Family with sequence similarity 13, member Aの欠損は内皮ー間葉転換を促進する結果、肺高血圧症を増悪させる

概要

Background and purpose:
Pulmonary hypertension is a progressive and fatal lung disease diagnosed by a sustained elevation of pulmonary arterial pressure more than 20 mmHg. Pulmonary hypertension is characterized by pathological pulmonary artery remodeling such as intimal and medial thickening of muscular arteries, vaso-occlusive lesions, and fully muscularized small diameter vessels that are normally non-muscular peripheral vessels. These vascular remodeling is a result from endothelial cell dysfunction, smooth muscle cell and endothelial cell proliferation, and also cellular transdifferentiation. Many pathogenic pathways in pulmonary arterial hypertension have been revealed; TGF-β signaling, inflammation, pericyte-mediated vascular remodeling, iron homeostasis, and endothelial-to-mesenchymal transition (EndMT). Nonetheless, detailed molecular mechanisms underlying pulmonary hypertension remain to be elucidated, especially to develop more effective therapeutic strategies to treat this life-threatening disease.

Recent genome-wide association studies identified family with sequence similarity 13, member A (FAM13A) gene as a genetic locus associated with chronic pulmonary disease including chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis. However, the role of FAM13A in the development of pulmonary hypertension remained to be unknown. The aim of this study is to investigate the possible role of FAM13A in the pathogenesis of pulmonary hypertension.

Methods:
For in vivo studies, wild-type (WT) and Fam13a-/- mice at 6-7 weeks old were put in the chamber with non-recirculating gas mixture of 10% O2 and 90% N2 for 3-6 weeks to induce pulmonary hypertension. Before the hemodynamic assessments, heart rate, fractional shortening, cardiac output, and pulmonary artery acceleration time were evaluated by echocardiography. Mice were anesthetized with ~2% isoflurane, and RVSP was measured by inserting catheter transducer into right ventricle through right jugular vein. Mice were then sacrificed, lung samples were collected for histological analysis, western blot, and quantitative PCR. Hearts were dissected and weighed to evaluate the right ventricular hypertrophy.

For in vitro studies, human pulmonary artery endothelial cells (PAECs) were cultured in Humedia-EG2. To induce endothelial-to-mesenchymal transition, PAECs were treated with 10 ng/mL TGF-β1 and 10 ng/mL IL-1β for 6 days in the medium supplemented with 2% FBS. The medium was changed every other day. For retrovirus infection, PAECs were grown to 70% confluency, and incubated with medium containing retrovirus carrying GFP or FAM13A gene in the presence of polybrene (8 µg/mL) for 24 hours. The medium was then replaced with a fresh growth medium, and cells were treated or used for functional assays 48 hours after initial infection.

Results:
In the lungs of mice with pulmonary hypertension induced by chronic exposure to hypoxia, Fam13a expression was remarkably reduced comparing to that in the control mice. We have generated mice with target deletion of FAM13A (Fam13a -/-) in which LacZ cassette was inserted into the intron of the Fam13a gene locus. By using LacZ-staining, the expression of Fam13a was found in endothelial cells of lung vasculatures.

The role of FAM13A in pulmonary hypertension was then further explored by using Fam13a-/- mice. Under normoxic condition, there was no significant difference in lung structures, hemodynamic, and pulmonary arterial pressure between wild-type (WT) and Fam13a -/- mice. When exposed to chronic hypoxia, Fam13a -/- mice showed deteriorated pulmonary hypertension assessed by higher right ventricular systolic pressure and augmented right heart ventricular hypertrophy. Histologically, fully muscularized small diameter vessels were increased, while peripheral capillaries decreased in the lungs of Fam13a-/- mice. Mechanistically, mesenchymal markers and transcription factors expression were found to be significantly increased both in mRNA and protein levels in Fam13a-/- mice. These data suggested the enhanced endothelial-to-mesenchymal-transition (EndMT) in the lungs of Fam13a-/- mice exposed to chronic hypoxia. Furthermore, EndMT assessed by the emergence of cells double positive for endothelial and mesenchymal marker was apparently enhanced in the lungs of Fam13a -/- mice compared with that in WT mice. These data strongly suggest that loss of FAM13A promotes EndMT, resulting in the deteriorated pulmonary vascular remodeling and consequent pulmonary hypertension.

To further analyze the role of FAM13A in EndMT, in vitro study was done by utilizing PAECs. When EndMT was induced by IL-1β and TGF-β1 treatment, FAM13A expression was significantly reduced in PAECs. By using retrovirus-mediated gene transfection, FAM13A was overexpressed in PAECs, and subsequently treated with IL-1β and TGF-β1 to induce EndMT. Overexpression of FAM13A inhibited the induction of mesenchymal markers, whereas reduction of endothelial markers was not affected. In contrast, endothelial angiogenic capacities such as tube-formation, migration, proliferation, and apoptosis were not affected by FAM13A-overexpression. Of note, overexpression of FAM13A significantly reduced the non-phosphorylated active β-catenin and its nuclear accumulation in PAECs overexpressing FAM13A as compared to the control cells. Considering a crucial role of β-catenin in promoting EndMT, FAM13A decelerates the EndMT process at least partially through inhibiting the β-catenin signaling.

Discussion:
In this manuscript, the previously undescribed role of FAM13A in the development of pulmonary hypertension was finally revealed. Given that Fam13a was reduced in the lungs of mice with pulmonary hypertension, and genetic loss of FAM13A exacerbated pulmonary hypertension, enhancing and/or preserving FAM13A in the lungs might have a therapeutic potential.

All forms of pulmonary arterial hypertension are characterized by vascular remodeling and dysfunction, of which EndMT is one of the potential factors. EndMT is a cell transdifferentiation process in which endothelial cells lose endothelial specific markers and acquire mesenchymal properties. It has been reported that EndMT is involved in a variety of cardio-pulmonary diseases such as atherosclerosis, cardiac fibrosis, pulmonary fibrosis, and pulmonary hypertension. In the remodeled vasculatures in pulmonary arterial hypertension, α-smooth muscle actin-expressing mesenchymal-like cells accumulate, especially in obstructive pulmonary vascular lesions, and EndMT derives significant number of these mesenchymal-like cells.

Previous genome-wide association studies strongly suggested a role of FAM13A in chronic lung diseases. FAM13A is expressed in various types of tissues and cells, including airway and alveolar epithelial cells in the lung, pulmonary vascular cells, and mature adipocytes in adipose tissue. Accordingly, FAM13A has been involved in multiple biological processes such as epithelial cell regeneration, tumor cell proliferation and survival, and insulin signaling. In the current study, a protective role of FAM13A in the progression of pulmonary hypertension have been identified by utilizing mice in which Fam13a was genetically deleted. To our knowledge, this is the first report that identifies Fam13a expression in the lung vasculature and FAM13A negatively regulates β-catenin activity in endothelial cell. β-catenin signaling has been involved in epithelial-to-mesenchymal transition in pulmonary disease and cancer. Also, β-catenin has been reported to promote EndMT through nuclear accumulation and subsequent activation of TCF/Lef transcription factors. In the current study, overexpression of FAM13A decelerates the EndMT process in association with reduced active β-catenin levels and its nuclear accumulation in endothelial cells. These data strongly suggest that FAM13A negatively regulates EndMT process at least partially through inhibiting β-catenin signaling.

Because FAM13A is expressed in variety types of cells in the lungs, other FAM13A- mediated cellular processes might be involved in the pathogenesis of pulmonary hypertension. Nonetheless, our in vivo data using Fam13a-/- mice clearly showed that loss of FAM13A exacerbated pulmonary hypertension, and thus FAM13A is an attractive pharmacotherapeutic target for the treatment of pulmonary hypertension.