Differential IL-1 signaling induced by BMPR2 deficiency drives pulmonary vascular remodeling

Abstract

Bone morphogenetic protein receptor type 2 (BMPR2) mutations are present in patients with heritable and idiopathic pulmonary arterial hypertension (PAH). Circulating levels of interleukin-1 (IL-1) are raised in patients and animal models. Whether interplay between BMP and IL-1 signaling can explain the local manifestation of PAH in the lung remains unclear. Cell culture, siRNA, and mRNA microarray analysis of RNA isolated from human pulmonary artery (PASMC) and aortic (AoSMC) smooth muscle cells were used. R899X^+/- BMPR2 transgenic mice fed a Western diet for six weeks were given daily injections of IL-1ß prior to assessment for PAH and tissue collection. PASMC have reduced inflammatory activation in response to IL-1ß compared with AoSMCs; however, PASMC with reduced BMPR2 demonstrated an exaggerated response. Mice treated with IL-1ß had higher white blood cell counts and significantly raised serum protein levels of IL-6 and osteoprotegerin (OPG) plasma levels recapitulating in vitro data. Phenotypically, IL-1ß treated mice demonstrated increased pulmonary vascular remodeling. IL-1ß induces an exaggerated pulmonary artery specific transcriptomic inflammatory response when BMPR2 signaling is reduced.

Keywords: BMPR-II; inflammation; interleukin-1ß; pulmonary hypertension.

Fig. 1.

Microarray analysis identifies tissue bed specific changes in of IL-1ß transcriptome. mRNA expression patterns in PASMC and AoSMC (n = 9) (a). Changes in mRNA showing a log2 fold-change of > 1 with false discovery rate (FDR)-adjusted P value ≤ 0.05. Pathway analysis was performed using signaling pathway impact analysis (SPIA) pathway analysis software on IL-1ß stimulated in PASMC by using the SPIA package in Limma using programming language R; all gene information from the PASMC arrays are taken into account and cross-referenced against known pathways. Altered pathways discovered using SPIA with significant changes to IL-1ß stimulation in PASMC were compared with the AoMSC IL-1ß responsive pathways changes to generate heatmaps indicating their activation or inhibition in each cell type (b). qRT-PCR validation was performed in PASMC and AoSMC showing increased expression of inflammatory proteins IL-6 and OPG being more pronounced in AoSMC than PASMC; PASMC have a sevenfold higher baseline level of VIPR1 which is reduced to IL-1ß. Receptors also showed PDGF receptors a and b being unaltered to IL-1ß in either cell type. (c) Ordinary one-way ANOVA with multiple comparison by Tukey’s post-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 2.

Microarray investigation in PASMC identifies BMPR2 specific changes in IL-1ß transcriptome. qRT-PCR was performed on pooled samples to confirm knockdown prior to arrays (n = 6 for BMPR2si and 3 for NTSi) (a). mRNA expression patterns in PASMC +/– BMPR2 siRNA (n = 9). Ordinary one-way ANOVA with multiple comparisons by Tukey’s post-test (****P < 0.0001). Changes in mRNA showing log2 fold-change of > 1 with adjusted P value ≤ 0.05 (b). Altered pathways discovered using SPIA with significant changes to IL-1ß stimulation in PASMCs +/– BMPR2 siRNA. Pathway changes to generate a heatmap indicating their activation or inhibition in each condition (c). t-SNE analysis was conducted on all arrays mock-transfected and BMPR2 si-transfected data-points for dimensionality reduction and shows data with multiple feature correlation in two dimensions (d). PASMC were co-transfected with Cignal NFkB reporter plasmid and NTsiRNA or BMPR2 siRNA and stimulated with BMP4 +/– IL-1ß for 48 h (e). Results were normalized to renila expression in same plasmid and then expressed as percentage of IL-1ß treated positive controls. Two-way ANOVA with Tukey post-test correction (*P < 0.05, **P < 0.01).

Fig. 3.

Comparison of the effect of reduced BMPR2 expression on IL-1-induced gene expression in commercial AoSMC and PASMC (a). IL-1ß stimulation in conditions of reduced BMPR2 expression in PASMC resulted in a greater induction of IL-6 compared to normal BMPR2 expression in PASMC but not in AoSMC or in PASMC taken from donors with and without known BMPR2 mutations (b). Expression of OPG is increased in PASMC upon loss of BMPR2 regardless of IL-1 ß stimulation. In AoSMC, BMPR2 loss causes a decrease of OPG which was reversed by IL-1ß stimulation; in donor cells increases were seen in OPG to IL-1ß stimulation regardless of their mutation status (c). VIPR1 expression is unaffected by loss of BMPR2 in aortic cells; however, PASMC lacking BMPR2 show baseline increases which normalize to IL-1ß. PASMC taken from donors display the same pattern as commercial PASMCs where loss of BMPR2 function induces a large increase in VIPR1 expression which can be normalized to an extent by IL-1ß (d). Array validation by qRT-PCR of receptor expression showed that expression of PDGF receptors in PASMC increased to loss of BMPR2 expression but normalized by IL-1ß; this trend is also the case in donor PAMSCs in PDGFRB, whereas PDGFRB is increased in mutant patient cells regardless of IL-1ß stimulation. In AoSMC, however, loss of BMPR2 induces loss of PDGFRa and b expression (e, f). Ordinary one-way ANOVA with multiple comparison by Tukey’s post-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 4.

Disease model protocol for IL-1ß dosing (a). No significant changes were seen in RVSP, whereas calculated PVR shows significant increase upon disruption of BMPR2 signaling by mutant overexpression and IL-1ß stimulation along with increases in WBC count indicating a greater inflammatory response to IL-1ß in the mutant mice (b). Smooth muscle actin and PCNA content of the small arterioles increases with a combination of IL-1ß increase and BMPR2 loss with representative IHC images (c). Increases in serum levels of IL-6 are seen in the IL-1ß treated mutant mice compared to the wild-type litter-mates and serum levels of OPG display significant increase to IL-1ß regardless of mutation status (d). Ordinary one-way ANOVA with multiple comparison by Tukey’s post-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 5.

A schematic diagram of the pathways discussed in this paper.