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
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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

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Figure 5

Ligand-receptor analysis demonstrates prominent role of altered BMP signaling in TNF-mediated PH.

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Ligand-receptor analysis demonstrates prominent role of altered BMP sign...
(A) BMPR2 signaling in TNF-Tg mice. MultiNicheNet was used to identify ligand-receptor pairs that were specific to either WT or TNF conditions. WT lungs demonstrated strong gCAP Bmpr2 interactions with Bmp4, Bmp6, Bmp7, and Nog in fibroblasts, which were absent TNF-Tg conditions. (B) Aberrant Bmp2 signaling in TNF-Tg lungs. TNF lungs demonstrated the presence of interactions between Bmp2 in a variety of cells interacting with Hjv in smooth muscle cells and Bmpr1a in Col13+ fibroblasts, and these interactions were absent in WT lungs. (C) Decreased expression of Bmpr2 in TNF-Tg gCAP cells. Violin plots demonstrate expression across cell types. (D) Increased expression of Bmp2 in TNF-Tg lungs. Lungs from TNF-Tg and C57BL/6 mice were stained with antibodies specific for BMP2, BMPR2, and nuclei labeled with DAPI. BMP2 expression was comparable in the bronchial regions in the lungs of (E) TNF-Tg and (I) C57BL/6 mice. In contrast, BMP2 expression increased in the vascularized areas in the lungs of (F) TNF-Tg mice compared with (J) C57BL/6 mice. BMPR2 expression decreased in the bronchial areas of (G) TNF-Tg mice compared with (K) C57BL/6 mice. BMP2 pulmonary expression was higher in vascularized regions of (H) TNF-Tg mice than (L) C57BL/6 mice. Asterisks depict lumen of blood vessels. Scale bars: 100 μm. Three random pictures were taken (original magnification, ×200) in bronchial and vascularized areas in the lungs of TNF-Tg (n = 2–3, 3–9 pictures) and C57BL/6 mice (n = 2–3, 6–9 pictures). Representative pictures (×200) are shown. Area of BMP2+ or BMPR2+ signal in ×200 random fields was measured with NIH ImageJ. Area covered by BMP2 in (M) bronchial areas was comparable in TNF-Tg and C57BL/6 mice, while BMPR2 area was significantly increased in (O) bronchial areas of C57BL/6 mice. Area covered by (N) BMP2 and (P) BMPR2 in vascularized regions in the lungs of TNF-Tg mice was significantly larger than areas of BMP2 and BMPR2 signal in C57BL/6 mice. Graphs represent the mean ± SEM. Statistical significance was calculated with a paired, 2-tailed Student’s t test. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.