The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted

Abstract

In pulmonary arterial hypertension (PAH), antiapoptotic, proliferative, and inflammatory diatheses converge to create an obstructive vasculopathy. A selective down-regulation of the Kv channel Kv1.5 has been described in human and animal PAH. The resultant increase in intracellular free Ca(2+) ([Ca(2+)](i)) and K(+) ([K(+)](i)) concentrations explains the pulmonary artery smooth muscle cell (PASMC) contraction, proliferation and resistance to apoptosis. The recently described PASMC hyperpolarized mitochondria and increased bcl-2 levels also contribute to apoptosis resistance in PAH. The cause of the Kv1.5, mitochondrial, and inflammatory abnormalities remains unknown. We hypothesized that these abnormalities can be explained in part by an activation of NFAT (nuclear factor of activated T cells), a Ca(2+)/calcineurin-sensitive transcription factor. We studied PASMC and lungs from six patients with and four without PAH and blood from 23 PAH patients and 10 healthy volunteers. Compared with normal, PAH PASMC had decreased Kv current and Kv1.5 expression and increased [Ca(2+)](i), [K(+)](i), mitochondrial potential (Delta Psi m), and bcl-2 levels. PAH but not normal PASMC and lungs showed activation of NFATc2. Inhibition of NFATc2 by VIVIT or cyclosporine restored Kv1.5 expression and current, decreased [Ca(2+)](i), [K(+)](i), bcl-2, and Delta Psi m, leading to decreased proliferation and increased apoptosis in vitro. In vivo, cyclosporine decreased established rat monocrotaline-PAH. NFATc2 levels were increased in circulating leukocytes in PAH versus healthy volunteers. CD3-positive lymphocytes with activated NFATc2 were seen in the arterial wall in PAH but not normal lungs. The generalized activation of NFAT in human and experimental PAH might regulate the ionic, mitochondrial, and inflammatory remodeling and be a therapeutic target and biomarker.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

NFATc2 and Kv1.5 in human normal and PAH pulmonary arteries (PA). Confocal immunohistochemistry of human PAs from both normal and PAH patients (see also SI Fig. 11). PAH patients show up-regulated and activated NFATc2 (colocalized with the nucleus, which is stained with DAPI) within the PA wall. The anatomy of the vessels (lumen, media) can be seen in the dissemintated intravascular coagulation windows. Examples of nuclear NFATc2 are shown by arrows in the merged “zoom” column. In the same PAs, Kv1.5 expression is significantly down-regulated. In contrast, in four patients with normal pulmonary circulation and one patient with thromboembolic pulmonary hypertension, Kv1.5 expression is strong and NFAT2c expression is low and not nuclear.

Fig. 2.

NFATc2 and Kv1.5 in PAH PASMC. (A) Compared with normal PASMCs, iPAH PASMCs have significantly decreased whole-cell K+ current density and 4-AP-sensitive K+ current, membrane potential (Em), cell capacitance, and [K+]i. All of these are normalized by VIVIT or cyclosporine (CSA) (n = 11–12 per group). (B) [Ca2+]i measured with fluo-3 acetoxymethyl ester is significantly increased in iPAH compared with normal PASMC and is normalized by VIVIT (n = 30–35 per group). ∗, P < 0.05 vs. iPAH. (C) Immunocytochemistry (n ≈ 70 cells per group per experiment, three experiments) for NFATc2 (green) and the nucleus (blue) reveals that compared with normal cells, the majority of iPAH PASMC have activated NFATc2 (green within the nucleus). VIVIT or CSA, but not antennapedia, reverse the NFATc2 activation. (D) iPAH PASMC have decreased levels of Kv1.5 protein, compared with healthy PASMC, which is reversed by 48 h of exposure to VIVIT or CSA. Immunoblots in cultured PASMC were performed in triplicate; representative and mean densitometry data are shown. ∗, P < 0.05 vs. healthy controls.

Fig. 3.

NFATc2 regulates mitochondrial function, Bcl-2, and apoptosis in PAH PASMC. (A) iPAH PASMC mitochondria are hyperpolarized compared with normal and normalized by VIVIT or CSA (n≈ 50 per group per experiment, three experiments). (B) Although in untreated iPAH and healthy cells cytochrome c (green) colocalizes with the mitochondria (stained by mitotracker red), in the VIVIT-treated cells, cytochrome c is cytoplasmic (diffuse staining pattern). (C) VIVIT and CSA, but not antennapedia, induce apoptosis (% TUNEL-positive cells; annexin but not propidium iodide-positive cells) and decrease proliferation (% PCNA-positive cells) in iPAH PASMCs. (n ≈ 70 per group per experiment, three experiments). (D) (Left) Bcl-2 expression is increased in iPAH compared with healthy cells, which is reversed by VIVIT and CSA. (Right) Although in iPAH bcl-2 colocalizes with mitotracker (yellow staining), after VIVIT treatment, bcl-2 dissociates from mitochondria. ∗, P < 0.05 vs. iPAH.

Fig. 4.

NFAT in circulating blood cells in PAH. (A) Control patients do not show any infiltration with CD-3-positive T cells (red), or NFATc2 expression (green) in PAs; in PAH patients there are cells positive for both CD3 and activated NFATc2 attached to the endothelium (Upper) and within the PA wall (Lower). (B) NFATc2 is active in most circulating white cells in PAH but in none of the healthy patients (representative immunocytochemistry is shown). Buffy coat NFATc2 mRNA levels are higher in PAH patients (particularly scleroderma-PAH) compared with healthy controls or secondary pulmonary hypertension despite similar levels of pulmonary vascular resistance (PVR) with the latter (SI Table 2). However, ther was no correlation between NFATc2 levels and pulmonary vascular resistance in these cohorts. ∗, P < 0.05 vs. healthy controls.

Fig. 5.

Cyclosporine A reverses PAH. (A) Three weeks after MCT injection, rats were treated with CSA (1 mg/kg per os) for 2 weeks. CSA decreased mean PA pressure, total pulmonary vascular resistance, and right ventricular hypertrophy; increased cardiac output; and improved vascular remodeling (% medial thickness) (n = 5–6 per group). (B) Both immunohistochemistry and immunoblots (resistance PAs from four rats pooled per lane) show that CSA decreases NFATc2 activation and expression and restores Kv1.5 expression, mimicking VIVIT. PASMCs with activated NFATc2 are shown by arrows in the merged/zoom panels. CSA increases activated caspase 9 and decreases the bcl-2/bax ratio (a representative immunoblot from pooled PAs and densitometry data from three experiments are shown). ∗, P < 0.05 vs. MCT-PAH.

Fig. 6.

An NFAT-based theory for the pathogenesis of PAH and the multiple sites that NFAT-based therapeutic strategies might affect (see Discussion).