Abstract
Introduction. Vascular complications associated with systemic sclerosis (SSc) including pulmonary arterial hypertension (PAH-SSc), result from endothelial damage and loss of barrier function. The causes of endothelial dysfunction are unclear, but the integrity of the endothelium is likely to be significantly diminished in SSc. Endothelial progenitor cells (EPCs) derived from peripheral blood mononuclear cells (PBMCs) express endothelial and haematopoietic markers. It is thought they home to sites of vascular injury and differentiate into endothelial cells and restore the barrier. In SSc patients circulating levels of EPCs are reduced. This study aimed to: (i) develop a robust method to isolate and grow healthy control (HC) and SSc EPCs from PBMCs. (ii) Compare the cellular functions of EPCs to mature endothelial cells. Methods. Peripheral blood was taken from HC (n=10) and SSc donors (n=10). EPCs were cultured from PBMCs, and EPC colonies grown to passage 4. EPCs and human pulmonary artery endothelial cells (hPAECs) were seeded into transwell inserts and grown to confluence. Cells were incubated with TNF-α (10ng/ S-33 3rd Systemic Sclerosis World Congress Poster Tours - Clinical ml), and their capacity to form biological barriers and support immune cell influx was assessed using FITC-albumin (0.5mg/ml) and neutrophil transmigration. We further assessed the responses of EPCs to TNF-α stimulation by ELISA to quantify pro-inflammatory cytokine release. Results. We demonstrate that EPCs form biological barriers with similar capabilities as mature hPAECs in vitro. TNF-α significantly enhanced permeability of EPCs (p<0.05) and hPAECs (p<0.05) monolayers. Consistent with EPCs possessing similar cellular activities as mature endothelial cells, TNFa stimulated neutrophil transmigration in monolayers of EPCs (p<0.05) and hPAECs (p<0.05) and enhanced the secretion of IL-8 in both EPCs (p<0.01) and hPAECs (p<0.05). We sought to determine the frequency of EPC colony formation from PBMCs and found no significant difference in the capacity to form EPC colonies in HC and SSc patient PBMCs. Discussion. We have developed a robust method for isolating EPCs from PBMCs. We have demonstrated that endothelial progenitors can maintain an endothelial barrier consistent with that observed by mature hPAECs in vitro. We have established that EPCs respond to TNF-α in a similar manner to mature PAECs, secreting pro-inflammatory cytokines such as IL-8 and supporting neutrophil transmigration. We have shown no significant difference in the capacity of PBMCs from SSc patients to form EPC colonies compared to healthy control donors. The biological function and importance of EPCs from SSc patients in vasculopathy, restoration and maintenance of the endothelial barrier function remains unclear.