Session Information
Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's - Pathogenesis, Animal Models and Genetics
Session Type: Abstract Submissions (ACR)
Background/Purpose: Endothelial-to-mesenchymal transition (EndoMT) may be a crucial pathway in generating activated myofibroblasts, cells that play a pivotal role in the development of tissue and organ fibrosis in diseases such as Systemic Sclerosis (SSc). It has been previously demonstrated that endothelin 1 (ET-1) synergistically enhances TGF-β-induced EndoMT in vitro in murine lung endothelial cells (ECs). The purpose of this study was to develop a mouse model expressing a green distinct fluorescent label in ECs (green fluorescent protein; GFP) and a red fluorescent label in fibroblasts (mCherry protein) to allow monitoring of EndoMT in vivo.
Methods: Homozygous Tie2GFP transgenic mice expressing GFP under control of the EC specific Tie2 promoter (Tie2GFP mice) were crossed with homozygous mice expressing a doxycycline inducible red fluorescent mCherry protein in mesenchymal cells and fibroblasts under control of the Col1a1 promoter (Col1mCherry mice) to generate heterozygous bifluorescent Tie2GFP-Col1mCherry mice. At 4 weeks of age, osmotic pumps containing either saline, 2.5 µg TGF-β, or 2.5 µg TGF-β+5.0 µg ET-1 were implanted subcutaneously in the right intrascapular region of the mice (2 mice per treatment group). The pumps deliver their contents at a rate of 0.5 µl/h over a 2 week period. Mice received IP injections of 1 mg/kg doxycycline every other day starting at 2 weeks post-implantation of the pump and were sacrificed one week later. Both lungs and two skin samples, one at the pump dispersal site and one opposite to the pump site, were isolated. A portion of each tissue was fixed in formalin and processed for histopathologic analysis (hematoxyllin/eosin and Masson’s trichrome stains) whereas another portion was frozen and sectioned for evaluation of fluorescence. Another sample portion of each tissue was hydrolyzed for measurement of hydroxyproline content.
Results: Histopathology studies in samples from TGF-β-treated mice showed mononuclear cell infiltration and peribronchial fibrosis and diffuse interstitial fibrosis in lungs. Dermal fibrosis was present in both samples of skin. ET-1 synergistically enhanced the severity of fibrosis in all three tissues. Hydroxyproline levels in skin taken from the site of the osmotic pump demonstrated a 61% increase in response to TGF-β alone and a 113% increase (~2.2 fold) in response to TGF-β+ET-1 whereas skin from the opposite side of the back displayed a 29% increase in response to TGF-β alone and a 102% increase (~2 fold) in response to TGF-β+ET-1. In the lung, TGF-β increased hydroxyproline levels by 38% and TGF-β+ET-1 increased hydroxyproline levels by 75%.
Conclusion: Tie2GFP-Col1mCherry mice represent a valuable resource for monitoring EndoMT in vivo. ET-1 plays an important in vivo role in regulating EndoMT by causing a synergistic potentiation of TGF-β1-induced EndoMT. Since ET-1 plays a crucial role in the pathogenesis of SSc-associated pulmonary arterial hypertension and may play a profibrotic role in skin and lung fibrosis, the results described here identify a novel mechanism supporting the concept that ET-1 plays a key pathogenetic role in vivo in SSc-associated tissue fibrosis.
Disclosure:
P. J. Wermuth,
None;
S. A. Jimenez,
None.
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