The CD 47‐binding peptide of thrombospondin‐1 induces defenestration of liver sinusoidal endothelial cells

Background & Aims A fenestrated phenotype is characteristic of liver sinusoidal endothelial cells ( LSEC s), but liver sinusoids become defenestrated during fibrosis and other liver diseases. Thrombospondin‐1 ( TSP 1) is a matrix glycoprotein with pro‐fibrotic effects, and the CD 47‐binding fragment of TSP 1 also has anti‐angiogenic effects in endothelial cells. We hypothesized that the CD 47‐binding fragment of TSP 1 could induce defenestration in LSEC s through the Rho‐Rho kinase ( ROCK )‐myosin pathway. Methods Freshly isolated rat LSEC s were treated with TSP 1 or CD 47‐binding peptides of TSP 1. LSEC fenestration was assessed with scanning electron microscopy, and myosin phosphorylation was assessed with immuno‐fluorescence. Results Treating LSEC s with TSP 1 caused a dose‐dependent loss of fenestrae, and this effect could not be blocked by SB ‐431542, the TGF ‐β1 receptor inhibitor. A CD 47‐binding fragment of TSP 1, p4N1, was able to induce defenestration, and a CD 47‐blocking antibody, B6H12, was able to suppress p4N1‐induced defenestration. The p4N1 fragment also caused contraction of fenestra size, correlated with an increase in myosin activation. Pretreatment with Y‐237642 (a ROCK inhibitor) prevented p4N1‐induced myosin activation and fenestrae decrease. Simvastatin has also been shown to antagonize Rho‐ ROCK signalling, and we found that simvastatin pretreatment protected LSEC s from p4N1‐induced myosin activation and defenestration. Conclusions We conclude that CD 47 signals through the Rho‐ ROCK ‐myosin pathway to induce defenestration in LSEC s. In addition, our results show that simvastatin and Y‐237642 have a beneficial impact on fenestration in vitro , providing an additional explanation for the efficacy of these compounds for regression of liver fibrosis.