dermatitis. Bar=50 μm. Quantification results of VWF/CD31 ratio are presented in scatter dot plots of n⩾6 per group. **P<0.01 and ***P<0.001.
Journal of Investigative Dermatology  , 77-86DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
The von Willebrand factor (VWF)-blocking antibodies reduce cutaneous inflammatory response. (a) Immune complex–mediated vasculitis (ICV) is induced in three spots of the back skin (dotted circles), whereas (d) irritative contact dermatitis (ICD) induction causes one homogenous spot as visualized by Evans Blue tissue leakage. (b, e) Impact of anti-VWF antibodies on myeloperoxidase (MPO) activity (left charts) and edema formation (right charts) during ICV and ICD is shown. Data are presented as mean values±SEM of relative increase to healthy control skin (n=8–12 mice per group; ***P<0.001). (c, f) Hematoxylin and eosin (H&E) staining of biopsies from the sites of inflammation permits analysis of recruited leukocytes (arrows) in the skin of anti-VWF-treated mice versus control (ctrl) animals. Bar=50 μm.
Journal of Investigative Dermatology  , 77-86DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Mice deficient for von Willebrand factor (VWF−/−) show reduced cutaneous inflammation. Evans Blue leakage after 4 hours of (a) immune complex–mediated vasculitis (ICV) or (b) irritative contact dermatitis (ICD) in the back skin of VWF−/− mice (right panels) and wild-type (WT) controls (left panels). Please note that during ICV, tissue hemorrhage was observed in VWF−/− mice that have a known bleeding disorder. (c, d) Neutrophil infiltration (myeloperoxidase (MPO) activity, upper charts) and edema formation (biopsy weight, lower charts) on inflammatory stimulation in VWF−/− mice as compared with WT mice. For quantification, the inflammatory response of control animals was set to 100%. Results are presented as mean values±SEM of n=8 mice per group. ***P<0.001.
Journal of Investigative Dermatology  , 77-86DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
In vivo imaging of leukocyte recruitment in the dorsal skinfold chamber (DSC) and permeability assays. Mice equipped with DSC received calcein green–labeled wild-type (WT) bone marrow cells (BMCs) and anti-von Willebrand factor (anti-VWF) or control (Ctrl) IgG. Irritative contact dermatitis (ICD) was induced on the backside of the DSC. (a) Representative images of recruited calcein green–labeled leukocytes (arrows) on control IgG treatment (upper right) or anti-VWF treatment (lower right). Bar=200 μm. (b) Quantification of recruited cells in the DSC. (c) Quantification of rolling cells. (d) Vascular permeability presented as Evans Blue leakage in response to intradermal injection of histamine or vascular endothelial growth factor (VEGF). Results are presented as mean values±SEM of n=6 mice per group. *P<0.05; ***P< NS, not significant; PBS, phosphate-buffered saline.
Journal of Investigative Dermatology  , 77-86DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Antibodies against von Willebrand factor (VWF) reduce ongoing cutaneous inflammation without interfering with hemostasis. (a) Bleeding times as assessed by tail bleeding assays. In case of continuous hemorrhage, experiments were terminated after 5 minutes (dotted line). Results are presented in a scatter dot plot (n⩾4 per group). (b) Time bar illustrating the experimental procedure of therapeutic anti-VWF treatment (filled arrow) 2 hours after immune complex–mediated vasculitis (ICV) or irritative contact dermatitis (ICD) induction (empty arrow). (c, d) Evans Blue leakage, myeloperoxidase (MPO) activity, and edema formation on anti-VWF therapy. (e) ICD-induced ear swelling on interference with VWF, measured at indicated time points. Results are presented as mean values±SEM of n⩾4 mice per group. *P<0.05; **P<0.01; ***P< Ctrl, control; GPIbα, glycoprotein Ibα; NS, not significant; WT, wild type.
Journal of Investigative Dermatology  , 77-86DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions