Increased thrombospondin-2 in human fibrosclerotic and stenotic aortic valves Virva Pohjolainen, Erja Mustonen, Panu Taskinen, Juha Näpänkangas, Hanna.

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Increased thrombospondin-2 in human fibrosclerotic and stenotic aortic valves Virva Pohjolainen, Erja Mustonen, Panu Taskinen, Juha Näpänkangas, Hanna Leskinen, Pauli Ohukainen, Tuomas Peltonen, Jani Aro, Tatu Juvonen, Jari Satta, Heikki Ruskoaho, Jaana Rysä Atherosclerosis Volume 220, Issue 1, Pages 66-71 (January 2012) DOI: 10.1016/j.atherosclerosis.2011.10.003 Copyright © 2011 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 (A) The proportion of calcified area in human aortic valve cusps to total aortic valve cusp area. (B) Mean number of vascular structures in square millimeter in human aortic valve cusps. Results are mean±SD; P-values are versus control group (PCtrl) and fibro(sclero)sis group (PFibr). Ctrl, control group (n=7); Fibr, fibro(sclero)sis group (n=5); AS, aortic stenosis group (n=19). Atherosclerosis 2012 220, 66-71DOI: (10.1016/j.atherosclerosis.2011.10.003) Copyright © 2011 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Gene expression of (A) TSP-1, (B) TSP-2, (C) TSP-3, and (D) TSP-4 in the different stages of calcific aortic valve disease. Values are mean±SD; P-values are versus control group (PCtrl). Ctrl, control group (n=8); Fibr, fibro(sclero)sis group (n=8); AS, aortic stenosis group (n=24). Atherosclerosis 2012 220, 66-71DOI: (10.1016/j.atherosclerosis.2011.10.003) Copyright © 2011 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Representative images demonstrating TSP-2 expression and semiquantitative immunoreactivity for TSP-2 in human aortic valve cusps. (A) Aortic valve endothelial cells (arrow) show positive TSP-2 immunostaining. (B) Secondary anti-goat antibody staining as negative control shows no staining in endothelium (arrow), same area as in A. (C) TSP-2 immunostaining showing endothelial immunoreactivity in neovessels of calcified aortic valve (arrow). However, not all of the vessels show positive staining in endothelium (double arrow). Images D, E and F show an area of myofibroblast proliferation in the same aortic valve as in C. (D) A distinct TSP-2 positivity in cells that are mainly myofibroblasts, confirmed by their immunoreactivity for αSMA (E). There are only a few macrophages, visualized by CD68 immunostaining (F). (G) The quantity of TSP-2 immunostaining in respective cell types: ++=moderate immunostaining. Ctrl, control group (n=3); Fibr, fibro(sclero)sis group (n=3); AS, aortic stenosis group (n=6). Atherosclerosis 2012 220, 66-71DOI: (10.1016/j.atherosclerosis.2011.10.003) Copyright © 2011 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 (A) NF-κB binding activity in human aortic valves. (B) Akt-pathway activation in human aortic valves. Results are mean±SD. Ctrl, control group (n=4–10); AS, aortic stenosis group (n=14–20). (C) Gel mobility shift assay of nuclear extracts from human aortic valves. Nuclear extracts were incubated with radiolabelled NF-κB oligonucleotide probe. (D) Representative western blots of phospho-Akt (P-Akt) and total-Akt (Tot-Akt) protein levels in human aortic valves. Ctrl, control group; AS, aortic stenosis group. Of note, considerable interindividual variability was seen in NF-κB binding activity and Akt-pathway activation in AS-group, most likely due to the board spectrum of phenotypic variation of aortic stenosis. Atherosclerosis 2012 220, 66-71DOI: (10.1016/j.atherosclerosis.2011.10.003) Copyright © 2011 Elsevier Ireland Ltd Terms and Conditions