Macrophage-derived PDGF-B induces muscularization in murine and human pulmonary hypertension
Aglaia Ntokou, … , W. Mark Saltzman, Daniel M. Greif
Aglaia Ntokou, … , W. Mark Saltzman, Daniel M. Greif
Published February 16, 2021
Citation Information: JCI Insight. 2021;6(6):e139067. https://doi.org/10.1172/jci.insight.139067.
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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 6

PDGF-B secreted by macrophages from PAH patients promotes hPASMC proliferation and migration.

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PDGF-B secreted by macrophages from PAH patients promotes hPASMC prolife...
Monocytes were isolated from PBMCs of human controls and IPAH or SSc-PAH patients and differentiated into macrophages in culture. (A) Macrophages derived from human control monocytes were cultured under normoxic or hypoxic (3% O2) conditions for 12 hours, and then Pdgfb mRNA levels were measured by qRT-PCR. n = 3 humans (2 females and 1 male, aged 30–60 years old) with qRT-PCR done in triplicate. (B) qRT-PCR was used to assay Pdgfb mRNA levels of macrophages from controls and PAH patients. n = 5 humans per PAH diagnostic class, and n = 9 controls (see Supplemental Table 4), with qRT-PCR done in triplicate. (C–E) hPASMCs were cultured for 24 hours with medium preconditioned by control and patient macrophages. BrdU was included in the last 10 hours of this incubation. Cells were then stained for BrdU and nuclei (PI). (D) The percentage of total cells (PI+ nuclei) expressing BrdU for control humans and patients was normalized to this percentage for controls. (E) Anti–PDGF-B blocking antibody or control IgG was added to the conditioned medium 1 hour prior to incubation with hPASMCs. Results are the ratio of the percentage of total (PI+) cells that are BrdU+ for anti–PDGF-B treatment relative to IgG treatment, stratified by patient diagnostic class. n = 3 humans per PAH diagnostic class and n = 6 controls (see Supplemental Table 5), with 10 microscopic fields per human, 30–60 cells per field. (F–I) Medium preconditioned by control or patient macrophages was treated with anti–PDGF-B blocking or control IgG antibody for 1 hour and then placed in the bottom chamber of a Boyden apparatus. hPASMCs were added to the top chamber to assess migration toward the conditioned medium for 8 hours. Migrated cells (i.e., on the membrane’s bottom surface) were stained with Crystal Violet. (I) Quantification of the migrated cells relative to control patients, IgG treatment is shown. n = 4 humans per PAH class and n = 3 controls (see Supplemental Table 7), with 5 microscopic fields per human, 8–90 cells per field. One-way ANOVA with Tukey’s multiple-comparison test (B, D, and I) and Student’s t test were used (A and E). #, ## vs. IPAH, P < 0.05, <0.01, and *, **, ns vs. corresponding IgG controls, P < 0.05, < 0.01, not significant, respectively. Scale bars: 25 μm (C) and 50 μm (F–H).