TNF drives aberrant BMP signaling to induce endothelial and mesenchymal dysregulation in pulmonary hypertension
Maria de la Luz Garcia-Hernandez, Javier Rangel-Moreno, Qingfu Xu, YeJin Jeong, Soumyaroop Bhattacharya, Ravi Misra, Stacey Duemmel, Ke Yuan, Benjamin D. Korman
Maria de la Luz Garcia-Hernandez, Javier Rangel-Moreno, Qingfu Xu, YeJin Jeong, Soumyaroop Bhattacharya, Ravi Misra, Stacey Duemmel, Ke Yuan, Benjamin D. Korman
View: Text | PDF
Research Article Inflammation Pulmonology Vascular biology

TNF drives aberrant BMP signaling to induce endothelial and mesenchymal dysregulation in pulmonary hypertension

Abstract

The pathobiology of pulmonary hypertension (PH) is complex and multiple cell types contribute to disease pathogenesis. We sought to characterize the molecular crosstalk between endothelial and mesenchymal cells that promote PH in the tumor necrosis factor α–transgenic (TNF-Tg) model of PH. Pulmonary endothelial and mesenchymal cells were isolated from WT and TNF-Tg mice and underwent single-cell RNA sequencing. Data were analyzed using clustering, differential gene expression and pathway analysis, ligand-receptor interaction, transcription factor binding, and RNA velocity assessments. Significantly altered ligand-receptor interactions were confirmed with immunofluorescent staining. TNF-Tg mice had increases in smooth muscle cells and Col14+ fibroblasts, and reductions in general capillary (gCAP) endothelial cells, Col13+ fibroblasts, pericytes, and myofibroblasts. Pathway analysis demonstrated NF-κB–, JAK/STAT-, and interferon-mediated inflammation, endothelial apoptosis, loss of vasodilatory pathways, increased TGF-β signaling, and smooth muscle cell proliferation. Ligand-receptor analysis demonstrated a loss of BMPR2 signaling in TNF-Tg lungs and establishment of a maladaptive BMP signaling cascade, which functional studies revealed stemmed from endothelial NF-κB activation and subsequent endothelial SMAD2/3 signaling. This system highlights a complex set of changes in cellular composition, cell communication, and cell fate driven by TNF signaling that lead to aberrant BMP signaling that is critical for development of PH.

Authors

Maria de la Luz Garcia-Hernandez, Javier Rangel-Moreno, Qingfu Xu, YeJin Jeong, Soumyaroop Bhattacharya, Ravi Misra, Stacey Duemmel, Ke Yuan, Benjamin D. Korman

×

Figure 7

Cell-specific effects of NF-κB activators and conditioned media from TNF-activated fibroblasts (FBs) on BMP2/BMPR2 and SMAD signaling.

Options: View larger image (or click on image) Download as PowerPoint
Cell-specific effects of NF-κB activators and conditioned media from TNF...
(A) Endothelial cells (ECs) from C57BL/6 mice (WT) or TNF-Tg mice (TNF) were cocultured with media from fibroblasts activated with TNF for 72 hours. RNA was isolated from cocultures 6 hours later and cDNA prepared for qPCR. Gene expression was normalized to 18s and gene expression in cocultures was compared to ECs from C57BL/6 mice stimulated with media from WT FBs stimulated with TNF. *P < 0.05 by 1-way ANOVA, and by Student’s 2-tailed t test. (B) Western blot analysis shows a global increase in Smad 2/3 in endothelial cells (left) of TNF-Tg mice stimulated with NF-κB agonists, which was not seen in fibroblasts (right). (C) BMP2, BMPR2, and ID2 in TNF-Tg ECs are downregulated by media from TNF-Tg FBs. FBs or ECs were stimulated with NF-κB agonists to determine their impact on BMP2 and BMPR2 expression. Gene expression was normalized to 18S compared to cells incubated with PBS (BL). LTβ increases BMP2 in ECs and FBs of TNF-Tg mice, while BMPR2 is globally reduced in stimulated fibroblasts of TNF-Tg mice. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.001 by Student’s 2-tailed t test.