Anti-osteopontin antibody treatment reduces osteopontin and CD44 receptor expression in microvascular endothelial cells and pericytes after acute ischemic stroke. (a,b) Representative images of immunofluorescence staining for osteopontin (OPN, red, inset), CD44 receptor (green, inset) and cell-specific markers (white, overlay) including podocalyxin for endothelial cells (a) and CD13 for pericytes (b) in the infarct core and peri-infarct region of Ctrl IgG and α-OPN antibody-treated mice 24 h post-ischemic stroke. White arrowheads indicate osteopontin and CD44 receptor expressing endothelial cells and pericytes. (c-f) Quantification of osteopontin and CD44 receptor expression intensity (arbitrary unit, a.u.) in endothelial cells (c,d) and pericytes (e,f) in the infarct core and peri-infarct region utilizing 3 images/region/animal, n = 8 and 8 for Ctrl IgG and α-OPN antibody treatment group, respectively; § P < 0.05, **/ § § P < 0.01, ***P < 0.001 and not significant (ns) P > 0.05. *Indicates two-tailed, unpaired t-test with Welch's correction when variances were significantly different based on F-test, comparing the two treatments groups for the same region, and § indicates two-tailed, paired t-test comparison of infarct core to peri-infarct region within the same treatment group. Scale bars 50 µm and 10 µm in insets (a,b).

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... cells and pericytes (Fig. 1a,b,e,f and Supplementary Fig. S2). A similar time-dependent expression pattern was observed for co-localized osteopontin and CD44 receptor, suggesting ligand-receptor interaction. The co-localization of osteopontin and CD44 receptor was most prominent 15 h post-tMCAO in all the individual peri-infarct NVU cells ( Supplementary Figs. S3 and S4), particularly in endothelial cells and pericytes. In the contralateral hemisphere, where osteopontin and CD44 receptor expression was low, osteopontin-CD44 receptor interaction was almost absent during the entire observation period (Supplementary Fig. S5). Moreover, the steep increase of osteopontin and CD44 receptor expression, and ...

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... of the glial activation markers GFAP in peri-infarct astrocytes and IBA1 in microglia/macrophages ( Supplementary Fig. S4) compared to those of the contralateral hemisphere ( Supplementary Fig. S5). A time-dependent increase of osteopontin and CD44 receptor expression ( Supplementary Fig. S1), and osteopontin-CD44 receptor co-localization ( Supplementary Figs. S3, S4) was also detected in NVU cells in the infarct core when compared to NVU cells in the contralateral hemisphere ( Supplementary Figs. S2, S5). The only exception being microglia/macrophages, where the expression pattern was less prominent in the infarct core ( Supplementary Figs. S2, S5). In this respect, immunofluorescence staining of ...

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... and significantly reduced hemorrhagic transformation of stroke lesions ( Fig. 2d), edema ( Fig. 2e) and infarct volumes (Fig. 2f). Importantly, improved outcome in mice receiving the combined anti-OPN antibody treatment in early and late acute phase was associated with a highly significant reduction of osteopontin and CD44 receptor expression (Figs. 3, 4), and osteopontin-CD44 receptor interaction (Supplementary Figs. S6, S7) in all the individual NVU cell types in the peri-infarct region compared to vehicle-treated animals. This was furthermore associated with significantly decreased expression of glial activation markers GFAP in astrocytes and IBA1 in microglia/macrophages ( ...

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... ( Supplementary Fig. S7), indicating an inhibitory effect of the anti-OPN antibody therapy on glial activation. In the infarct core, combined anti-OPN antibody treatment in early and late acute phase led to significant reduction of CD44 expression in endothelial cells and astrocytes, osteopontin expression in microglia/macrophages (Figs. 3, 4), and osteopontin-CD44 interaction in astrocytes and microglia/macrophages ( Supplementary Fig. S7). However, the therapeutic efficacy of the neutralizing antibody was lower than in the peri-infarct region. Osteopontin and CD44 receptor expression ( Supplementary Fig. S8), and consequently their co-localization in NVU cells in the ...