The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells

Liver Int. 2013 Oct;33(9):1386-97. doi: 10.1111/liv.12231. Epub 2013 Jun 26.


Background & aims: A fenestrated phenotype is characteristic of liver sinusoidal endothelial cells (LSECs), but liver sinusoids become defenestrated during fibrosis and other liver diseases. Thrombospondin-1 (TSP1) is a matrix glycoprotein with pro-fibrotic effects, and the CD47-binding fragment of TSP1 also has anti-angiogenic effects in endothelial cells. We hypothesized that the CD47-binding fragment of TSP1 could induce defenestration in LSECs through the Rho-Rho kinase (ROCK)-myosin pathway.

Methods: Freshly isolated rat LSECs were treated with TSP1 or CD47-binding peptides of TSP1. LSEC fenestration was assessed with scanning electron microscopy, and myosin phosphorylation was assessed with immuno-fluorescence.

Results: Treating LSECs with TSP1 caused a dose-dependent loss of fenestrae, and this effect could not be blocked by SB-431542, the TGF-β1 receptor inhibitor. A CD47-binding fragment of TSP1, p4N1, was able to induce defenestration, and a CD47-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 LSECs from p4N1-induced myosin activation and defenestration.

Conclusions: We conclude that CD47 signals through the Rho-ROCK-myosin pathway to induce defenestration in LSECs. 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.

Keywords: capillarization; discontinuous endothelium; mechanical signal transduction; myosin light chain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Analysis of Variance
  • Animals
  • CD47 Antigen / genetics
  • CD47 Antigen / pharmacology*
  • Endothelial Cells / drug effects*
  • Endothelial Cells / ultrastructure
  • Fluorescent Antibody Technique
  • Image Processing, Computer-Assisted
  • Liver / cytology*
  • Microscopy, Electron, Scanning
  • Molecular Sequence Data
  • Myosins / metabolism
  • Phosphorylation
  • Rats
  • Signal Transduction / physiology*
  • rho-Associated Kinases / metabolism


  • CD47 Antigen
  • rho-Associated Kinases
  • Myosins