Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

The Role of 12-Lipoxygenase in Hypoxia-Induced Rat Pulmonary Artery Smooth

Muscle Cell Proliferation

IOANA R. PRESTON, NICHOLAS S. HILL, ROD R. WARBURTON, BARRY L.

FANBURG

Pulmonary, Critical Care and Sleep Division, Tufts-New England Medical Center,

Tufts University School of Medicine, Boston, Massachusetts 02111

Running Head: 12-LO and pulmonary hypertension

Contact information: Ioana R. Preston, MD

Pulmonary, Critical Care and Sleep Division

Tufts-New England Medical Center

750 Washington Street, Box #257

Boston, MA 02111

Tel. 617-636-7609

Fax. 617-636-5953

Email. [email protected]

Articles in PresS. Am J Physiol Lung Cell Mol Physiol (September 30, 2005). doi:10.1152/ajplung.00114.2005

Copyright © 2005 by the American Physiological Society.

12-LO and pulmonary hypertension LCMP—00114-2005.R1

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ABSTRACT

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates

cell growth and metastasis of various cancer cells and the 12-LO metabolite,

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic

smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have

not been previous ly studied. We sought evidence for a role of 12-LO and 12-

HETE in the development of hypoxia-induced pulmonary hypertension. We found

that 12-LO gene and protein expression is elevated in lung homogenates of rats

exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody

revealed intense staining in endothelial cells of large pulmonary arteries, SMCs

(and possibly endothelial cells) of medium and small size pulmonary arteries, and

in alveolar walls of hypoxic lungs. 12-LO protein expression is increased in

hypoxic cultured rat pulmonary artery SMCs. 12-HETE at concentrations as low

as 10-5 µM stimulated proliferation of pulmonary artery SMCs. 12-HETE induced

ERK 1/ERK 2 phosphorylation, but had no effect on p38 kinase expression as

assessed by Western blotting. 12-HETE-stimulated SMC proliferation was

blocked by the MEK inhibitor, PD98059, but not by the p38 MAPK inhibitor,

SB202190. Hypoxia (3%) - stimulated pulmonary artery SMC proliferation was

blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We

conclude that 12-LO and its product, 12-HETE, are important intermediates in

hypoxia-induced pulmonary artery SMC proliferation and may participate in

hypoxia-induced pulmonary hypertension.

Keywords: 12-HETE, mitogen activated protein-kinases, ERK1/ERK2.


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INTRODUCTION

Pulmonary hypertension occurs in animals and humans exposed to acute or

sustained hypoxia (26) and this is a well-established in vivo experimental model

of pulmonary hypertension. The initial event is acute hypoxic pulmonary

vasoconstriction, followed by remodeling of small and medium-sized pulmonary

arteries (46). Muscular intrapulmonary arteries become fur ther muscularized,

and distal, non-muscular arteries become newly muscularized (26). Pulmonary

artery smooth muscle cell (PASMC) proliferation is a key feature of pulmonary

vascular remodeling. The mechanism of PASMC proliferation is thought to

involve an imbalance between mediators of apoptosis and cell proliferation.

Local paracrine mediators induced by hypoxia may also participate in the

proliferative response. Among these local mediators, metabolites of arachidonic

acid derived via the lipoxygenase pathway are known to exert vascular effects.

In particular, the leukocyte-type 12-lipoxygenase (12-LO) pathway has been

shown to stimulate cell proliferation and participate in inflammatory reactions in

aortic SMCs (4, 27, 29-32, 35, 36) and in migration and metastasis of various

cancer cell lines (6, 11, 18, 23, 24, 28, 38, 39, 41, 42, 44, 45). In addition, the 12-

LO product, 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), has a direct

hypertrophic effect on systemic vascular SMCs (29) and mediates hypertrophic

effects of angiotensin II (29) and chemotactic effects of platelet-derived growth

factor (27).

The 12-LO pathway has also been implicated in the proliferative response of

vascular SMCs in vivo. 12-LO gene expression is enhanced in SMCs of balloon-


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injured rat carotid arteries and 12-LO inhibition by ribozyme-mediated cleavage of

the 12-LO mRNA significantly reduces neointimal proliferation in this model (17).

Despite the evidence that 12-LO and its products contribute to SMC

proliferation in the systemic vasculature, no studies have yet addressed the role

of 12-LO on SMC proliferation in the pulmonary circulation exposed to hypoxia.

We hypothesized that hypoxia stimulates pulmonary artery smooth muscle cell

proliferation through a mechanism that involves the 12-LO pathway. We sought

to study the effects of hypoxia on PASMCs obtained from adult rats and to

determine if the 12-LO pathway participates in this process.

MATERIALS AND METHODS

Materials. Dulbecco’s modified Eagle’s medium (DMEM) was from Sigma (St.

Louis, MO). Fetal bovine serum (FBS) was from Hyclone (Logan, UT).

Antibodies to ERK and phospho-ERK were from Sigma, and to p38 MAPK,

phospho-p38 MAPK and secondary HRP-conjugated rabbit anti-mouse

antibodies were from Cell Signaling (Beverly, MA). The specific antibody against

leukocyte type 12-LO was a gift from Dr. Jerry Nadler, University of Virginia (2).

12-S-HETE, 15-S-HETE, SB202190, PD98059 and U0126 were purchased from

Biomol Research Laboratories (Plymouth Meeting, PA). [3H]Thymidine was

purchased from Perkin-Elmer (Boston, MA) and the PCR kit was purchased from

Quiagen (Santa Clarita, CA).

Exposure of animals to chronic hypoxia. Adult male Sprague-Dawley rats

were randomized to 3 weeks of normoxia or hypobaric hypoxia (0.5 atm).


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Hypobaric chambers were briefly opened 3 times weekly for cleaning and to

replace food and water. Twelve hour light exposure cycles, standard rat chow

and water ad libitum were provided to all rats. Normoxic rats were kept in the

same room adjacent to the hypobaric chamber. At the end of the 3-week

exposure period, rats were anesthetized and hemodynamic measurements were

obtained as previously described (22). After euthanasia using pentobarbital

injection (120 mg/kg ip), the thorax was opened immediately and the heart and

lungs were removed. The hearts were dissected into RV free wall and left

ventricle plus septum (LV+S) and the ratio of RV/(LV+S) was used as an index of

RV hypertrophy. The left lung was frozen in liquid nitrogen and stored at -80°C

for further measurements. Double fixation of the right lung was achieved in the

distended state by infusion of 4% aqueous buffered formalin into the trachea at

25 cm H2O pressure and into the pulmonary trunk at 5 cm H2O pressure. The

right lung was then processed for paraffin embedding. 5 µm sections were

immunostained with the leukocyte-type 12-LO antibody and counterstained with

hematoxylin-eosin. Vessels with a layer of smooth muscle cells comprising ≥ 3/4

of the vessel perimeter were categorized as fully muscularized. Vessels with a

smooth muscle layer comprising <3/4 of the vessel perimeter were categorized

as partially muscularized and vessels that had no identifiable smooth muscle

cells were categorized as nonmuscularized vessels.

Immunohistochemical detection of leukocyte-type 12-LO in rat lungs. The

immunohistochemical methods were carried out using the technique described

previously (31). Briefly, 5 µm paraffin-embedded tissue sections were mounted


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on Probe-on slides (Biotek Solutions) and dried for 1h in 56°C oven and

overnight at 45-50°C, deparaffinized in xylene and rehydrated in graduated

alcohol to distilled water. The slides were loaded into a Techmate Slide holder

and placed into 0.1 mol/l citrate buffer solution for HIER. The slides were

steamed in 0.1M citrate buffer for 20 min and then allowed to cool for 5 min.

After treatment with the first and second antibody, slides were stained using a

modified ABC technique using 3,3’-diaminobenzidine tetrahydrochloride (DAB)

as the chromogen and conterstained using Mayer’s hematoxylin. Staining was

performed using a Bioteck Techmate 1000 Immunostainer (Teckmate, Santa

Barbara, CA) with Biotek solutions and an ABC detection system (Teckmate).

We used the 12-LO antibody at 1:500 concentration. Parallel controls were run

without primary antibody.

Isolation and culture of pulmonary artery smooth muscle cells. For isolation

of PASMCs, freshly excised lobar pulmonary arteries obtained from adult male

Sprague Dawley rats were stripped of adventitia. Vascular segments were then

cut open and endothelium was removed by gentle scraping of the luminal surface

of the vessel. Rat PASMCs were cultured from explants as previously described

(1, 9). SMC phenotype was assessed by the hill and valley morphology (1).

Cells were grown in DMEM supplemented with 10%FBS, penicillin (100 units/ml),

streptomycin (100 units/ml), and fungizone (1.25 µg/ml) and were passaged

every 1-2 weeks at a 1:3 ratio using trypsin. Passage 3-8 cells at 80%

confluence were used in all reported experiments. Before exposure to hypoxia or

treatment with different agents, cells were starved in 0.1% FBS DMEM with


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antibiotics for 72 hours. Cells were exposed to hypoxia or treated with different

agents in 0.1% FBS DMEM with antibiotics, unless otherwise specified. Medium

was changed every 2 -3 days.

Exposure of PASMCs to hypoxia. PASMCs were exposed to 3% hypoxia in a

humidified modular incubator chamber (Billups-Rothenberg, Del Mar, CA) that

was maintained at 37°C. The incubator chamber was sealed and purged with

3%O2, 5%CO2, balance N2 for 15 minutes. Normoxic control PASMCs were

exposed to 95% ambient air, 5%CO2 for the entire incubation period. A portable

gas analyzer (Hudson Ventronics Division) was used to ensure that the O2

concentration inside the chamber was 3%. Hypoxia had no effect on the pH of

the medium, which was maintained at 7.36 ± 0.3.

[3H]Thymidine incorporation. PASMCs were plated in complete growth media

containing 10% FBS in 96-well plates at a density of 4 x 103 cells/cm2. After

plates were 80% confluent, cells were starved in 0.1% FBS for 72 hours. Cells

were then either exposed to hypoxia or normoxia, or treated with different agents

30 minutes before the beginning of the hypoxia exposure. Twenty four hours

before the end of the experiment, [3H]thymidine (0.1 mCi/ml) was added.

Incorporation of labeled thymidine was stopped by aspirating the medium and

trypsinizing the cells for 15 minutes at 37ºC, after which cells were transferred

onto a filter membrane using a cell harvester. Radioactivity was counted in a

microplate liquid scintillation counter (Perkin-Elmer) after 20 µl of scintillation

liquid was added to each well.

Cell proliferation. PASMCs were plated at 2 x 104 cells/well in 6-well plates in


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DMEM and 10%FBS with antibiotics, growth-arrested for 72 hours, then exposed

to normoxia or hypoxia for various times. Cells were removed from the wells by

trypsin digestion. Cells were counted using a Fischer hemocytometer

(Pittsburgh, Pa). In each of the three experiments, the results from 3 wells were

averaged to obtain a single cell count (± SD) for each time point.

Western blot analysis. Protein determinations from both lung homogenates

and cell lysates were made using the Bradford method (3). To prepare lysates,

cells were washed in phosphate-buffered saline and solubilized with 50 mM

Hepes solution (pH 7.4) containing 1% (v/v) Triton X-100, 4 mM EDTA, 1 mM

sodium fluoride, 0.1 mM sodium orthovanidate, 1 mM tetrasodium

pyrophosphate, 2 mM pheylmethylsulsonyl fluoride, 10 µg/ml leupeptin and 10

µg/ml aprotinin. Cell lysates (20 µg of protein) were electrophoresed through

12% SDS-polyacrylamide gel and electroblotted onto a polyvinylidine difluoride

membrane. The membrane was blocked and incubated with primary antibodies

for phospho-specific ERK1/2, ERK, phospho-specific p38 MAPK, p38 MAPK or

12-LO. The polyclonal antibody against amino acids 646 to 662 peptide

sequence of porcine leukocyte 12-LO protein was raised in rabbits and has been

shown to have excellent cross-reactivity with rat 12-LO (49). The levels of

proteins and phosphoproteins were detected with horseradish peroxide-linked

secondary antibodies and the ECL System (Amersham Biosciences).

RT-PCR for 12-LO. Lungs were homogenized and total RNA was extracted

from the lung tissues by using the RNeasy Total RNA Isolation Kit (Quiagen,

Valencia, CA). The PCR method is specific for the leukocyte-type 12-LO and


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does not cross-react with platelet-type 12-LO (17), which is the product of a

separate, linked gene. The primers for the rat 12-LO gene were: sense, 5’-TGG

GGC AAC TGG AAG G-3’, located at the 324 to 339 position of rat 12-LO, and

antisense, 5’-AGA GCG CTT CAG CAC CAT–3’ (2), located at the 718 to 735

position. PCR reaction was carried out under the following conditions: 38 cycles

of denaturation at 94ºC for 45 seconds, annealing at 68ºC for 60 seconds, and

extension at 72ºC for 60 seconds. A final extension was performed at 72ºC for

10 minutes. PCR products were separated by 3% polyacrylamide gel

electrophoresis in Tri-borate-EDTA buffer. For internal control, we performed RT-

PCR, then PCR for tubulin.

Statistical Analysis. Data are expressed as mean (± SD). Statistical analysis

was performed using the unpaired Student’s t-test, or analysis of variance, as

indicated. Differences were considered to be significant when p < 0.05.

RESULTS

12-LO mRNA and protein expression is increased in hypoxic rat lungs. Adult

rats exposed to 3 weeks of hypobaric hypoxia (0.5 atm) developed pulmonary

hypertension, as demonstrated by an increase in systolic pulmonary artery

pressures (27.5 ± 0.8 mmHg in normoxic rats and 42 ± 2 mmHg in hypoxic rats,

p < 0.05) and RV hypertrophy (RV/LV+S was 0.28 ± 0.005 in normoxic rats and

0.46 ± 0.02 in hypoxic rats, p < 0.05, N=5 animals per group). Lung

homogenates from these animals showed increases in both 12-LO mRNA (Fig 1

A) and protein (Fig 1 B, C) as compared to homogenates from normoxic controls.


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Histologic examination (hematoxylin-eosin staining) revealed thickening of the

small pulmonary arterioles and no evidence of inflammation (Fig 2).

Evidence of increased 12-LO expression in hypoxic rat lung. In normoxic

rats, medium size and small pulmonary arteries were nonmuscularized (Fig 2 C).

After 3 weeks of exposure to hypoxia, many of these arteries became either

partially, or fully muscularized (Fig 2 F). Immunohistochemistry of rat lungs

showed intense 12-LO staining of bronchial epithelial cells and alveolar

macrophages that remained unchanged with hypoxia (Fig 2 A, C, D, F). In

normoxic lungs, there was no 12-LO staining of the pulmonary vasculature (Fig 2

B, C). In contrast, there was intense 12-LO immunostaining of endothelial cells

of large pulmonary arteries (Fig 2 D, E), SMCs (and possibly endothelial cells) of

small pulmonary arteries, and alveolar pneumocytes of hypoxic lungs (Fig 2 F).

Hypoxia stimulates rat PASMC proliferation. To provide an in vitro correlate

for the hypoxia-induced medial hypertrophy of pulmonary arteries seen in vivo,

we determined whether cultured SMCs proliferate during exposure to hypoxia.

PASMCs were growth-arrested in 0.1%FBS prior to being exposed to hypoxia or

normoxia with or without 10% serum. Hypoxia stimulated DNA synthesis, as

assessed by incorporation of [3H]thymidine into cells in both 0.1% and 10 %

serum (Fig 3 A, B). The proliferative effect of hypoxia was validated by

demonstrating an increase in cell number in hypoxic compared to control SMCs

(see Fig 3 C for representative experiment).

12-LO is up-regulated in hypoxic rat PASMCs in culture. Because PASMCs

are believed to participate in the pulmonary vascular response to hypoxia, we


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next evaluated the influence of hypoxia on 12-LO protein expression in vitro.

Hypoxia up-regulated 12-LO protein expression in PASMCs at 2 hours and this

was maintained at 48 hours (Fig 4 A,B).

12-HETE stimulates PASMC proliferation through activation of ERK. Having

established that hypoxia enhances 12-LO expression and stimulates SMC

proliferation, we next sought to evaluate the effects of the 12-LO product, 12-

HETE, on SMC proliferation. Figure 5 A shows that 12-HETE directly stimulates

PASMC proliferation at concentrations as low as 10-5 µM. In contrast, 15-HETE,

a product resulting from arachidonic acid metabolism by 15-LO, did not have a

proliferative effect. In addition, 12-HETE-induced PASMC proliferation was

completely blocked by PD98059, a specific MEK inhibitor (an upstream factor of

ERK) (Fig 5 B). These results suggest that ERK and 12-LO are linked in the

proliferative pathway and that 12-LO up-regulation and 12-HETE formation

precede activation of ERK.

12-HETE stimulates ERK activation. In order to establish if ERK is

downstream of 12-HETE, we measured ERK activation in PASMCs stimulated

with 12-HETE. Fig 6 A and B demonstrate that 12-HETE directly activates the

ERK pathway by inducing phosphorylation of ERK1/ERK2. This is a biphasic

action with peaks at 10 and 90 minutes. Another signaling pathway that has

been shown to be activated during cell proliferation is p38 MAPK (10, 13). We

found that 12-HETE did not induce p38 MAPK phosphorylation (Fig 6 C, D).


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Hypoxia –induced PASMC proliferation is blocked by the MEK-1 inhibitor,

PD98059. In order to determine the cellular pathways that may be activated

during cell exposure to hypoxia, we evaluated the MAPK signaling pathway, in

particular ERK1/2 MAPK and p38 MAPK activation. PASMCs were exposed to

72 hours of hypoxia (3%O2) in the presence or absence of a specific MEK

inhibitor, U0126, as well as in the presence or absence of a specific p38 MAPK

inhibitor, SB202190. Fig 7 demonstrates inhibition of hypoxia-stimulated cell

proliferation by the MEK inhibitor and lack of effect by the p38 MAPK inhibitor.

Hypoxia-induced PASMC proliferation is blocked by 12-LO inhibition. A

partial blockade of hypoxia-stimulated SMC proliferation was obtained with the

12-LO inhibitor, baicalein (Fig 8). Studies using trypan blue exclusion

demonstrated that concentrations of baicalein from 0.5-10 µM had no effect on

cell viability (data not shown).

DISCUSSION

It is now generally accepted that hypoxia induces pulmonary hypertension

that is associated with pulmonary vascular remodeling and, in particular, smooth

muscle cell hyperplasia and hypertrophy. A large number of intermediate cell

signaling pathways by which this occurs have been proposed (1, 7-9, 14, 15, 19,

20, 22). The present study provides evidence that the 12-LO pathway via its

metabolite, 12-HETE, contributes to hypoxia-induced pulmonary arterial smooth

muscle cell remodeling. We found up-regulation of 12-LO mRNA and protein

expression in lung homogenates of rats exposed to chronic hypoxia and that 12-


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HETE, but not 15-HETE, stimulates smooth muscle cell proliferation in vitro. We

also found increased 12-LO immunostaining of pulmonary arteries and

pneumocytes of hypoxic, but not normoxic rat lungs. Furthermore, we

substantiated the contribution of the 12-LO pathway to hypoxia-induced

pulmonary vascular SMC proliferation by demonstrating that inhibition of 12-LO

blunts the proliferative response.

The LOs comprise a family of non-heme iron-containing dioxygenases that

catalyze the stereospecific oxygenation of the 5-, 12- or 15-carbon atoms of

arachidonic acid. There are four functionally distinct 12-LOs that vary in cell and

tissue distribution and catalytic activity and each is a product of linked, but

separate genes. These consist of “platelet-type” 12-LO (P-12LO), “leukocyte-

type” 12-LO (L-12LO), epidermal-type 12-LO (e-12LO) (25) and 12(R)-LO. The

12-LO isoforms and their metabolite 12-HETE have been described in a variety

of rat, mouse and human tissues (37). Although macrophages have the capacity

to synthesize the leukocyte-type 12-LO in rat lungs (5), we do not believe that

macrophages are the source of the increased 12-LO in our in vivo rat model

because histological studies did not reveal an inflammatory pattern (unpublished

data), although this does not exclude the possibility.

12-LO products such as 12-HETE have been implicated in tumor cell

proliferation, particularly that of prostatic, pancreatic and breast cancers (11, 12,

23, 28). At least some of these effects may be due to inhibition of apoptosis (41).

No cellular receptor for 12-HETE has yet been identified, but some potential

pathways by which it may stimulate cellular activities have been explored. For


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example, the mechanisms implicated in tumor invasion and metastasis that are

due, at least in part, to generation of 12-HETE, are believed to involve activation

of MAPK signaling pathways, in particular the ERK pathway (11, 39, 40, 44). In

addition, ERK activation has been demonstrated in the normal growth of

mesangial cells in association with the stimulation of the 12-LO pathway (34) and

in systemic vascular SMCs stimulated by arachidonic acid and its metabolites

(33).

Although we believe that ours is the first study exploring the role of 12-LO

and 12-HETE in pulmonary vascular smooth muscle cell proliferation, previous

studies have explored the influence of 12-LO and its product, 12-HETE, on

systemic vascular cells. 12-HETE is a known mitogenic factor for microvascular

endothelial cells (43) and systemic smooth muscle cells (29). In addition, 12-LO

participates in angiotensin II – induced vascular smooth muscle cell hypertrophy

(29, 30) and 15-LO has been shown to activate MAPK in systemic vascular

smooth muscle cells (33). The previously reported proliferative effects of 12-

HETE on systemic SMCs are consistent with our results demonstrating that 12-

HETE has a direct stimulatory effect on PASMC growth. This effect, similar to

the hypoxic stimulus, seems to involve ERK MAPK activation, as evidenced by

the increased phosphorylation of ERK that we observed during 12-HETE

treatment. What seems to be unique for the PASMCs is the biphasic activation of

ERK, which has not been reported in other cell systems. This possibly leads to a

positive feedback mechanism involving activation of the ERK pathway at multiple

steps. Furthermore, the proliferative effect of both hypoxia and 12-HETE on


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PASMCs was completely blocked by the ERK inhibitor, PD98059, suggesting

that the ERK pathway is critical in 12-HETE-induced PASMC proliferation.

Although 15-HETE and 12-HETE have similar effects in tumor cells,

stimulating tumor invasion and metastasis, we found that, unlike 12-HETE, 15-

HETE exerts no proliferative effects in PASMCs. This indicates that some actions

of the various lipoxygenases are cell-specific.

Prior studies have observed activation of lipoxygenases in the lungs of

animals exposed to hypoxia, but these focused on pulmonary vasoconstriction

and examined gene regulation only to a limited extent. For example, Zhu et al.

(52) found activation of 15-lipoxygenase in the lungs of neonatal rabbits exposed

to hypoxia and enhanced constriction of pulmonary vascular rings from these

animals to 15-HETE. These data suggest that different lipoxygenases may play

different roles in various species.

The effects of hypoxia on proliferation of cultured PASMCs has been less

extensively studied than those on pulmonary vessels in intact experimental

models, and results have been less consistent. Whereas fibroblasts from

pulmonary arteries consistently proliferate in response to hypoxia (48), smooth

muscle cells behave differently. For example, bovine PAs have 2 distinct

populations of cells, only one of which proliferates in hypoxia (16). On the other

hand, hypoxia stimulates proliferation of cultured human PASMCs, even in the

absence of serum (50). In addition, when combined with serum, moderate

hypoxia (5%O2) synergistically stimulates human PASMC growth (9). Consistent

with human PASMCs, the rat PASMCs isolated from lobar PAs in our study


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proliferated in response to hypoxic exposure (3%O2) and manifested synergy in

response to the combination of hypoxia and serum.

Prior studies on pulmonary arteries from chronically hypoxic rats have shown

increases in JNK, ERK and p38 MAPK activities, indicating involvement of

MAPKs in hypoxia-induced remodeling (19). Hypoxia also increases

phosphorylated-MAPK immunostaining in both large and small intrapulmonary

arteries (19). Our finding that a MEK inhibitor blocks hypoxia-stimulated PASMC

growth supports the idea that MAPKs are involved in the response. On the other

hand, p38 MAPK does not appear to be involved, as suggested by the lack of a

change in expression of phosphorylated p38 and lack of effect of a specific p38

MAPK inhibitor on PASMC proliferation. In contrast, p38 MAPK is activated in

hypoxic cultured PA endothelial cells (21), suggesting that signaling pathways

responding to hypoxic exposure are cell-specific.

Limitations of our study include the fact that our focus was on the role of 12-

LO in the pulmonary vasculature during hypoxia and we did not examine the

roles of other lipoxygenases, such as 5-LO, or 15-LO. The 5-LO pathway

appears to participate in the development of hypoxia-induced pulmonary

hypertension, because inhibition of 5-lipoxygenase-activating protein (FLAP)

reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic

rats (47), with the most intense FLAP immunoreactivity in pulmonary arterial

endothelial cells. Also, chronic hypoxia activates 15-LO in neonatal rabbit lungs,

with increased 15-LO being found in both pulmonary arterial endothelial cells and

SMCs (52). Thus, it is possible that an array of lipoxygenases is increased by


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hypoxia and each of them has different cellular effects on different cell types. It is

also possible that the type of lipoxygenase that participates in hypoxia is species-

specific, since rabbit 15-LO has 70% homology with rat 12-LO (51).

We conclude that the 12-LO pathway and its metabolic product, 12-HETE,

participate in hypoxia-induced rat PASMC proliferation in vitro, most likely via the

ERK1/ERK2 MAPK pathway and apparently without the involvement of p38

MAPK. We speculate that the 12-LO pathway also contributes to the

development of hypoxia-induced pulmonary hypertension in vivo, but further

studies will be necessary to substantiate this.

Grants: This study was supported by NIH/NHLBI HL32723-15 (BLF) and a

Charlton Award from Tufts University School of Medicine (IRP).


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Figure legends:

Fig 1. Chronic hypoxia up-regulates 12-LO in rat lung homogenates. A) RT-

PCR for 12-LO mRNA shows up-regulation in hypoxic rat lungs. For internal

control, we performed RT-PCR for tubulin, together with an internal standard for

tubulin (N=normoxia, H=hypoxia). B) Western blot analysis demonstrates that 12-

LO protein is up-regulated in hypoxic rat lung homogenates compared with

normoxic controls. C) Densitometric analysis of Western blot. N = 5 animals per

group, *p<0.05.

Fig 2. Increased 12-LO immunostaining in hypoxic rat lung. Immunostaining

with an antibody to leukocyte-type 12-LO and H and E counterstaining of normoxic

lung (A, B, C) compared with hypoxic lung sections (D, E, F) (100X magnification).

There is intense immunostaining of bronchial epithelial cells at baseline (Fig 2 A,

arrow), with no change by hypoxia (Fig 2 B, arrow). Hypoxia was associated with

increased immunostaining of endothelial cells of large pulmonary arteries (Fig 2 B,

arrow head and C, arrow), smooth muscle cells and possibly endothelial cells of

medium size and small pulmonary arteries (Fig 2 F, arrows) and pneumocytes of

alveolar walls. Arrow heads from fig 2 C and F depict intense staining of alveolar

macrophages, unchanged by hypoxia.

Fig 3. Hypoxia stimulates PASMC proliferation. Hypoxia stimulated cell


23

proliferation as demonstrated by increased thymidine uptake in 0.1% and 10%

serum (A and B) and cell count (C) in rat PASMCs exposed to hypoxia, compared

with their normoxic controls. N = 6, *p<0.05.

Fig 4. Hypoxia up-regulates 12-LO protein in rat PASMCs in culture. A) 12-LO

immunoblot was compared with Western blot for actin. B) Densitometric ratio

demonstrated that 12-LO is up-regulated by hypoxia starting at 2 hours. N = 5,

*p<0.05. Normoxic PASMCs did not show an increase in 12-LO expression with

time in culture (results not shown).

Fig 5. 12-HETE stimulates cell proliferation of cultured rat PASMCs. A)

PASMCs were treated for 24 hours with 12-HETE at 10-7-1µM. In contrast, 15-

HETE has no effect on PASMCs. B) Pretreatment with PD98059 at 10µM 30 min

before the 12-HETE treatment (0.001-1µM), completely blocked cell proliferation.

N = 6, *p<0.05.

Fig 6. 12-HETE stimulates phosphorylation of ERK. PASMCs were treated with

0.1 µM 12-HETE for 5 to 90 minutes. Western blot and densitometric ratio of

phophorylated ERK and total ERK (A and B) showed that ERK phosphorylation

has a biphasic pattern. p38 MAPK phosphorylation is not affected by 12-HETE (C

and D). N = 5, *p<0.05 compared with time 0.

Fig 7. Hypoxia-induced PASMC proliferation is dependent on ERK. Hypoxia-


24

induced stimulation of DNA synthesis was blocked by the MEK inhibitor (U0126,

5µM), while the p38 MAPK inhibitor (SB202190 10µM) had no effect. N = 6,

*p=0.02 compared with vehicle-treated hypoxic PASMCs.

Fig 8. 12-LO participates in hypoxia-induced PASMC proliferation. Hypoxia-

induced PASMC proliferation was blocked by the specific 12-LO inhibitor baicalein.

N = 6, *p<0.05 between normoxia and hypoxia, †p<0.05 between baicalein-treated

and vehicle-treated hypoxic experiments.


25

Figure 1 A, B, C

- 12-LO immunoblot - IgG

- 12-LO PCR

- Tubulin - Internal Standard

N N N H H A

B

Normoxia Hypoxia

Den

sito

met

ric

Rat

io(1

2-L

O/Ig

G)

0

1

2

3

(3 weeks)

*C


26

Figure 2 A, B, C, D, E, F


27

Figure 3 A, B, C

A

B

C

Days

0 1 2 3 4 5 6 7 8 9 10

Cel

l Nu

mb

erx1

000/

cm2

0

10

20

30

40Normoxia, 0.1%SerumHypoxia 3%O2, 0.1%Serum

*

* * * *

[3H

]Th

ymid

ine

Up

take

CP

M/w

ell (

x100

0)

0

4

8

12

16Normoxia, 0.1%SerumHypoxia (3%O2), 0.1%Serum

Day 1 Day 3

*

*

[3H

]Th

ymid

ine

Up

take

CP

M/w

ell (

x100

0)

0

20

40

60

80Normoxia, 10%SerumHypoxia 3%O2, 10%Serum

Day 1 Day 3

*

*

**


28

Figure 4 A, B

0

Den

sito

met

ric

Rat

io(1

2-L

O/A

ctin

)

0

2

4

6 Rat PASMCs

0 1h 2h 24h 48h

* **

- 12-LO immunoblot - Actin

A

B

0 1 1 2 2 24 24 48 hours


29

Figure 5 A, B

[3H

]Thy

mid

ine

Upt

ake

CP

M/w

ell (

x100

0)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 0.1 1 10-3 10 -2 0.1

12-HETE (µM) 15-HETE (µM)

Cont

rol

* ** * *

*

A

B

12 HETE (µM)

[3H

]Th

ymid

ine

Up

take

CP

M/w

ell (

x100

0)

0

2

4

6

ControlPD98059

0 0.001 0.01 0.1 1

* * **


30

Figure 6 A, B, C, D

- pERK1/2 immunoblot - ERK1/2 immunoblot

- p p38 MAPK immunoblot - p38 MAPK immunoblot

A

B

C

0 5 10 15 30 60 90 min

Minutes

0 5 10 15 30 60 90

Rat

io(p

P38

MA

PK

/P38

MA

PK

)

0.0

0.4

0.8

1.2D

Minutes

0 5 10 15 30 60 90

Rat

io(p

ER

K/E

RK

)

0.0

0.4

0.8

1.2

1.6

2.0

* *

* *

*

0 5 1 15 30 60 90 min


31

Figure 7

0.1%Serum Vehicle U0126 SB202190

[3 H]

Thym

idin

e U

ptak

eC

PM

/wel

l (x1

000)

0.0

0.2

0.4

0.6

0.8

1.0

1.2 NormoxiaHypoxia

*


32

Figure 8

Baicalein (µM)

0 0.5 1 2 3

[3 H]T

hym

idin

e U

pta

keC

PM

/wel

l (x

1000

)

0

1

2

3

4

5

6NormoxiaHypoxia

**

** *

†

Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

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