Stem Cells Defects in Systemic Sclerosis.
Authors: Quirici Nadia , Del Papa Nicoletta , Scavullo Cinzia , Cortiana Michela , Borsotti Chiara , Maglione Wanda , Comina Denise , Cortelezzi Agostino , Deliliers Giorgio Lambertenghi , Soligo Davide 
Published: April 2020

Abstract
Systemic Sclerosis (SSc) is a connective tissue disease characterized by early generalized microangiopathy and culminating in systemic fibrosis. Recent studies have provided evidence that SSc is associated with a reactive but ineffective angiogenesis, so that the disease finally leads to the irreversible loss of capillaries. Aim of the study was to investigate whether impaired vasculogenesis in SSc is due to defective characteristics in BM microenvironment. Peripheral blood (PB) samples were collected from 70 patients (pts): circulating endothelial progenitors (CEPs) were characterized as CD45−/CD133+ and evaluated by flow cytometry. BM samples were collected from 14 SSc pts and hematopoiesis evaluated by various assays. CD133+ cells were isolated by immunomagnetic sorting (IMS) and grown in order to induce endothelial differentiation. Long-term bone marrow cultures (LTBMC) were assessed and the number of stromal clonogenic precursors evaluated by a CFU-F (colony-forming unit fibroblast) assay. Mesenchymal stem cells (MSC) were separated by IMS for the expression of the nerve growth factor-receptor (NGF-R+) and grown in order to assess the clonogenic potential and the proliferative capacity, while their multipotential differentiation ability was determined after culture in different conditioned media. Phenotypic analysis of BM mononuclear cells showed a greater expression of the surface markers P1H12 and CD105 TGF-β receptor (1.2%±0.6 vs 0.5%±0.1 in normal controls, p=0.01 and 9.9%±5 vs 4.7%±3, p=0.02 respectively), but lower percentages of NGF-R+ stromal cell precursors (0.73±0.5 vs 1.61±0.6, p=0.02) and CD133+ cells (0.36%±0.4 vs 1.2%±0.8, p=0.05). On the contrary, the absolute number of CEPs in PB was higher in patients with SSc than in healthy controls (mean values 2.1 cells/μL vs 0.26 cells/μL, p=0.04). When BM CD133+ cells were grown in the presence of VEGF, only 3/12 cases gave endothelial differentiation, but always with a reduced proliferative ability. All pts showed a defective stromal compartment and a reduced number of BM stromal precursors, as detected by the LTBMC and by the lower CFU-F frequency (4%±3.2 vs 43%±19.8/1x10(e)6 LDMNCs, p=0.002 and 7±12.8 vs 69±61/1x10(e)5 NGF-R+ cells, p=0.01). Interestingly, NGF-R+ MSC overexpressed KDR and CD117 (26.4%±7.4 vs 4.6%±1.7, p=0.01 and 87.7%±5.1 vs 57.6%±11, p=0.03 respectively): when grown in the presence of VEGF they gave rise to endothelial colonies, only in 2/8 cases they formed a confluent layer with fibroblastic morphology but a reduced proliferative ability, while in the presence of adipogenic or osteogenic inductive media they failed to origin specific differentiation. Moreover, all “in vitro” differentiated endothelial cells even before activation showed high levels of CD62-E, VCAM-1 and CD105 expression, suggestive of the presence of increased levels of proangiogenic factors in BM. The results of this study provide evidence that patients with SSc have a stem cell defect involving both the hematopoietic and the stromal cells compartments. The higher expression of KDR on NGF-R+ cells suggests a role for VEGF in inducing endothelial differentiation of MSC, so resulting in a depletion of stromal precursors. The continuous recruitment of endothelial progenitors to sites of vascular injury, suggested by the high numbers of CEPs in PB, might lead to the irreversible BM damage we observed.