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
View: Text | PDF
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

×

Figure 4

NKX2-5 expression locks SMCs into the synthetic phenotype.

Options: View larger image (or click on image) Download as PowerPoint
NKX2-5 expression locks SMCs into the synthetic phenotype. (A) An immort...
(A) An immortalized HPASMC (iPASMC) cell line was transduced with an NKX2-5 lentiviral vector (iPASMC-NKX) or a control vector (iPASMC-control). The transduced cells were cultured for 7 days in growth media with 1% FBS and various concentrations of TGF-β to induce contractile marker expression. Evaluation of these results by 2-way ANOVA (Supplemental Figure 10) confirmed that the differences between control and transduced cells were highly significant (P < 0.01). Control and NKX2-5 samples were always run on the same blot. For all proteins, the same samples were run on different, but concurrent, Western blots. (B) Primary HPASMCs were transduced with either an NKX2-5 lentiviral vector (NKX) or a control vector. After transduction and selection, the cells were cultured for 7 days in media either at 10% FBS and low density to induce the synthetic phenotype or 1% FBS and high density to activate the contractile phenotype. Two-tailed Student’s t test analysis (n = 2) showed reduction in ACTA2 and TAGLN protein expression after NKX2-5 transduction was significant in both 1% and 10% FBS (P < 0.01), but the CNN1 reduction was only significant in 1% FBS (P < 0.01). (C) Western blot analysis of NKX2-5 expression in nuclear extracts from iPASMC-NKX and iPASMC-control (iPASMC-ctrl). Contractile HPASMCs are also included as a reference. In B and C, different genes were analyzed in the same samples run on different, but concurrent, blots.