Endothelin receptor blockade ameliorates vascular fragility in endothelial cell-specific Fli-1-knockout mice by increasing Fli-1 DNA binding ability.

Abstract

OBJECTIVE:

It is generally accepted that blockade of endothelin receptors has potentially beneficial effects on vasculopathy associated with systemic sclerosis (SSc). The aim of this study was to clarify the molecular mechanism underlying these effects using endothelial cell-specific Fli-1-knockout (Fli-1 ECKO) mice, an animal model of SSc vasculopathy.

METHODS:

Levels of messenger RNA for target genes and the expression and phosphorylation levels of target proteins were determined in human and murine dermal microvascular endothelial cells by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. The binding of Fli-1 to the target gene promoters was evaluated using chromatin immunoprecipitation. Expression levels of Fli-1 and α-smooth muscle actin in murine skin were evaluated using immunohistochemistry. Vascular structure and permeability were evaluated in mice injected with fluorescein isothiocyanate-dextran and Evans blue dye, respectively.

RESULTS:

In human dermal microvascular endothelial cells, endothelin 1 induced phosphorylation of Fli-1 at Thr(312) through the sequential activation of c-Abl and protein kinase Cδ, leading to a decrease in Fli-1 protein levels as well as a decrease in binding of Fli-1 to the target gene promoters, whereas bosentan treatment reversed those effects. In Fli-1 ECKO mice, 4 weeks of treatment with bosentan increased endothelial Fli-1 expression, resulting in vascular stabilization and the restoration of impaired leaky vessels.

CONCLUSION:

The vascular fragility of Fli-1 ECKO mice was improved by bosentan through the normalization of Fli-1 protein levels and activity in endothelial cells, which may explain, in part, the mechanism underlying the beneficial effects of endothelin receptor blockade on SSc vasculopathy.

• Authors: Kaname Akamata, Yoshihide Asano, Takashi Yamashita, Shinji Noda, Takashi Taniguchi, Takehiro Takahashi, Yohei Ichimura, Tetsuo Toyama, Maria Trojanowska, Shinichi Sato
• Journal: Arthritis & rheumatology (Hoboken, N.J.) (Arthritis Rheumatol) Vol. 67 Issue 5 Pg. 1335-44 (May 2015) ISSN: 2326-5205 [Electronic] United States
• PMID: 25707716 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2015, American College of Rheumatology.

Chemical References

• Actins
• Endothelin Receptor Antagonists
• Endothelin-1
• Fli1 protein, mouse
• Proto-Oncogene Protein c-fli-1
• RNA, Messenger
• Receptors, Endothelin
• Sulfonamides
• alpha-smooth muscle actin, mouse
• Proto-Oncogene Proteins c-abl
• Protein Kinase C-delta
• Bosentan

Topics

• Actins (metabolism)
• Animals
• Bosentan
• Capillary Permeability
• Cell Line
• Cells, Cultured
• Chromatin Immunoprecipitation
• Endothelial Cells (metabolism)
• Endothelin Receptor Antagonists (pharmacology)
• Endothelin-1 (metabolism)
• Gene Expression
• Humans
• Immunoblotting
• Immunohistochemistry
• Mice
• Mice, Knockout
• Microvessels (cytology)
• Phosphorylation
• Promoter Regions, Genetic
• Protein Kinase C-delta (metabolism)
• Proto-Oncogene Protein c-fli-1 (genetics, metabolism)
• Proto-Oncogene Proteins c-abl (metabolism)
• RNA, Messenger (metabolism)
• Receptors, Endothelin (metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• Scleroderma, Systemic (metabolism)
• Sulfonamides (pharmacology)
• Vascular Diseases