Effect of 5-Lipoyxgenase on the Development of Pulmonary Hypertension in Rats

John E. Jones1, Jennifer L. Walker2, Yanli Song2, Norbert Weiss2#, Wellington V. Cardoso3, Rubin M. Tuder4, Joseph Loscalzo2, and Ying-Yi Zhang2*

1Department of Surgery, 2Whitaker Cardiovascular Institute and Evans Department of Medicine, and 3The Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, 4Division of Cardiopulmonary Pathology and Pulmonary and Critical Care Medicine, Department of Pathology and Medicine, The Johns Hopkins University School of Medicine #Current address: Klinikum der Universität Munchen, Medizinische Poliklinik-Innenstadt, D-80336 Münnich, Germany

Running title: 5-Lipoxygenase and pulmonary hypertension

Abbreviations: 5LO-5-lipoxygenase; MCT-monocrotaline; Ad5LO-adenovirous expressing 5LO;

AdGFP-adenovirus expressing green fluorescence protein; PAAT-pulmonary artery acceleration

time; RVSP-right ventricular systolic pressure; PBS-Dulbecco’s phosphate-buffered saline.

Corresponding author:

Ying-Yi Zhang, Ph.D. Boston University School of Medicine Whitaker Cardiovascular Institute 715 Albany St., W-507 Boston, MA 02118 Telephone: (617) 638-4896 Fax: (617) 638-4066 e-mail: yyzhang@bu.edu

Articles in PresS. Am J Physiol Heart Circ Physiol (January 15, 2004). 10.1152/ajpheart.00281.2003

Copyright (c) 2004 by the American Physiological Society.

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Abstract

5-Lipoxygenase (5LO) and its downstream leukotriene products have been implicated in the

development of pulmonary hypertension. In this study we examined the effects of 5LO

overexpression in rat lungs on pulmonary hypertension, using a recombinant adenovirus

expressing 5LO (Ad5LO). Transthoracic echocardiography and right heart catheterization data

showed that 5LO overexpression in lung did not cause pulmonary hypertension in normal rats;

however, it markedly accelerated the progression of pulmonary hypertension in rats treated with

monocrotaline (MCT). An increase in pulmonary artery pressure occurred earlier in the rats

treated with MCT+Ad5LO (7-10 days) as compared to those treated with control vector,

MCT+AdGFP, or MCT alone (15-18 days). The weight ratio of right ventricle-to-left ventricle plus

septum was higher in the MCT+Ad5LO group than that of MCT+AdGFP or MCT alone group

(0.45±0.08 vs. 0.35±0.03 or 0.33±0.06). Lung tissue histological sections from MCT+Ad5LO

rats exhibited more severe inflammatory cell infiltration and pulmonary vascular muscularization

than those from MCT+AdGFP- or MCT-treated rats. Administration of 5LO inhibitors, zileuton or

MK-886, to either MCT- or MCT+Ad5LO-treated rats prevented the development of pulmonary

hypertension. These data suggest that 5LO plays a critical role in the progression of pulmonary

hypertension in rats, and that the detrimental effect of 5LO is manifest only in the setting of

pulmonary vascular endothelial cell dysfunction.

Key words: 5-lipoxygenase, inflammation, pulmonary hypertension, monocrotaline.

3

Introduction

5-Lipoxygenase (5LO) catalyzes two consecutive reactions that convert arachidonic acid

to leukotriene A4 (6, 32). The latter is a crucial precursor for the formation of leukotriene B4, a

potent chemotactic agent (14), and for the formation of leukotrienes C4, D4, and E4 (collectively

referred to as cysteinyl leukotrienes) (26), substances that cause an increase in vascular

permeability (9, 13) and constriction of bronchi (10, 17) and certain types of blood vessels (4, 5).

The substrate for 5LO is generated by phospholipase A2 (11), and is presented to 5LO by 5LO-

activating protein (FLAP) (12).

5-Lipoxygenase has also been found to participate in other cellular processes, including

cell proliferation (3, 39), tyrosine kinase signaling (21), and F-actin polymerization (21, 27, 29).

In addition, 5LO was found to bind to a TGF-β receptor I-associated protein (TRAP-1) (30),

although the effect of this association is currently unclear.

Increased 5LO expression has been found in the lung tissue of patients with primary

pulmonary hypertension, within infiltrating perivascular alveolar macrophages and in small

pulmonary artery endothelial cells (41). Several studies also suggested that 5LO may play a

role in the development of pulmonary hypertension. For example, cysteinyl leukotrienes were

found in the lung lavage fluid of neonates with persistent pulmonary hypertension (33), patients

with chronic obstructive pulmonary disease (28), and animals subjected to acute hypoxia (25).

Targeted disruption of the 5LO gene (38), or treatment of animals with diethylcarbamazine (24),

MK886 (an inhibitor of FLAP) (38), or leukotriene antagonists (1, 16, 23, 31), reduced hypoxia-

induced pulmonary hypertension. Diethylcarbamazine, an anti-parasitic drug that also inhibits

5LO, was shown to ameliorate the development of pulmonary hypertension caused by

monocrotaline in rats (34).

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In the present study, we explored two questions: 1) does 5LO overexpression cause

pulmonary hypertension? and 2) does 5LO facilitate the development of pulmonary

hypertension caused by other factors? To address the first question, we expressed an

exogenous 5LO (human 5LO) in rat lungs by adenovirus-mediated gene transfer, and monitored

the development of pulmonary hypertension in the animal. Human 5LO has about 93% protein

sequence identity to rat 5LO (GenBank accession # J03600 and J03960, respectively), and can

be expressed well during transfection in various mammalian cell lines, such as COS-1 and

NIH3T3 cells. We have previously expressed human 5LO in porcine pulmonary artery

endothelial cells and shown the recombinant enzyme was able to utilize endogenous

arachidonic acid to generate leukotrienes (43).

To address the second question, we used monocrotaline-treated rats. Monocrotaline

(MCT) is a plant alkaloid that is dehydrogenated in rat liver to form a highly reactive pyrrole

derivative (8). The latter interacts with the first vascular bed encountered, causing pulmonary

endothelial cell injury and lung inflammation (2, 8, 40). These events lead to progressive

pulmonary vascular remodeling. Elevation of pulmonary arterial pressure occurs in rats in ~2.5

weeks after MCT injection (7, 15, 22). In this study, we examined whether 5LO accelerates the

development of pulmonary hypertension and whether specific 5LO inhibitors prevent the

disease process.

5

Methods

Animals

Male Sprague-Dawley rats weighing 225-250 grams were obtained from Hilltop Lab

Animals, Inc. (Scottdale, PA). Rats were acclimated for four days before initial treatment.

Drug preparation and treatment

Monocrotaline (Sigma) was prepared at the concentration of 60 mg/ml by dissolving in

HCl-acidified PBS and adjusting the pH to 7.2 with NaOH. The compound was administered to

rats at 60 mg/kg via intraperitoneal injection. MK886 (CalBiochem) was dissolved in

dimethylsulfoxide (DMSO), and administered to rats at 20 mg/kg via subcutaneous injection.

Zileuton (Abbott) was prepared by grinding the tablet into powder and suspending it in water at

50 mg/ml. The drug was administered to rats by oral gavage at 100 mg/kg/day.

Delivery of adenovirus

Replication-deficient adenovirus expressing human 5LO (Ad5LO) was prepared as

previously described (43). Adenovirus expressing green fluorescence protein (AdGFP) was a

kind gift from Dr. John Gray at the Harvard Gene Therapy Initiative. To administer the

recombinant adenovirus to rat lungs, rats were anesthetized with ketamine (100 mg/kg) and

xylazine (7.5 mg/kg). Respiratory tubing was fitted to the snout of the rat, and connected to the

aerosol chamber of a MISTY nebulizer (Allegiance Health Co., McGaw Park, IL) containing 2 x

109 plaque forming units of Ad5LO or AdGFP in 1 ml saline. The chamber was also connected

to a room air source at a flow rate of 8 L/min, causing aerosolization of the virus, and to a tank

6

of 21% oxygen with balanced nitrogen at a flow rate of 1.5 L/min to deliver the adenovirus. The

aerosolized delivery lasted 20 minutes for each rat.

RNA isolation and Northern blotting

Rat lungs were gently perfused with 40 ml of saline through the pulmonary artery, and

~300 mg of lung tissue was homogenized immediately in 10 ml TRIzol Reagent (Invitrogen)

followed by chloroform extraction for total RNA. Northern blotting was carried out by separating

20 µg RNA in 1.2 % agarose gels containing 4% formaldehyde. The RNA was transferred onto

nitrocellulose membranes and hybridized with an [α-32P]-dCTP-labeled 5LO probe that

encompasses nucleotides 45-290 of human 5LO cDNA (GenBank accession #J03600).

Hybridization was carried out at 68°C overnight in MiracleHyb solution (Stratagene) containing

10 µg/ml sheared salmon sperm DNA. The membrane was washed with 2 X SSC/0.1% SDS at

room temperature for 15 min and then in 0.1 X SSC/0.1% SDS at 68°C for 20 min before

exposure to X-ray film. The membrane was then washed in 0.01 X SSC/0.1% SDS at 100°C

twice for 15 min to strip the bound 5LO probe, and hybridized with a [α-32P]-dCTP-labeled

mouse GAPDH probe (Ambion), which crossreacts with rat GAPDH, to estimate total RNA

loading.

Echocardiography

Transthoracic echocardiography was performed on rats according to a previously

described method (18). Rats were anesthetized with a ketamine/xylazine mixture as described

above, and their chests shaved prior to examination. Two-dimensional and Doppler imaging

was performed using an Acuson C256 ultrasonographic system. Pulmonary flow acceleration

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time (PAAT), pulmonary flow waveform, right ventricular free wall thickness, and tricuspid

regurgitation were examined on all rats on days 3, 7, 10, 15, 18, and 24.

Hemodynamic measurements

Rats underwent right heart catheterization at various time points during the study. Prior

to catheterization, rats were anesthetized with the same ketamine/xylazine mixture as above.

After dissection to expose the right jugular vein, a 1.4F Millar Mikro-Tip pressure catheter (Millar

Instruments) was inserted into the vein and advanced to the right ventricle. The catheter was

connected to a transducer unit (Millar Instruments) and interfaced with a signal amplifier and

recorder (Gould Instrument Systems), and the right ventricular systolic pressure (RVSP) was

recorded. On day 24, the systemic blood pressure of rats from each group was also measured.

The left carotid artery was exposed and cannulated with a 1.4F Millar pressure catheter.

Histology

Rat lungs were inflated with 10% phosphate buffered formalin at a pressure of 20 cm

H2O and fixed for 20 hr at 4°C. The lung tissue was processed and paraffin-embedded using

Hypercenter XP System and Embedding Center (Shandon Inc, Pittsburgh, PA) and cut into 5-

µm sections. The tissue sections were then heat-dried on slides at 56ºC for 1 hr before being

deparaffinized and rehydrated.

For hematoxylin and eosin staining, tissue sections were incubated in Gill-2 hematoxylin

(Thermo Shandon) for 2 min, rinsed with water for 2 min, dipped once in acid alcohol (70%

ethanol and 1% concentrated HCl), rinsed with water for 1 min, dipped in 1% NH4OH for 15 sec,

rinsed with water for 1 min, and dipped twice in 1% Eosin Y (Fisher). The stained sections were

8

dehydrated by incubation in 95% ethanol for 2 x 30 sec, 100% ethanol for 2 x 30 sec, and

xylene for 2 x 2.5 min, and mounted with Cytoseal60 (Richard-Allan Scientific).

For trichrome staining, paraffin-embedded tissue sections were deparaffinized,

rehydrated, and stained with a Masson’s Trichrome staining kit (Poly Scientific R&D Corp)

according to the manufacturer’s instruction. For immunohistochemistry, paraffin embedded lung

tissues were cut into 5-µm sections, heat-dried onto slides, deparaffinized, and rehydrated as

described above. Antigen retrieval was carried out in 10 mM citric acid, pH 6, by heating for 4 x

2 min at 400 watts in a microwave oven and cooling at room temperature for 20 min. The

sections were washed with deionized water twice for 5 min and with PBS for 5 min before

blocking with 10% goat serum in PBS at room temperature for 30 min.

For α-actin staining, the sections were incubated with a mouse monoclonal antibody

against smooth muscle cell α-actin (Sigma, 1:800 dilution in 1% immunohistochemical grade

BSA/PBS) at 4°C overnight. The sections were washed in PBS twice for 5 min and incubated

with the secondary antibody, biotinylated goat anti-mouse IgG (Jackson Immunoresearch), at a

dilution of 1:500 in 1% BSA/PBS for 30 min at room temperature. The sections were washed

with PBS twice for 5 min and incubated with complexes of avidin DH and biotinylated-alkaline

phosphatase H (provided in Vectastain ABC-AP kit, Vector Lab, Burlingame, CA) for 30 min.

The sections were washed with PBS twice for 5 min and incubated with alkaline phosphatase

substrate (provided in Vector Red Substrate kit, Vector Lab) for 20 min. The slides were

washed under running tap water for 5 min before being counterstained with hematoxylin, which

was carried out by dipping the slides in Harris Modified Hematoxylin for 10 sec, washing with

water, dipping in acid alcohol, and rinsing with water.

For monocyte/macrophage staining, the sections were incubated with a mouse

monoclonal antibody against calprotectin (Clone MCA387, Chemicon, Temecula, CA, 1:50

9

dilution in 1% immunohistochemical grade BSA/PBS) at 4ºC overnight. Calprotectin is

expressed in monocytes, macrophages, and granulocytes. The sections were washed and

stained with the secondary antibody as described above. The sections were developed by

washing with PBS twice for 5 min, incubated with avidin and biotinylated horseradish peroxidase

complex (Vectastatin ABC kit) for 30 min, washed with PBS twice again for 5 min, and incubated

with peroxidase substrate (DAB substrate kit, Vector Lab) for 7 min. The sections were washed

with tap water and counterstained with methyl green for 4 min at 60ºC, followed by rinsing in

water for 1 min and in acetone containing 0.05% acetic acid for 6 min. The stained sections

were dehydrated, and the slides were mounted as described above.

For 5LO staining, the sections were incubated with rabbit anti-human 5LO antiserum

(1551-4b) at 1:1600 dilution or pre-bleed serum at 1:1600 dilution (43) at 4°C overnight and

developed with the same procedure as described above for α-actin staining.

Statistical analysis

Data are presented as the mean ± standard deviation of the mean. Statistical analysis

was performed by two-way ANOVA, with the Tukey test used for post-hoc analysis. P<0.05

indicates statistical significance.

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Results

Overexpression of 5LO in rat lungs

To examine the role of 5LO in the development of pulmonary hypertension, recombinant

human 5LO was expressed in rat lungs via adenovirus-mediated gene transfer. Approximately

2 x 109 plaque forming units of adenovirus expressing human 5LO (Ad5LO) were aerosolized

and delivered to the rat lung through inhalation. The time course of the transgene expression in

the lungs was analyzed by Northern blotting. Three groups of rats were examined: rats given

vehicle - Dulbecco’s phosphate-buffered saline (PBS), MCT, or MCT+Ad5LO. As shown in

Figure 1, rats in the last group expressed human 5LO mRNA; the expression peaked between

7-10 days and decreased by day 15 after Ad5LO delivery.

The localization of the expressed 5LO protein in rat lungs was examined by

immunohistochemical staining. As shown in Figure 2, the expression occurred in a wide range

of cell types, including alveolar pneumocytes, bronchial epithelial cells, alveolar macrophages,

and small pulmonary vessels. The anti-human 5LO antiserum used for the study crossreacts

with rat 5LO, which is normally expressed in leukocytes and macrophages.

.

Effect of 5LO overexpression on the development of pulmonary hypertension

To determine the effect of 5LO on the development of pulmonary hypertension in rats,

five groups of rats were studied: rats given PBS, Ad5LO, MCT, MCT+AdGFP, and MCT+Ad5LO.

All the treatments were performed on day 0.

On days 3, 7, 10, 15, 18, and 24, rats were examined by echocardiography to assess

pulmonary artery acceleration time (PAAT), pulmonary flow waveform, and right ventricular free

wall thickness. As shown in Figure 3, the decrease in PAAT occurred significantly earlier in the

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rats treated with MCT+Ad5LO than those treated with MCT alone (7-10 days vs. 15-18 days).

Administration of MCT+AdGFP had the same effect as that of MCT alone. Rats that received

Ad5LO alone did not have decreased PAAT over the entire study period, which was similar to

that observed in rats given a PBS injection. Pulmonary flow waveform change (development of

a midsystolic notch) occurred also earlier in the rats treated with MCT+Ad5LO than those

treated with MCT only or MCT+AdGFP (data not shown).

Right ventricular systolic pressures, measured by right heart catheterization, are shown in

Figure 4 for all groups of rats on days 0, 10, 15, 18, and 24. Consistent with the

echocardiographic findings, rats treated with MCT+Ad5LO had a significantly higher right

ventricular systolic pressure on day 10 compared to that of rats treated with MCT. Rats treated

with MCT+AdGFP behaved similarly to rats treated with MCT alone. Administration of Ad5LO

alone or PBS did not cause increases in right ventricular systolic pressure. By day 24, no

significant difference in right ventricular systolic pressure was found among the MCT+Ad5LO,

MCT+AdGFP, and MCT alone treatment groups.

Changes in right ventricle to left ventricle plus septum weight ratio

Rats in the above treatment groups were sacrificed at day 26. Their systolic pressures

and body weights were measured, and hearts dissected. The weight ratio of right ventricle-to-left

ventricle plus septum (RV/LV+S) was calculated. As shown in Figure 5, the ratio was

significantly higher in the group treated with MCT+Ad5LO than in the groups treated with

MCT+AdGFP or MCT alone (0.45±0.08 vs. 0.35±0.03 or 0.33±0.06, P<0.02). In addition, the

ratios in the groups treated with MCT were significantly higher than those without MCT. As the

MCT+Ad5LO-treated group were exposed to a significantly longer period of increased

pulmonary artery pressure, it may explain the higher RV/LV+S found in this group, even though

12

its right ventricular systolic pressure was no longer higher than that of the MCT and

MCT+AdGFP treatment groups by day 24. There was no significant difference in systolic blood

pressure or heart rate among the 5 groups at day 26 (data not shown).

Histological changes

Lungs from the treated rats were harvested at various time points and were fixed in

formalin. After paraffin embedding and sectioning, the tissues were stained with hematoxylin

and eosin. Three major histological changes were found in the MCT-treated groups:

inflammatory cell infiltration, muscularization of the distal pulmonary vessels, and fibrosis of the

interstitium. These changes in the MCT+Ad5LO group were more severe than those in the

MCT+AdGFP or MCT alone groups. The sections also show scattered post-inflammatory

extension of bronchiolar epithelium to thickened septa, which appeared to be similar in all MCT-

treated groups (data not shown). To compare the degree of inflammation and muscularization of

distal vessels, lung tissue sections were stained with specific antibodies against

monocytes/macrophages or smooth muscle cell α-actin, and the numbers of the antibody-

stained cells or vessels were counted. In addition, Masson’s trichrome staining was performed

on tissue sections collected at day 26 after treatment to monitor the fibrosis in these sections.

Figure 6A shows the photomicrographs of hematoxylin and eosin-stained lung sections

prepared from rats treated with PBS, MCT, MCT+AdGFP, and MCT+Ad5LO at day 15. The

staining revealed the presence of inflammatory cells, predominantly monocytes/macrophages

(include enlarged foam cells) and lymphocytes. The distribution of the infiltrated inflammatory

cells in the lung is patchy, many being in the proximity of blood vessels. To estimate the degree

of inflammation, lung sections from MCT, MCT+AdGFP, and MCT+Ad5LO treatment groups

collected at days 7, 15, and 22 were stained with anti-monocyte/macrophage antibody and the

13

number of stained cells was counted. As shown in Figure 6B, the number of macrophages in

the MCT+AdGFP and MCT+Ad5LO groups was the highest at day 15 and was reduced by day

22. The number of macrophages in the MCT group was increased by day 22. The

MCT+Ad5LO group had significantly more macrophage infiltration than the MCT+AdGFP group

at day 15. The MCT only group had noticeably less macrophage infiltration than the

MCT+Ad5LO and MCT+AdGFP groups, indicating that part of the inflammation was caused by

adenovirus infection.

Shown in Figure 7A are photomicrographs of an anti-smooth muscle cell α-actin stained

lung sections prepared from rats treated with PBS, MCT+AdGFP, and MCT+Ad5LO at day 22.

The muscularization and thickening of distal pulmonary vessels is more visible in the

MCT+Ad5LO group than the MCT+AdGFP group. The numbers of the muscularized distal

vessels were counted in groups treated with MCT+Ad5LO and MCT+AdGFP, at days 7, 15, 22,

and 26. As shown in Figure 7B, the number of muscularized distal pulmonary vessels

increased significantly at 15 day and reached a plateau by day 22. The MCT+Ad5LO treatment

group displayed a trend toward higher numbers of muscularized vessels than those of the

MCT+AdGFP treatment group on days 22 and 26, but the difference did not reach statistical

significance.

Figure 8 shows representative photomicrographs of lung sections stained with Masson’s

trichrome stain from rats treated with PBS, MCT+AdGFP, or MCT+Ad5LO at day 26. As

demonstrated, the lung sections had extensive collagen deposition, consistent with fibrosis. The

MCT+Ad5LO treatment group appeared to have more alveolar thickening and collagen

deposition than the MCT+AdGFP treatment group.

14

Effect of 5LO inhibition on the development of pulmonary hypertension

To examine whether these effects of 5LO is dependent on its activity, two specific

inhibitors, one for 5LO (zileuton) and one for FLAP (MK886), were utilized. Rats were treated

with MCT+Ad5LO on day 0, and DMSO (solvent for MK886), MK886, or zileuton was

administered on day 1 and daily thereafter. The one-day delay of the administration of inhibitors

was designed to avoid potential interference of the activation of MCT in the liver. Right heart

catheterization was performed on all groups at days 10, 15, and 26. As shown in Figure 9A,

both inhibitors prevented the increase of right ventricular pressure in the MCT+Ad5LO-treated

rats at day 10 (15±2 mmHg and 16±4 mmHg for MCT+Ad5LO+ MK886 and

MCT+Ad5LO+zileuton, respectively, vs. 25±2 mmHg for MCT+Ad5LO; p<0.05). Interestingly,

MK886 and zileuton also prevented the late development of pulmonary hypertension in the rats

(day 26: MCT+Ad5LO = 42±4 mmHg vs. 25±5 mmHg and 22±8 mmHg for

MCT+Ad5LO+MK886 and MCT+Ad5LO+zileuton rats, respectively; p<0.05 vs. MCT+Ad5LO).

To examine the possibility that 5LO plays a role in MCT-induced pulmonary hypertension

in rats, rats were treated with MCT only and received daily administration of vehicle (DMSO),

MK886, or zileuton as described above. Right heart catheterization was performed on all

groups at day 35. As shown in Figure 9B, both MK886 and zileuton prevented the development

of pulmonary hypertension in the MCT-treated rats, which suggests that endogenous 5LO

played an important role in mediating MCT-induced pulmonary hypertension in rats.

15

Discussion

This study examined the role of 5LO in the development of pulmonary hypertension by

overexpressing the enzyme in rat lungs through adenovirus-mediated gene transfer, and by

administrering specific 5LO inhibitors to MCT-treated rats. The results show that

overexpression of 5LO in normal lungs did not cause pulmonary hypertension; however, it

markedly accelerated the development of pulmonary hypertension in rats treated with MCT. 5LO

inhibitors inhibited the effect of 5LO, and also prevented MCT-induced pulmonary hypertension.

Two questions arise from the results of this study: 1) why did the overexpression of 5LO

have an adverse effect on the pulmonary vasculature in MCT-treated rats but not in normal rats?

and 2) why did the 5LO inhibitors prevent the pulmonary hypertension caused by MCT?

The first question can be explained by two possibilities. One is that the normal

pulmonary vasculature is somewhat protected from leukotriene-dependent vasoconstriction, but

in the MCT-injured pulmonary vessels, this protection is weakened. The source of the

protection has been implicated by Bäck and colleagues who reported that cysteinyl leukotrienes

stimulate prostacyclin production in the isolated human pulmonary artery, and that endothelium-

denuded or indomethacin-treated human pulmonary artery is significantly more sensitive to

cysteinyl leukotriene-induced vasoconstriction (in an organ chamber) than endothelialized

pulmonary arteries (4). The second possibility is that MCT-treated rats had more pulmonary

inflammation than normal rats, and the inflammation provided a supportive environment for 5LO-

catalyzed leukotriene biosynthesis. Leukotriene biosynthesis consists of three major steps:

release of arachidonic acid from cell membranes by phospholipase A2, conversion of

arachidonic acid to leukotriene A4 by 5LO, and transformation of leukotriene A4 to leukotrienes

B4 or cysteinyl leukotrienes. Both phospholipase A2 and 5LO require calcium for their activity,

and intracellular calcium release is related to the state of cell activation. Inflammatory stimuli

16

can cause activation of a variety of cells. Further analysis of prostacyclin and leukotriene

release in normal and dysfunctional pulmonary vasculature in animals expressing 5LO in the

lung would help to confirm these hypotheses.

The finding that 5LO inhibitors prevented the MCT-induced pulmonary hypertension

suggests endogenous 5LO is essential for MCT-induced pulmonary hypertension. MCT-

induced pulmonary endothelial injury is known to be followed by significant pulmonary vascular

and alveolar inflammation (34, 40), and the latter is thought to be important in subsequent

pulmonary vascular remodeling and pulmonary hypertension. Previous studies have shown that

inhibition of inflammation by interleukin-1 receptor antagonists (37) or by an antibody that

neutralizes monocyte chemotactic and activating factor/monocyte chemoattractant protein-1

(19) alleviate MCT-induced pulmonary hypertension. As 5LO is predominantly expressed in

inflammatory cells, infiltration of these cells in the lung results in an increase in 5LO in the

tissue. Downstream leukotriene products of 5LO could markedly amplify the inflammatory

process by attracting more inflammatory cells to the lung (leukotriene B4 ) and by increasing

vascular permeability to facilitate cell infiltration. It is possible that 5LO inhibitors prevented the

MCT-induced pulmonary hypertension via a similar mechanism as that by other inflammatory

inhibitors.

In addition to the facilitation of inflammatory processes, 5LO could exert its effect through

several other possible mechanisms. These include: 1) causing pulmonary vasoconstriction by

increasing cysteinyl leukotriene production; 2) promoting vascular cell proliferation by its nuclear

localization or through interaction with cytoskeletal proteins (21, 27, 29); and 3) altering TGF-β

superfamily signaling through binding to a TGF-β receptor I-associated protein (TRAP-1). To

address these mechanisms, further studies need to explore 1) the types of cysteinyl leukotriene

receptors on pulmonary arteries or arterioles and the potential influence of endothelial

17

dysfunction on the function of these receptors; 2) the role of the cytoskeleton, actin, or tyrosine

kinase signaling in vascular remodeling during the development of pulmonary arterial

hypertension; and 3) the effect of the 5LO-TRAP-1 interaction on TGF-β signaling. This latter

interaction may be of particular relevance to familial and sporadic forms of primary pulmonary

hypertension in which imbalanced TGF-β superfamily signaling is believed to play a role in the

disease development (20, 35, 36, 42). Since 5LO overexpression has been demonstrated in

primary pulmonary hypertension, examining these possibilities in the future would help clarify

the specific role of 5LO in pulmonary hypertension, as well as the mechanism of the

development of pulmonary hypertension, in general.

18

Acknowledgements:

The authors thank Stephanie Tribuna for expert secretarial assistance, Antoinette Hayes

for invaluable contribution with animal work, and Ray Qian for excellent assistance in

morphometric analysis of histology slides.

This work is supported by NIH Grants P01 HL66254 project III (RMT), P50 HL55993,

HL58976, HL61795 (JL), and AHA National Grant-in-Aid 0256282N (YYZ).

19

References

1. Ahmed, T., and W. Oliver, Jr. Does slow-reacting substance of anaphylaxis mediate

hypoxic pulmonary vasoconstriction? Am Rev Respir Dis 127: 566-71., 1983.

2. Allen, J. R., and L. A. Carstens. Pulmonary vascular occlusions initiated by endothelial

lysis in monocrotaline-intoxicated rats. Exp Mol Pathol 13: 159-71., 1970.

3. Avis, I. M., M. Jett, T. Boyle, M. D. Vos, T. Moody, A. M. Treston, A. Martinez, and J. L.

Mulshine. Growth control of lung cancer by interruption of 5-lipoxygenase- mediated growth

factor signaling. J Clin Invest 97: 806-13, 1996.

4. Back, M., X. Norel, L. Walch, J. Gascard, V. de Montpreville, S. Dahlen, and C. Brink.

Prostacyclin modulation of contractions of the human pulmonary artery by cysteinyl-

leukotrienes. Eur J Pharmacol 401: 389-95., 2000.

5. Berkowitz, B. A., B. Zabko-Potapovich, R. Valocik, and J. G. Gleason. Effects of the

leukotrienes on the vasculature and blood pressure of different species. J Pharmacol Exp Ther

229: 105-12., 1984.

6. Borgeat, P., and B. Samuelsson. Arachidonic acid metabolism in polymorphonuclear

leukocytes: unstable intermediate in formation of dihydroxy acids. Proceedings of the National

Academy of Sciences of the United States of America 76: 3213-7, 1979.

7. Bruner, L. H., K. S. Hilliker, and R. A. Roth. Pulmonary hypertension and ECG changes

from monocrotaline pyrrole in the rat. Am J Physiol 245: H300-6., 1983.

8. Butler, W. H., A. R. Mattocks, and J. M. Barnes. Lesions in the liver and lungs of rats

given pyrrole derivatives of pyrrolizidine alkaloids. J Pathol 100: 169-75., 1970.

9. Dahlen, S. E., J. Bjork, P. Hedqvist, K. E. Arfors, S. Hammarstrom, J. A. Lindgren, and B.

Samuelsson. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary

20

venules: in vivo effects with relevance to the acute inflammatory response. Proc Natl Acad Sci U

S A 78: 3887-91., 1981.

10. Dahlen, S. E., P. Hedqvist, S. Hammarstrom, and B. Samuelsson. Leukotrienes are

potent constrictors of human bronchi. Nature 288: 484-6., 1980.

11. Dennis, E. A. Phospholipase A2 in eicosanoid generation. American Journal of

Respiratory & Critical Care Medicine 161: S32-5, 2000.

12. Dixon, R. A., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and

D. K. Miller. Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis.

Nature 343: 282-4, 1990.

13. Drazen, J. M., K. F. Austen, R. A. Lewis, D. A. Clark, G. Goto, A. Marfat, and E. J. Corey.

Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro. Proc

Natl Acad Sci U S A 77: 4354-8., 1980.

14. Ford-Hutchinson, A. W., M. A. Bray, M. V. Doig, M. E. Shipley, and M. J. Smith.

Leukotriene B, a potent chemokinetic and aggregating substance released from

polymorphonuclear leukocytes. Nature 286: 264-5, 1980.

15. Ghodsi, F., and J. A. Will. Changes in pulmonary structure and function induced by

monocrotaline intoxication. Am J Physiol 240: H149-55., 1981.

16. Goldberg, R. N., C. Suguihara, T. Ahmed, B. D. de Cudemus, P. Barrios, E. S. Setzer,

and E. Bancalari. Influence of an antagonist of slow-reacting substance of anaphylaxis on the

cardiovascular manifestations of hypoxia in piglets. Pediatr Res 19: 1201-5., 1985.

17. Hanna, C. J., M. K. Bach, P. D. Pare, and R. R. Schellenberg. Slow-reacting substances

(leukotrienes) contract human airway and pulmonary vascular smooth muscle in vitro. Nature

290: 343-4., 1981.

21

18. Jones, J. E., L. Mendes, M. A. Rudd, G. Russo, J. Loscalzo, and Y. Y. Zhang. Serial

noninvasive assessment of progressive pulmonary hypertension in a rat model. Am J Physiol

Heart Circ Physiol 283: H364-71., 2002.

19. Kimura, H., Y. Kasahara, K. Kurosu, K. Sugito, Y. Takiguchi, M. Terai, A. Mikata, M.

Natsume, N. Mukaida, K. Matsushima, and T. Kuriyama. Alleviation of monocrotaline-induced

pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte

chemoattractant protein-1. Lab Invest 78: 571-81., 1998.

20. Lane, K. B., R. D. Machado, M. W. Pauciulo, J. R. Thomson, J. A. Phillips, 3rd, J. E.

Loyd, W. C. Nichols, and R. C. Trembath. Heterozygous germline mutations in BMPR2,

encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. The

International PPH Consortium. Nat Genet 26: 81-4., 2000.

21. Lepley, R. A., and F. A. Fitzpatrick. 5-Lipoxygenase contains a functional Src homology

3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins.

J Biol Chem 269: 24163-8, 1994.

22. Meyrick, B., and L. Reid. Development of pulmonary arterial changes in rats fed

Crotalaria spectabilis. Am J Pathol 94: 37-51., 1979.

23. Morganroth, M. L., J. T. Reeves, R. C. Murphy, and N. F. Voelkel. Leukotriene synthesis

and receptor blockers block hypoxic pulmonary vasoconstriction. J Appl Physiol 56: 1340-6.,

1984.

24. Morganroth, M. L., K. R. Stenmark, K. G. Morris, R. C. Murphy, M. Mathias, J. T. Reeves,

and N. F. Voelkel. Diethylcarbamazine inhibits acute and chronic hypoxic pulmonary

hypertension in awake rats. Am Rev Respir Dis 131: 488-92., 1985.

22

25. Morganroth, M. L., K. R. Stenmark, J. A. Zirrolli, R. Mauldin, M. Mathias, J. T. Reeves, R.

C. Murphy, and N. F. Voelkel. Leukotriene C4 production during hypoxic pulmonary

vasoconstriction in isolated rat lungs. Prostaglandins 28: 867-75., 1984.

26. Murphy, R. C., S. Hammarstrom, and B. Samuelsson. Leukotriene C: a slow-reacting

substance from murine mastocytoma cells. Proceedings of the National Academy of Sciences of

the United States of America 76: 4275-9, 1979.

27. Peppelenbosch, M. P., L. G. Tertoolen, W. J. Hage, and S. W. de Laat. Epidermal growth

factor-induced actin remodeling is regulated by 5- lipoxygenase and cyclooxygenase products.

Cell 74: 565-75., 1993.

28. Piperno, D., Y. Pacheco, R. Hosni, P. Moliere, C. Gharib, M. Lagarde, and M. Perrin-

Fayolle. Increased plasma levels of atrial natriuretic factor, renin activity, and leukotriene C4 in

chronic obstructive pulmonary disease. Chest 104: 454-9., 1993.

29. Provost, P., J. Doucet, T. Hammarberg, G. Gerisch, B. Samuelsson, and O. Radmark. 5-

Lipoxygenase interacts with coactosin-like protein. J Biol Chem 276: 16520-7., 2001.

30. Provost, P., B. Samuelsson, and O. Radmark. Interaction of 5-lipoxygenase with cellular

proteins. Proc Natl Acad Sci U S A 96: 1881-5., 1999.

31. Schreiber, M. D., M. A. Heymann, and S. J. Soifer. Leukotriene inhibition prevents and

reverses hypoxic pulmonary vasoconstriction in newborn lambs. Pediatr Res 19: 437-41., 1985.

32. Shimizu, T., O. Radmark, and B. Samuelsson. Enzyme with dual lipoxygenase activities

catalyzes leukotriene A4 synthesis from arachidonic acid. Proceedings of the National Academy

of Sciences of the United States of America 81: 689-93, 1984.

33. Stenmark, K. R., S. L. James, N. F. Voelkel, W. H. Toews, J. T. Reeves, and R. C.

Murphy. Leukotriene C4 and D4 in neonates with hypoxemia and pulmonary hypertension. N

Engl J Med 309: 77-80., 1983.

23

34. Stenmark, K. R., M. L. Morganroth, L. K. Remigio, N. F. Voelkel, R. C. Murphy, P. M.

Henson, M. M. Mathias, and J. T. Reeves. Alveolar inflammation and arachidonate metabolism

in monocrotaline- induced pulmonary hypertension. Am J Physiol 248: H859-66., 1985.

35. Thomson, J. R., R. D. Machado, M. W. Pauciulo, N. V. Morgan, M. Humbert, G. C. Elliott,

K. Ward, M. Yacoub, G. Mikhail, P. Rogers, J. Newman, L. Wheeler, T. Higenbottam, J. S.

Gibbs, J. Egan, A. Crozier, A. Peacock, R. Allcock, P. Corris, J. E. Loyd, R. C. Trembath, and

W. C. Nichols. Sporadic primary pulmonary hypertension is associated with germline mutations

of the gene encoding BMPR-II, a receptor member of the TGF- beta family. J Med Genet 37:

741-5., 2000.

36. Trembath, R. C., J. R. Thomson, R. D. Machado, N. V. Morgan, C. Atkinson, I. Winship,

G. Simonneau, N. Galie, J. E. Loyd, M. Humbert, W. C. Nichols, N. W. Morrell, J. Berg, A.

Manes, J. McGaughran, M. Pauciulo, and L. Wheeler. Clinical and molecular genetic features of

pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med

345: 325-34., 2001.

37. Voelkel, N. F., R. M. Tuder, J. Bridges, and W. P. Arend. Interleukin-1 receptor

antagonist treatment reduces pulmonary hypertension generated in rats by monocrotaline. Am J

Respir Cell Mol Biol 11: 664-75., 1994.

38. Voelkel, N. F., R. M. Tuder, K. Wade, M. Hoper, R. A. Lepley, J. L. Goulet, B. H. Koller,

and F. Fitzpatrick. Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary

vascular reactivity and pulmonary hypertension in hypoxic rats. J Clin Invest 97: 2491-8., 1996.

39. Walker, J. L., J. Loscalzo, and Y. Y. Zhang. 5-Lipoxygenase and human pulmonary artery

endothelial cell proliferation. Am J Physiol Heart Circ Physiol 282: H585-93., 2002.

24

40. Wilson, D. W., H. J. Segall, L. C. Pan, and S. K. Dunston. Progressive inflammatory and

structural changes in the pulmonary vasculature of monocrotaline-treated rats. Microvasc Res

38: 57-80., 1989.

41. Wright, L., R. M. Tuder, J. Wang, C. D. Cool, R. A. Lepley, and N. F. Voelkel. 5-

Lipoxygenase and 5-lipoxygenase activating protein (FLAP) immunoreactivity in lungs from

patients with primary pulmonary hypertension. Am J Respir Crit Care Med 157: 219-29., 1998.

42. Yeager, M. E., G. R. Halley, H. A. Golpon, N. F. Voelkel, and R. M. Tuder. Microsatellite

instability of endothelial cell growth and apoptosis genes within plexiform lesions in primary

pulmonary hypertension. Circ Res 88: E2-E11., 2001.

43. Zhang, Y. Y., J. L. Walker, A. Huang, J. F. Keaney, C. B. Clish, C. N. Serhan, and J.

Loscalzo. Expression of 5-lipoxygenase in pulmonary artery endothelial cells. Biochem J 361:

267-76., 2002.

25

Figure Legends

Figure 1. Northern blotting analysis of the time course of 5LO transgene expression. Rats were

treated with PBS, MCT, or MCT+Ad5LO, and total RNA was isolated from the lung tissues of

the rats at days 3, 7, 10, and 15. Northern blotting analysis was carried out using an [α-32P]-

dCTP-labeled human 5LO cDNA probe (upper panel) and a probe against rat GAPDH (lower

panel). RNA samples from three individual rats were combined and examined at each time

point.

Figure 2. Distribution of 5LO transgene protein in rat lungs delivered by intratracheal

aerosolization. Rats were treated with PBS (panel A) or MCT+Ad5LO (panels B and C), and the

lung tissues were collected at day 10 after treatment. Paraffin embedded lung sections were

stained with rabbit anti-human 5LO antiserum (1:1600 dilution) (panels A and C) and rabbit pre-

bleed serum (1:1600 dilution) (panel B), and developed with biotinylated-alkaline phosphatase

and Vector Red Substrate. The slides were counterstained with hemotoxylin.

Figure 3. Pulmonary artery acceleration time (PAAT). Rats were treated with MCT+Ad5LO ( ),

MCT+AdGFP ( ), MCT only ( ), Ad5LO only ( ), or PBS ( ) and followed by transthoracic

echocardiography over time. The PAAT was significantly shorter in the MCT+Ad5LO group

from day 10 to day 18 as compared to all other groups. No difference existed between the

MCT+AdGFP and the MCT only groups. The Ad5LO alone did not decrease the PAAT as

compared to the PBS treatment group. *p<0.05 vs. PBS treatment and Ad5LO only groups.

#p<0.05 vs. MCT+AdGFP and MCT only groups. All values expressed as mean ± standard

deviation; n=3-10 per group.

26

Figure 4. Right ventricular systolic pressure (RVSP). Rats were treated with MCT+Ad5LO ( ),

MCT+AdGFP ( ), MCT only ( ), Ad5LO only ( ), or PBS ( ) and right heart catheterization

was performed on rats from each group on days 0, 10, 15, 18, and 24. The RVSP was

significantly higher in the MCT+Ad5LO group from day 10 to day 18 as compared to all other

groups. No difference existed between the MCT+AdGFP and the MCT only groups. Treatment

with Ad5LO alone did not increase the RVSP as compared to controls. *indicates p<0.05 vs.

PBS treatment and Ad5LO only groups. #indicates p<0.05 vs. the MCT+AdGFP and MCT only

groups. All values expressed as mean ± standard deviation. n=3-8 per group.

Figure 5. Right ventricle to left ventricle plus septum (RV/LV+S) weight ratio. Rats were treated

with PBS, Ad5LO, MCT, MCT+AdGFP, or MCT+Ad5LO, and sacrificed at day 26. The

RV/LV+S weight ratio was measured immediately after sacrifice. * indicates p<0.01 vs. the PBS

groups and # indicates p<0.02 vs. MCT+AdGFP group. All values are expressed as mean ±

standard deviation. n=3-8 per group.

Figure 6. Inflammatory cell infiltration in lungs. Rats were treated with PBS, MCT,

MCT+AdGFP, or MCT+Ad5LO. Paraffin-embedded lung tissue sections were prepared at days

7, 15, and 22 after treatment. Figure 6A shows hematoxylin and eosin-stained lung sections

from rats treated with PBS (A), MCT (B), MCT+AdGFP (C), and MCT+Ad5LO (D) harvested on

day 15. Figure 6B shows the number of monocytes/macrophages in lung sections prepared

from rats treated with MCT (white), MCT+AdGFP (gray), and MCT+Ad5LO (cross hatch), and

prepared at days 7, 15, and 22. The lung sections were stained with anti-monocyte/

macrophage antibody, and the cells in each section were counted in twenty consecutive fields

(200x). Data represent mean ± standard deviation of the number of cells in the lung tissue

27

sections obtained from 3-5 rats at each time point. * indicates p<0.05 vs. MCT+AdGFP group

and # indicates p<0.05 vs. MCT group.

Figure 7. Muscularized distal pulmonary vessels. Rats were treated with PBS, MCT+AdGFP,

and MCT+Ad5LO on day 0, and the lung tissues were collected on days 7, 15, 22, and 26 after

treatment. Tissue sections were stained with anti-smooth muscle cell α-actin antibody and

developed with biotinylated-alkaline phosphatase and Vector Red Substrate. The numbers of

muscularized vessels with diameter of 10-50 µm, and located distal to terminal bronchioles were

counted in twenty consecutive fields (200x) per section. Figure 7A shows representative

photomicrographs (400x) of stained lung section from rats treated with PBS (A), MCT+AdGFP

(B), and MCT+Ad5LO (C) at day 22. Figure 7B shows the average number of the muscularized

distal vessels in the lung tissue sections obtained from 3-5 rats at each time point. Open bars

represent treatment with MCT+AdGFP, and solid bars represent treatment with MCT+Ad5LO.

All values are expressed as mean ± standard deviation.

Figure 8. Fibrosis in lung tissues of rats treated with MCT+Ad5LO and with MCT+AdGFP.

Lung sections from day 26 were stained with Masson’s trichrome stain in order to visualize

collagen deposition. Panels A-C show representative photomicrographs of lung sections from a

rat treated with PBS (A), a rat treated with MCT+AdGFP (B), and a rat treated with MCT+Ad5LO

(C).

Figure 9. Inhibition of 5-lipoxygenase reduces the development of pulmonary hypertension in

MCT+Ad5LO- and MCT-treated rats. A) Rats were treated with MCT+Ad5LO on day 0 and

received vehicle (DMSO) (gray bar), MK886 (black bar), or zileuton (white bar) on day 1 and

28

daily thereafter. Right heart catheterization was performed on days 10, 15, and 26. B) Rats

were treated with MCT on day 0 and received DMSO, MK886, or zileuton on day 1 and daily

thereafter. Right heart catheterization was performed on day 35. Data represent mean ±

standard deviation of values obtained from 4 (panel A) and 5 rats (panel B), *indicates p<0.05

vs. MCT+DMSO (MCT) group.

Human 5LO

3 7 10 153 7 10 153 7 10 15Day

PBS MCT MCT+Ad5LO

GAPDH

Figure 1

Figure 2

A

B

C

0 3 7 10 15 18 2420

25

30

35

40

45#*

Figure 3

* *

*

*

p<0.05 vs PBS or Ad5LOp<0.05 vs AdGFP+MCT

*

**

##

#

***

MCT+Ad5LO MCT+AdGFP MCT Ad5LO PBS

PA

AT

(mse

c)

Days after treatment

0 5 10 15 20 2510

20

30

40

50

60#*

Figure 4

p<0.05 vs. PBS or Ad5LOp<0.05 vs. AdGFP+MCT or MCT

**

*

****

#

##

*

**

MCT+Ad5LO MCT+AdGFP MCT Ad5LO PBS

RV

SP

(mm

Hg)

Days after treatment

PBS

Ad5LO MCT

MCT+AdG

FPMCT+A

d5LO

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Figure 5

p< 0.02 vs. MCT+AdGFP group#p<0.01 vs. PBS group*

**

#

*

RV

/LV

+S w

eigh

t rat

io

A

C D

B

Figure 6A

7 15 220

100

200

300

400

500

600

MCT MCT+AdGFP MCT+Ad5LO

Figure 6B

*# p<0.05 vs. MCT

p<0.05 vs. MCT+AdGFP

#

#*

# m

onoc

ytes

/Mac

roph

ages

Day

A

B

C

Figure 7A

7 15 22 260

20

40

60

80

100

Figure 7B

# M

uscu

lariz

ed d

ista

l ves

sels

Day

MCT+AdGFP MCT+Ad5LO

Figure 8

A

B

C

Day 10 Day 15 Day 260

10

20

30

40

50

* p<0.05 vs. MCT+Ad5LO

**

**

**

Figure 9A

RV

SP

(mm

Hg)

MCT+Ad5LO MCT+Ad5LO+MK886 MCT+Ad5LO+zileuton

MCT MCT+MK886 MCT+zileuton0

10

20

30

40

50

60

Figure 9B

*

p<0.05 vs. MCT*

*

RV

SP

(mm

Hg)