NFATc3 is required for chronic hypoxia-induced pulmonary ...

Am J Physiol Lung Cell Mol Physiol 301: L872–L880, 2011. First published September 9, 2011; doi:10.1152/ajplung.00405.2010.

NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice R. Bierer, C. H. Nitta, J. Friedman, S. Codianni, S. de Frutos, J. A. Dominguez-Bautista, T. A. Howard, T. C. Resta, and L. V. Gonzalez Bosc Departments of Pediatrics and Cell Biology and Physiology, School of Medicine, University of New Mexico, Albuquerque, New Mexico Submitted 11 November 2010; accepted in final form 5 September 2011

Bierer R, Nitta CH, Friedman J, Codianni S, de Frutos S, Dominguez-Bautista JA, Howard TA, Resta TC, Gonzalez Bosc LV. NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice. Am J Physiol Lung Cell Mol Physiol 301: L872–L880, 2011. First published September 9, 2011; doi:10.1152/ajplung.00405.2010.—Pulmonary hypertension occurs with prolonged exposure to chronic hypoxia in both adults and neonates. The Ca2⫹-dependent transcription factor, nuclear factor of activated T cells isoform c3 (NFATc3), has been implicated in chronic hypoxia-induced pulmonary arterial remodeling in adult mice. Therefore, we hypothesized that NFATc3 is required for chronic hypoxiainduced pulmonary hypertension in adult and neonatal mice. The aim of this study was to determine whether 1) NFATc3 mediates chronic hypoxia-induced increases in right ventricular systolic pressure in adult mice; 2) NFATc3 is activated in neonatal mice exposed to chronic hypoxia; and 3) NFATc3 is involved in chronic hypoxiainduced right ventricular hypertrophy and pulmonary vascular remodeling in neonatal mice. Adult mice were exposed to hypobaric hypoxia for 2, 7, and 21 days. Neonatal mouse pups were exposed for 7 days to hypobaric chronic hypoxia within 2 days after delivery. Hypoxiainduced increases in right ventricular systolic pressure were absent in NFATc3 knockout adult mice. In neonatal mice, chronic hypoxia caused NFAT activation in whole lung and nuclear accumulation of NFATc3 in both pulmonary vascular smooth muscle and endothelial cells. In addition, heterozygous NFATc3 neonates showed less right ventricular hypertrophy and pulmonary artery wall thickness in response to chronic hypoxia than did wild-type neonates. Our results suggest that NFATc3 mediates pulmonary hypertension and vascular remodeling in both adult and neonatal mice. arterial remodeling; right ventricular hypertrophy; nuclear factor of activated T cells reporter activity; pulmonary arterial smooth muscle; pulmonary arterial endothelial cells SECONDARY PULMONARY HYPERTENSION (PH) is caused by a variety of obstructive pulmonary diseases and residence at high altitude, two conditions associated with chronic hypoxia (CH) (20). Pulmonary vascular resistance rises in these settings due to pulmonary vasoconstriction, arterial remodeling, and polycythemia, which results in increased right ventricular systolic pressure (RVSP), right ventricular (RV) hypertrophy, and often heart failure (28). However, there is currently limited availability of novel therapies (34). Persistent PH of the newborn (PPHN) is a cause of significant morbidity and mortality in both term and preterm neonates. The incidence is reported to be 1–2 per 1,000 live births, with a mortality rate from 10 to 20% (22). PPHN is a disorder

Address for reprint requests and other correspondence: L. V. Gonzalez Bosc, Associate Professor, Cell Biology and Physiology, School of Medicine, Univ. of New Mexico, MSC08 4750, Albuquerque, NM 87131 (e-mail: lgonzalezbosc @salud.unm.edu). L872

of vascular transition from fetal to neonatal circulation, which manifests as hypoxemic respiratory failure (22). Mechanisms that interplay in the pathogenesis of PPHN include endothelial dysfunction, inflammation, mechanical strain, and hypoxia (22). PH can be very effectively developed in rodents by exposing them to simulated altitude in a hypobaric chamber; therefore, altitude exposure in this species provides an excellent model of secondary PH and PPHN (5, 6, 9, 13, 30). Postnatally, air-exposed mice demonstrate a regression of muscularization or a reduction in arterial wall thickness of pulmonary arteries (5, 6). This process is inhibited by hypoxia, leading to persistent fetal-like, thicker walled arteries (5, 6). Conversely, in adult animals, hypoxic pulmonary arterial remodeling is an active process. It is characterized by pulmonary arterial smooth muscle (PASMC) proliferation and hypertrophy, increased muscularity of the pulmonary arteries, and extended muscularity to more distal arteries (31). NFAT (nuclear factor of activated T cells) is a transcription factor that interlinks Ca2⫹ signaling, via a calcineurin-dependent pathway, with other signaling pathways to induce specific genetic programs. The NFAT family of proteins includes four isoforms, NFATc1 to NFATc4 (reviewed in Ref. 29). NFAT activates transcription of a large number of genes during an effective immune response and regulates cell differentiation in nonimmune cells (19). NFATc3 has specifically been implicated in vasculature development (18), regulation of smooth muscle contractile phenotype (11, 13, 16, 17), and modulation of vascular smooth muscle contractility (7, 25). In smooth muscle, NFATc3 nuclear accumulation and transcriptional activity are increased on activation of Gq/11-coupled receptors (1, 15, 17, 33). Our group has demonstrated that CH activates NFATc3 in mouse pulmonary arteries (13), which is dependent on endothelin-1 (ET-1) upregulation and Rho kinase (ROK) activation (12). Additionally, our group has demonstrated that NFATc3 is required for CH-induced RV hypertrophy and pulmonary arterial remodeling, suggesting that NFATc3 is necessary for the development of CH-induced PH (13) in adult mice. Consistent with these findings, Bonnet et al. (8) have implicated a role for NFATc2 in monocrotaline-induced PH in rats and in idiopathic human pulmonary arterial hypertension. However, it has not been previously demonstrated that NFATc3 is required for CH-induced increases in pressure in the pulmonary circulation in adult mice, and nothing is known about the role of NFATc3 in CH-induced PH in neonatal mice. Based on this information, we hypothesized that NFATc3 activation is required for CHinduced PH in both adult and neonatal mice.

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NFATC3 IN ADULT AND NEONATAL PULMONARY HYPERTENSION

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EXPERIMENTAL PROCEDURES

Luciferase Activity

All protocols employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico, School of Medicine (Albuquerque, NM).

Adult male 9x-NFAT-luciferase reporter mice were provided by Dr. Jeffery D. Molkentin (Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, OH). NFATc3 knockout (KO) mice have been kindly provided by Dr. Laurie Glimcher (Harvard University). The background strain is Balb/C. Heterozygous (HET) and HET or wild-type (WT) mice were bred to obtain age-matched WT, HET, and KO.

In adult mice, luciferase activity was measured from isolated left pulmonary arteries, as previously described (13). In contrast to adult mice, luciferase activity in neonatal mice was measured in whole lung due to technical limitations associated with obtaining sufficient vascular tissue from neonatal mouse lungs for analysis. One lobe of lung was isolated and lysed using tissue lysis buffer (Promega), according to the manufacturer’s protocol. Lysate was centrifuged for 10 min at 10,000 relative centrifugal force. Both luciferase activity and protein content were determined in the supernatant. Luciferase activity was measured using a Luciferase Assay System kit (Promega), and light was detected with a luminometer (TD20/20, Turner). Protein content was determined by the Bradford method (Bio-Rad) and used to normalize luciferase activity per sample.

Normoxia or Hypobaric Hypoxia Exposure

NFATc3 Immunofluorescence Confocal Microscopy

Adults. Mice designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ⬃380 mmHg for 2, 7, or 21 days. The chamber was opened one time per week to provide animals with fresh food, water, and clean bedding. Control animals were housed at ambient barometric pressure (normoxia, ⬃630 mmHg). All animals were maintained on a 12:12-h light-dark cycle. Neonates. Within 48 h of delivery, the dam and pups designated for CH exposure were placed in the hypobaric chamber while control litters remained in normoxia (ambient barometric pressure) for 7 days. Two days before euthanasia, mice were administered 5-ethynyl-2=deoxyuridine (EdU, Invitrogen) (see Cell Proliferation Assay) daily. All animals were maintained on a 12:12-h light-dark cycle. Only NFATc3 WT and HET were used for these experiments. Neonatal NFATc3 KO mice could not be studied due to maternal cannibalism in the CH group and because these mice were born at a lower than expected Mendelian ratio (12% instead of 25%).

Isolated lungs were fixed with Histochoice (Amrefco). Lungs were cryoprotected with 30% sucrose in PBS, embedded in optimum cutting temperature medium, and frozen. Cryostat sections (10 ␮m) were stained with primary rabbit polyclonal anti-NFATc3 (Santa Cruz Biotechnology) and mouse anti-␣-actin (Sigma), followed by donkey anti-rabbit Cy5 and anti-mouse Cy3 (Jackson ImmunoResearch Laboratories), as previously described (10, 13). Nuclei were stained using SYTOX green (Molecular Probes). Sections were examined using a ⫻40 objective on a Zeiss 510 laser scanning confocal microscope. Specificity of immune staining was confirmed by the absence of fluorescence in tissues incubated with primary or secondary antibodies alone. For scoring of NFATc3-positive nuclei, multiple fields for each vessel were imaged and counted by two independent observers using MetaMorph software (Universal Imaging). The software was programmed so that individual pixels would appear white instead of yellow, if the green nucleic acid stain and red NFATc3 stain colocalized. Thus a cell was considered positive if colocalization (white) was uniformly distributed in the nucleus and negative if no colocalization (green only) was observed.

Animal Models

In Vivo Assessment of RVSP and Hypertrophy in Adult Mice Adult mice were anesthetized with isoflurane in 100% inspired O2 fraction. A 23-gauge needle attached to a pressure transducer was inserted into the abdomen below the xiphoid process and directed it into the thoracic cavity toward the RV of the heart. Entry into the RV was confirmed by monitoring the pressure waveform. Peak RVSP and heart rate were obtained using Windaq data-acquisition software (Dataq Instruments). For the purposes of this study, an elevation in RVSP was taken as indicative of PH, since RVSP closely parallels pulmonary arterial pressure (32). The heart was isolated, and the atria and major vessels were removed. The RV was dissected from the left ventricle (LV) and septum (S) (LV⫹S). The degree of RV hypertrophy was expressed as the %ratio of RV to LV⫹S.

Vascular Morphometry

Tissue Harvesting from Neonatal Mice

Cell Proliferation Assay

After euthanasia with pentobarbital sodium (Sleepaway, 200 mg/kg ip), a midsternal incision was made to expose the chest cavity. Blood was withdrawn from the RV for hematocrit measurement. The trachea, lungs, and heart were isolated and removed en bloc and placed in HEPES- physiological salt solution solution. The right lobes of the lung were separated after the bronchi were tied off. The largest lobe was fixed in Histochoice (EMS), cryoprotected, and frozen in optimum cutting temperature. The left lung was placed in Ca2⫹-free HEPES solution to dilate the pulmonary vasculature before fixation. Next, the trachea was cannulated with a 24-gauge angiocath, and the left lung was fixed by inflation with a 4% formaldehyde solution to a pressure of 20 mmHg. After immersion in formaldehyde for 24 h, the lung was processed for morphological analysis. RV hypertrophy was assessed similarly to that of the adult mice. Also, a segment of tail was collected for genotyping of the pups.

Adult and neonatal pups were injected with EdU (3.3 mg/kg ip) daily for 2 days before euthanasia. EdU is a nucleoside analog to thymidine that is incorporated into DNA during active DNA synthesis. Frozen lung tissue sections (10 ␮m) were stained with a Click-iT EdU Alexa Fluor 488 imaging kit (Invitrogen) and the fluorescent DNA dye Hoechst 33342 (Invitrogen) to detect proliferating cells and determine total cell number, respectively. The %ratio of EdU-positive PASMC nuclei to total PASMC nuclei per artery were averaged per animal. Approximately 12 arteries/animal were analyzed.

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Neonate paraffin lung sections (5 ␮m) were stained with hematoxylin and eosin. Vessels were examined with a ⫻40 objective on a Zeiss 200M microscope with a Hamamatsu C4742–95 camera. Vessel images were acquired with Metamorph imaging system hardware and software (Universal Imaging) and analyzed with Image J (NIH). Vessels sectioned at oblique angles were excluded from analysis. Percent wall thickness was calculated and compared between groups using the following equation: {[2 ⫻ (luminal radius ⫺ external radius)]/external diameter} ⫻ 100. Luminal and external radii were calculated from vessel inner and outer circumferences, respectively. Approximately 10 arteries/animal were analyzed.

Cell Apoptosis Assay Neonate paraffin lung sections (5 ␮m) were stained with an In Situ Cell Death Detection Kit, POD (Roche). The staining is based on the terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-

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ing (TUNEL) reaction, which uses terminal deoxynucleotidyl transferase to label DNA strand breaks (caused by apoptosis), by incorporating FITC-labeled nucleotides to 3=-OH-free DNA ends in a template-independent manner. Arterial nuclei were counterstained with the DNA dye DAPI. Some sections were preincubated with DNase I (1 U/100 ␮l, Ambion) for 10 min at RT to degrade DNA and generate a positive control. Images were acquired using a Zeiss Axiovert 200M microscope with a Photometrics CoolSnap EZ camera run by MetaMorph. Activated caspase-3 was detected using a rabbit polyclonal antibody against cleaved caspase-3 (Cell Signaling Technology) and horseradish peroxidase-donkey anti-rabbit (Jackson ImmunoResearch). Secondary antibody-horseradish peroxidase was detected by incubating ⬃10 min in 0.5 mg/ml DAB in 1⫻ PBS and 0.025% H2O2. Images were acquired on a Nikon Eclipse E400 microscope with a DS-Fi1 camera (Nikon) run by NIS-Elements software. All the arteries in each section were analyzed. Statistical Analysis Results are expressed as the mean ⫾ SE. Statistical significance was tested at the 95% (P ⬍ 0.05) confidence level using Student’s t-test or two-way ANOVA, followed by Bonferroni’s post hoc test, as appropriate. RESULTS

NFATc3 Is Required for CH-induced PH in Adult Mice Our laboratory has previously demonstrated that CH increases NFAT activity in pulmonary arteries, and that pharmacological inhibition of calcineurin or genetic deletion of NFATc3 attenuates CH-induced RV hypertrophy and increased pulmonary arterial wall thickness in adult mice (13). To determine whether NFATc3 is indeed involved in CHinduced PH, RVSP and the degree of RV hypertrophy were measured in NFATc3 WT, HET, and KO adult mice. Consistent with our hypothesis, CH induced an increase in RVSP in WT and HET mice, but not in KO mice (Fig. 1A). Furthermore, RVSP in CH HET and KO mice was significantly less than RVSP in CH NFATc3 WT mice. There was no significant difference in RVSP between genotypes in normoxia. The RV hypertrophy seen in WT mice exposed to CH was not present in KO mice, as previously shown (13), but also HET mice displayed no RV hypertrophy (%RV/LV⫹S) in response to CH (Fig. 1B). There was no significant difference in RV hypertrophy between genotypes in normoxia. The same results were obtained whether we analyzed RV weight, %RV/ body weight (BW), or %RV/LV⫹S (Table 1 and Fig. 1B). In addition, there was no significant effect of CH or genotype on LV⫹S weight (Table 1). However, if LV⫹S weight is expressed as a percentage of BW, then there is a significant increase in %LV⫹S/BW in CH HET and CH KO compared with the same genotype exposed to normoxia. These results indicate that NFATc3 is required for increased RVSP and subsequent RV hypertrophy, which are signs of PH. PASMC Proliferation is Mediated by NFATc3 in Adult Mice In WT adult mice, PASMC proliferation increased after 2 days of CH (Fig. 2). However, at 7 and 21 days, this increase in cell proliferation was no longer seen compared with normoxic controls. No such proliferation was observed in KO mice, indicating that NFATc3 is required for proliferation of PASMC in response to short-term exposure to CH. AJP-Lung Cell Mol Physiol • VOL

Fig. 1. Nuclear factor of activated T cells isoform c3 (NFATc3) is required for chronic hypoxia (CH)-induced increases in right ventricular (RV) systolic pressure (RVSP) and RV hypertrophy in adult mice. WT, NFATc3 wild type; HET, NFATc3 heterozygote; KO, NFATc3 knockout mice exposed to normoxia or CH for 21 days. A: RVSP. B: RV hypertrophy expressed as the %ratio of RV to left ventricle (LV) and septum (S) (%RV/LV⫹S). Values are means ⫾ SE; n ⫽ no. of mice (see bars). *P ⬍ 0.05 vs. normoxia, #P ⬍ 0.05 vs. CH WT (two-way ANOVA followed by Bonferroni’s post hoc test).

CH Activates NFATc3 in Pulmonary Arteries of Neonatal Mice Luciferase activity (a measure of NFAT activity) was significantly increased in whole lungs from neonatal mice exposed to CH for 7 days compared with normoxic controls (Fig. 3A), indicating that NFAT is activated with hypoxic exposure in neonates similarly to what has been seen in pulmonary arteries from adult mice (13). Additionally, an increase in nuclear accumulation of NFATc3 was observed in both endothelial cells and PASMC of the neonatal pulmonary arteries compared with normoxic controls (Fig. 3, B and C). NFATc3 Is Required for CH-induced RV Hypertrophy in Neonatal Mice RV hypertrophy in response to CH exposure was observed only in NFATc3 WT neonatal mice (Fig. 4) similar to adult mice (Fig. 1B). RV hypertrophy in CH HET was significantly less than in CH WT mice (Fig. 4). Although CH produced a corresponding increase in %RV/BW in WT mice (Table 2), the tendency for %RV/BW to be less in CH HET vs. CH WT mice did not achieve statistical significance. However, because an-

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Table 1. Adult parameters Normoxia

BW, g RV, mg %RV/BW, g/g LV⫹S, mg %LV⫹S/BW, g/g n

CH

WT

HET

KO

WT

HET

KO

24.5 ⫾ 1.0 27.1 ⫾ 2.5 0.11 ⫾ 0.01 82.4 ⫾ 2.8 0.33 ⫾ 0.01 9

24.8 ⫾ 1.3 31.8 ⫾ 2.6 0.12 ⫾ 0.01 80.6 ⫾ 5.9 0.33 ⫾ 0.02 10

22.4 ⫾ 1.9 28.7 ⫾ 2.2 0.13 ⫾ 0.01 66.6 ⫾ 6.0 0.29 ⫾ 0.02 6

20.4 ⫾ 1.3* 40.9 ⫾ 3.0* 0.21 ⫾ 0.03* 73.4 ⫾ 5.2 0.38 ⫾ 0.01 9

22.9 ⫾ 1.5 32.5 ⫾ 1.6 0.14 ⫾ 0.01# 88.1 ⫾ 4.7 0.39 ⫾ 0.01* 10

20.6 ⫾ 1.3 34.8 ⫾ 2.7 0.18 ⫾ 0.02 82.5 ⫾ 4.1 0.42 ⫾ 0.03* 8

Values are means ⫾ SE; n, no. of animals. Nuclear factor of activated T cells isoform c3 (NFATc3) wild type (WT), heterozygote (HET), and knockout (KO) adult mice were exposed to normoxia or chronic hypoxia (CH) for 21 days. Body weight (BW), right ventricular (RV), and left ventricular (LV) plus septum (S) weight were measured and used to calculate %RV/BW and %LV⫹S/BW. *P ⬍ 0.05 vs. normoxia, #P ⬍ 0.05 vs. WT (two-way ANOVA followed by Bonferroni’s post hoc test).

imals exposed to CH exhibited lower BW than normoxic neonatal mice, regardless of genotype (Table 2), the parameter %RV/LV⫹S likely provides a more accurate measure of RV hypertrophy. These results thus demonstrate less CH-induced RV hypertrophy in neonatal mice, expressing only one allele of the NFATc3 gene. As expected (13), the polycythemic response to CH did not differ between the two genotypes (Table 2). LV⫹S and RV weight were lower in HET compared with WT mice, regardless of treatment (Table 2). No differences were observed in %LV⫹S/BW between groups. NFATc3 Is Required for CH-induced Increases in Pulmonary Arterial Wall Thickness in Neonatal Mice As previously reported for adult mice (13), a significantly thicker small pulmonary arterial wall was seen in neonatal CH WT compared with normoxia WT mice (Fig. 5). However, this response to CH was absent in HET mice. CH-induced Increase in Pulmonary Arterial Wall Thickness is Not Associated with Increased Cell Proliferation or Decreased Apoptosis

PASMC proliferation had occurred earlier in the exposure to CH. Unfortunately, we were not able to measure proliferation at earlier time points due to increased maternal cannibalism caused by neonate handling. Detection of cellular apoptosis was performed by TUNEL and activated caspase-3 staining. Figure 7A shows representative images of pulmonary arteries from lung sections stained for activated caspase-3 from NFATc3 WT and HET exposed to normoxia and CH. No apoptotic cells were found in the pulmonary arterial wall of any of the groups. All of the arteries present in three consecutive lung sections from four animals per group were analyzed. However, activated caspase-3-positive cells were detected in lung parenchyma, demonstrating that the staining was indeed working. TUNEL staining also showed no positive cells in the arterial wall of any of the experimental groups. Figure 7B shows a TUNEL-positive cell in lung parenchyma and a lung section incubated with DNAse before TUNEL staining as positive control. DISCUSSION

The observed thicker arterial wall in CH WT neonate mice was not associated with increased PASMC proliferation (Fig. 6). On the contrary, the %EdU-positive PASMC was less in arteries from CH-exposed mice compared with normoxic controls, regardless of genotype. However, it is possible that

The aim of this study was to determine whether: 1) NFATc3 mediates CH-induced increase in RVSP in adult mice; 2) NFATc3 is activated in neonatal mice exposed to CH; and 3) NFATc3 is involved in CH-induced PH in neonatal mice. The main finding of our study is that NFATc3 is required for CH-induced PH in both adult and neonatal mice. NFATc3 Is Required for CH-induced PH in Adult Mice

Fig. 2. NFATc3 is required for early CH-induced pulmonary arterial smooth muscle (PASMC) proliferation in adult mice. NFATc3 KO mice were exposed to normoxia (N) or CH for 2, 7, and 21 days. Values are means ⫾ SE of %5-ethynyl-2=-deoxyuridine (EdU)-positive PASMC; n ⫽ no. of mice (see bars). *P ⬍ 0.05 vs. N WT, #P ⬍ 0.05 vs. CH 2d WT (two-way ANOVA followed by Bonferroni’s post hoc test). Twelve arteries/animal were analyzed. AJP-Lung Cell Mol Physiol • VOL

We have previously shown that CH induces NFATc3 activation in pulmonary arteries of adult mice (13). Furthermore, NFATc3 KO mice do not develop RV hypertrophy in response to CH (13), suggesting NFATc3 is required for CH-induced PH. However, our previous study did not address whether NFATc3 was indeed involved in CH-induced increases in pulmonary arterial pressure per se. Our present findings indicate that NFATc3 activation is required for the increased RVSP seen in adult mice exposed to CH, demonstrating that it is required for CH-induced PH. Our results also support our laboratory’s previous findings that RV hypertrophy is only observed in NFATc3 WT mice exposed to CH (13). RV weight, %RV/BW, and %RV/LV⫹S significantly increased only in NFATc3 WT mice. However, %LV⫹S/BW was significantly greater in CH HET and CH KO compared with normoxic controls. These differences seem to be due to a slight but not significant reduction in BW in

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Fig. 3. CH activates NFATc3 in lungs from neonatal mice. A: CH increases luciferase activity in whole lungs from neonatal mice. Values are means ⫾ SE; n ⫽ 7 mice/group. *P ⬍ 0.05, Student’s t-test. RLU, relative light units. B: representative images of NFATc3 nuclear accumulation in endothelial cells (EC) and PASMC from pulmonary arteries of neonatal mice. Arrows indicate NFATc3 nuclear-positive cells. The white arrow depicts an EC, and the yellow arrow a PASMC. Red, NFATc3 staining; blue, ␣-actin staining; green, nuclear staining. ␣-Actin staining was used to distinguish PASMC from EC. Scale bar ⫽ 20 ␮m. C: summary showing CH-induced increase in %NFATc3 nuclear-positive EC and PASMC. Values are means ⫾ SE; n ⫽ no. of arteries (see bars) from 7 mice/group. *P ⬍ 0.05, Student’s t-test. 100 PASMC and 50 EC nuclei were counted per animal.

response to CH more than due to changes in LV⫹S weight. Systemic blood pressure was not measured; therefore, we cannot discard the possibility that they might be systemic hypertensive. However, it is unlikely because it has been previously demonstrated by us and other that NFATc3 KO mice do not respond with an increase in systemic blood pressure after exposing them to a hypertensive stimulus like intermittent hypoxia (10) or angiotensin II infusion (25). Most likely the RV hypertrophy is a consequence of increased pulmonary pressure. It is interesting that NFATc3 HET mice do not develop RV hypertrophy in response to CH, even though RVSP is slightly but significantly elevated. Therefore, we cannot discard the participation of myocardial NFATc3 in CH-induced RV hypertrophy, particularly because NFATc3 AJP-Lung Cell Mol Physiol • VOL

has been implicated in pressure overload-induced LV hypertrophy (24). Previous work from our laboratory has demonstrated that NFATc3 plays a significant role in CH-induced pulmonary vascular remodeling in adult mice by mediating increased pulmonary arterial wall thickness and upregulation of smooth muscle ␣-actin contractile protein (13). In the present study, we found that, in adult mice, there is a modest but significant early proliferative response of PASMC to CH (2 days) that is dependent on NFATc3. Longer exposure to CH, however, did not affect PASMC proliferation rate. A similar time course of CH-induced PASMC proliferation was reported previously by Paddenberg et al. (26). Our results are consistent with our laboratory’s previous findings showing downregulation of the

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Fig. 4. NFATc3 is required for CH-induced RV hypertrophy in neonatal mice. NFATc3 HET neonatal mice were exposed to normoxia or CH for 7 days. %RV/LV⫹S is expressed as means ⫾ SE; n ⫽ no. of mice (see bars). *P ⬍ 0.05 vs. normoxia, #P ⬍ 0.05 vs. CH WT (two-way ANOVA followed by Bonferroni’s post hoc test).

smooth muscle differentiation marker soluble guanlyl cyclase at 2 days of CH (11). In that same study, we also demonstrated that the expression of soluble guanlyl cyclase recovered after 21 days of CH, a time at which the hypertrophic marker ␣-actin was significantly upregulated through an NFATc3-dependent mechanism (13). Therefore, CH-induced pulmonary arterial remodeling seems to be due to initial proliferation (de-differentiation), followed by differentiation and hypertrophy of PASMC, and NFATc3 is required for the entire process. NFATc3 Activation in CH-induced PH in Neonatal Mice Neonatal mice develop RV hypertrophy and pulmonary arterial remodeling in response to CH (5, 6), both of which are indexes of PH. In a newborn pig model, elevated pulmonary vascular resistance after prolonged hypoxia is largely due to remodeling and fibrosis with a minimal component of PASMC contraction, as demonstrated by failure of vascular relaxation upon treatment with papaverine (14). However, in two different chronic PH neonatal rat models, ROK-mediated vasoconstriction appears to play a key role, demonstrated by a reduction in pulmonary vascular resistance with both acute (23) and chronic administration of a ROK inhibitor (37). Currently, there is no information of the differential contribution of

PASMC contraction and pulmonary arterial remodeling to PH in neonatal mice. We have focused on determining the role of NFATc3 on CH-induced pulmonary arterial remodeling in neonatal mice because our laboratory has previously demonstrated that this transcription factor is required for CH-induced pulmonary arterial remodeling in adult mice (11, 13). Our study demonstrates, for the first time, that NFATc3 is activated in the lungs of neonatal mice exposed to CH. Although NFAT activity was measured in whole lung rather than isolated pulmonary arteries, immunofluorescence microscopy confirmed increased nuclear accumulation of NFATc3 in endothelial cells and PASMC of neonatal pulmonary arteries in response to CH. The mechanism of NFATc3 activation in lungs of neonatal mice exposed to CH is currently unknown. It is possible, however, that ET-1 is a contributing factor, given the established role of ET-1 in neonatal hypoxic pulmonary vascular remodeling (2, 3) and our laboratory’s recent finding that ET-1 contributes to CH-induced NFATc3 activation in mouse pulmonary arteries (12). Findings from the present study also demonstrate that CHinduced activation of NFATc3 appears to be required for the development of PH, because neonatal NFATc3 HET mice do not respond to CH with RV hypertrophy. Based on our results in adult mice, RV hypertrophy is most likely due to increased PA pressure and RVSP. However, the participation of NFATc3 in myocardial hypertrophy cannot be ruled out. The reduced RV and LV⫹S weights in neonatal NFATc3 HET compared with WT mice are consistent with the lower BW of these mice. Our data in neonate mice (Table 2) and previous reports show that NFATc3 KO mice have lower BW than WT littermates. It has been demonstrated that this reduction is due to reduced muscle mass compared with WT mice (21). Even though HET mice were not analyzed in that study, it is possible that a small reduction in muscle mass is also present in neonatal NFATc3 HET mice. This reduction in BW is not present in adult NFATc3 HET or KO mice. It has been shown that, postnatally, pulmonary arteries become thinner in air-exposed mice. This process is inhibited by hypoxia, leading to persistent fetal-like, thicker-walled arteries (6). The effects of hypoxia are most evident in smaller resistance pulmonary arteries of newborn mice, similar to what is seen in human neonates (5). Our results are consistent with the latter study in that the walls of small pulmonary arteries from NFATc3 WT CH-exposed neonates were significantly thicker than those of normoxic NFATc3 WT mice. More importantly, NFATc3 HET mice did not show signs of pulmo-

Table 2. Neonate parameters Normoxia

BW, g RV, mg %RV/BW, g/g LV⫹S, mg %LV⫹S/BW, g/g %Hct n

CH

WT

HET

WT

HET

6.40 ⫾ 0.29 11.85 ⫾ 1.36 0.21 ⫾ 0.03 28.25 ⫾ 1.88 0.45 ⫾ 0.03 35.6 ⫾ 0.5 11

5.54 ⫾ 0.42 8.53 ⫾ 0.44# 0.17 ⫾ 0.02 22.10 ⫾ 0.97# 0.42 ⫾ 0.03 35.6 ⫾ 1.3 10

4.87 ⫾ 0.26* 13.84 ⫾ 1.46 0.30 ⫾ 0.02* 24.79 ⫾ 2.24 0.53 ⫾ 0.03 39.0 ⫾ 1.2* 7

4.36 ⫾ 0.31* 8.21 ⫾ 1.01# 0.25 ⫾ 0.03* 19.08 ⫾ 2.15# 0.48 ⫾ 0.03 39.8 ⫾ 0.6* 12

Values are means ⫾ SE; n, no. of animals. NFATc3 WT and HET neonatal mice were exposed to normoxia or CH for 7 days. Hematocrit (%Hct), BW, and LV⫹S were measured and used to calculate %LV⫹S/BW. *P ⬍ 0.05 vs. normoxia, #P ⬍ 0.05 vs. WT (two-way ANOVA followed by Bonferroni’s post hoc test). AJP-Lung Cell Mol Physiol • VOL

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Fig. 5. Greater pulmonary arterial wall thickness in CH neonatal mice requires NFATc3. NFATc3 HET neonatal mice were exposed to normoxia or CH for 7 days. A: representative images. Scale bar ⫽ 50 ␮m. B: summary of results. External diameter ranged from 25 to 60 ␮m. Values are means ⫾ SE; n ⫽ no. of mice (see bars). *P ⬍ 0.05 vs. normoxia, #P ⬍ 0.05 vs. CH WT (two-way ANOVA followed by Bonferroni’s post hoc test). Ten arteries per animal were analyzed.

nary arterial remodeling after CH exposure, demonstrating that, as in adult mice, NFATc3 is required for CH-induced pulmonary arterial remodeling. There is evidence that hypoxia leads to proliferation of PASMC in the newborn calf (35) and mouse models of PH (36). However, our results indicate decreased PASMC proliferation at the same time point that previous studies have shown increased proliferation in response to hypoxia (35), suggesting that the thicker arterial wall is not related to increased PASMC proliferation. The difference between our study and previous reports could be due to species and/or strain differences and the method used to determine cell proliferation. Additional mechanisms that could lead to increased arterial wall thickness include reduced apoptosis, PASMC hypertrophy, and/or accuAJP-Lung Cell Mol Physiol • VOL

mulation of extracellular matrix (2– 6, 27, 36). However, we have found no apoptotic cells in the pulmonary arterial wall in any of the animals, regardless of the experimental treatment or genotype. Apoptosis was determined by two different methods, activated caspase-3 and TUNEL. This negative result is not due to a deficiency in the detection methods, because apoptotic cells were detected in lung parenchyma in many of the sections analyzed from multiple animals. Limitations of our study include the use of a newborn mouse model, which may not closely resemble the human equivalent, and the analysis of NFAT activity in whole lung homogenates, which does not identify the cells responsible for the abnormalities in arterial remodeling. However, our findings that CH resulted in NFATc3 nuclear accumulation in both endothelial

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Fig. 6. NFATc3 is not required for CH-induced decreases in PASMC proliferation in neonatal mice. Incorporation of EdU in PASMC nuclei from NFATc3 WT and HET neonatal mice exposed to normoxia or CH for 7 days was expressed as %EdU-positive PASMC nuclei. Values are means ⫾ SE; n ⫽ no. of mice (see bars). *P ⬍ 0.05 vs. normoxia (two-way ANOVA followed by Bonferroni’s post hoc test). Twelve arteries per animal were analyzed.

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mechanism by which NFATc3 mediates this response remains to be determined. In conclusion, our results demonstrate that NFATc3 is required for CH-induced PH and the initial PASMC proliferative response to CH in adult mice. Furthermore, in neonatal mice, NFATc3 is activated by CH in pulmonary arteries, and NFATc3 is required for both CH-induced pulmonary arterial remodeling and associated RV hypertrophy. Therefore, our findings suggest that NFATc3 is involved in the vascular changes that underlie the development of PH in both adult and neonatal mice. More studies are needed to determine the cellular mechanisms and/or gene expression patterns regulated by NFATc3 that are involved in the development of hypoxiainduced PH. A clear understanding of these mechanisms may enable the identification of new therapeutic strategies for the management of pulmonary vascular remodeling in both infants and adults with PH. GRANTS

cells and PASMC suggest that NFAT activation occurs at the level of small pulmonary arteries that exhibit remodeling in response to CH. Additionally, the high mortality seen in KO neonates limited the analysis of complete NFATc3 gene deletion in the neonate and the effects seen with hypoxia. Similarly, the high mortality of younger neonates limited the determination of whether PASMC proliferation occurs at shorter CH exposure times. Therefore, while it is clear that NFATc3 is required for CH-induced arterial remodeling in the neonate, the

Funding support was from National Heart, Lung, and Blood Institute Grants R01HL088151 and R01HL088192 and the Department of Pediatrics, Division of Neonatology, University of New Mexico. Images in this paper were generated in the University of New Mexico & Cancer Center Fluorescence Microscopy Shared Resource, funded as detailed on: http://hsc.unm.edu/crtc/ microscopy/Facility.html. DISCLOSURES No conflicts of interest, financial or otherwise are declared by the author(s).

Fig. 7. CH does not induce pulmonary arterial cellular apoptosis in neonatal mice. A: representative images of activated caspase-3 detected by immunohistochemistry microscopy in lung sections from NFATc3 WT and HET neonatal mice exposed to normoxia or CH for 7 days. Activated caspase-3 was not detected in ECs or PASMC of any of the pulmonary arteries present in one section per animal in any of the experimental groups. Arrows depict positive activated caspase-3 cells, demonstrating apoptotic cells in lung parenchyma. Scale bar ⫽ 50 ␮m. B: representative images of a TUNEL-positive cell (depicted by arrow) present in lung parenchyma: first image, total nuclei were stained with DAPI; second image, positive TUNEL nuclei; third image, composite of DAPI and TUNEL channels; fourth image, DNAse-treated positive control. No TUNEL-positive cells were found in pulmonary arteries from the different experimental groups. All of the arteries present in three consecutive lung sections from four animals per group were analyzed. AJP-Lung Cell Mol Physiol • VOL

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