Abstract

Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC) treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

Highlights

  • Pulmonary arterial hypertension (PAH) is a disease characterized by progressively increasing pulmonary vascular resistance, leading to right heart failure and death

  • We found that the dominant pulmonary phenotype caused by suppression of SMAD signaling, through BMPR2 in all cell types in Rosa26-Bmpr2delx4+ mice, was strong inflammation in the airways associated with increased recruitment of macrophages (Figures 1,2)

  • By crossing the Rosa26-Bmpr2delx4+ mice onto two different nuclear factor of kappa B (NF-kB) reporter lines and examining effects in whole animal, tissue, and cells, we were able to narrow the source of NFkB-dependent inflammation in Bmpr2delx4+ mice to constitutive activation of tissue macrophages (Figures 3, 4)

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Summary

Introduction

Pulmonary arterial hypertension (PAH) is a disease characterized by progressively increasing pulmonary vascular resistance, leading to right heart failure and death. We have previously shown that the Bmpr2delx4+ mutation, which truncates the receptor just after the transmembrane domain, acts as a dominant negative for SMAD signaling and causes PAH, secondary to smooth muscle dedifferentiation, when expressed only in smooth muscle [9,10]. It causes PAH, secondary to inflammatory and clotting difficulties, when expressed only in endothelium [11]. We have never previously expressed the Bmpr2delx4+ mutation in all tissue types, and the initial motivation for the current study was to determine whether universal expression had a phenotype distinct from smooth muscle- or endothelium- specific expression

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