PB1050 - Blockade of K-ras signalling prevents coagulatory phenotype of alternatively activated macrophages.

Background: Alternatively acitvated macrophages are characterized by a procoagulatory phenotype including increased expression of tissue factor (TF) and reduced activation of urokinase-type plasminogen activator (uPA) due to increased expression of plasminogen activator inhibitor 1 (PAI-1).

Aims: To identify signaling pathways required for a procoagulatory phenotype of alternatively activated macrophages.

Methods: We studied the role of K-ras in human macrophages as well as in a lung fibrosis model in mice, by inhibiting K-ras signalling pharmacologically using fendiline (key experiments confirmed with a second K-RAS inhibitor BAY-293). Lung fibrosis was induced in mice by inhalative bleomycin application

Results: Alternative activation of macrophages (AAM) by IL-4 and IL-13 led to a significant phosphorylation of ERK1/2 in vitro and in vivo. K-RAS inhibition significantly reduced the phosphorylation of ERK1/2 in macrophages leading to a significant reduction in AAM markers including TF and PAI-1. K-ras treated AAM showed significantly prolonged coagulation times as indicated by ROTEM measurements. Tissue Factor, uPA, PAI-1 as well as collagen-6 was reduced in K-RAS inhibited AAM. In bleomycin-induced lung fibrosis, mice treated with fendiline (K-RAS inhibitor) showed significantly less pulmonary fibrosis. After initial weight loss associated with pulmonary inflammation, treated mice gained weight from day 9 on, whereas untreated mice lingered at their reduced weight.

Conclusion(s): Blocking intracellular K-ras signalling in mice and human macrophages, reduced the fibrotic and coagulatory phenotype associated with pulmonary fibrosis. Mechanistically, fendiline led to a reduction of phosphorylated ERK1/2 and a downregulation of the coagulatory proteins TF, uPA, PAI-1 and collagen- 6. These cellular alterations led to a prolonged coagulation potential. Treatment of mice with fendiline reduced bleomycin-induced lung fibrosis.