Early Endothelial Progenitor Cells (eEPCs) in systemic sclerosis (SSc) - dynamics of cellular regeneration and mesenchymal transdifferentiation

Abstract

Background: Patients with systemic sclerosis (SSc) are endagered by tissue fibrosis and by microvasculopathy, with the latter caused by endothelial cell expansion/proliferation. SSc-associated fibrosis potentially results from mesenchymal transdifferentiation of endothelial cells. Early Endothelial Progenitor Cells (eEPCs) act proangiogenic under diverse conditions. Aim of the study was to analyze eEPC regeneration and mesenchymal transdifferentiation in patients with limited and diffuse SSs (lSSc and dSSc).

Methods: Patients with both, lSSc and dSSc were included into the study. The following parameters were evaluated: eEPC numbers and regeneration, concentrations of vasomodulatory mediators, mesenchymal properties of blood-derived eEPC. Serum samples of healthy subjects and SS patients were used for stimulation of cultured human eEPC, subsequently followed by analysis of mesenchymal cell characteristics and mobility.

Results: Twenty-nine patients were included into the study. Regenerative activity of blood-derived eEPCs did not differ between Controls and patients. Circulating eEPC were significantly lower in all patients with SSc, and in limited and diffuse SSc (lSSc/dSSc). Serum concentrations of promesenchymal TGF-b was elevated in all patients with SSc. Cultured mononuclear cells from SS patients displayed higher abundances of CD31 and of CD31 and aSMA combined. Finally, serum from SSc patients inhibited migration of cultured eEPCs and the cells showed lower sensitivity towards the endothelin antagonist Bosentan.

Conclusions: The eEPC system, which represents an essential element of the endogenous vascular repair machinery is affected in SSc. The increased appearance of mesenchymal properties in eEPC may indicate that alterations of the cells potentially contribute to the accumulation of connective tissue and to vascular malfunction.

Fig. 1

Percentages of peripheral circulating eEPC and eEPC regeneration in SSc. a shows CD133+/Flk-1+ cells (eEOCs). Percentages of double-positive cells significantly differed between Controls and all SSc categories (SSc all, lSSc, and dSSc) with lower values in SSc patients, respectively. b shows eEPC colony formation. Colony Forming Unit-Endothelial Cells (CFU-Ecs) were not different between the categories (Data as mean ± SEM, ✻ : p < 0.05 - for exact p-values see text)

Fig. 2

Mesenchymal transdifferentiation of blood-derived eEPCs in SSc. Expression CD31 was significantly higher in all patients with SSc and in lSSc, dSSc did not differ from Controls (a). b displays aSMA in CD31+ cells with higher percentages in SSc all and in lSSc. c shows results of additional marker staining (FSP-1), the results were comparable to those aquired with aSMA (Data as mean ± SEM, ✻ : p < 0.05 - for exact p-values see text)

Fig. 3

Pro-mesenchymal TGF-b in SSc. All Patients with SSc (SSc all) showed higher concentrations of TGF-b as compared to healthy controls (Data as mean ± SEM, ✻: p < 0.05 - for exact p-values see text)

Fig. 4

eEPC migration and EndoMT in vitro. Cultured human eEPCs were treated with serum samples from healthy controls, and from patients with SSc with versus without simultaneous administration of Bosentan to the culture medium. Analysis were performed at 5 h after beginning of the incubation. In general, migration of eEPC treated with patient serum was significantly slower (a). Bosentan reduced cellular migration in the ‘healthy subjects’ category. This effect exclusively occurred under the following experimental conditions: serum:medium-ratio 1:5, Bosentan concentration 2000 ng/ml. Expression of aSMA by the cells was comparable in all categories with no significant alteration by Bosentan (b – experimental conditions: serum:medium-ratio 1:5, Bosentan concentration 2000 ng/ml) (bos.: Bosentan; ‘serum’ represents the serum:medium-ratio; Data as mean ± SEM, ✻: differences between healthy subjects and SSc patients within a group significant with p < 0.05; #: differences between healthy subjects between two groups significant with with p < 0.05)