Treprostinil Inhibits the Adhesion and Differentiation of Fibrocytes via the Cyclic Adenosine Monophosphate–Dependent and Ras-Proximate Protein–Dependent Inactivation of Extracellular Regulated Kinase

Abstract

Fibrocytes comprise a recently described cell type of blood-derived, fibroblast-like cells that are recruited from the circulation to sites of wound repair, vascular remodeling, or fibrotic tissue remodeling. We recently showed that the stable prostacyclin analogue treprostinil, a clinically approved drug for pulmonary arterial hypertension (PAH), significantly reduced the recruitment of fibrocytes to sites of vascular remodeling in experimental hypoxic pulmonary hypertension. Here we report on the molecular mechanism underlying the inhibitory action of treprostinil on the adhesion and differentiation of human fibrocytes. Human fibrocytes expressed the prostanoid receptors, prostaglandin I (IP) receptors and prostaglandin E subtype receptors (EP2 and EP4). The generation of intracellular cyclic adenosine monophosphate (cAMP) by treprostinil reduced the expression of the integrins CD49 and CD29 when freshly isolated human peripheral blood mononuclear cells were treated with treprostinil. Cell-matrix adhesion was significantly impaired by treatment with treprostinil. We present evidence for a treprostinil/cAMP-induced downstream suppression of extracellular regulated kinase (ERK) that is transmitted via a protein kinase A-independent pathway through Rap proteins, which sequester Ras. The resulting dephosphorylated state of c-Raf limits the activity of ERK. The cell-matrix adhesion assay with the ERK inhibitor further confirmed that the adhesion of fibrocytes was impaired. Thus our data suggest that treprostinil inhibits the adhesion and differentiation of fibrocytes by limiting the activity of ERK via the cAMP-Rap axis.