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Am J Physiol Lung Cell Mol Physiol 290: L367-L374, 2006. First published September 30, 2005; doi:10.1152/ajplung.00114.2005
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Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

Ioana R. Preston, Nicholas S. Hill, Rod R. Warburton, and Barry L. Fanburg

Pulmonary, Critical Care and Sleep Division, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts

Submitted 14 March 2005 ; accepted in final form 27 September 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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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 previously studied. We sought evidence for a role of 12-LO and 12(S)-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 was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10–5 µM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. 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(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.

12(S)-hydroxyeicosatetraenoic acid; mitogen-activated protein kinases; extracellular signal-regulated kinase-1/ extracellular signal-regulated kinase-2

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 further muscularized, and distal, nonmuscular 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 smooth muscle cells (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-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 PASMC 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
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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 those to p38 MAPK and phospho-p38 MAPK and secondary horseradish peroxidase-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, SB-202190, PD-98059, 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 Qiagen (Santa Clarita, CA).

Exposure of animals to chronic hypoxia. Animal protocol was approved by the Institutional Animal Care and Use Committee at Tufts-New England Medical Center. Adult male Sprague-Dawley rats were randomized to 3 wk of normoxia or hypobaric hypoxia (0.5 atm). Hypobaric chambers were briefly opened three 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-wk exposure period, rats were anesthetized, and hemodynamic measurements were obtained as previously described (22). After killing the rats by pentobarbital injection (120 mg/kg ip), we immediately opened the thorax and removed the heart and lungs. The hearts were dissected into right ventricle (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 cmH2O pressure and into the pulmonary trunk at 5 cmH2O pressure. The right lung was then processed for paraffin embedding. Sections of 5 µm were immunostained with the leukocyte-type 12-LO antibody and counterstained with hematoxylin-eosin. Vessels with a layer of smooth muscle cells comprising ≥ of the vessel perimeter were categorized as fully muscularized. Vessels with a smooth muscle layer comprising < 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 by the technique described previously (31). In brief, 5-µm paraffin-embedded tissue sections were mounted on Probe-on slides (Biotek Solutions) and dried for 1 h in a 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 heat-induced epitope retrieval. The slides were steamed in 0.1 M citrate buffer for 20 min and then allowed to cool for 5 min. After treatment with the first and second antibody, slides were stained by a modified avidin-biotin complex (ABC) technique using 3,3'-diaminobenzidine tetrahydrochloride as the chromogen and counterstained with 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 PASMCs. 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 wk at a 1:3 ratio with 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 antibiotics for 72 h. 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 min. 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 h. Cells were then either exposed to hypoxia or normoxia or treated with different agents 30 min before the beginning of the hypoxia exposure. Twenty-four hours before the end of the experiment, [3H]thymidine (0.1 mCi/ml) was added. We stopped incorporation of labeled thymidine by aspirating the medium and trypsinizing the cells for 15 min at 37°C, after which cells were transferred onto a filter membrane with a cell harvester. Radioactivity was counted in a microplate liquid scintillation counter (Perkin-Elmer) after 20 µl of scintillation liquid were added to each well.

Cell proliferation. PASMCs were plated at 2 x 104 cells/well in six-well plates in DMEM and 10% FBS with antibiotics, growth arrested for 72 h, and then exposed to normoxia or hypoxia for various times. Cells were removed from the wells by trypsin digestion. Cells were counted by a Fischer hemocytometer (Pittsburgh, PA). In each of the three experiments, the results from three 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 by 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% (vol/vol) Triton X-100, 4 mM EDTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 2 mM phenylmethylsulfonyl 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 polyvinylidene 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 646–662 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 (Qiagen, Valencia, CA). The PCR method is specific for the leukocyte-type 12-LO and 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 324–339 position of rat 12-LO, and antisense, 5'-AGA GCG CTT CAG CAC CAT-3' (2), located at the 718–735 position. PCR reaction was carried out under the following conditions: 38 cycles of denaturation at 94°C for 45 s, annealing at 68°C for 60 s, and extension at 72°C for 60 s. A final extension was performed at 72°C for 10 min. 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 means ± SD. Statistical analysis was performed by the unpaired Student's t-test or analysis of variance, as indicated. Differences were considered to be significant when P < 0.05.


   RESULTS
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12-LO mRNA and protein expression is increased in hypoxic rat lungs. Adult rats exposed to 3 wk 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. 1A) and protein (Fig. 1, B and C) compared with homogenates from normoxic controls. Histological examination (hematoxylin-eosin staining) revealed thickening of the small pulmonary arterioles and no evidence of inflammation (Fig. 2).



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  		Fig. 1. Chronic hypoxia upregulates 12-lipoxygenase (12-LO) in rat lung homogenates. A: RT-PCR for 12-LO mRNA shows upregulation 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 upregulated in hypoxic rat lung homogenates compared with normoxic controls. C: densitometric analysis of Western blot. *P < 0.05; n = 5 animals per group.

 


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  		Fig. 2. Increased 12-LO immunostaining in hypoxic rat lung. Immunostaining with an antibody to leukocyte-type 12-LO and hematoxylin and eosin counterstaining of normoxic lung (A–C) compared with hypoxic lung sections (D–F) (x100 magnification). There is intense immunostaining of bronchial epithelial cells at baseline (arrow, A), with no change by hypoxia (arrow, D). Hypoxia was associated with increased immunostaining of endothelial cells of large pulmonary arteries (arrowhead in D and arrow in E), smooth muscle cells, and possibly endothelial cells of medium-size and small pulmonary arteries (arrows, F) and pneumocytes of alveolar walls. Arrows in B and C reveal a normal pulmonary artery. Arrowheads in C and F depict intense staining of alveolar macrophages, unchanged by hypoxia.

 
Evidence of increased 12-LO expression in hypoxic rat lung. In normoxic rats, medium-size and small pulmonary arteries were nonmuscularized (Fig. 2C). After 3 wk of exposure to hypoxia, many of these arteries became either partially or fully muscularized (Fig. 2F). 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, and F). In normoxic lungs, there was no 12-LO staining of the pulmonary vasculature (Fig. 2, B and C). In contrast, there was intense 12-LO immunostaining of endothelial cells of large pulmonary arteries (Fig. 2, D and E), SMCs (and possibly endothelial cells) of small pulmonary arteries, and alveolar pneumocytes of hypoxic lungs (Fig. 2F).

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 before 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 and B). The proliferative effect of hypoxia was validated by demonstrating an increase in cell number in hypoxic compared with control SMCs (see Fig. 3C for representative experiment).



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  		Fig. 3. Hypoxia stimulates pulmonary artery smooth muscle cell (PASMC) proliferation. Hypoxia stimulated cell 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. cpm, Counts per minute. *P < 0.05 between normoxia and hypoxia; **P < 0.05 between day 1 and 3 of normoxia, n = 6.

 
12-LO is upregulated in hypoxic rat PASMCs in culture. Because PASMCs are believed to participate in the pulmonary vascular response to hypoxia, we next evaluated the influence of hypoxia on 12-LO protein expression in vitro. Hypoxia upregulated 12-LO protein expression in PASMCs at 2 h, and this was maintained at 48 h (Fig. 4, A and B).



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  		Fig. 4. Hypoxia upregulates 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 upregulated by hypoxia starting at 2 h. *P < 0.05; n = 5. Normoxic PASMCs did not show an increase in 12-LO expression with time in culture (results not shown).

 
12(S)-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(S)-HETE, on SMC proliferation. Figure 5A shows that 12(S)-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(S)-HETE-induced PASMC proliferation was completely blocked by PD-98059, a specific MEK inhibitor (an upstream factor of ERK) (Fig. 5B). These results suggest that ERK and 12-LO are linked in the proliferative pathway and that 12-LO upregulation and 12(S)-HETE formation precede activation of ERK.



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  		Fig. 5. 12-hydroxyeicosatetraenoic acid (12(S)-HETE) stimulates cell proliferation of cultured rat PASMCs. A: PASMCs were treated for 24 h with 12(S)-HETE at 10–7-1 µM. In contrast, 15-HETE has no effect on PASMCs. B: pretreatment with PD-98059 at 10 µM 30 min before the 12(S)-HETE treatment (0.001–1 µM) completely blocked cell proliferation. *P < 0.05; n = 6.

 
12(S)-HETE stimulates ERK activation. To establish whether ERK is downstream of 12(S)-HETE, we measured ERK activation in PASMCs stimulated with 12(S)-HETE. Figure 6, A and B, demonstrates that 12(S)-HETE directly activates the ERK pathway by inducing phosphorylation of ERK1/ERK2. This is a biphasic action with peaks at 10 and 90 min. Another signaling pathway that has been shown to be activated during cell proliferation is p38 MAPK (10, 13). We found that 12(S)-HETE did not induce p38 MAPK phosphorylation (Fig. 6, C and D).



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  		Fig. 6. 12(S)-HETE stimulates phosphorylation of ERK. PASMCs were treated with 0.1 µM 12(S)-HETE for 5–90 min. Western blot and densitometric ratio of phosphorylated (p) ERK and total ERK (A and B) showed that ERK phosphorylation has a biphasic pattern. p38 MAPK phosphorylation is not affected by 12(S)-HETE (C and D). *P < 0.05 compared with time 0; n = 5.

 
Hypoxia-induced PASMC proliferation is blocked by the MEK-1 inhibitor PD-98059. 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 h 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, SB-202190. Figure 7 demonstrates inhibition of hypoxia-stimulated cell proliferation by the MEK inhibitor and lack of effect by the p38 MAPK inhibitor.



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  		Fig. 7. Hypoxia-induced PASMC proliferation is dependent on ERK. Hypoxia-induced stimulation of DNA synthesis was blocked by the MEK inhibitor (U0126, 5 µM), whereas the p38 MAPK inhibitor (SB-202190, 10 µM) had no effect. *P = 0.02 compared with vehicle-treated hypoxic PASMCs; n = 6.

 
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 to 10 µM had no effect on cell viability (data not shown).



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  		Fig. 8. 12-LO participates in hypoxia-induced PASMC proliferation. Hypoxia-induced PASMC proliferation was blocked by the specific 12-LO inhibitor baicalein. *P < 0.05 between normoxia and hypoxia, {dagger}P < 0.05 between baicalein-treated and vehicle-treated hypoxic experiments; n = 6.

 

   DISCUSSION
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It is now generally accepted that hypoxia induces pulmonary hypertension that is associated with pulmonary vascular remodeling and, in particular, SMC 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(S)-HETE, contributes to hypoxia-induced PASMC remodeling. We found upregulation of 12-LO mRNA and protein expression in lung homogenates of rats exposed to chronic hypoxia and that 12(S)-HETE, but not 15-HETE, stimulates SMC 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 lipoxygenases comprise a family of nonheme 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, leukocyte-type 12-LO, epidermal-type 12-LO (25), and 12(R)-LO. The 12-LO isoforms and their metabolite 12(S)-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 (see Fig. 2), although this does not exclude the possibility.

12-LO products such as 12(S)-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(S)-HETE has yet been identified, but some potential pathways by which it may stimulate cellular activities have been explored. For example, the mechanisms implicated in tumor invasion and metastasis that are due, at least in part, to generation of 12(S)-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(S)-HETE in pulmonary vascular SMC proliferation, previous studies have explored the influence of 12-LO and its product, 12(S)-HETE, on systemic vascular cells. 12(S)-HETE is a known mitogenic factor for microvascular endothelial cells (43) and systemic SMCs (29). In addition, 12-LO participates in angiotensin II-induced vascular SMC hypertrophy (29, 30), and 15-LO has been shown to activate MAPK in systemic vascular SMCs (33). The previously reported proliferative effects of 12(S)-HETE on systemic SMCs are consistent with our results demonstrating that 12(S)-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(S)-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(S)-HETE on PASMCs was completely blocked by the ERK inhibitor, PD-98059, suggesting that the ERK pathway is critical in 12(S)-HETE-induced PASMC proliferation.

Although 15-HETE and 12(S)-HETE have similar effects in tumor cells, stimulating tumor invasion and metastasis, we found that, unlike 12(S)-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-LO 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), SMCs behave differently. For example, bovine pulmonary arteries have two 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 pulmonary arteries in our study 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 an 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 pulmonary artery 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-LO-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 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(S)-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.


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This study was supported by National Heart, Lung, and Blood Institute Grant HL-32723-15 (B. L. Fanburg) and a Charlton Award from Tufts University School of Medicine (I. R. Preston).


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. R. Preston, Pulmonary, Critical Care and Sleep Division, Tufts-New England Medical Center, 750 Washington St., Box #257, Boston, MA 02111 (e-mail: ipreston{at}tufts-nemc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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  1. Arjona AA, Hsu CA, Wrenn DS, and Hill NS. Effects of natriuretic peptides on vascular smooth-muscle cells derived from different vascular beds. Gen Pharmacol 28: 387–392, 1997.[ISI][Medline]
  2. Bleich D, Chen S, Gu JL, Thomas L, Scott S, Gonzales N, Natarajan R, and Nadler JL. Interleukin-1 beta regulates the expression of a leukocyte type of 12-lipoxygenase in rat islets and RIN m5F cells. Endocrinology 136: 5736–5744, 1995.[Abstract]
  3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  4. Brinkman HJ, van Buul-Wortelboer MF, and van Mourik JA. Involvement of cyclooxygenase- and lipoxygenase-mediated conversion of arachidonic acid in controlling human vascular smooth muscle cell proliferation. Thromb Haemost 63: 291–297, 1990.[ISI][Medline]
  5. Chen XS, Kurre U, Jenkins NA, Copeland NG, and Funk CD. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases. J Biol Chem 269: 13979–13987, 1994.[Abstract/Free Full Text]
  6. Chen Y, Hagmann W, and Honn KV. Regulation of 12(S)-HETE production in tumor cells. In: Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation, and Radiation Injury, edited by Konn KV. New York: Plenum, 1997, p. 159–166.
  7. Christou H, Morita T, Hsieh CM, Koike H, Arkonac B, Perrella MA, and Kourembanas S. Prevention of hypoxia-induced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat. Circ Res 86: 1224–1229, 2000.[Abstract/Free Full Text]
  8. Cogo A, Napolitano G, Michoud MC, Barbon DR, Ward M, and Martin JG. Effects of hypoxia on rat airway smooth muscle cell proliferation. J Appl Physiol 94: 1403–1409, 2003.[Abstract/Free Full Text]
  9. Cooper AL and Beasley D. Hypoxia stimulates proliferation and interleukin-1{alpha} production in human vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 277: H1326–H1337, 1999.[Abstract/Free Full Text]
  10. Denhardt DT. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem J 318: 729–747, 1996.
  11. Ding XZ, Tong WG, and Adrian TE. 12-lipoxygenase metabolite 12(S)-HETE stimulates human pancreatic cancer cell proliferation via protein tyrosine phosphorylation and ERK activation. Int J Cancer 94: 630–636, 2001.[CrossRef][ISI][Medline]
  12. Ding XZ, Tong WG, and Adrian TE. Cyclooxygenases and lipoxygenases as potential targets for treatment of pancreatic cancer. Pancreatology 1: 291–299, 2001.[CrossRef][ISI][Medline]
  13. Duan C. The chemotactic and mitogenic responses of vascular smooth muscle cells to insulin-like growth factor-I require the activation of ERK1/2. Mol Cell Endocrinol 206: 75–83, 2003.[CrossRef][ISI][Medline]
  14. Eddahibi S, Fabre V, Boni C, Martres MP, Raffestin B, Hamon M, and Adnot S. Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ Res 84: 329–336, 1999.[Abstract/Free Full Text]
  15. Fanburg BL and Lee SL. A role for the serotonin transporter in hypoxia-induced pulmonary hypertension. J Clin Invest 105: 1521–1523, 2000.[ISI][Medline]
  16. Frid MG, Aldashev AA, Dempsey EC, and Stenmark KR. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities. Circ Res 81: 940–952, 1997.[Abstract/Free Full Text]
  17. Gu JL, Pei H, Thomas L, Nadler JL, Rossi JJ, Lanting L, and Natarajan R. Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries. Circulation 103: 1446–1452, 2001.[Abstract/Free Full Text]
  18. Herbertsson H and Hammarstrom S. Cytosolic 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid binding sites in carcinoma cells. Adv Exp Med Biol 400A: 287–293, 1997.
  19. Jin N, Hatton N, Swartz DR, Xia X, Harrington MA, Larsen SH, and Rhoades RA. Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries. Am J Respir Cell Mol Biol 23: 593–601, 2000.[Abstract/Free Full Text]
  20. Karamsetty MR, Pietras L, Klinger JR, Lanzillo JJ, Leiter JC, Ou LC, and Hill NS. The role of endothelin-1 in strain-related susceptibility to develop hypoxic pulmonary hypertension in rats. Respir Physiol 128: 219–227, 2001.[CrossRef][ISI][Medline]
  21. Kayyali US, Pennella CM, Trujillo C, Villa O, Gaestel M, and Hassoun PM. Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2. J Biol Chem 277: 42596–42602, 2002.[Abstract/Free Full Text]
  22. Klinger JR, Warburton RR, Pietras L, and Hill NS. Brain natriuretic peptide inhibits hypoxic pulmonary hypertension in rats. J Appl Physiol 84: 1646–1652, 1998.[Abstract/Free Full Text]
  23. Liu B, Maher RJ, Hannun YA, Porter AT, and Honn KV. 12(S)-HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha. J Natl Cancer Inst 86: 1145–1151, 1994.[Abstract/Free Full Text]
  24. Liu B, Timar J, Howlett J, Diglio CA, and Honn KV. Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C. Cell Regul 2: 1045–1055, 1991.[ISI][Medline]
  25. McDonnell M, Davis W Jr, Li H, and Funk CD. Characterization of the murine epidermal 12/15-lipoxygenase. Prostaglandins 63: 93–107, 2001.[ISI][Medline]
  26. Meyrick B and Reid L. The effect of chronic hypoxia on pulmonary arteries in young rats. Exp Lung Res 2: 257–271, 1981.[ISI][Medline]
  27. Natarajan R, Bai W, Rangarajan V, Gonzales N, Gu JL, Lanting L, and Nadler JL. Platelet-derived growth factor BB mediated regulation of 12-lipoxygenase in porcine aortic smooth muscle cells. J Cell Physiol 169: 391–400, 1996.[CrossRef][ISI][Medline]
  28. Natarajan R, Esworthy R, Bai W, Gu JL, Wilczynski S, and Nadler J. Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor. J Clin Endocrinol Metab 82: 1790–1798, 1997.[Abstract/Free Full Text]
  29. Natarajan R, Gonzales N, Lanting L, and Nadler J. Role of the lipoxygenase pathway in angiotensin II-induced vascular smooth muscle cell hypertrophy. Hypertension 23: 142–147, 1994.
  30. Natarajan R, Gu JL, Rossi J, Gonzales N, Lanting L, Xu L, and Nadler J. Elevated glucose and angiotensin II increase 12-lipoxygenase activity and expression in porcine aortic smooth muscle cells. Proc Natl Acad Sci USA 90: 4947–4951, 1993.[Abstract/Free Full Text]
  31. Natarajan R, Pei H, Gu JL, Sarma JM, and Nadler J. Evidence for 12-lipoxygenase induction in the vessel wall following balloon injury. Cardiovasc Res 41: 489–499, 1999.[Abstract/Free Full Text]
  32. Natarajan R, Rosdahl J, Gonzales N, and Bai W. Regulation of 12-lipoxygenase by cytokines in vascular smooth muscle cells. Hypertension 30: 873–879, 1997.[Abstract/Free Full Text]
  33. Rao GN, Baas AS, Glasgow WC, Eling TE, Runge MS, and Alexander RW. Activation of mitogen-activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells. J Biol Chem 269: 32586–32591, 1994.[Abstract/Free Full Text]
  34. Reddy MA, Adler SG, Kim YS, Lanting L, Rossi J, Kang SW, Nadler JL, Shahed A, and Natarajan R. Interaction of MAPK and 12-lipoxygenase pathways in growth and matrix protein expression in mesangial cells. Am J Physiol Renal Physiol 283: F985–F994, 2002.[Abstract/Free Full Text]
  35. Reddy MA, Kim YS, Lanting L, and Natarajan R. Reduced growth factor responses in vascular smooth muscle cells derived from 12/15-lipoxygenase-deficient mice. Hypertension 41: 1294–1300, 2003.[Abstract/Free Full Text]
  36. Reddy MA, Thimmalapura PR, Lanting L, Nadler JL, Fatima S, and Natarajan R. The oxidized lipid and lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid induces hypertrophy and fibronectin transcription in vascular smooth muscle cells via p38 MAPK and cAMP response element-binding protein activation. Mediation of angiotensin II effects. J Biol Chem 277: 9920–9928, 2002.[Abstract/Free Full Text]
  37. Reddy RG, Yoshimoto T, Yamamoto S, Funk CD, and Marnett LJ. Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization. Arch Biochem Biophys 312: 219–226, 1994.[CrossRef][ISI][Medline]
  38. Szekeres CK, Tang K, Trikha M, and Honn KV. Eicosanoid activation of extracellular signal-regulated kinase 1/2 in human epidermoid carcinoma cells. J Biol Chem 275: 38831–38841, 2000.[Abstract/Free Full Text]
  39. Szekeres CK, Trikha M, and Honn KV. 12(S)-HETE, pleiotropic functions, multiple signaling pathways. Adv Exp Med Biol 507: 509–515, 2002.[ISI][Medline]
  40. Szekeres CK, Trikha M, Nie D, and Honn KV. Eicosanoid 12(S)-HETE activates phosphatidylinositol 3-kinase. Biochem Biophys Res Commun 275: 690–695, 2000.[CrossRef][ISI][Medline]
  41. Tang DG, Chen YQ, and Honn KV. Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis. Proc Natl Acad Sci USA 93: 5241–5246, 1996.[Abstract/Free Full Text]
  42. Tang DG, Grossi IM, Tang KQ, Diglio CA, and Honn KV. Inhibition of TPA and 12(S)-HETE-stimulated tumor cell adhesion by prostacyclin and its stable analogs: rationale for their antimetastatic effects. Int J Cancer 60: 418–425, 1995.[ISI][Medline]
  43. Tang DG, Renaud C, Stojakovic S, Diglio CA, Porter A, and Honn KV. 12(S)-HETE is a mitogenic factor for microvascular endothelial cells: its potential role in angiogenesis. Biochem Biophys Res Commun 211: 462–468, 1995.[CrossRef][ISI][Medline]
  44. Tang DG, Tarrien M, Dobrzynski P, and Honn KV. Melanoma cell spreading on fibronectin induced by 12(S)-HETE involves both protein kinase C- and protein tyrosine kinase-dependent focal adhesion formation and tyrosine phosphorylation of focal adhesion kinase (pp125FAK). J Cell Physiol 165: 291–306, 1995.[CrossRef][ISI][Medline]
  45. Timar J, Bazaz R, Kimler V, Haddad M, Tang DG, Robertson D, Tovari J, Taylor JD, and Honn KV. Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the alpha IIb beta 3-integrin in melanoma cells. J Cell Sci 108: 2175–2186, 1995.[Abstract]
  46. Voelkel N and Reeves J. Primary pulmonary hypertension. In: Pulmonary Vascular Diseases, edited by Moser K. New York: Dekker, 1997, p. 573–649.
  47. Voelkel NF, Tuder RM, Wade K, Hoper M, Lepley RA, Goulet JL, Koller BH, and Fitzpatrick F. Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats. J Clin Invest 97: 2491–2498, 1996.[ISI][Medline]
  48. Welsh DJ, Scott P, Plevin R, Wadsworth R, and Peacock AJ. Hypoxia enhances cellular proliferation and inositol 1,4, 5-triphosphate generation in fibroblasts from bovine pulmonary artery but not from mesenteric artery. Am J Respir Crit Care Med 158: 1757–1762, 1998.[Abstract/Free Full Text]
  49. Wen Y, Gu J, Liu Y, Wang PH, Sun Y, and Nadler JL. Overexpression of 12-lipoxygenase causes cardiac fibroblast cell growth. Circ Res 88: 70–76, 2001.[Abstract/Free Full Text]
  50. Yang X, Sheares KK, Davie N, Upton PD, Taylor GW, Horsley J, Wharton J, and Morrell NW. Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol 27: 688–696, 2002.[Abstract/Free Full Text]
  51. Yoshimoto T, Suzuki H, Yamamoto S, Takai T, Yokoyama C, and Tanabe T. Cloning and sequence analysis of the cDNA for arachidonate 12-lipoxygenase of porcine leukocytes. Proc Natl Acad Sci USA 87: 2142–2146, 1990.[Abstract/Free Full Text]
  52. Zhu D, Medhora M, Campbell WB, Spitzbarth N, Baker JE, and Jacobs ER. Chronic hypoxia activates lung 15-lipoxygenase, which catalyzes production of 15-HETE and enhances constriction in neonatal rabbit pulmonary arteries. Circ Res 92: 992–1000, 2003.[Abstract/Free Full Text]



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