NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension
Ioannis Papaioannou, Athina Dritsoula, Ping Kang, Reshma S. Baliga, Sarah L. Trinder, Emma Cook, Xu Shiwen, Adrian J. Hobbs, Christopher P. Denton, David J. Abraham, Markella Ponticos
Ioannis Papaioannou, Athina Dritsoula, Ping Kang, Reshma S. Baliga, Sarah L. Trinder, Emma Cook, Xu Shiwen, Adrian J. Hobbs, Christopher P. Denton, David J. Abraham, Markella Ponticos
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Research Article Pulmonology Vascular biology

NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension

Abstract

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Authors

Ioannis Papaioannou, Athina Dritsoula, Ping Kang, Reshma S. Baliga, Sarah L. Trinder, Emma Cook, Xu Shiwen, Adrian J. Hobbs, Christopher P. Denton, David J. Abraham, Markella Ponticos

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Figure 8

Nkx2-5 deletion ameliorates symptoms of chronic hypoxia–induced pulmonary vascular remodeling.

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Nkx2-5 deletion ameliorates symptoms of chronic hypoxia–induced pulmonar...
Nkx2-5–null (Nkx2-5flox Cre+ 4OH-T) or control male mice were exposed to hypoxia or kept under normoxia for 21 days (see Figure 7 and Methods). (A) Sections of the entire left lobe of the mouse lungs were immunostained for ACTA2 to visualize vessels. Representative images of small (20–50 μm), medium (40–70 μm), and large arteries (>70 μm) are shown. Scale bar: 100 μm. (B) Muscularization (thickness/circumference ratio) of the arterial wall of pulmonary vessels was analyzed (n = 5 mice per group and 300–500 vessels per group) and quantified under hypoxia and normoxia. *P < 0.05 by unpaired, 2-tailed Student’s t test. Data presented as mean ± SD. (C) RSVP measurements in Nkx2-5–null and control mouse groups under hypoxia and normoxia (n = 8 mice per group). **P < 0.01 by unpaired, 2-tailed Student’s t test. Data presented as mean ± SD. (D) Cumulative concentration-response curves to phenylephrine (PE) or sodium nitroprusside (SNP) in endothelium-intact first- and second-order pulmonary artery segments from Nkx2-5–null (red, n = 4) or control (black, n = 5) mice. Contraction in response to PE concentration is expressed as mean percentage ± SEM. Relaxation is expressed as mean ± SEM percentage reversal of PE-induced tone. Statistical significance at P < 0.05 was confirmed by 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 by post hoc unpaired, 2-tailed Student’s t test. (E) Mean RV/LV ratio as a standard measure of RV hypertrophy. **P < 0.01 by unpaired, 2-tailed Student’s t test. (F) Expression of Nkx2-5 in whole heart tissue. Representative blot from 3 different experiments. No statistically significant differences observed. Nkx2-5 and Gapdh were run on different, but concurrent, blots.