The International Journal of Biochemistry & Cell Biology 54 (2014) 137–148

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Endothelin-1 driven proliferation of pulmonary arterial smooth muscle cells is c-fos dependent Valentina Biasin a , Karolina Chwalek b , Jochen Wilhelm b , Johannes Best b , Leigh M. Marsh a , Bahil Ghanim a,c , Walter Klepetko c , Ludger Fink b,d , Ralph T. Schermuly b , Norbert Weissmann b , Andrea Olschewski a,e , Grazyna Kwapiszewska a,e,∗ a

Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria Department of Internal Medicine II, Universities of Giessen and Marburg Lung Center (UGMLC), Justus-Liebig University, Member of the German Lung Center (DZL), Giessen, Germany c Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria d Institute of Pathology and Cytology, Wetzlar, Germany e Department of Experimental Anaesthesiology, Medical University of Graz, Graz, Austria b

a r t i c l e

i n f o

Article history: Received 11 February 2014 Received in revised form 8 June 2014 Accepted 30 June 2014 Available online 10 July 2014 Keywords: Pulmonary hypertension Proliferation Endothelin-1 Transcription factor c-Fos

a b s t r a c t Pulmonary hypertension (PH) is characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation leading to vascular remodeling. Although, multiple factors have been associated with pathogenesis of PH the underlying mechanisms are not fully understood. Here, we hypothesize that already very short exposure to hypoxia may activate molecular cascades leading to vascular remodeling. Microarray studies from lung homogenates of mice exposed to only 3 h of hypoxia revealed endothelin-1 (ET-1) and connective tissue growth factor (CTGF) as the most upregulated genes, and the mitogen-activated protein kinase (MAPK) pathway as the most differentially regulated pathway. Evaluation of these results in vitro showed that ET-1 but not CTGF stimulation of human PASMCs increased DNA synthesis and expression of proliferation markers such as Ki67 and cell cycle regulator, cyclin D1. Moreover, ET-1 treatment elevated extracellular signal-regulated kinase (Erk)-dependent c-fos expression and phosphorylation of c-fos and c-jun transcription factors. Silencing of c-fos with siRNA abrogated the ET-1-induced proliferation of PASMCs. Expression and immunohistochemical analyses revealed higher levels of total and phosphorylated c-fos and c-jun in the vessel wall of lung samples of human idiopathic pulmonary arterial hypertension patents, hypoxia-exposed mice and monocrotaline-treated rats as compared to control subjects. These findings shed the light on the involvement of c-fos/c-jun in the proliferative response of PASMCs to ET-1 indicating that already very short hypoxia exposure leads to the regulation of mediators involved in vascular remodeling underlying PH. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Increased intrapulmonary pressure leading to right heart hypertrophy, pressure overload and eventually to right heart failure are the hallmarks of pulmonary hypertension (PH) (Olschewski et al.,

Abbreviations: PASMCs, pulmonary artery smooth muscle cells; IPAH, idiopathic pulmonary arterial hypertension; ET-1, endothelin-1. ∗ Corresponding author at: Ludwig Boltzmann Institute for Lung Vascular Research, c/o ZMF; Stiftingtalstrasse 24, 8010 Graz, Austria. Tel.: +43 316 385 72918; fax: +43 316 385 72058. E-mail address: [email protected] (G. Kwapiszewska). http://dx.doi.org/10.1016/j.biocel.2014.06.020 1357-2725/© 2014 Elsevier Ltd. All rights reserved.

2001). The increase in pulmonary pressure is mainly due to a persistent vasoconstriction of the pulmonary arteries and thickening of the vessel wall, which contribute to the reduction of lumen, further increasing blood pressure (Schermuly et al., 2011). All three layers of the vessel wall (intima, media and adventitia) are involved in the remodeling processes with the most prominent contribution of smooth muscle cells (SMCs). Different animal models such as chronic hypoxia exposure in mice and monocrotaline-induced injury in rats have been established in order to study the development and molecular mechanisms underlying PH (Stenmark et al., 2009). Although, none of the mentioned animal models can fully reproduce the complexity of human disease (Bauer et al., 2007), they can recapitulate several aspects of PH such as the increased

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intrapulmonary pressure and the increased thickening of vessel wall due to the hyperproliferation of SMCs. The increased expansion of SMCs may be caused by a cumulative effect of several growth factors acting in an autocrine and/or paracrine manner (Toshner et al., 2010). Many of these factors are secreted by SMCs (platelet-derived growth factor (PDGF)-BB) (Perros et al., 2008), by fibroblasts (transforming growth factor (TGF-␤) (Kelley et al., 1991) or endothelial cells (endothelin-1(ET-1)) (Vane and Botting, 1992). ET-1 is a 21 amino acid peptide released from endothelial cells under hypoxic conditions (Kourembanas et al., 1991) that can bind to two receptors: endothelin receptor A (ETA ) expressed only by SMCs or endothelin receptor B (ETB ) expressed by both endothelial and SMCs (Hall et al., 2011). The binding of ET-1 to ETA induces calcium influx, contraction and proliferation of SMCs (Wagner et al., 1992). Moreover, increase in calcium has been demonstrated to enhance expression of activator protein (AP)-1 family members which can, in a positive loop, be responsible for transcription of ET-1(Yamashita et al., 2001) and other growth factors such as TGF␤ and connective tissue growth factor (CTGF) (Moritani et al., 2003; Gonzalez-Ramos et al., 2012). AP-1 protein complex is a family of transcription factors, including jun (c-jun, junB and junD) and fos (c-fos, fosl2, fosl1 and fosB) components (Shaulian and Karin, 2001). These transcription factors are also called the immediate early genes, due to their capability to be activated transiently and rapidly in response to many different stimuli (Shaulian and Karin, 2001). C-fos and c-jun were first identified as oncogenic genes activated by the FBJ murine osteosarcoma virus (Silbermann et al., 1987) and avian sarcoma virus (Nishimura and Vogt, 1988), respectively. Consequently, they were shown to be highly relevant for cancer development and progression (Healy et al., 2013). While c-fos and c-jun are the most studied transcription factors in cancer field, their role and contribution to PH is still not fully addressed. Therefore, in the present study we focused on the possible role of c-fos and c-jun in proliferation of SMCs and development of PH in response to ET-1. 2. Materials and methods 2.1. Animal models Adult male Sprague-Dawley rats (300–350 g in body weight; Charles River Laboratories) were randomized for treatment 28 days after as.c. injection of saline or 60 mg/kg monocrotaline (MCT; Sigma-Aldrich) to induce pulmonary hypertension (PH). BALB/c mice were exposed to normobaric normoxia (inspiratory O2 fraction (FiO2 ) 0.21) or normobaric hypoxia (FiO2 of 0.10) for 3 h and C57BL/6 for 21 and 35 days. The left lung was fixed in 4% neutral buffered formalin for histology, while the right lung was snapfrozen in liquid nitrogen for molecular biology analysis. All animal experiments were approved by the local authorities (Graz, Austria and Giessen, Germany).

(hypoxia/normoxia) were performed for 18 h in UltraHyb buffer (Ambion, Austin, TX) at 42 ◦ C on 60-mer oligonucleotide microarrays (MWG 30K mouse) using the GeneTAC hybridization station (PerkinElmer, Waltham, MA). The data were analyzed using the limma package in R (Dean and Nielsen, 2007; Smyth and Speed, 2003). Intensity values were background subtracted; log ratios were normalized using a loess correction on the MA-plot values (Smyth and Speed, 2003). Regulated genes were filtered by using moderated t-statistics and adjusting the false discovery rate at 10% (Smyth and Speed, 2003). Experimental design is provided in Supplementary Fig. 1. Pathways were analyzed using PathwayExpress and OntoExpress of the Onto-Tools. The whole microarray data have been uploaded in GEO database with the record GSE56698. 2.3. Cell culture, transfection and cell stimulation Primary human pulmonary artery smooth muscle cells (hPASMCs) were isolated from pulmonary arteries from nontransplanted donor lungs. The purity of hPASMCs cultures was confirmed using immunofluorescent antibody staining for smooth muscle-specific isoforms of ␣-actin (minimum 95% of cells stained positive). All experiments were performed with cells between passage one to six (Supplementary Fig. 2). Growth arrest was performed by serum deprivation for 12 h. Pre-designed, commercially available siRNA sequence directed against human c-fos and c-jun were purchased from Darmacon (Thermo Scientific) and were used at the concentration of 100 nM. To control for non-specific gene inhibition a universal negative-control siRNA sequence was employed (Ambion). Cells were transfected using the Effectene Transfection Reagent (Qiagen). The siRNA-mediated down-regulation of the target proteins was assessed 48 h after transfection by real-time PCR and Western blotting. Recombinant ET-1 (Sigma Aldrich) was used at a concentration of 500 nM for indicated time points. For inhibitor studies, cells were treated with: 5 ␮M SP600126, 50 ␮M U0126, 80 ␮M Wortmannin, 8 ␮M SB203580, 5 ␮M BAPTA, 1 ␮M BQ123, and 1 ␮M BQ788 (all from Sigma Aldrich) and 10 ␮M U73122 (TOCRIS bioscience) for 1 h before addition of the ET-1. 2.4. Proliferation For proliferation studies, siRNA against c-fos and c-jun was applied to PASMCs. After 48 h, 1 × 104 hPASMCs were seeded on 96-well plates, and starved (VascuLife® Basal Medium, 0% FCS, 0.2% antibiotic/antimycotic) for 12 h. Afterwards 500 nM ET-1 was added for 24 h, and the proliferation of PASMCs was determined by [3H]-thymidine (BIOTREND Chemikalien GmbH) incorporation, as an index of DNA synthesis and measured as radioactivity by a scintillation counter (Wallac 1450 MicroBetaTriLux Liquid Scintillation Counter & Luminometer). All experiments were performed in quadruplicates. 2.5. RNA isolation and real-time PCR

2.2. Microarray analysis Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) from lung homogenate of 18 BALB/c mice per group exposed for 3 h to normobaric normoxia (FiO2 of 0.21) and to normobaric hypoxia (FiO2 of 0.10) (Veith et al., 2013). RNA from 6 mice each was used as a pooled sample for labeling and hybridization. Labeled cDNA was generated using Superscript II to reverse-transcribe 50 ␮g of RNA incorporating of Cy3and Cy5-dCTP (all reagents from Invitrogen, Karlsruhe, Germany). The labeled cDNA was purified using a PCR purification kit (Qiagen). The volume of elute was reduced from 50 ␮l to 10 ␮l using a centrifugal vacuum concentrator. Competitive hybridizations

RNA was isolated using the peqGOLD Total RNA isolation kit (PeqLab). Quantity of RNA was determined by measurement of absorbance at 260 nm and the quality of RNA was determined by measurement of the absorbance ratio at 280/260 nm (Nanodrop). Total RNA was reverse transcribed using iScript kit (BioRad) according to manufacturer’s instructions. Real-time PCR was performed using a LightCycler 480 (Roche). The PCR reactions were set up using QuantiFast SYBR PCR kit (Qiagen). Cycling conditions were as follows: 5 min at 95 ◦ C, [5 s at 95 ◦ C, 5 s at 60 ◦ C, and 10 s at 72 ◦ C] x45. Due to the non-selective double strand DNA binding of the SYBR® Green I dye, melting curve analysis and gel electrophoresis were performed to confirm the specific

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amplification of the expected PCR product. The Ct values were normalized to internal control hydroxymethylbilane synthase (HMBS) or beta-2-macroglobulin (B2M) using the following formula:Ct = Ct reference gene-Ct gene of interest. Therefore higher values of Ct correspond to higher relative expression of the gene of interest. Primer sequences are provided in Supplementary Table 1. 2.6. Protein isolation and Western blotting Proteins were isolated by applying cold complete RIPA buffer (supplemented with protease inhibitor and phosphatase inhibitor from Thermo Scientific) on the cell monolayers. Protein extracts were separated on a 12% SDS polyacrylamide gel, followed by electro-transfer to a nitrocellulose membrane. After blocking with 5% non-fat dry milk in TBS-T buffer, the membrane was incubated overnight at 4 ◦ C with one of the following antibodies: anti c-fos (Novus Biological),anti-p-c-fos (Biosource), anti-c-jun, anti-p-c-jun, anti-p-Erk1/2, anti-Erk1/2, anti-p-Jnk, anti-Jnk and anti-␣-tubulin (all antibodies were purchased from Cell Signaling and used in 1:1000 dilution). After 1 h incubation with peroxidaselabeled secondary antibody (Pierce), proteins were detected using ECL Prime Kit (GE Healthcare). 2.7. Immunohistochemistry and tissue staining Formalin-fixed, paraffin embedded lung tissues were cut to 2 ␮m thick sections and immunohistochemistry was performed using ZytoChemPlus AP-Fast Red Kit (Zymed Laboratories) or ImmPACTTM VIP Kit (Vector Laboratories) according to the manufacturer instructions. The following dilutions of primary antibodies were used: anti-c fos (1:100 in human, Novus Biologicals, 1:50 in mouse and rat, Atlas antibody no. HPA018531), anti-␣-SMA (1:100; Sigma-Aldrich no. 2228), anti-p-c-fos (1:50 Cell Signaling, phospho-serine 32 no. 5348), anti-c-jun (1:100 Cell Signaling no. 9165), and anti-p-c-jun (1:100 Cell Signaling, phospho-serine 73 no. 3270). Negative controls were performed with the omission of the primary antibody. Slides were scanned with an Aperio slide scanner and images were captured with Image Scope software (Aperio). 2.8. Immunofluorescence For immunofluorescence analysis, hPASMCs grown on 8-well chamber slides were fixed for 10 min in cold methanol and rinsed three times in PBS. Afterwards, the cells were blocked for 1 h with 3% BSA with 0.02% TritonX-100 in PBS, and then incubated overnight at 4 ◦ C with anti-c-fos (1:100; Atlas antibody no. HPA018531), anti-c-jun antibodies (1:100; Cell Signaling no. 9165) and anti-␣-SMA(1:150, SIGMA no. 2228). Cells were washed 3× with PBS buffer, incubated with anti-rabbit conjugated with Alexa Fluor® Dye 488 and/or Alexa Fluor® Dye 555 (Life Technologies) and mounted with fluorescence vectashield mounting medium (Vector). Cell nuclei were counterstained with 4 ,6-diamidino-2phenylindole dihydrochloride (DAPI, Sigma-Aldrich). For double immunofluorescence staining, the two primary and secondary antibodies were mixed. For microscopic inspection, an Olympus fluorescence Filters on Basic BX51 microscope was used.

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tissue were approved by the ethics committee of the Medical Faculty, Justus-Liebig-Universitaet Giessen (Germany). The patients’ characteristics have been reported previously (Veith et al., 2013; Kwapiszewska et al., 2012a,b). 2.10. Statistical analysis Data are presented in scattered dot plot, estimates are shown as median for all experiments. Min to max bars with median line has been used in the in vitro inhibitor studies for reasons of clarity. Statistical analysis was performed with GraphPad Prism 5 software. Significance of differences between two group medians was assessed by Student’s t test. p-values of differences between more than two groups were derived by Dunnett’s multiple-to-one comparison tests. For in vitro inhibitor studies, p-values of differences between multiple groups medians were derived by Bonferroni test, comparing selected pairs of medians. p-Values less than 0.05 were considered significant for all analysis. Significance is indicated by an asterisk on the graphs. All experiments were designed with control conditions. 3. Results 3.1. ET-1 is upregulated in microarray analysis from lung homogenate of mice exposed to 3 h hypoxia Chronic hypoxia is probably the most commonly used animal model to study PH and vascular remodeling. Therefore, the gene analysis of animals exposed to hypoxia might help to decipher molecular mechanism leading to the remodeling processes. For this reason we performed a microarray analysis on lung homogenates from mice exposed only to 3 h hypoxia to delineate genes and pathways which might trigger these processes. Our analysis revealed that the most differentially regulated genes belonged to MAPK signaling pathway (Supplementary Fig. 3). Out of the top 100 regulated genes 80 genes were upregulated and 20 were downregulated (Table 1). Many of them were identified as growth factors (Table 1, bold genes) (e.g. endothelin-1 (Edn1/ET-1), connective tissue growth factor (ctgf/CTGF), platelet derived growth factor (Pdgfb/PDGF-BB), proheparin-binding EGF-like growth factor (hbegf/HB-EGF) which act intracellularly through MAP kinases. As ET-1, was one of the most upregulated genes (Fig. 1) and is a wellestablished factor in PH (Rubin et al., 2002), we decided to delineate its role in the induction of the remodeling process. 3.2. ET-1 induces proliferation of human pulmonary artery smooth muscle cells (hPAMSCs) As the ET-1 is produced by endothelial cells and the receptors are mostly expressed in smooth muscle cells (Hall et al., 2011) we examined the relevance of ET-1 on hPASMCs. ET-1 stimulation significantly enhanced the mRNA expression of proliferative markers such as Ki67 (Fig. 2A) and cell cycle progression protein, cyclin D1 (Fig. 2B) but not PCNA (Supplementary Fig. 4). Accordingly, dose dependent stimulation experiment revealed that 500 nM ET-1 increased DNA synthesis as indicated by thymidine incorporation (Fig. 2C), suggesting a weak but statistically significant effect of ET-1 on hPASMCs proliferation.

2.9. Human samples

3.3. ET-1 induces c-fos expression in hPASMCs

Lung tissues were obtained from idiopathic pulmonary arterial hypertension (IPAH) patients (n = 8) who underwent lung transplantation. Non-transplanted donor lungs served as controls (n = 10). Samples were either snap-frozen in liquid nitrogen or fixed in 4% (m/v) paraformaldehyde. The investigations on human

Several studies revealed the connection between ET-1 and AP-1 transcription factors (Huang et al., 1993; Fantozzi et al., 2003) and both c-fos and c-jun, were upregulated in our 3 h hypoxia microarray (c-fos adjusted p-value = 0.032465787, p-value = 0.000454843, c-jun, adjusted p-value = 0.08238952, p-value = 0.002268897) (GEO

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Table 1 Top 100 genes differentially regulated between normoxic and hypoxic mouse lung homogenate. (A) Sequential order of the 80 most upregulated genes. (B) Sequential order of the most downregulated genes. Symbol A Mt2 Edn1 Edn1 Ctgf Ctgf Cdkn1a Mt1 Tnfrsf12a Thbs1 Ier3 Bnip3 Fkbp5 Map3k6 1190002H23Rik Fst Pde4b Lox 2310016C08Rik Rcan1 F3 Als2cr13 Angptl4 Lpl Lpl Hbegf Fas Gbe1 Tinagl Bcl2l1 Cyr61 Sgk1 Tspyl4 Errfi1 Sult1a1 Anxa1 Acot1 Tiam1 Sgk1 March7 Serpine1 Ccl17 Bcl2l1 Sgk1 Dusp6 1810011O10Rik Arg2 Tinagl Plat Flt1 Nt5e 9430020K01Rik Add3 Tmem37 Eln Bcar3 Serpina3n Adamts1 Ifrd1 Acot9 Rcan1 Rapgef4 Tagln Pdgfb Ptprr Ldha Avpi1 Pdk4 Higd1a EG546723 Mgll Ldha Ctsh Klf9 Ccbp2

Coefficients 2.971 2.168 2.127 2.046 2.039 2.031 1.978 1.956 1.884 1.709 1.705 1.687 1.672 1.653 1.581 1.536 1.491 1.412 1.384 1.354 1.343 1.335 1.23 1.216 1.19 1.14 1.129 1.127 1.082 1.079 1.068 1.033 1.025 1.023 1.021 1 0.997 0.995 0.991 0.988 0.977 0.948 0.944 0.916 0.903 0.899 0.889 0.885 0.876 0.857 0.846 0.838 0.835 0.831 0.83 0.829 0.817 0.804 0.792 0.783 0.761 0.76 0.723 0.718 0.715 0.711 0.691 0.649 0.646 0.632 0.631 0.622 0.611 0.611

Amean

Sem

10.45 9.7 9.83 10.19 10.71 10.64 10.83 9.83 8.83 10.32 10.54 9 9.26 11.82 8.58 9.48 8.83 11.45 8.12 9.3 8.77 9.54 10.65 10.86 8.74 9.01 8.97 10.74 9.68 9.8 10.69 11.77 9.42 9.59 11.6 10.29 8.48 11.66 8.48 7.83 8.03 10.76 9.97 10.21 9.9 8.38 9.25 8.76 9.28 9.12 10.54 10.17 9.73 9.16 9.76 9.28 8.98 10.58 9.27 9 7.88 12.05 11.24 7.4 11.68 10 8.38 9.77 11.2 11.73 11.42 11.53 10.8 8.66

0.159 0.138 0.071 0.125 0.064 0.102 0.104 0.089 0.114 0.099 0.073 0.095 0.087 0.127 0.073 0.089 0.123 0.094 0.097 0.102 0.106 0.105 0.087 0.087 0.072 0.115 0.07 0.065 0.114 0.092 0.075 0.119 0.103 0.077 0.111 0.102 0.08 0.094 0.076 0.079 0.088 0.081 0.083 0.076 0.079 0.073 0.076 0.097 0.078 0.079 0.069 0.083 0.067 0.081 0.087 0.106 0.077 0.092 0.101 0.07 0.083 0.074 0.088 0.066 0.077 0.085 0.075 0.072 0.079 0.075 0.069 0.074 0.07 0.07

t 18.71 15.75 30.06 16.4 32.07 19.99 19.09 21.91 16.49 17.22 23.46 17.71 19.24 13.04 21.53 17.27 12.15 14.96 14.22 13.25 12.68 12.76 14.09 14.01 16.55 9.92 16.09 17.43 9.53 11.79 14.2 8.71 9.96 13.23 9.16 9.77 12.51 10.57 13.04 12.46 11.08 11.75 11.35 12.12 11.45 12.39 11.64 9.17 11.27 10.8 12.31 10.15 12.44 10.2 9.54 7.83 10.62 8.75 7.83 11.16 9.17 10.24 8.17 10.8 9.35 8.38 9.16 9.01 8.17 8.47 9.13 8.4 8.76 8.74

p

Adjusted (p)

Lods

2.33E − 08 1.01E − 07 6.14E − 11 2.02E − 08 3.29E − 11 3.08E − 09 4.79E − 09 1.29E − 09 1.92E − 08 1.27E − 08 6.68E − 10 9.76E − 09 4.45E − 09 4.95E − 07 1.52E − 09 1.24E − 08 3.31E − 07 4.80E − 08 7.72E − 08 1.49E − 07 2.24E − 07 2.11E − 07 2.59E − 07 8.88E − 08 1.85E − 08 2.08E − 06 2.42E − 08 1.14E − 08 2.99E − 06 4.37E − 07 7.83E − 08 6.57E − 06 2.01E − 06 1.51E − 07 8.95E − 06 2.39E − 06 2.54E − 07 1.18E − 06 1.72E − 07 2.63E − 07 7.67E − 07 4.49E − 07 6.17E − 07 3.40E − 07 5.71E − 07 2.77E − 07 4.92E − 07 4.20E − 06 6.59E − 07 9.70E − 07 2.93E − 07 1.70E − 06 2.66E − 07 1.63E − 06 2.96E − 06 1.65E − 05 1.13E − 06 6.36E − 06 1.65E − 05 7.19E − 07 4.18E − 06 1.58E − 06 1.15E − 05 9.70E − 07 7.63E − 06 9.20E − 06 4.23E − 06 1.02E − 05 1.15E − 05 8.39E − 06 4.36E − 06 9.00E − 06 6.25E − 06 6.37E − 06

3.41E − 05 0.0001099 7.35E − 07 3.22E − 05 7.35E − 07 1.23E − 05 1.43E − 05 7.28E − 06 3.22E − 05 2.54E − 05 5.33E − 06 2.54E − 05 1.43E − 05 0.00027566 7.28E − 06 2.54E − 05 2.14E − 04 6.39E − 05 9.37E − 05 0.00015072 1.85E − 04 0.00018023 1.93E − 04 0.0001012 3.22E − 05 0.00080327 3.41E − 05 2.54E − 05 1.04E − 03 0.00026819 9.37E − 05 0.00199101 7.89E − 04 0.00015072 2.48E − 03 0.00087617 0.00019304 0.00054106 0.00016507 0.00019304 0.00038263 0.00026853 0.00032824 0.00021427 0.0003105 0.00019477 0.00027566 0.00138719 0.00034261 0.00046447 0.00019477 0.00070418 0.00019304 0.00069703 0.00103679 0.00402827 0.00053227 0.0019538 0.00402827 0.00036612 0.00138719 0.00069703 0.00293103 0.00046447 0.00222685 0.00250231 0.00138719 0.00272463 0.00293103 0.00241931 0.00141097 0.00247612 0.0019538 0.0019538

8.7 7.8 13.3 9.6 13.6 10.7 10.7 11.6 9.6 9.7 12 10.2 10.7 6.3 11.5 10 7.2 8.7 8.5 7.9 7.6 7.6 7.1 8.2 9.7 5.5 9.4 10 5.2 7 8.5 4.4 5.5 7.9 4.1 5.4 7.5 6 7.8 7.4 6.4 6.9 6.6 7.2 6.7 7.4 6.9 4.8 6.5 6.2 7.3 5.7 7.4 5.7 5.2 3.5 6.1 4.4 3.5 6.4 4.8 5.8 3.8 6.2 4.2 4.1 4.8 4 3.8 4.2 4.8 4.1 4.4 4.4

V. Biasin et al. / The International Journal of Biochemistry & Cell Biology 54 (2014) 137–148

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Table 1 (Continued) Symbol Pla2g12a Add3 Cnn1 Lims2 1110018M03Rik Hpca Xpnpep1 B Dbp Sox18 Wisp2 Cxcr4 Plac8 Plxnd1 Rtkn2 BC057170 Sox19 Pitpnc1 Tnfsf10 Pygb Foxf1a Gpr182 Hlx Wnt2 Elf2 Irf1 Stat1

Coefficients

Amean

Sem

0.61 0.609 0.609 0.599 0.592 0.573 0.505

9.21 9.67 10.25 11.25 9.28 9.81 9.21

0.072 0.07 0.077 0.063 0.072 0.069 0.064

−1.314 −1.176 −0.953 −0.943 −0.89 −0.85 −0.786 −0.731 −0.709 −0.696 −0.689 −0.679 −0.669 −0.658 −0.64 −0.63 −0.607 −0.602 −0.583

10.33 9.69 8.65 9.21 9.69 9.68 10.6 8.49 10.04 9.53 8.12 8.82 11.29 8.87 8.82 7.77 7.91 8.94 8.59

0.151 0.081 0.091 0.073 0.077 0.069 0.077 0.074 0.073 0.074 0.069 0.066 0.068 0.079 0.076 0.062 0.078 0.066 0.075

t

p

Adjusted (p)

Lods

8.44 8.69 7.95 9.58 8.18 8.26 7.84

8.64E − 06 6.74E − 06 1.45E − 05 2.85E − 06 1.13E − 05 1.04E − 05 1.63E − 05

0.00246056 0.00199101 0.00365096 0.00101745 0.00293103 0.00273399 0.00402827

4.1 4.4 3.6 5.2 3.9 3.9 3.5

−8.7 −14.54 −10.51 −12.97 −11.64 −12.32 −10.15 −9.87 −9.76 −9.43 −10.05 −10.22 −9.86 −8.37 −8.4 −10.1 −7.81 −9.11 −7.79

6.68E − 06 1.99E − 07 1.24E − 06 1.81E − 07 4.93E − 07 2.91E − 07 1.71E − 06 2.18E − 06 2.42E − 06 3.29E − 06 1.86E − 06 1.60E − 06 2.21E − 06 9.31E − 06 8.99E − 06 1.78E − 06 1.70E − 05 4.44E − 06 1.73E − 05

0.00199101 0.00017635 0.00056021 0.00016703 0.00027566 0.00019477 0.00070418 0.00082564 0.00087617 0.00112342 0.00074121 0.00069703 0.00082564 0.00250292 0.00247612 0.0007232 0.00410339 0.00141795 0.00412165

4.4 7.5 6 7.7 6.8 7.3 5.7 5.5 5.4 5.1 5.6 5.8 5.4 4.1 4.1 5.7 3.5 4.8 3.4

together these results indicate that ET-1 mediated the increased c-fos expression through ETA and subsequent activation of ERK pathway. Accordingly, phosphorylation of ERK was observed after 5 min stimulation with ET-1, returning to its basal level already after 15 min (Fig. 4B). The expression level of c-jun was not affected by any of the aforementioned inhibitors and phosphorylation of Jnk was unchanged upon ET-1 stimulation (Supplementary Fig. 5A and B). These findings indicate a major role of MEK/ERK1/2 pathway in regulating ET-1 driven c-fos expression. 3.4. ET-1 stimulation leads to c-fos and c-jun phosphorylation

Fig. 1. ET-1 is upregulated in hypoxia. Volcano plot showing ET-1 as one of the most upregulated gene in the microarray analysis: log 2 fold-change versus log 2-fold regulation.

record GSE56698). For the aforementioned reasons we examined whether ET-1 signaling was dependent on c-fos and c-jun transcription factors. Stimulation of hPASMCs with recombinant ET-1 led to very rapid and transient increase in c-fos mRNA levels (Fig. 3A). Accordingly, the maximal level of c-fos protein was achieved within 3- to 10-h post-stimulation, and then returned to basal level (Fig. 3B). In contrast, in this investigated time points expression of c-jun was unchanged on both mRNA (Fig. 3C) and protein levels (Fig. 3D). Pretreatment of hPASMCs with the ETA inhibitor, BQ123 attenuated the ET-1 dependent increase of c-fos (Fig. 4A). On the other hand, no significant effect on c-fos expression was observed with ETB inhibitor, BQ788. This finding suggests that the ET-1 dependent regulation of c-fos is mediated exclusively via ETA . Moreover, the pre-treatment of hPASMCs with U0126, a MEK/Erk1/2 inhibitor, abolished ET-1-dependent increase of cfos, while inhibition of the Jnk, Akt, p38, PLC and extracellular Ca2+ pathways did not exert any effect on c-fos expression. Taken

As transcription factors can be activated or stabilized by phosphorylation, (Okazaki and Sagata, 1995; Fuchs et al., 1996) we next investigated the direct effect of ET-1 on the phosphorylation of c-fos and c-jun. Upon ET-1 stimulation c-fos phosphorylation of serine 32 was increased after 45 min and remained highly phosphorylated at 60 min (Fig. 5A). In contrast, weak phosphorylation of c-jun of serine 73 was already apparent after 30 min of ET-1 stimulation. This finding suggests that ET-1 does not only affect the total protein amount but also the phosphorylation level of both c-jun and c-fos, enhancing their stability and therefore their transcriptional activity. To assess whether the ET-1-driven proliferation is mediated by c-fos and c-jun, we transfected cells with specific siRNAs against c-fos and c-jun. Proliferation was affected when c-fos was silenced, however, silencing of c-jun did not significantly decrease proliferation (Fig. 5B). Simultaneous knock-down of c-fos and c-jun did not have additive inhibitory effect on ET-1-driven proliferation of hPASMCs (Fig. 5B). Silencing efficiency is shown in Supplementary Fig. 6. Taken together this data suggest that ET-1 induced proliferation of hPASMCs is mediated via c-fos transcription factor. 3.5. CTGF does not affect Jnk pathway and c-jun expression level CTGF was the second most up-regulated gene in our microarray analysis. As ET-1 has been shown to increase CTGF and TGF-␤ levels (Lambers et al., 2013), we then aimed to evaluate whether expression of these growth factors is affected by ET-1 and if they

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Fig. 2. ET-1 induces proliferation of hPASMCs in a c-fos dependant manner: (A) Ki67 and (B) cyclin D1 mRNA relative expression quantified by real-time-PCR from ET-1 (500 nM) stimulated and untreated (nt) hPASMCs. * Significant difference (p < 0.05) compared to untreated cells. (C) Relative thymidine incorporation of hPASMCs treated with dose dependence ET-1. * Significant difference (p < 0.05) compared to untreated cells (nt) = reference group.

Fig. 3. ET-1 is upregulated in hypoxia and regulates c-fos: (A) mRNA expression quantified by real-time-PCR and (B) Western blot with respective quantification of c-fos level from ET-1 stimulated and untreated (nt) hPASMCs. * Significant difference (p < 0.05) compared to untreated cells. n = 7 individual experiments. (C) mRNA expression quantified by real-time-PCR and (D) Western blot with respective quantification of c-jun level from ET-1 stimulated and untreated (nt) hPASMCs. * Significant difference (p < 0.05) compared to untreated cells. n = 4 individual experiments.

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Fig. 4. ET-1 mediated c-fos upregulation is dependent on Erk. Relative (A) c-fos mRNA expression quantified by real-time-PCR from ET-1 (500 nM) stimulated and untreated (nt) hPASMCs. Cells were treated 1 h prior ET-1 stimulation with BQ123 (ETA antagonist), BQ788 (ETB antagonist), BQ123 + BQ788 (ETA + ETB antagonists), BAPTA (Ca2+ chelator), U73122 (PLC inhibitor), Wortmannin (AKT inhibitor) SP600126 (Jnk inhibitor), U0126 (Erk1/2 inhibitor), SB203580 (p-38 inhibitor) and DMSO. * Significant difference (p < 0.05) (n = 4 individual experiments). Min to max bars with median line were used for reasons of clarity (B) p-Erk protein level (Western blot) after stimulation with ET-1 (500 nM), and respective quantification. * Significant difference (p < 0.05) n = 4 individual experiments.

Fig. 5. ET-1 induces phosphorylation of c-fos and c-jun and c-fos-dependent proliferation: (A) c-fos and c-jun phosphorylation state (Western blot) after stimulation with ET-1 (500 nM) with respective quantification n = 5 and n = 3 individual experiments. (B) Relative thymidine incorporation of hPASMCs treated with si c-fos and si c-jun separate or in combination upon ET-1 stimulation. * Significant difference (p < 0.05) compared to ET-1 treated cells transfected with si-ctrl = reference group.

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Fig. 6. CTGF stimulation does not affect c-fos/c-jun and hPASMC proliferation: (A) c-jun (B) c-fos and mRNA relative expression quantified by real-time-PCR from CTGF (10 ␮g/ml) stimulated and untreated (nt) hPASMCs. * Significant difference (p < 0.05) compared to untreated cells. (C) p-c-jun and (D) p-Jnk protein level (Western blot) after stimulation with CTGF (10 ␮g/ml) with the respective quantification. * Significant difference (p < 0.05) n = 3 individual experiments. (E) Relative thymidine incorporation of hPASMCs treated with PDGF-BB (10 ng/ml) ET-1 (500 nM), CTGF (10 ng/ml) and CTGF + ET-1. * Significant difference (p < 0.05) compared to untreated cells nt= reference group.

can influence c-fos/c-jun levels in hPASMCs. As shown in Supplementary Fig. 7A and B, ET-1 treatment of hPASMCs increased TGF-␤ mRNA expression but not CTGF. Moreover, CTGF stimulation did not increase ET-1 (Supplementary Fig. 7C) as well as c-jun/c-fos mRNA expression levels (6A and B) or phosphorylation of c-jun and Jnk proteins (Fig. 6C and D) at the indicated time points. In addition, we did not observe any effect on the expression levels of proliferation markers (Supplementary Fig. 8A–C). CTGF did not increase hPASMCs proliferation as assessed by thymidine incorporation, neither alone nor in combination with ET-1, suggesting no cumulative role of CTGF with ET-1 induced proliferation (Fig. 6E). 3.6. c-Fos is upregulated in mouse hypoxia model of PH To confirm our finding in animal models of PH we analyzed whether c-fos and c-jun expression are upregulated in an acute and chronic hypoxic mouse model of PH. There was statistically significant change in c-fos mRNA expression level in mouse lung homogenate upon short hypoxia exposure but not upon long hypoxia exposure (Fig. 7A and B). Moreover, significantly increased mRNA expression of c-fos was observed in isolated pulmonary arteries after chronic hypoxia (21 days, Fig. 7C). On the other hand, c-jun expression levels were unchanged both in lung homogenates and isolated pulmonary arteries in investigated time points (Fig. 7D–F). Of note, TrkB expression levels, a gene reported to be highly regulated in hypoxia exposed mice (Kwapiszewska et al., 2012a,b) were elevated in all investigated time-points

(Supplementary Fig. 9A–C). Immunohistochemical staining on serial sections clearly demonstrated presence of c-fos and c-jun in the vessel wall (Fig. 7G). Importantly, phosphorylated c-fos/c-jun were localized in the smooth muscle cells layer of intrapulmonary arteries in hypoxic mice (Fig. 7G). To evaluate whether hypoxia has a direct effect on c-fos/c-jun expression, primary human hPASMCs were exposed to 1% of hypoxia for up to 24 h. Both c-fos and c-jun levels were unchanged by hypoxia exposure in our experimental set up (Supplementary Figs. 10A and 9B). These findings indicate that expression of c-fos and c-jun are not directly regulated by hypoxia but rather by factors such as ET-1 which expression is hypoxia-dependent. In line with this notion, in hypoxia mouse lung homogenates, ET-1 mRNA levels were upregulated at 3 h but not at 3 and 5 weeks when the remodeling is fully established (Supplementary Fig. 11A).

3.7. Expression levels of c-fos and c-jun are enhanced in monocrotaline rat model of PAH We then investigated expression levels of c-fos and c-jun in another model of PH the monocrotaline (MCT) rat model. Real-time PCR analysis of rat lung homogenates demonstrated elevated c-fos and c-jun levels in 4 weeks MCT-treated rats as compared to salinetreated controls (Fig. 8A and B). Immunostaining of adjacent tissue sections confirmed expression of both c-fos and c-jun and their phosphorylated forms in the smooth muscle cells layer (Fig. 8C).

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Fig. 7. c-fos and c-jun are upregulated in hypoxic mouse lungs: Relative mRNA levels of c-fos in (A) short time (B) long time hypoxia exposure and (C) hypoxic mouse isolated pulmonary artery. Relative mRNA levels of c-jun in (D) short time (E) long time hypoxia exposure and (F) hypoxic mouse isolated pulmonary arteries. * Significant difference (p < 0.05) compared to normoxia treatment. Normoxia samples values were pulled as no difference between different normoxia time points were observed. (G) Representative lung sections from hypoxic (5 weeks) and normoxic mice stained against ␣-sma, c-fos, c-jun, phospho c-fos and phospho c-jun. neg. ctrl-negative control, was performed with omission of the primary antibody.

3.8. c-Fos and c-jun mRNA levels are upregulated in IPAH lung samples Finally, we examined the relevance of c-fos and c-jun in human IPAH disease. Both c-fos and c-jun mRNA levels were elevated in human IPAH lung samples as compared to donors (Fig. 9A and B). Immunohistochemical staining localized both proteins to smooth muscle cells layer within the pulmonary vasculature (Fig. 9C) as demonstrated by co-staining against the ␣-SMA (Supplementary Fig. 12). Interestingly, stronger immunoreactivity for c-fos, c-jun and their phosphorylation forms were observed in remodeled vessels of IPAH patients (Fig. 9C). In accordance with mouse data, ET-1 mRNA levels were unchanged in established PH in MCT-treated rats as compared to controls as well as in IPAH patients versus donor samples (Supplementary Fig. 11B and C). 4. Discussion The molecular mechanisms leading to vascular remodeling in PH remain poorly understood. The hypoxic mouse model is a widely used to study and recapitulate some of the features of PH, such as vasoconstriction, remodeling and muscularization of the intrapulmonary arteries (Stenmark et al., 2009). Recently, we

have demonstrated that already a very short exposure of mice to hypoxia as well as their reoxygenation followed by unbiased screening technologies can lead to discovery of potential molecules involved in vascular remodeling (Kwapiszewska et al., 2012a; Veith et al., 2013; Weisel et al., 2014). Here, we showed that already very short hypoxia exposure (3 h) reveals many genes being regulated in the mouse lungs. Many of them have been described to be involved in processes such as vasoconstriction (e.g. guanine nucleotide exchange factors (Rap) and of the Rho kinase substrate, calponin) (Wang et al., 2001) and vascular remodeling (e.g. elastin, transgelin) (Merklinger et al., 2005; Zhang et al., 2014). Moreover, some of the regulated genes have been implicated to be hypoxia inducible factors (HIF)-dependent such as ET-1 (Hu et al., 1998), CTGF (Higgins et al., 2004) and lox (Erler et al., 2006). In our microarray screening HIF transcription factors were not found to be regulated which is in agreement with the notion that HIFs are mainly regulated at the protein level, thorough their stabilization (Lee et al., 2004). Additionally, at this very early time point many growth factors and mediators were found to be significantly upregulated, exemplarily PDGF-BB, known factor in PH (ten Freyhaus et al., 2011; Schermuly et al., 2005; Perros et al., 2008), plasminogen activator inhibitor (PAI) and tissue plasminogen activator (tPA) (Martin et al.,

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Fig. 8. c-fos and c-jun are upregulated in monocrotaline (MCT) rat lungs: Relative mRNA levels of (A) c-fos and (B) c-jun in MCT and control rat lung homogenate. * Significant difference (p < 0.05) compared to saline treated rats. (C) Representative lung sections from MCT treated and control rats stained against ␣-sma, c-fos, c-jun, phospho c-fos and phospho c-jun neg. ctrl-negative control, was performed with omission of the primary antibody.

Fig. 9. c-Fos and c-jun are up-regulated in IPAH patient lungs. Relative mRNA levels of (A) c-fos and (B) c-jun in IPAH and donors patient lung homogenate. * Significant difference (p < 0.05) compared to donor lung homogenate. (C) Representative lung sections from IPAH and donor lung stained against and ␣-sma, c-fos, c-jun, phospho c-fos and phospho-c-jun neg. ctrl-negative control, was performed with omission of the primary antibody.

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2002), CTGF, and ET-1 (Morrell et al., 2009). ET-1 is one of the most effective vasoconstrictors and an important regulator of vascular tone. It is produced by endothelial cells and has been shown to be elevated in PH patients (Nootens et al., 1995). Even though ET-1 is a current therapeutic target, its role in mediating smooth muscle cell proliferation is controversial. A recent study revealed that ET-1 by itself does not lead to proliferation of PASMCs (Lambers et al., 2013), whereas our data and others (Kunichika et al., 2004) showed that high concentration of ET-1 significantly affects DNA synthesis in starved hPASMCs. This is further supported in our study by the ET-1 induced expression of proliferating markers such as Ki67 and cyclin D1. Stimulation of tyrosine kinases or G protein coupled receptors lead to MAPK induction and consequently to transcription factor activation, which can influence the proliferative or migratory state of the cells (Pearson et al., 2001). Transcription factors are the convergent molecules of multiple pathways activated by different growth factors. Therefore, analysis of downstream transcription factors may help to understand the molecular crosstalk between different stimuli such as growth factors, stressor hypoxia. We further investigated the role of AP-1 components, c-jun and c-fos, in PH and their contribution to the smooth muscle cell proliferation in response to ET-1. We showed that ET-1 treatment did not change c-jun levels but increased c-fos in hPASMCs. Accordingly, ET-1 treatment of hPASMCs led to phosphorylation of ERK1/2 and blockage of ERK1/2 attenuated ET-1 dependent increase in c-fos. These finding supports our array result where MAPK as the most differentially regulated pathway, might play an important role in vascular remodeling. Furthermore, recently it has been reported that ET-1 affects smooth muscle cell proliferation only in combination with others growth factors such as TGF-␤ (Lambers et al., 2013) revealing that milieu of different factors can act in a cumulative manner. Additionally, ET-1 leads to the release of TGF-␤ and CTGF from hPASMCs, which can then act in a paracrine or autocrine manner (Lambers et al., 2013). As next to ET-1, CTGF was the second most up-regulated gene in our microarray screening, we speculated that ET-1 could increase CTGF release, which then in turn might induce proliferation of hPASMCs in a cumulative manner. However, we did not observe any effect of CTGF on c-jun and no increased proliferation of hPASMCs upon co-stimulation with ET1 and CTGF. In contrast, other growth factors involved in vascular remodeling such as IL-6 (Lin et al., 2011) or PDGF-BB (Biasin et al., 2014) have been reported to lead to increased c-jun expression. We have recently shown that PDGF-BB stimulation of hPASMCs transiently increased c-jun mRNA and protein levels (Biasin et al., 2014). These findings cumulatively indicate cell-type specific regulation of AP-1 family members. We observed that c-jun and c-fos were highly expressed in the smooth muscle layer of the vessel wall in lungs in both animal models and human PH samples. Our observations are supported by a recent study, showing that in isolated hPASMCs from IPAH patients; c-fos mRNA and protein levels were increased (White et al., 2011). Here, we additionally showed that hypoxia exposure of mice led to enhanced expression of c-fos after only 3 h in the lung homogenate and in isolated pulmonary arteries, but not in the primary hPASMCs. This implies that the changes in c-fos are not a direct effect of hypoxia, but rather a secondary response. Therefore, presumably hypoxia activates c-fos expression in hPASMCs by intermediate factors. Hypoxia has been reported to increase levels of factors such as PDGF-BB, TGF-␤, CTGF and ET-1 both in vitro and in vivo (Stenmark et al., 2006). Accordingly, we found that very short hypoxia exposure of mice (3 h) led to up-regulation of ET-1 mRNA in mouse lung homogenates which was not evident in chronic conditions. In contrast, expression of c-jun was neither altered in pulmonary arteries from hypoxia-exposed mice nor in hPASMCs exposed to hypoxia or ET-1. However, similarly to c-fos, increased c-jun

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expression was observed in the IPAH human lungs. This is in accordance with the notion that multiple stimuli are involved in the development of PH, and hypoxia is only one of them (Yuan and Rubin, 2005). Accordingly, in the MCT rat model, which is also an inflammatory and lung injury model (Stenmark et al., 2009), c-jun was up-regulated, further supporting the need of more initiating stimuli to develop vascular remodeling. In this study we have delineated the ET-1/c-fos dependent axis leading to hPASMC proliferation. Although we could not observe a significant effect of c-jun in ET-1 driven proliferation, we cannot exclude importance of c-jun in hPASMC proliferation. Accordingly, it has been demonstrated that overexpression of c-jun can indeed, lead to increase proliferation of hPASMCs (Yu et al., 2001). Additionally to the increased expression of c-fos, we could observe an ET-1 induced phosphorylation of both c-fos and c-jun. It is known that phosphorylation of c-fos in ser32, and phosphorylation of cjun in ser73 and 63 enhance their activity and stability (Sasaki et al., 2006). Thus, we postulate that ET-1 has a double wave effect on smooth muscle cells. The immediate effect is an increase of c-fos expression followed by the enhanced stabilization and activation of c-fos and c-jun, consequently leading to transcription of target genes (Okazaki and Sagata, 1995; Tanos et al., 2005). At a later time point the effect of ET-1 can influence c-fos protein expression and cell proliferation. In conclusion, we have demonstrated that c-fos transcription factor might play a role in ET-1 driven proliferation of smooth muscle cells. ET-1, which is highly up-regulated under hypoxia, increases expression of proliferative markers and DNA synthesis in a c-fos dependent manner. Moreover, ET-1 by inducing phosphorylation of c-fos and c-jun, probably enhances their activity. Even though we cannot exclude that other transcription factor play a role and other mechanisms are involved in the ET-1 driven remodeling, these findings add a new piece of information on the mechanism of the ET-1 effect on smooth muscle cells and on vascular remodeling underlying PH. Acknowledgements We would like to thank Julia Schittl, Ida Niklasson, Sabrina Reinisch and Lisa Oberreiter for excellent technical support. This work was supported by the Deutsche Forschungsgemeinschaft (WE 1978/4-1), Excellence Cluster Cardio-Pulmonary System EU FP6 “PULMOTENSION” (LSHM-CT-2005-018725) and the Medical University of Graz, Austria (PhD Program Molecular Medicine to V. Biasin). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel. 2014.06.020. References Bauer NR, Moore TM, et al. Rodent models of PAH: are we there yet? Am J Physiol Lung Cell Mol Physiol 2007;293(3):L580–2. Biasin V, Marsh LM, et al. Meprin beta, a novel mediator of vascular remodeling underlying pulmonary hypertension. J Pathol 2014;233(1):7–17. Dean CB, Nielsen JD. Generalized linear mixed models: a review and some extensions. Lifetime Data Anal 2007;13(4):497–512. Erler JT, Bennewith KL, et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 2006;440(7088):1222–6. Fantozzi I, Zhang S, et al. Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 2003;285(6):L1233–45. Fuchs SY, Dolan L, et al. Phosphorylation-dependent targeting of c-Jun ubiquitination by Jun N-kinase. Oncogene 1996;13(7):1531–5. Gonzalez-Ramos M, Mora I, et al. Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-beta1 regulation. Int J Biochem Cell Biol 2012;44(6):963–71.

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