Introduction and objectives Bone morphogenetic protein receptor type 2 (BMPR2) mutations are found in heritable and idiopathic pulmonary arterial hypertension however penetrance is incomplete implying necessity for a ‘second hit’. IL-1ß and IL-6 are increased in PAH patients and animal models and are thought to have a role in disease. We aimed to determine pulmonary specific interplay between BMPR2 and IL-1ß signalling through assessing IL-1ß responsiveness of pulmonary artery and aortic smooth muscle cells (Ao/PA SMC) and determine the effects of reduced BMPR2.
Methods Microarray analysis of PASMC and AoSMC mRNA was performed using microarray on mRNA isolated from cells cultured in SMGM-2 (Lonza) +/- functional BMPR2 (by use of siRNA) and stimulation with 10 ng/ml IL-1ß for 6 h. Subsequent bioinformatics was performed using R. Findings were validated using quantitative PCR and western blotting. Furthermore R899X+/- BMPR2 transgenic mice were fed western diet for six weeks and injected daily with IL-1ß then assessed for inflammatory activation and PAH phenotype (catheter/echo). mRNA and protein changes were measured by TaqMan PCR, western blotting and serum ELISA. Immuno-staining of paraffin embedded lung sections assessed pulmonary vascular remodelling.
Results Array data shows reduced inflammatory activation in response to IL-1ß in PASMC compared with AoSMCs, analysis of cells lacking functional BMPR2 identified an exaggerated inflammatory response to IL-1ß in PASMC lacking BMPR2 (siRNA). Significant up-regulation of IL-6, IL-1α and adhesion molecules (>2-fold) shown by array analysis was validated by qPCR. In the absence of BMPR2 a 1.5 fold increase in proliferation was observed in response to IL-1ß compared to PASMC with functional BMPR2. Mice treated with IL-1ß show higher white blood cell counts (1.7-fold), and protein levels of OPG and IL-6 (serum) matching in vitro data.
Conclusion IL-1ß induces a pulmonary specific transcriptome altered by suppression of BMPR2 signalling indicating cross-talk between the pathways. In the presence of BMPR2, PASMCs show limited response to IL-1ß however reducing BMPR2 exacerbated this response increasing the likelihood of a PAH phenotype in PASMCs. This highlights a mechanism that increased IL-1ß may provide “second hit” to reduced BMPR2 to stimulate development of PAH.
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