Abstract Vascular complications associated with systemic sclerosis (SSc) including pulmonary arterial hypertension (PAH-SSc), result from endothelial damage and loss of barrier function. Endothelial progenitor cells (EPCs) express endothelial (VEGFR2+, CD31+ ) and haematopoietic (CD133+ ) markers. They home to sites of vascular injury and differentiate into endothelial cells restoring the endothelium. In SSc patients circulating levels of EPCs are reduced. This study aimed to develop a robust method to grow EPCs from peripheral blood mononuclear cells (PBMCs) and to compare cellular functions to mature endothelial cells.
Methods EPCs and human pulmonary artery endothelial cells (hPAECs) were seeded into transwell inserts and grown to confluence. Cells were incubated with TNFa (50ng/ml), and their capacity to form biological barriers assessed using FITC-albumin (5mg/ml). FITC-albumin ‘leak’ was quantified by fluorescent absorbance over time. We further assessed the responses of EPCs to TNFa stimulation by ELISA to quantify pro-inflammatory cytokine release.
Results EPCs form a biological exclusion barrier with similar capabilities as mature hPAECs. TNFa significantly enhanced the permeability of EPCs (P < 0.05) and hPAECs (P < 0.05) monolayers. There is no difference in EPC colony formation between HC and SSc EPCs.
Discussion We developed a robust method for isolating EPCs from PBMCs. We have demonstrated that EPCs can maintain an endothelial barrier consistent with that observed by mature hPAECs in vitro. We have established that EPCs respond to TNFa in a similar manner to mature PAECs. We have shown no significant difference in the capacity of PBMCs from SSc patients to form EPC colonies compared to HCs.