PBX transcription factors drive pulmonary vascular adaptation to birth
David J. McCulley, … , Licia Selleri, Xin Sun
David J. McCulley, … , Licia Selleri, Xin Sun
Published February 1, 2018; First published December 18, 2017
Citation Information: J Clin Invest. 2018;128(2):655-667. https://doi.org/10.1172/JCI93395.
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Research Article Development Genetics

PBX transcription factors drive pulmonary vascular adaptation to birth

Abstract

A critical event in the adaptation to extrauterine life is relaxation of the pulmonary vasculature at birth, allowing for a rapid increase in pulmonary blood flow that is essential for efficient gas exchange. Failure of this transition leads to pulmonary hypertension (PH), a major cause of newborn mortality associated with preterm birth, infection, hypoxia, and malformations including congenital diaphragmatic hernia (CDH). While individual vasoconstrictor and dilator genes have been identified, the coordination of their expression is not well understood. Here, we found that lung mesenchyme–specific deletion of CDH-implicated genes encoding pre–B cell leukemia transcription factors (Pbx) led to lethal PH in mice shortly after birth. Loss of Pbx genes resulted in the misexpression of both vasoconstrictors and vasodilators in multiple pathways that converge to increase phosphorylation of myosin in vascular smooth muscle (VSM) cells, causing persistent constriction. While targeting endothelin and angiotensin, which are upstream regulators that promote VSM contraction, was not effective, treatment with the Rho-kinase inhibitor Y-27632 reduced vessel constriction and PH in Pbx-mutant mice. These results demonstrate a lung-intrinsic, herniation-independent cause of PH in CDH. More broadly, our findings indicate that neonatal PH can result from perturbation of multiple pathways and suggest that targeting the downstream common effectors may be a more effective treatment for neonatal PH.

Authors

David J. McCulley, Mark D. Wienhold, Elizabeth A. Hines, Timothy A. Hacker, Allison Rogers, Ryan J. Pewowaruk, Rediet Zewdu, Naomi C. Chesler, Licia Selleri, Xin Sun

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Figure 5

Abnormal VSM contraction in Pbx1/2-CKO mice is reversed by treatment with a Rho-kinase inhibitor.

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Abnormal VSM contraction in Pbx1/2-CKO mice is reversed by treatment wit...
(A) Treatment of P0 to P7 mice with captopril or the endothelin receptor-A antagonist BQ-123 did not reduce the PVR of Pbx1/2-CKO mice compared with controls (untreated, *P = 0.004; captopril-treated, **P = 0.003; BQ-123–treated, ***P = 0.0004). (B) Acute treatment with BQ-123 at P7 increased the PAAT/PAET ratio in control (†P = 0.03) but not Pbx1/2-CKO (P = 0.80) mice. (C–E) Phosphorylated MLC-S20 staining was increased in VSM cells from Pbx1/2-CKO mice (§P = 0.013). (F–H) Lung perfusion with the Rho-kinase inhibitor Y-27632 decreased MLC-S20 staining compared with PBS in Pbx1/2-CKO mice (‡P = 0.03). (I) Y-27632 perfusion at P3 increased VSM cell size in Pbx1/2-CKO (#P = 0.001) but not control (P = 0.14) mice. VSM cell size was similar following Y-27632 perfusion in control and Pbx1/2-CKO lungs (P = 0.97). VSM cell size was similar between Y-27632–treated Pbx1/2-CKO and PBS-treated control (P = 0.07) mice. (J) PVR computed from the histological small artery size revealed increased resistance in Pbx1/2-CKO mice relative to controls (††P = 0.008). Perfusion with Y-27632 significantly decreased the PVR in Pbx1/2-CKO lungs (†††P = 0.02) but not in control lungs (P = 0.36). The PVR of Y-27632–treated Pbx1/2-CKO mice was similar to that of untreated controls (P = 0.96). (K) Measurement at P14 showed increased RVSP following central venous injection of PBS (‡‡P = 0.0006) and decreased RVSP following injection into lungs of Y-27632 compared with the RVSP before treatment (§§P = 0.008) and after treatment with PBS (##P < 0.0001). (L) Acute treatment with Y-27632 increased the PAAT/PAET ratio in Pbx1/2-CKO mice (§§§P = 0.04) but not in controls (P = 0.08) and restored the PAAT/PAET ratio in Pbx1/2-CKO mice to that of the untreated controls (P = 0.22). For all statistical analyses, 4 samples were included from each group. Statistical analysis was performed using a Student’s t test for comparisons between 2 groups, 1-way ANOVA with Tukey’s method for comparisons among 3 groups, or 2-way ANOVA with Tukey’s method for comparisons between 2 groups under 2 or more conditions. Data are presented as the mean ± SEM. Scale bars: 200 μm.