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Abstract Number: 1493

Transforming Growth Factor-β and Endothelin-1 Induce Endothelial-to-Mesenchymal Transition in Cultured Human Endothelial Cells

Stefano Soldano1, Paola Montagna1, Renata Brizzolara1, Barbara Villaggio2, Alberto Sulli3 and Maurizio Cutolo4, 1Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy, Genova, Italy, 2Research Laboratory of Nephrology, Department of Internal Medicine, University of Genova, Genova, Italy, 3Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy, 4University of Genova, Genova, Italy

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: endothelial cells

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Session Information

Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud’s – Pathogenesis, Animal Models and Genetics

Session Type: Abstract Submissions (ACR)

Background/Purpose:

The endothelial/microvascular injury and the myofibroblast activation are crucial events that seem to contribute to the development of fibrosis in connective tissue diseases such as systemic sclerosis (SSc), which is characterized by increased local transforming growth factor-β (TGF-β) and endothelin-1 (ET-1) levels (1,2). Recently it was shown that myofibroblast activation from altered microvasculature may arise through the transition of endothelial to mesenchymal cells (EndoMT), thus expressing α-smooth muscle actin (α-SMA), vimentin and fibrillar collagens (3).

To investigate the possible involvement of TGF-β and ET-1 in the early step of EndoMT in cultures of human endothelial cells.

Methods:

Human umbilical vein endothelial cells (HUVEC) were cultured in collagen-coated dishes with EGM-2 medium and used between the third and fifth passages. The cells were treated with ET-1 (100 nM) or TGF-β (10 ng/ml) for 1, 3 and 6 days. Untreated endothelial cells were used as controls (cnt). Cell proliferation was evaluated by methyl-tetrazolium salt test (MTT) after 1 day. The expression of α-SMA as marker of myofibroblast phenotype, and platelet endothelial cell adhesion molecule (PECAM-1 or CD31), as marker of endothelial phenotype, was evaluated after 3 and 6 days of treatment by immunofluorescence (IF) and western blot analysis (WB) according to recent evidences (4). Data were obtained by eight different experiments and statistical analysis was carried out by a non-parametric Friedman test.

Results:

After 6 days of treatment, TGF-β induced the α-SMA expression in cultured human endothelial cells, which maintain their capability to express CD31.

Interestingly, also ET-1 was able to stimulate the endothelial cells to express α-SMA after 6 days of treatment. The data were obtained by IF and confirmed by WB analysis.

In addition, MTT test showed that both TGF-β and ET-1 induced a statistically significant increase of proliferation of human endothelial cells vs. cnt (p<0.001).

Conclusion: These preliminary results show that both ET-1 and TGF-β seem to share similar effects in inducing the α-SMA expression and cell proliferation in human endothelial cells thus supporting a possible direct involvement in promoting the transition from endothelial to myofibroblast phenotype (2, 4-6). The implications in the fibrotic process that characterize SSc are matter of further evaluations.

References. 1.Wynn TA. J Pathol 2008;214:199-210. 2 Batthacharyya S. et al. Nat Rev Rheumatol 2012;8:42-54. 3.Piera-Velazquez S et al. Am J Pathol 2011;179:1074-80 4.Widyantoro B et al. Circulation 2010;8:2407-18. 5.Kitao A et al. Am J Phatol 2009;175:616-26. 6. Abraham D et al. Arthrit Res Ther 2007;9:doi10.1186/ar2186.


Disclosure:

S. Soldano,
None;

P. Montagna,
None;

R. Brizzolara,
None;

B. Villaggio,
None;

A. Sulli,
None;

M. Cutolo,
None.

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