Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction
Christa F. Gaskill, Erica J. Carrier, Jonathan A. Kropski, Nathaniel C. Bloodworth, Swapna Menon, Robert F. Foronjy, M. Mark Taketo, Charles C. Hong, Eric D. Austin, James D. West, Anna L. Means, James E. Loyd, W. David Merryman, Anna R. Hemnes, Stijn De Langhe, Timothy S. Blackwell, Dwight J. Klemm, Susan M. Majka
Christa F. Gaskill, Erica J. Carrier, Jonathan A. Kropski, Nathaniel C. Bloodworth, Swapna Menon, Robert F. Foronjy, M. Mark Taketo, Charles C. Hong, Eric D. Austin, James D. West, Anna L. Means, James E. Loyd, W. David Merryman, Anna R. Hemnes, Stijn De Langhe, Timothy S. Blackwell, Dwight J. Klemm, Susan M. Majka
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Research Article Pulmonology

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction

Abstract

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Authors

Christa F. Gaskill, Erica J. Carrier, Jonathan A. Kropski, Nathaniel C. Bloodworth, Swapna Menon, Robert F. Foronjy, M. Mark Taketo, Charles C. Hong, Eric D. Austin, James D. West, Anna L. Means, James E. Loyd, W. David Merryman, Anna R. Hemnes, Stijn De Langhe, Timothy S. Blackwell, Dwight J. Klemm, Susan M. Majka

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Figure 2

Isolated murine and human ABCG2+ lung MPCs demonstrate increased pericyte lineage specification in response to decreased BMPR2 or increased Wnt/β-catenin signaling.

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Isolated murine and human ABCG2+ lung MPCs demonstrate increased pericyt...
(A–F) BMPR2 knockdown and canonical Wnt activation in murine ABCG2+ lung MPCs increased pericyte lineage commitment. Sixteen weeks after induction, WT, Bmpr2fl/+, and βOE MPCs from lineage-labeled mice were isolated by flow sorting to detect eGFP fluorescence. (A) Following expansion, qRT-PCR analysis was performed to examine the expression levels of pericyte lineage–specific genes and canonical Wnt targets. (B) Western blot analysis was performed to quantitate the expression of NG2 and WISP1 proteins. A representative blot from two independent blots presented. (C) Isolated MPCs were stained with antibodies to detect cell-surface determinants characteristic of MPCs and pericytes. Gates were set based on fluorescence minus one control (FMO) controls. SSC-A, side scatter, area scaling. (D) Immunostaining was performed to localize β-catenin or cyclin D1 (Ccnd1) (red) in isolated cells (blue = DAPI-stained nuclei). Scale bars: 100 μm. (E and F) BMP signaling was decreased in isolated murine WT lung MPCs using the small-molecule inhibitor DMH1, independently 2 times. (E) Canonical Wnt signaling activity was measured 48 hours after transfection using a TCF/LEF dual-luciferase reporter assay and repeated independently 3 times. (F) Canonical Wnt target and pericyte lineage gene expression levels were evaluated after 48 hours by qRT-PCR. (G and H) Human PAH ABCG2+ lung MPCs. (G) qRT-PCR analyses of control, HPAH, and IPAH human ABCG2+ lung MPCs were performed to quantitate the relative levels of gene expression for the pericyte lineage markers CSPG4, ACTA2, RGS5, the Wnt pathway targets DKK1, SFRP1, and WISP1, as well as the matrix proteins COL1A1 and COL1A3 and the cell-cycle regulator CCND1 (n = 3–4). (H) Protein levels of NG2 and the β-catenin target cyclin D1 were quantified by Western blotting (n = 3–5). Each assay was performed independently 2 times. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and #P < 0.001, by 1-way ANOVA followed by Tukey’s post-hoc test (for murine qRT-PCR) and a nonparametric Wilcoxon-Kruskal-Wallis test and χ2 approximation (for patients’ samples).