Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Role of Endothelial-to-mesenchymal transition in the pathogenesis of Systemic Sclerosis

ABSTRACT Background: Endothelial-to-mesenchymal transition (EndoMT), a newly recognized type of cellular trans differentiation, 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) (42,44). Circulating endothelial progenitors cells (EPCs) derive from bone marrow stem cells and contribute to de novo vessels 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. Objectives: to assess phenotypic characteristics of EPCs from SSc 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: 1. Circulating Early (CD34+KDR+CD 133+) and Late EPCs (CD34+KDR+) in the peripheral blood using flow cytometry 2. Cultured EPCs using Western blot analysis 3. Cultured EPCs from HC with serum from SSc and VEDOSS patients and CXCL4 and HC EPCs with and without the challenge with hydroxychloroquine using flow cytometry 4. Cultured EPCs using Immunofluorescence Materials and Methods: we enrolled 41 patients (25 SSc and 16 VEDOSS), classified according to the classification criteria for SSc (16) and for VEDOSS (22) not fulfilling SSc criteria and 20 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 HC were also cultured with 20% SSc patient serum, CXCL4 with or without hydroxychloroquine (HCQ). Collected EPCs were analyzed with flow cytometry and immunocytochemistry to asses expression of a-SMA (anti-alpha smooth muscle Actin antibody [1A4]-Abcam) and fibroblasts specific protein-1 (FSP-1) (Abcam ab196380). A part of culture EPCs were lysed and a Western blot analysis for a-SMA detection was performed Results: we found a significant higher percentage of α-SMA positive Early EPCs in all patients respect to HC particularly in VEDOSS patients. In cultured EPCs from patients and HC, we found an higher percentage of -SMA positive EPCs in SSc (p=0.0003) and in VEDOSS patients (p=0.0016) and a lower level of FSP-1 (p=0.0237 and p=0.0112 respectively) compare to HC. WB analysis showed a significant higher expression of α-SMA protein in cultured EPCs from all patients and VEDOSS patients respect to HC. Immunocytochemistry on cultured EPCS confirm the higher expression of FSP-1 in HC. Only SSc serum seems to increase the α-SMA positive EPCs expression respect to same HC cultured without SSc serum but not when cultured with HCQ. Conclusions: we found higher percentage of α-SMA positive EPCs in SSc and in VEDOSS patients respect to HC and this could be considered the expression of the involvement of EPCs in the EndoMT process. Surprisingly, FSP-1 seems to be higher in HC. A recent work shows that a subtype of activated fibroblasts expressing FSP-1 show angiogenetic properties respect to a-SMA positive fibroblasts. So we hypothesized a similar behavior of EPCs that can be a source of this kind of fibroblast through EndoMT process. Moreover, this study suggest a potential effect on EPCs of pharmacological therapy and the importance to find out the first step of the complex mechanism underlying EndoMT and SSc pathogenesis to find out a real disease modifying drug for these severe condition.