P4493Microvesicles from patients with pulmonary arterial hypertension modulate bFGF from endothelial cells

Abstract

Abstract Background Pulmonary Arterial Hypertension (PAH) is a progressive condition with remodeling of precapillary arteries. Cytokines involved in angiogenesis have been found elevated in PAH but the role of proangiogenic factors and their regulation is far from fully understood. Microvesicles (MVs) can act as intercellular messengers and modify cell production of proteins. Purpose To evaluate whether MVs from patients with PAH modulate proangiogenic factors in plasma and endothelial cells. Methods 62 patients with PAH and 20 healthy controls were enrolled in the study. The number of MVs were determined in whole blood by flow cytometry. For in vitro studies, MVs were isolated from plasma and a primary endothelial cell line of human pulmonary artery endothelial cells between passages 3 to 7 were used. Protein levels in plasma and conditioned media were analyzed by electrochemiluminescence whereas protein levels in cell lysates were analyzed by western blot (WB). Results The majority of the MVs in whole blood were derived from platelets and patients with PAH had higher concentrations compared to healthy controls (p<0.0001). In plasma both basic Fibroblast Growth Factor (bFGF) and vascular endothelial growth factor receptor 1 (Flt-1) were increased in the patients as compared to healthy controls (median in pg/ml (IQR) Pat. vs Ctrl: bFGF 4.2 (2.5–9.2) vs 1.2 (0.9–2.1); Flt-1 143.0 (88.5–234.5) vs 53.9 (47.7–59.67); p<0.001 for both). Endothelial cells were incubated with or without MVs for 18 hours and effects were monitored by analyzing eNOS and ICAM-1 levels by WB. Both markers were significantly altered, i.e. an about 50% decrease of eNOS and a 50% increase of ICAM-1, by patient MVs but not by the control MVs. The endothelial cells were then incubated with MVs for 6h, 12h, 24h, and 48h and bFGF and Flt-1 were measured in the conditioned media. The presence in media of both proteins were increased over time, but only bFGF was significantly regulated by MVs from PAH patient as compared to MVs from the control group at 24h (2.8 fold increase) and at 48h (12.7 fold increase). Conclusions An increase of two important proangiogenic proteins were found in PAH patients. The MVs from these patients modulate the endothelial response and contribute to a release of bFGF, whereas the Flt-1 levels are regulated by other currently unknown mechanisms. Further studies of the MVs from PAH patients are needed to elucidate how they are involved in the angiogenic process. Acknowledgement/Funding Swedish Research Council, Science For Life Laboratory Sweden