AB0170 PHENOTYPIC CHARACTERIZATION OF ENDOTHELIAL PROGENITORS CELLS OF SYSTEMIC SCLEROSIS (SSC) PATIENTS: ROLE IN ENDOTHELIAL-TO-MESENCHIMAL TRANSITION PROCESS

Abstract

Background:Endothelial-to-mesenchymal transition (EndoMT), a newly recognized type of cellular transdifferentiation, seems to be involved in Systemic Sclerosis (SSc) pathogenesis. In this process endothelial cells lose their specific markers, and acquire a mesenchymal phenotype, thus expressing cell products such as alpha smooth muscle actin (α-SMA) (1,2).Circulating endothelial progenitors cells (EPCs) derive from bone marrow stem cells and contribute tode novovessels formation. Several studies, although with conflicting results, have shown that EPCs in the peripheral blood of patients with SSc are impaired in their number and function (3).Objectives:to assess phenotypic characteristics of EPCs fromSSc patients and from patients with Very Early Diagnosis of SSc (VEDOSS) compared with healthy controls (HC). In particular we want to evaluate the expression of α-SMA, as marker of a pro-mesenchymal switch (EndoMT) in:Circulating Early (CD34+KDR+CD 133+) and Late EPCs(CD34+KDR+) in the peripheral blood using flow cytometryCultured EPCs using Western blot analysisMethods:we enrolled 11 patients (6 SSc and 5 VEDOSS), classified according to the classification criteria for SSc (4) and for VEDOSS not fulfilling SSc criteria (5), and 5 HC. Phenotypic characterization was performed as previously described by Vasa et al. using a FACS Calibur (BD Immunocytometry Systems). EPCs number was expressed as a percentage of cells within the lymphocyte gate. 5*106 PBMCs were plated on human fibronectin-precoated (10 μg/ml Sigma-Aldrich) 6-well plates and cultured for 7-12 days to obtain EPCs. PBMCs from one HC were also cultured with 20% SSc patient serum. Collected EPCs were lysed and a Western blot analysis for α-SMA detection was performed.Results:we found a significant higher percentage of α-SMA positive Early EPCs in all patients respect to HC (0,06% ±0,03 vs 0,03% ± 0,01; p=0,0149) particularly in VEDOSS patients (0,07%±0,01 vs 0,03%±0,01 p=0,008). Similarly we found a significant higher expression of α-SMA protein in all patients and VEDOSS patients respect to HC (0,1895±0,16 vs 0,07± 0,06 p= 0,0342; 0,3075 ± 0,14 vs 0,07± 0,06 p=0,0159). After the incubation of HC PBMCs with SSc serum, the α-SMA protein expression seems to be increased respect to its expression in thePBMCs of the same HC cultured without SSc serum (0,33 vs 0,1).Conclusion:we found higher percentage of Early α-SMA positive EPCs and a higher expression of α-SMA protein in cultured EPCs in patients group (SSc and VEDOSS) than in HC. So we hypothesized a predominant pro-mesenchymal phenotype of this kind of EPCs. This could be considered the expression of the involvement of EPCs in the EndoMT process and it better explain the controversial role of EPCs in SSc pathogenesis. Moreover the modified expression of α-SMA in HC EPCs co-cultured with 20% SSc serum could suggest the presence of a factor inducing the EndoMT process in the disease.