Effect of defribrotide on in vitro activation of human pulmonary microvascular endothelial cells

Abstract

Hematopoietic stem cell transplantation causes development of such complications as venoocclusive disease (VOD), thrombotic microangiopathy (TMA) and idiopathic pneumonia syndrome (IPS). The key pathophysiological mechanism in VOD and TMA is endothelial cell (EC) damage, and a role of EC damage in IPS is presumed. Defibrotide demonstrated effects on activated/damaged EC cultures and efficacy in VOD treatment and prophylaxis. To demonstrate in vitro effects of defibrotide on human pulmonary microvascular endothelial cells (HPMEC), the cells were activated with TNF alpha (TNFa), and treated with defibrotide before and after TNFa activation. The doses 10-100 ng/mL of TNFa and 20-100 µg/mL of defibrotide were applied for 4 and 24 hr. Changes in HPMEC gene expression were measured by polymerase chain reaction (PCR) for VCAM1, NOS3, VWF, CASP3, BAX and BCL2, and upregulation effects of TNFa and downregulation effects of defibrotide were demonstrated. For the final experiments 20 ng/mL of TNFa and 20 µg/mL of defibrotide were chosen, and the experiments were performed in 3 different donors cell cultures. HPMEC viability estimated by lactate dehydrogenase cytotoxicity assay, was not affected by TNFa and defibrotide. From the PCR results of TNFa effects on EC biology genes NOS3, SERPINE, CCL2, VCAM1, IL1B, MMP9, PLAT, SELE, CASP3, BCL2 were selected. The effects of defibrotide were observed in a few genes in single cell cultures. Immunofluorescent detection of eNOS and VCAM1 showed that defibrotide treatment before and after TNFa activation reduced protein expression in cultures from 2 of the 3 donors. Enzyme-linked immunosorbent assay was used to measure PAI1, MCP1, tPA, E-selectin, MMP9 and IL1B in HPMEC supernatants, and only few effects in single cultures were shown. To conclude, the results demonstrate downregulatory effects of defibrotide on inflammatory gene and protein expression in activated with TNFa HPMEC and suggest a possible mechanism for the effect of defibrotide on IPS.