Macrophage-derived PDGF-B induces muscularization in murine and human pulmonary hypertension
Aglaia Ntokou, Jui M. Dave, Amy C. Kauffman, Maor Sauler, Changwan Ryu, John Hwa, Erica L. Herzog, Inderjit Singh, W. Mark Saltzman, Daniel M. Greif
Aglaia Ntokou, Jui M. Dave, Amy C. Kauffman, Maor Sauler, Changwan Ryu, John Hwa, Erica L. Herzog, Inderjit Singh, W. Mark Saltzman, Daniel M. Greif
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Research Article Pulmonology Vascular biology

Macrophage-derived PDGF-B induces muscularization in murine and human pulmonary hypertension

Abstract

Excess macrophages and smooth muscle cells (SMCs) characterize many cardiovascular diseases, but crosstalk between these cell types is poorly defined. Pulmonary hypertension (PH) is a lethal disease in which lung arteriole SMCs proliferate and migrate, coating the normally unmuscularized distal arteriole. We hypothesized that increased macrophage platelet-derived growth factor–B (PDGF-B) induces pathological SMC burden in PH. Our results indicate that clodronate attenuates hypoxia-induced macrophage accumulation, distal muscularization, PH, and right ventricle hypertrophy (RVH). With hypoxia exposure, macrophage Pdgfb mRNA was upregulated in mice, and LysM‑Cre mice carrying floxed alleles for hypoxia-inducible factor 1a, hypoxia-inducible factor 2a, or Pdgfb had reduced macrophage Pdgfb and were protected against distal muscularization and PH. Conversely, LysM‑Cre von-Hippel Lindaufl/fl mice had increased macrophage Hifa and Pdgfb and developed distal muscularization, PH, and RVH in normoxia. Similarly, Pdgfb was upregulated in macrophages from human idiopathic or systemic sclerosis–induced pulmonary arterial hypertension patients, and macrophage-conditioned medium from these patients increased SMC proliferation and migration via PDGF-B. Finally, in mice, orotracheal administration of nanoparticles loaded with Pdgfb siRNA specifically reduced lung macrophage Pdgfb and prevented hypoxia-induced distal muscularization, PH, and RVH. Thus, macrophage-derived PDGF-B is critical for pathological SMC expansion in PH, and nanoparticle-mediated inhibition of lung macrophage PDGF-B has profound implications as an interventional strategy for PH.

Authors

Aglaia Ntokou, Jui M. Dave, Amy C. Kauffman, Maor Sauler, Changwan Ryu, John Hwa, Erica L. Herzog, Inderjit Singh, W. Mark Saltzman, Daniel M. Greif

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Figure 7

Nanoparticle-mediated knockdown of Pdgfb attenuates distal arteriole muscularization, myofibroblast accumulation, and PH.

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Nanoparticle-mediated knockdown of Pdgfb attenuates distal arteriole mus...
(A and B) Nanoparticles (diameter 400 nm) loaded with the dye DiD were administered orotracheally to normoxic mice, and 12 hours later, cells from BALF and residual lung were stained for CD64 and subjected to flow cytometric analysis. (C) Quantification of experiments from A and B and Supplemental Figure 10, A and B, showing percentage of BALF or residual lung (RL) cells containing DiD+ nanoparticles (diameter 400 or 200 nm as indicated) expressing CD64. n = 3 mice per treatment. (D) BALF cells were harvested from normoxic mice, cultured with DiD-loaded 400 nm nanoparticles for 6 hours, and then stained for nuclei (DAPI). (E–M) Nanoparticles of 400 nm diameter were loaded with siRNA targeted against Pdgfb or scrambled (Scr) RNA, then administered to mice at the onset of hypoxia and twice per week thereafter. (E and F) Lungs were isolated from mice at hypoxia day 3, stained for Ly6G and CD64, and subjected to flow cytometry, and the percentage of CD64+Ly6G– macrophages was quantified (G). n = 3 mice per treatment. (H) Pdgfb RNA levels of CD64+Ly6G– macrophages isolated as in E and F were quantified by qRT-PCR. n = 3 mice per treatment with qRT-PCR done in triplicate. (I–M) Mice were treated with hypoxia for 21 days or maintained in normoxia. For hypoxic mice, sections containing distal arterioles in the L.L1.A1 area (I) or alveolar region (L) were stained for CD31 and SMA. Boxed regions are shown below as close-ups. RVSP (J), Fulton index (K), and number of myofibroblasts (arrowheads) per 100 alveoli were measured. More than 500 alveoli per mouse were quantified. Arterioles are labeled with “a” in L. n = 3 mice per treatment group. One-way ANOVA with Tukey’s multiple-comparison test (C, J, and K) and Student’s t test were used (G, H, and M). * vs. normoxia, P < 0.05. Scale bars: 10 μm (D) and 25 μm (I and L).