Diethylcarbamazine: A potential treatment drug for pulmonary hypertension?

Accepted Manuscript Diethylcarbamazine: A potential treatment drug for pulmonary hypertension?

Edlene Lima Ribeiro, Ingrid Tavares Fragoso, Fabiana Oliveira dos Santos Gomes, Amanda Costa Oliveira, Amanda Karoline Soares e Silva, Patrícia Martins e Silva, Bianca Torres Ciambarella, Isalira Peroba Rezende Ramos, Christina Alves Peixoto PII: DOI: Reference:

S0041-008X(17)30354-X doi: 10.1016/j.taap.2017.08.015 YTAAP 14034

To appear in:

Toxicology and Applied Pharmacology

Received date: Revised date: Accepted date:

5 June 2017 9 August 2017 25 August 2017

Please cite this article as: Edlene Lima Ribeiro, Ingrid Tavares Fragoso, Fabiana Oliveira dos Santos Gomes, Amanda Costa Oliveira, Amanda Karoline Soares e Silva, Patrícia Martins e Silva, Bianca Torres Ciambarella, Isalira Peroba Rezende Ramos, Christina Alves Peixoto , Diethylcarbamazine: A potential treatment drug for pulmonary hypertension?. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2017), doi: 10.1016/j.taap.2017.08.015

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ACCEPTED MANUSCRIPT 1 Diethylcarbamazine: a potential treatment drug for pulmonary hypertension?

Edlene Lima Ribeiroa,b, Ingrid Tavares Fragosoa,b, Fabiana Oliveira dos Santos Gomesa,b, Amanda Costa Oliveira a,b, Amanda Karoline Soares e Silva a,b, Patrícia Martins e Silva c , Bianca Torres Ciambarella c , Isalira Peroba Rezende

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a

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Ramosd, Christina Alves Peixoto a*

Laboratory of Ultrastructure, Aggeu Magalhães Research Center – CPqAM,

Pernambuco, Brazil. Federal University of Pernambuco, Brazil.

c

Laboratory of Inflammation – FIOCRUZ, Rio de Janeiro, Brazil. National Center Structural Biology and Bio-imaging, Carlos Chagas Filho

Biophysics Institute and

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b

Department of Radiology - University Hospital

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Clementino Fraga Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

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*Corresponding author: Dr Christina Alves Peixoto. aLaboratory of

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Ultrastructure, Aggeu Magalhães Research Center, Avenida Moraes Rego s/n, Cidade Universitária, 50670-420, Recife, PE, Brazil. Fax: 55-81-21012500,

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Phone: 55-81-21012557.

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ABSTRACT

The present study demonstrated the potential effects of diethylcarbamazine

DEC for 28 days.

50 mg/kg body weight of

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(600mg/kg) was administered once per week, and

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(DEC) on monocrotaline (MCT)-induced pulmonary hypertension. MCT solution

Three C57Bl/6 male mice groups (n=10) were studied:

Control; MCT28, and MCT28/DEC. Echocardiography analysis was performed

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and lung tissues were collected for light microscopy (haematoxylin-eosin and Masson's trichrome staining), immunohistochemistry (αSMA, FADD, caspase 8,

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caspase 3, BAX, BCL2, cytochrome C and caspase 9) western blot (FADD, caspase 8, caspase 3, BAX, BCL2, cytochrome C and caspase 9) and qRt-PCR

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(COL-1α and αSMA). Echocardiography analysis demonstrated an increase in the pulmonary arterial blood flow gradient and velocity in the systole and RV

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area in the MCT28 group, while treatment with DEC resulted in a significant

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reduction in these parameters. Deposition of collagen fibers and αSMA staining around the pulmonary arteries was evident in the MCT28 group, while treatment

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with DEC reduced both. Western blot analysis revealed a decrease in BMPR2 in the MCT28 group, in contrast DEC treatment resulted in a significant increase in the level of BMPR2. DEC also significantly reduced the level of VEGF compared to the MCT28 group. Apoptosis extrinsic and intrinsic pathway markers were reduced in the MCT28 group. After treatment with DEC these levels returned to baseline. The results of this study indicate that DEC

ACCEPTED MANUSCRIPT 3 attenuates PH in an experimental monocrotaline-induced model by inhibiting a series of markers involved in cell proliferation/death.

Key

words:

Diethylcarbamazine,

monocrotaline,

hypertension,

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apoptosis.

pulmonary

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INTRODUCTION Pulmonary hypertension (PH) is a life-threatening progressive disorder associated with abnormally elevated pulmonary pressure and right heart failure.

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It is a disease of a complex etiology and pathobiology that results from

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interactions between the genetic make-up of an individual and the surrounding environment [1, 2].

The initial pathological events of the pulmonary artery dysregulation

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involve the proliferation of the smooth muscle cells. Several lines of evidence suggested that increased proliferation and decreased apoptosis of the

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pulmonary arterial smooth muscle cells can mediate thickening of the pulmonary vasculature, which would subsequently lead to a reduced inner

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diameter and increased pulmonary vascular resistance [3]. Endothelial cell (EC) apoptosis and apoptosis resistance seems to play

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crucial roles in the development of plexiform lesions that feature in the

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pathogenesis of PH. Subsequently, EC injury associated with smooth muscle cell (SMC) proliferation facilitates vascular remodeling and eventually leads to

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narrowed vascular lumen, increased pulmonary vascular resistance, increased pulmonary arterial pressure, and right heart failure [4]. The imbalance between cell death and proliferation occurs in every stage of pulmonary vascular remodeling and the pathogenesis of PH, and involves every cell type in the vasculature including, but not limited to ECs, SMCs, and fibroblasts [5]. Intriguingly, PH pathogenesis involves both inappropriate apoptosis and over-proliferation. Apoptosis in ECs, after initial environmental insults, has been

ACCEPTED MANUSCRIPT 5 recognized as one of the crucial events that trigger pulmonary vascular remodeling in PH [6]. Despite extensive studies, a detailed understanding of the cellular and molecular mechanisms involved in the transition from initial apoptosis to apoptosis resistant proliferation of ECs and SMCs has yet to be established [4].

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The MCT model is considered by some to be a toxic model, as it is

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suggested that MCT rats die from hepatic veno-occlusive disease with liver failure, instead of right ventricle failure [7]. MCT is known to cause pulmonary endothelial injury and pulmonary hypertension in humans and rats [8, 9], but

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has little effect on mice [10].

The drug diethylcarbamazine (DEC) is used throughout the world against

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lymphatic filariasis. However, in recent years many studies have described other pharmacological activities of DEC. It has been established that DEC

eicosanoid

production

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interferes with the cyclooxygenase and lipoxygenase pathways, reducing and

acting

as

an

anti-inflammatory

drug

[11].

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Furthermore, DEC inhibits the activation of NF-kB, suppressing target genes

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involved in pulmonary inflammatory response [12]. DEC has also been shown to be effective in different models of lung inflammation, such as tropical eosinophilia,

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pulmonary

pulmonary

hypertension,

eosinophilic

pulmonary

inflammation and asthma [13, 14, 15, 16]. Queto et al. (2010) [15] reported that DEC suppressed the pulmonary and bone marrow eosinophilia by CD95L/CD95 signaling. Since the CD95L (FasL) is a ligant for the apoptosis inducing receptor CD95 (Fas), these results suggest that DEC can possibly act as an apoptosis inducer. The aim of the present study was to evaluate the cell death markers and action of DEC on a monocrotaline-induced pulmonary hypertension model.

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MATERIALS AND METHODS Animals Thirty male C57BL/6 mice, weighing 26-30 g (11 weeks), were used in all

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experiments. The mice were examined to determine their health status and

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acclimated to a laboratory environment of 23-24°C. They were kept in a 12/12 h day/night cycle photoperiod, housed in metal cages and fed a standard diet with water ad libitum [17, 18]. All experimental procedures were approved by the Committee

for

Animal

Experimentation

(Prot.

63/2014

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Ethics

Drugs and experimental Design

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FIOCRUZ/CPqAM).

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Diethylcarbamazine citrate (DEC) was obtained from Sigma (St. Louis, MO, D8765) and dissolved in distilled water. The lymphatic filariasis therapeutic

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dose regimens recommended by the World Health Organization of 6 mg/kg

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were used. Considering that the total metabolism rate of a mouse is approximately seven times that of a human, the present study used 50 mg/kg

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body weight of DEC, adjusted according to the body weight of mice, administered through drinking water for 28 days [12]. The mice received an intraperitoneal (i.p) injection of MCT (600mg/kg SigmaAldrich, St. Louis, MO, USA) dissolved in saline solution and administered once per week (0, 7, 14 and 21 th days) as described elsewhere [19, 20, 21]. Mice were randomly allocated into three groups of ten (N = 10) animals each:

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7

Determination of right ventricular hypertrophy (RVH), pulmonary artery and left ventricular function

Canada)

on

day

28.

During

the

procedure,

isofluorane/O2

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Toronto,

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Echocardiography analysis was performed using a VisualSonic Vevo770,

administration was administered using a facemask to keep the mice lightly

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anesthetized, with heart rates in the range of 300-350 bpm. The right ventricle was visualized from the right parasternal long axis view with a 704 RMV

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scanhead. The right ventricular wall thickness was measured from images produced in M-mode, using the depth interval (mm) generic measurement tool

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(Vevo770 v3.0 software, VisualSonics). Doppler flow images were recorded

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from the left parasternal long axis view with a 707 B scanhead pointing slightly towards the left shoulder to visualize the pulmonary artery. Volume was measured at the level of the pulmonary valve, and several indices of pulmonary artery blood flow (velocity time integral, peak and mean pressure gradient and peak and mean velocity) were assessed using the pulmonary valve protocol measurement tool [19, 22].

Histological examination

ACCEPTED MANUSCRIPT 8 The lung fragments were washed twice in PBS pH 7.2 and fixed in Bouin solution for 8 hours (1% saturated picric acid, formaldehyde and 40% glacial acetic acid), before being dehydrated in an increasing ethanol series, cleared in xylene, embedded and included in purified paraffin (VETEC, São Paulo, SP, Brazil). Tissue sections of 5 μm were cut using a microtome (Leica RM

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hematoxylin/eosin and studied using light microscopy [23].

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2125RT) and deparaffinized with xylene. They were then stained with

Immunohistochemical Localization

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The paraffin sections of lung tissue were mounted onto slides. After being deparaffinized, the tissues were incubated overnight at 4°C with primary

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antibody anti- α-SMA (1:100 cat. ab 5694) anti-FADD (1:50 cat. Sc 6036), anticaspase 8 (1:50 cat. Sc5263), anti-caspase 3 (1:100 cat. Ab4011), anti-BAX

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(1:50 cat. Ab7977), anti-BCL2 (1:100 cat. Ab7973), anti-cytochrome C (1:50 cat. Sc13156), anti-caspase 9 (1:50 cat. sc56076). The antigen-antibody reaction

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was visualized with avidin-biotin peroxidase (Dako Universal LSAB + Kit,

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Peroxidase), using 3,3-diaminobenzidine as a chromogen. Imaging was performed by light microscopy. Five pictures at the same magnification were

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quantitatively analyzed using the Gimp 2.8 software program (GNU Image Manipulation Program, UNIX platforms) [24]. RNA extraction and quantitative real-time polymerase chain reaction (q RTPCR) Total RNA from mouse tissues was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The forward and reverse primers used for each

gene

were

as

follows:

Collagen

Type

1α

(COL-1α):

5’-

ACCEPTED MANUSCRIPT 9 GAACGGTCCACGATTGCATG-3’ and 5’-GGCATGTTGCTAGGCACGAAG-3’, αSMA:

5’-ATCTGGCACCACTCTTTCTA-3’

GTACGTCCAGAGGCATAGAG-3’,

GAPDH

and

(endogenous

control):

5’5′-

AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA3′. All reactions were performed in triplicate and included the following: 1 μL of

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cDNA; 5 μM of each primer; 2x SYBR Green PCR Master Mix (Applied

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Biosystems); and water added to give a final volume of 25 µL. The relative amount of mRNA was determined using the comparative threshold (Ct) method by normalizing target cDNA Ct values to those of GAPDH. Fold increase ratios

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were calculated relative to control (basal conditions) for each group using the

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formula 2e-ΔΔCt [24].

Western Blot Analysis

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The lungs were submerged in liquid nitrogen and the total proteins were

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extracted using an extraction cocktail (10 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM sodium fluoride

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(NaF), 10 mM sodium pyrophosphate, 10 mM sodium orthovanadate (NaVO4),

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10 mg of aprotinin, and 100 mM Tris(hydroxymethyl)aminomethane, pH 7.4). Western blotting and the subsequent quantification of each blot were performed as previously described [25]. The primary antibodies for anti-BAX, anti-Bcl2 and anti-caspase 3 were obtained from Abcam (CA, USA), while anti-caspase 8, anti-FADD, anti-caspase 9 and anti-cytochrome c were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Secondary antibodies and β-actin were acquired from Sigma-Aldrich (USA).

ACCEPTED MANUSCRIPT 10 Data Analysis GraphPad Prism software (version 6) was used for statistical analysis. Data was expressed as mean ± standard deviation. Differences between the control and treatment groups were analyzed using analysis of variance (ANOVA), prior to Tukey’s post hoc test or the Student's t-test being carried out.

RESULTS

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Effect of DEC on pulmonary hypertension

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Probability values less than 0.05 were considered significant.

In humans, transthoracic echocardiography is an excellent noninvasive

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screening test for patients with symptoms or risk factors for PH by providing direct and/or indirect signs of elevated pulmonary artery pressure (PAP).

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Doppler analysis at the pulmonary valve level, recorded by ultrasonography in lightly anesthetized mice (heart rate 300-350 bpm) demonstrated an increased

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in the pulmonary arterial blood flow gradient and velocity in the systole and RV

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area in the MCT28 group, while treatment with DEC resulted in a significant reduction in these parameters (Table 1).

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Tissue lesions of pulmonary hypertension are characterized by changes in all components of the pulmonary arterial walls. The MCT28 group exhibited remarkable changes in the pulmonary arteries, arterioles and in the pulmonary parenchyma, as well as congestion and atelectasis. Treatment with DEC reduced the lung damage (Figure 1A).

Effect of DEC on pulmonary arteriole muscularization

ACCEPTED MANUSCRIPT 11 Vascular remodeling and fibrosis are among the key pathological features in PAH. One of the main features of vascular remodeling seen in PAH is collagen deposition in the remodeled pulmonary vessels. Masson’s trichrome staining revealed a significant increase in collagen deposition in the pulmonary interstitium, around the arteries, vessels and bronchioles. In contrast, after

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treatment with DEC there was an evident reduction of collagen (Figure 1B), a

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finding also observed in the gene expression of COL-1α mRNA (Figure 1E). αSMA is a marker of the expression of a smooth-muscle phenotype, expressed by the PSMCs of existing vessel walls in both the normal and

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hypertensive lung. However, an increase in αSMA expression can lead to a thickening of the middle layer of the pulmonary arteries. Immunolabeling of the

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lung sections with αSMA revealed a significant increase in the MCT28 group, in comparison with the control group, mainly around the arteries and vessels,

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demonstrating the muscularization process. In contrast, the DEC group decreased the expression of αSMA (Figures 1C,D). Those same results were

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observed in the gene expression of αSMA (Figure 1F).

Effect of DEC on growth factors

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Studies have identified the bone morphogenetic protein (BMP) pathway, a member of the TGF-superfamily of receptors, as having particular importance in PAH pathogenesis, suggesting that this pathway might be important in the pathogenesis of a variety of common clinical situations in which pulmonary hypertension is a feature. Western blot analysis revealed a decrease in BMPR2 in the MCT28 group, compared to the control group. In contrast, treatment with

ACCEPTED MANUSCRIPT 12 DEC increased the BMPR2 level significantly, returning to levels observed in the control group (Figures 2A,B). Models of pulmonary hypertension have been shown to be associated with increased levels of vascular endothelial growth factor (VEGF) transcripts. Dysregulation can cause increased vascular permeability and stimulate

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neovascularization in the physiological and pathological processes. Western

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blot analysis revealed an increase in VEGF in the MCT28 group, compared to the control group, whereas treatment with DEC significantly reduced the VEGF

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expression (Figures 2A,B).

Effect of DEC on expression of Apoptotic Pathways proteins

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Apoptosis is triggered and modulated by two pathways. The intrinsic pathway involves the mitochondria in response to stress, such as reactive

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oxygen species, nutrient deprivation or DNA damage, whereas the extrinsic pathway is induced by receptor binding to pro-apoptotic death ligands such as

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tumor necrosis factor–α (TNF-α) and Fas.

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The extrinsic pathway markers FADD, caspase 8 and caspase 3 were analyzed by immunohistochemistry and western blotting, through which a

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significant reduction of these markers was observed in the MCT group. In contrast, treatment with DEC significantly increased levels of FADD, caspase 8 and caspase 3 (Figures 3 A-E and G-H). The intrinsic pathway markers BAX, cytochrome C and caspase 9 displayed reduced levels of expression, other than Bcl2 which did not show a significant change. Treatment with DEC increased the levels of expression of BAX, cytochrome C and caspase 9, demonstrating that this drug exerts an

ACCEPTED MANUSCRIPT 13 effect on both the intrinsic and the extrinsic pathways of apoptosis (Figure 4 A-H and I-J).

DISCUSSION In the present study, we presented evidence that MCT promotes

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inactivation of the apoptosis pathway, as well as interfering with growth factors.

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The pathobiological mechanisms of PH have been extensively studied. The PH “phenotype” is characterized by endothelial dysfunction, a decreased ratio of apoptosis/proliferation in PASMCs, and a thickened, disordered adventitia in

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which there is excessive activation of adventitial metalloproteases. Like cancer and atherosclerosis, PAH does not have a single origin, but a variety of

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underlying causes [26].

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While recent advances have led to greater recognition and new therapies, relatively few therapies are still used for PH. The MCT model

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continues to be a frequently investigated model of PH, as it offers technical simplicity, reproducibility, and low cost compared with other models of PH.

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Elucidating the pathobiology of PH continues to be critical for the design of new

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and effective therapeutic strategies, and animal models are fundamental to achieving this objective [27]. The C57BL/6J strain has displayed the advantage of presenting a knockout series for several genes, which allows the functional analyze of different cytokines and growth factors in PH development. DEC is a drug that is used against lymphatic filariasis all over the world, however, in recent years many studies have described other pharmacological activities of DEC [11]. Ribeiro et al. (2014) [23] showed that DEC had an antiinflammatory effect in acute lung injury, and Fragoso et al. (2017) [24] found

ACCEPTED MANUSCRIPT 14 that DEC prevented inflammatory cells accumulation and accelerated the inflammation resolution by stimulating apoptosis. Interestingly, a 1985 study tested the hypothesis that monocrotaline would activate arachidonic acid metabolism in rats. These authors described that the arachidonate metabolism was activated before pulmonary hypertension developed, and that inflammatory

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cell infiltration in the alveolus followed the hypertensive process. Furthermore,

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DEC treatment attenuated both monocrotaline-induced inflammatory response and the pulmonary hypertension [28].

Experimental models are important tools for the study of the pathogenic

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mechanisms of PH, and for the development of new therapeutic strategies. Established models of PH include chronic exposure to hypoxia, monocrotaline

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(MCT), overexpression or knockout mice (IL-6 overexpression, BMPR2 model). Perivascular inflammation is common in the remodeling of blood vessels, both

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in animal models and in human PH. The monocrotaline acute toxic model is by

peripheral

accumulation

of

mononuclear

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characterized

vascular

damage

inflammatory

caused

cells,

by

without

a

massive

formation

of

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obstructive intimal lesions [29]. Our studies expanded on monocrotalineinduced PH previous results by demonstrating that DEC also had an action on

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growth and apoptotic factors. Pulmonary arterial hypertension (PAH) is a particularly fatal form of PH that is characterized by increased pulmonary vascular pressure, caused by pathological remodeling [30]. The pathology is associated with abnormal connective tissue deposition and characterized by structural and functional changes in the pulmonary vasculature, including vascular smooth muscle cell

ACCEPTED MANUSCRIPT 15 proliferation hypertrophy and excess collagen formation and remodeling pulmonary vessels [29]. Circulating levels of N-terminal propeptide of type III procollagen (PIIINP), Carboxyterminal telopeptide

of type

I collagen (CITP), matrix

metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP)

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are elevated in PAH patients, with the results suggesting that the elevated

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levels were markers of the state of the disease rather than of the etiology of PAH. Furthermore, circulating markers of new collagen formation, type 1 collagen degradation, elastase (MMP9) activity, and inhibition of matrix

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metalloproteinase by a ubiquitous MMP inhibitor (TIMP1) may be indicative of active vascular remodeling and be clinically relevant [31].

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The sequence of morphologic changes that lead to neomuscularization of the microvessels has also been seen, and there is a series of studies that

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characterize the evolving phenotype of smooth-muscle cells (PSMCs). We studied α-SMA expression, a recognized first marker of developing smooth-

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muscle cells during microvessel wall remodeling in both an experimental and

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human model [32, 33]. We described that DEC promoted a reduction in the αSMA expression, inhibiting the development of morphological changes in the

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pulmonary arteries.

A number of studies have examined the effect of BMPR2 and VEGF in inducing pulmonary artery smooth muscle cell (PASMC) apoptosis in human and experimental models [34, 35, 36]. Bone Morphogenetic Proteins (BMPs) are synthesized and secreted from a variety of cell types including pulmonary vascular smooth muscle and endothelial cells and play an important role in regulating cell proliferation, apoptosis and differentiation [5, 34, 37]. Mutations

ACCEPTED MANUSCRIPT 16 in the gene encoding a bone morphogenetic protein type II receptor (BMPRII) are the most frequent cause of PH. Emerging studies suggest that modulation of BMPRII signaling is a promising alternative that could prevent and reverse pulmonary vascular remodeling [38]. In monocrotaline-induced PH, there was a decrease in levels of BMPR2 expression, consistent with PH development. After

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treatment with DEC significant increased levels of BMPR2 were evident. Reduced levels of vascular endothelial growth factor (VEGF) in both hypoxia‑ and MCT‑ induced PH models have been described in literature [39,40]. VEGF has been known to confer a potent protective effect on ECs from

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apoptosis through the extrinsic pathway [41]. In a variety of experimental PH

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models, EC apoptosis has been shown to be associated with reduced levels of VEGF [39,40]. The MCT28 group exhibited high levels of VEGF in protein analysis, demonstrating an increase in these proteins. After DEC treatment a

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significant reduction of VEGF levels was observed. Another interesting fact was

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imbalance of the extrinsic apoptosis pathway markers, where it was observed that levels of FADD, active caspase-8 and caspase-3 decreased in the MCT28

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group in comparison with the control group, whereas after treatment with DEC

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these apoptotic proteins had a similar expression to the control group. Similar results were observed in the intrinsic pathway. Some hypotheses have been discussed in relation to apoptosis and lung cells. The lung is the most vascular organ in the body and the massive pulmonary endothelial surface area is almost directly exposed to the environment through the air we breathe [42]. Thus, it is very likely that even in healthy individuals, there are episodes of endothelial cell (EC) injury induced by environmental triggers resulting in waves of apoptosis, but the normal

ACCEPTED MANUSCRIPT 17 reparative mechanisms involving the proliferation and migration of neighboring ECs and possibly the homing of circulating endothelial progenitor cells are sufficient to restore vascular continuity and maintain the integrity of pulmonary circulation [43]. Endothelial cells show an apoptosis-resistant phenotype in idiopathic

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pulmonary hypertension [4]. Recently, a study using a thromboembolic

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pulmonary hypertension rodent model confirmed the imbalance between proapoptotic and anti-apoptotic proteins. The thromboembolism led to the upregulation of Bad and the down-regulation of Bcl-2, associated to decreased

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mRNA and protein levels of FoxO1, a member of the FoxO family that plays important role in cell cycle, proliferation, apoptosis, and tumorigenesis [44].

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It has been demonstrated that increased PASMC proliferation and/or inhibited PASMCS apoptosis both contribute to the inducing of pulmonary

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vascular medial hypertrophy. However, the precise mechanisms involved in the regulation of PASMC proliferation and apoptosis in PH are still incompletely

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understood. Another important factor is that the BMPR2 pathway prevents EC

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apoptosis and maintains the integrity of the lung microvascular cells, and thus inactivating BMPR2 mutations play a crucial role in triggering pathological

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vascular remodeling [5].

The present work has some limitations, although a monocrotalineinduced pulmonary hypertension has been effectively established in C57Bl-6 mice in this study, ECS and PASMCS were difficult to isolate and in vitro assays have not been performed. Therefore, functional assays to characterize the mechanism of DEC on apoptosis/proliferation and pulmonary remodeling have been left to be investigated further.

ACCEPTED MANUSCRIPT 18 In conclusion, the results of the present study indicate that DEC attenuates PH in an experimental induced-monocrotaline model by inhibiting a series of markers involved in apoptosis resistance that plays an important role in pulmonary vascular remodeling.

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ACKNOWLEDGEMENTS: The present study was supported by the following

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Brazilian fostering agencies: Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), Centro de Pesquisas Aggeu Magalhães

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(CPqAM/FIOCRUZ) and National Center Structural Biology and Bio-imaging.

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Table 1: Pulmonary artery blood velocity after DEC treatment: Velocity-time integral (VTI, cm) mean and peak gradient (mmHg) and mean and peak velocity

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imaging. N=5. Mean±SD; p<0.05 vs SHAM, #p<0.05 vs MCT28

Fig. 1: Effect of DEC treatment on histological alterations in lung after MCT-induced pulmonary hypertension. A: Representative images of H&E staining demonstrating, interstitial edema with thickening of the septum alveolar, infiltrates of inflammatory cells with the presence of activated macrophages and plexiform lesions of pulmonary arteries and emphysema in MCT28 groups. Administration of DEC significantly attenuated the lung damage. Histological analysis of the control group did not reveal any morphological changes. B:

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significantly increased collagen deposition and MCT28 group. Administration of DEC significantly reduced collagen. C: Immunohistochemical localization of αSMA labeling around the arteries, vessels and bronchioles and MCT28 group. After treatment with DEC there was a reduction of immunostaining for α-SMA.

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Fig. 2: Effect of DEC treatment on expression of BMPR2 and VEGF: A:

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Fig. 3: Effect of DEC treatment on the extrinsic pathway of apoptosis: Immunohistochemical localization for FADD, C8 and C3 (A, B and C respectively). D-F: Quantitative densitometry analysis (GIMP2 analyzed). Data is expressed as mean ± S.D. from n = 5 mice for each group. G: Representative

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VTI, cm

2.27 ± 0.4 4.28 ± 0.35* 3.15 ± 0.24#

Peak Gradient, mmHg

Mean Velocity, mm/s

Peak velocity, mm/s

Area RV (mm2 )

0.84 ± 0.22 3.40 ± 0.69* 2.42 ± 0.45#

268.0 ±49.49 565.5± 42.39* 461.8± 40.48#

473.4± 86.49 959.2± 67.87* 775.1± 75.80#

11.40 ± 0.52 17.57 ± 0.97* 12.78 ± 1.63#

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SHAM MCT28 MCT28/DEC

Mean Gradient, mmHg 0.29 ± 0.1 1.19 ± 0.22* 0.84 ± 0.15#

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First study on the therapeutic effects of diethylcarbamazine in lung hypertension. Lung hypertension exhibited reductions apoptosis extrinsic and intrinsic pathways Diethylcarbamazine inhibited markers involved in cell proliferation/death.

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