Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease affecting skin and internal organs. It is characterized by vascular damage, inflammation, and fibrosis. Microvascular injury and insufficient angiogenesis lead to tissue ischemia and clinical manifestations such as fingertip ulcers. UCD-206 chickens are the only animal model showing all hallmarks of the human disease, including microvascular damage and insufficient angiogenesis. The major inducer of angiogenesis is vascular endothelial growth factor (VEGF). Uncontrolled expression of VEGF and its receptors VEGFR-1 and VEGFR-2 has been shown in both, human and avian SSc. Another set of molecules that critically regulate angiogenesis are the endothelial cell-specific receptor tyrosine kinase 2 (Tie-2) and its ligands angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). Although abnormal plasma levels of soluble sTie-2, sAng-1, and sAng-2 have been reported in SSc patients, expression in lesional tissues has not been examined.
Objectives To study the expression of Tie-2, Ang-1 and Ang-2 in lesional and non-lesional skin of UCD-206 chickens.
Methods The expression of Tie-2, Ang-1 and Ang-2 was studied by indirect immunofluorescence tests on frozen tissue sections from lesional and non-lesional neck skins from 5 weeks old UCD-206 chickens. Age matched H.B15 chickens were used as healthy controls. Samples were double stained with monoclonal anti-Tie-2 antibody and the endothelial cell marker anti-von Willebrand factor (vWF), monoclonal anti-Ang-1 antibody and anti-alpha smooth muscle actin antibody, as a marker for mural cells, or with monoclonal anti-Ang-2 antibody and anti-vWF. The tissue cytometry software TissueQuest® was used for quantitative analyses. Statistical significance was calculated using the Mann-Whitney-U test.
Results The number of Tie-2 expressing endothelial cells was significantly increased in lesional skin compared to non-lesional UCD-206 skin and healthy controls. Expression of Ang-2 was significantly decreased in lesional compared to non-lesional UCD-206 skin and to healthy H.B15 skin. Whereas no significant difference was seen in the numbers of Ang-1 expressing mural cells, other Ang-1 expressing cells were reduced in lesional skin compared to non-lesional and healthy controls. The altered endothelial expression of Ang-2 and Tie-2 resulted in significantly decreased Ang-2:Tie-2 ratios in lesional (median 0.9, IQR 0.3 – 1.8) compared to non-lesional UCD-206 skin (median 6.9, IQR 3.4 – 14.2, p≤0.0005) and healthy controls. (median 11.2, IQR 2.4 – 25.2, p≤0.0005). The Ang-2:Ang-1 ratio was also significantly diminished in lesional (median 0.4, IQR 0.1 – 0.5) compared to non-lesional UCD-206 skin (median 0.7, IQR 0.4 – 1.3, p≤0.01) and healthy controls. (median 1.0, IQR 0.6 – 1.6, p≤0.002).
Conclusions Altered expression of Ang-1, Ang-2 and Tie-2 might contribute to the vasculopathy in SSc.
Acknowledgements This work was funded by the Austrian Science Fund (FWF): P23230-B13.
Disclosure of Interest None declared