Abstract 596: Increased Migration of Neutrophils Across Endothelial Cells from Patients with Pulmonary Arterial Hypertension is Related to Reduced Platelet Endothelial Cell Adhesion Molecule -1 and is Prevented by the Neutrophil Elastase Inhibitor Elafin

Abstract

Pulmonary arterial hypertension (PAH) is a devastating progressive disease associated with a high mortality despite current vasodilator therapies. Perivascular inflammation and high levels of neutrophil elastase are thought to play a pivotal role in the adverse vascular remodeling of small pulmonary arteries that causes PAH. Despite this, the function of neutrophils and in particular, their interaction with the pulmonary arterial endothelium has not been studied in PAH. We hypothesized that neutrophil functions such as adhesion to a substratum or to endothelial cells and transmigration across a substratum or trans-endothelial migration (TEM) are abnormal in PAH on the basis of dysfunction of both cell types. Using a neutrophil like cell line dHL-60 and isolated human neutrophils from donors and PAH patients, we demonstrated no significant difference in adhesion to PAH vs. control PAECs when stimulated with TNF-α (100μg/mL). However, TEM of both IL-8 (100ng/ml) and fMLP (100nM) stimulated dHL-60 cells and donor neutrophils was enhanced in PAH vs. control PAECs (p<0.01) likely related to reduced expression of Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1) in PAH PAECs (p <0.001). We therefore further hypothesized that inhibition of neutrophil elastase via recombinant human elafin would be sufficient to reverse TEM in both PAH and control PAEC by reducing neutrophil adhesion, a feature known to be elastase-dependent. Administration of elafin (1μg/mL) attenuated fMLP and IL-8 induced dHL-60 and neutrophil adhesion to fibrinogen and fibronectin (p<0.05 for both) as well as transmigration across both substrates (p<0.05 for both). Elafin reduced adhesion of neutrophil similarly in PAH and control PAEC (p <0.05 for both). Furthermore, in a dose dependent manner, elafin inhibited TEM in PAH PAEC by 40% in control PAECs, by 20% (p<0.001 and p<0.01 respectively) bringing values to within the same range of suppressing but not eliminating TEM. We therefore conclude that despite the reduction in PECAM-1 in PAH PAEC, inhibition of neutrophil adhesion with the elastase inhibitor elafin is sufficient to prevent the enhanced TEM. Elafin may therefore be of benefit in suppressing the deleterious impact of neutrophil in enhanced inflammation seen in PAH.