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 6

Signaling pathways that result in NKX2-5 activation.

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Signaling pathways that result in NKX2-5 activation. (A) HPASMCs culture...
(A) HPASMCs cultured for 2 days, in synthetic conditions (10% FBS, low density), in hypoxia (1% oxygen), or normoxia, with or without TGF-β1 (4 ng/mL). Total NKX2-5 protein was analyzed by densitometry (n = 3). au, arbitrary units. (B) Synthetic HPASMCs expressing NKX2-5 were treated with inhibitors for ASK1 (TS ASK10), ERK5 (ERK5-IN-1), and PI3K (GSK2126458). (C) Densitometry quantification (n = 3) for B. (D) HPASMCs grown under synthetic conditions, treated with a TGF-β receptor II inhibitor (SD208). (E) Western blotting was performed using nuclear extracts from HPASMCs grown under synthetic conditions and treated with vehicle, siRNA (50 nM final concentration) against ERK5 (siERK5), or control siRNA (siCTRL) and quantified by densitometry (n = 3). Data presented as mean ± SD. For all blots with multiple proteins, the same samples were run on different, but concurrent, Western blots. (F) Potential signaling mechanism for the activation of NKX2-5 in HASMCs: Combinations of hypoxia and injury aided by TGF-β reactivate NKX2-5 via the PI3K and ERK5 pathways. Statistical significance was assessed by paired, 2-tailed Student’s t test. *P < 0.05.