NFATc3 and VIP in Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease

Abstract

Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are both debilitating lung diseases which can lead to hypoxemia and pulmonary hypertension (PH). Nuclear Factor of Activated T-cells (NFAT) is a transcription factor implicated in the etiology of vascular remodeling in hypoxic PH. We have previously shown that mice lacking the ability to generate Vasoactive Intestinal Peptide (VIP) develop spontaneous PH, pulmonary arterial remodeling and lung inflammation. Inhibition of NFAT attenuated PH in these mice suggesting a connection between NFAT and VIP. To test the hypotheses that: 1) VIP inhibits NFAT isoform c3 (NFATc3) activity in pulmonary vascular smooth muscle cells; 2) lung NFATc3 activation is associated with disease severity in IPF and COPD patients, and 3) VIP and NFATc3 expression correlate in lung tissue from IPF and COPD patients. NFAT activity was determined in isolated pulmonary arteries from NFAT-luciferase reporter mice. The % of nuclei with NFAT nuclear accumulation was determined in primary human pulmonary artery smooth muscle cell (PASMC) cultures; in lung airway epithelia and smooth muscle and pulmonary endothelia and smooth muscle from IPF and COPD patients; and in PASMC from mouse lung sections by fluorescence microscopy. Both NFAT and VIP mRNA levels were measured in lungs from IPF and COPD patients. Empirical strategies applied to test hypotheses regarding VIP, NFATc3 expression and activity, and disease type and severity. This study shows a significant negative correlation between NFAT isoform c3 protein expression levels in PASMC, activity of NFATc3 in pulmonary endothelial cells, expression and activity of NFATc3 in bronchial epithelial cells and lung function in IPF patients, supporting the concept that NFATc3 is activated in the early stages of IPF. We further show that there is a significant positive correlation between NFATc3 mRNA expression and VIP RNA expression only in lungs from IPF patients. In addition, we found that VIP inhibits NFAT nuclear translocation in primary human pulmonary artery smooth muscle cells (PASMC). Early activation of NFATc3 in IPF patients may contribute to disease progression and the increase in VIP expression could be a protective compensatory mechanism.

Fig 1. VIP attenuates NFAT activation.

A) VIP administration inhibited CH-induced NFAT activation in isolated mouse intrapulmonary arteries. RLU = relative luciferase units. N = normoxia, CH V = 2 days chronic hypoxia. CH VIP = VIP (0.166 mg/kg/day) in osmotic pump one day prior and during CH exposure for 2 days. *p<0.05 vs N and CH VIP, n = 3–5 mice. ANOVA followed by Newman-Keuls. B) VIP attenuated ET-1-induced NFATc3-EGFP nuclear import in human PASMC. *p<0.05, n = 5 cells. t-test.

Fig 2. PASMC NFATc3 nuclear accumulation is enhanced in VIP KO mice.

Top: representative immunofluorescence confocal microscopy images. Nuclei depicted in green (SYTOX), NFATc3 in red and α-actin in blue. NFATc3 nuclear co-localization in white (see white arrows). Bottom: summary of percent of PASMC with NFATc3 in the nucleus. n = 15 to 28 arteries from 6 mice each group. *p<0.001

Fig 3. Airway and vascular remodeling graphed based on severity of histology.

Group 1 COPD FEV1<50%; Group 2 COPD FEV 50–80%; Group 3 COPD, control, FEV1>80%; Group 4 IPF FVC<50%. One-way analysis of variance (ANOVA) determined that means of airway remodeling scores were significantly different (p<0.002), and Post hoc Tukey’s Multiple Comparison Test found that Group 4 compared to each of the other three groups was significant for differences in airway remodeling (p<0.05 for each comparison). n = 5–7

Fig 4. NFATc3 activity and expression in pulmonary artery endothelial and smooth muscle cells from lungs from COPD and IPF patients.

A) Representative images of NFATc3 (red) and smooth muscle alpha-actin (blue) immunostaining of lung sections of patients from groups 1–4. Nuclei were stained with Sytox green. NFATc3 nuclear co-localization is shown in white. Scale bar = 50 μm. B) Summary results of endothelial cell (EC) NFATc3 staining intensity, % NFATc3+ EC nuclei, vascular smooth muscle (VSMC) NFATc3 staining intensity and % NFATc3+ VSMC nuclei. N = 7–14 patients. ANOVA followed by Newman-Keuls.

Fig 5. NFATc3 activity and expression in airway epithelial and smooth muscle cells from lungs from COPD and IPF patients.

A) Representative images of NFATc3 (red) and smooth muscle alpha-actin (blue) immunostaining of lung sections of patients from groups 1–4. Nuclei were stained with Sytox green. NFATc3 nuclear co-localization is shown in white. Scale bar = 50 μm. B) Summary results of airway epithelial cells (AEPC) NFATc3 staining intensity, % NFATc3+ AEPC nuclei, airway smooth muscle (ASMC) NFATc3 staining intensity and % NFATc3+ ASMC nuclei. N = 5–7 patients. ANOVA followed by Newman-Keuls.

Fig 6. NFATc3 and VIP mRNA levels in lungs from COPD and IPF patients.

NFATc3 and VIP mRNA levels were determined by real time PCR in lungs from patients from groups 1–4.