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 2

NKX2-5 expression is associated with the synthetic phenotype in HPASMCs.

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NKX2-5 expression is associated with the synthetic phenotype in HPASMCs....
HPASMCs were cultured in vitro under conditions favoring the contractile or synthetic phenotype over 7 days. (A) Protein expression levels of NKX2-5, synthetic cell markers COL1, and FN1, and contractile cell markers ACTA2, CNN1, and TAGLN were determined by Western blotting. A representative blot from 3 independent experiments is shown. Statistical significance was examined by 2-way ANOVA (Supplemental Figure 8). The effect of FBS and the effect of time were both significant at P < 0.05 for all genes. All genes come from the same samples run on different, but concurrent, blots, except for COL1, which was run on a separate occasion. (B) Nuclear extracts were used to quantify NKX2-5 expression via Western blotting with the E1Y8H antibody. A representative blot from 2 independent experiments is shown. Substantial expression of NKX2-5 was only observed after prolonged culture in serum (P < 0.05 via 2-tailed Student’s t test). NKX2-5 and TBP were run on different, but concurrent, blots. (C) Immunofluorescence was carried out on contractile (7 days in 1% FBS) or synthetic (7 days in 10% FBS) HPASMCs using specific antibodies for NKX2-5 (Alexa Fluor 488, green), intracellular pro-collagen type I (Procol1, Alexa Fluor 488, green), FN1 (Alexa Fluor 594, red), ACTA2 (Cy3, orange), and DAPI (blue). Nuclear expression of NKX2-5 was concomitant with high COL1 and FN1 expression in synthetic, but not contractile, HPASMCs. High levels of organized ACTA2 expression were only visible in contractile cells. Scale bars: 50 μm. All protein molecular weights are given as kDa in parentheses.