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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 688-696, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0067OC

Hypoxic Induction of Cox-2 Regulates Proliferation of Human Pulmonary Artery Smooth Muscle Cells

Xudong Yang, Karen K. K. Sheares, N. Davie, Paul D. Upton, Graham W. Taylor, Jo Horsley, John Wharton and Nicholas W. Morrell

Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge; and Section on Clinical Pharmacology, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, United Kingdom

Address correspondence to: Dr. Nicholas W. Morrell, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Hills Road, Cambridge CB2 2QQ, UK. E-mail: nwm23{at}cam.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Therefore, we examined the effect of hypoxia on growth of pulmonary artery smooth muscle cells (PASMCs) from human distal pulmonary arteries. Initial studies identified that serum-induced proliferation of explant-derived PASMCs was inhibited under hypoxic conditions (3–4 kPa in medium). However, selection of hypoxia-stimulated cells was achieved by culturing cells at low density under conditions of prolonged hypoxia (1–2 wk). In hypoxia-inhibited and -stimulated cells, Western blotting revealed hypoxic induction of cyclooxygenase (COX)-2, which was dependent on the activation of p38MAPK, but not COX-1, inducible nitric oxide synthase (iNOS), or hemoxygenase-1 (HO-1). Hypoxic induction of COX-2 was also observed in the media of pulmonary arteries in lung organ culture. Hypoxia induced a 4- to 5-fold increase (P < 0.001) in prostaglandin (PG)E2, PGD2, PGF2{alpha}, and 6-keto-PGF1{alpha} release from PASMCs. Hypoxic inhibition of proliferation was attenuated by incubation with indomethacin (10 µM), or the COX-2 antagonist, NS398 (10 µM), but not by the COX-1 antagonist, valeryl salicylate (0.5 mM). In conclusion, we have isolated cells from human peripheral pulmonary arteries that are either inhibited or stimulated by culture under hypoxic conditions. In both cell types hypoxia modulates cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins. Induction of COX-2 may contribute to the inhibition of hypoxia-induced pulmonary vascular remodeling.

Abbreviations: carbon monoxide, CO • cyclooxygenase, COX • Dulbecco's modified Eagle's medium, DMEM • fetal bovine serum, FBS • heme oxygenase-1, HO-1 • interleukin, IL • inducible nitric oxide synthase, iNOS • nitric oxide, NO • pulmonary artery smooth muscle cells, PASMCs • prostaglandin, PG • S-Nitroso-N-acetylpenicillamine, SNAP • valeryl salicylate, VS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prolonged exposure to alveolar hypoxia leads to structural changes in the walls of small pulmonary arteries, known as pulmonary vascular remodeling, and a sustained elevation of pulmonary vascular resistance (1). One of the earliest and consistent features of hypoxia-induced pulmonary vascular remodeling in animals (2) and in humans is the extension of smooth muscle into previously nonmuscular pulmonary arterioles and increased wall thickness of muscular arteries. These processes occur as a result of smooth muscle cell hypertrophy (3), hyperplasia (4), and migration.

Regulation of pulmonary vascular structure under hypoxic conditions may involve the interaction between locally derived growth factors, circulating hormones, and differences in genetic susceptibility (5). In addition, the pulmonary vascular smooth muscle cell responds directly to hypoxia by changes in ion channel activity, activation of protein kinases, including p38MAPK (6), and expression of transcription factors such as hypoxia-inducible factor-1, all of which may impact on growth signals.

In vivo rodent models of hypoxia-induced pulmonary hypertension have demonstrated the potential importance of nitric oxide (NO) (7) and carbon monoxide (CO) (8) in the regulation of pulmonary vascular tone and growth. Enzymes responsible for the production of NO and CO, inducible nitric oxide synthase (iNOS), and hemoxygenase-1 (HO-1), respectively, are induced by hypoxia in vitro (9) and influence cell proliferation. Another enzyme induced by hypoxia in endothelial cells is cyclooxygenase (COX)-2 (10) leading to increased production of prostaglandins (11). Vasodilating prostaglandins such as prostacyclin (PGI2) and prostaglandin E2 (PGE2) are critical in the regulation of pulmonary vascular remodeling. For example, mice overexpressing prostaglandin synthase are protected from the development of hypoxia-induced pulmonary hypertension (12).

Despite the importance of hypoxia to the development of pulmonary hypertension, few studies have directly examined the regulation of pulmonary artery smooth muscle cell (PASMC) growth by hypoxia. In this study, we have attempted to characterize the effect of hypoxia on growth of human PASMCs isolated from peripheral vessels. In addition, we have examined whether hypoxia regulates the expression of key enzymes (iNOS, HO-1, and COX-2) involved in the generation of vasoactive factors. Our results suggest that smooth muscle cells from human peripheral pulmonary arteries comprise at least two cell types that may be inhibited or stimulated under hypoxic conditions. In both cell types, hypoxia modulates cell proliferation by induction of COX-2, which is partly dependent on the activation of p38MAPK, and production of antiproliferative prostaglandins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Human lung tissue was obtained from patients undergoing lobectomy or pneumonectomy for bronchial carcinoma. Lung tissue distant from the tumor was selected. Only lungs judged by the pathologist to have no significant pulmonary vascular disease by light microscopy of tissue sections were used. Distal PASMCs were isolated from peripheral segments of artery (0.3–1.0 mm external diameter) by microdissection, as previously described (13). Briefly, arterial segments and attached branches were carefully separated from the parenchyma and adventitia, washed in phosphate-buffered saline and cut into 2-mm segments. Segments were left to adhere overnight and then maintained in Dulbecco's modified Eagle media (DMEM) in 20% fetal bovine serum (FBS) containing antibiotic-antimycotic solution. The cells were passed after 2 wk into a single 75 cm2 flask and grown to confluence in DMEM/10% FBS. Approval for studies was obtained from Papworth Hospital Ethical Review Board. For experiments PASMCs were plated in DMEM containing 10% FBS and used between passages 3 and 10. The smooth muscle lineage of cells was confirmed by positive immunofluorescence staining using antibodies to {alpha}-smooth muscle actin and smooth muscle myosin (Sigma-Aldrich Co Ltd, Poole, UK) (13).

Exposure of Cells to Hypoxia
In initial experiments PASMCs were grown under standard normoxic conditions in a CO2 incubator, before exposure to hypoxic conditions. Hypoxia was induced by pre-gassing cell culture medium (DMEM + 25 mM HEPES) with a gas mixture containing 95%N2/5%CO2 for 30 min inside a gas-tight isolator. The hypoxic culture medium was then added to cells plated in 24- or 48-well plates. Plates were then maintained inside specially designed perspex chambers (Bellco Glass, Inc., Vineland, NJ), gassed with 95%N2/5%CO2. Chambers were re-gassed daily. The pH, PO2, and PCO2 in the medium was checked at the beginning and end of each experiment using a blood gas analyzer (ABL5; Radiometer Ltd, West Sussex, UK). Hypoxic cells were not reoxygenated at any stage of the experimental procedure.

In some experiments, freshly isolated cells or first passage cells were plated in 20%FBS/DMEM in 96-well plates at an approximate density of 10 cells/well and left to adhere overnight. Plates were then placed under hypoxic conditions as described above and maintained for up to 2–3 wk. Viable, proliferating cells were trypsinized and transferred sequentially to 24- then 6-well plates before passage into T75 cm2 flasks, while maintaining hypoxic conditions throughout.

Cell Growth and Proliferation Assays
The effect of hypoxia on growth and proliferation of PASMCs was determined by [3H]thymidine incorporation as an index of DNA synthesis, and cell proliferation was assessed with a hemocytometer. In addition, the effect of hypoxia on the cell cycle was studied by incorporation of propidium iodide and flow cytometry, as described (13). For [3H]thymidine and proliferation assays cells were plated at 104 cells/well in 10%FBS/DMEM in 48-well plates and left to adhere overnight under normoxic conditions. At the beginning of the experimental period, fresh hypoxic or normoxic medium was added either alone or containing COX inhibitors. [3H]thymidine (0.25 µCi/well) was added for the final 6 h. Cells were exposed to 1,2, 3, or 6 d of normoxia or hypoxia.

The effect of COX inhibition on growth responses to hypoxia and was tested by coincubating with the nonselective COX inhibitor indomethacin, the selective COX-1 inhibitor valeryl salicylate (VS), or the selective COX-2 inhibitor NS398 (Alexis Corporation, Notts, UK).

Western Blotting
Expression of COX isoforms, iNOS, and HO-1 protein in PASMCs was assessed by Western blotting, following exposure to normoxia, hypoxia, interleukin (IL)-1ß, or S-Nitroso-N-acetylpenicillamine (SNAP) for up to 72 h. Quiescent cells were exposed to experimental conditions in 0.1%FBS/DMEM in 6-well plates. Cells were lysed and extracts were boiled at a 1:1 ratio with 2x protein loading buffer for 5 min. Samples (5 µg) were loaded onto 10% (for HO-1) or 7.5% (for iNOS and COX-1/COX-2) SDS-PAGE gels and separated by electrophoresis for 1–2 h. The gels were transferred to nitrocellulose membrane, incubated with blocking buffer, then incubated with a specific mouse monoclonal anti-HO-1 (1:1,000) (OSA-110; Stressgen Biotechnologies Corp., York, UK), rabbit polyclonal anti-iNOS (1:1,000), goat polyclonal anti–COX-1 or anti–COX-2 (1:2,000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4°C. The blots were then incubated with an appropriate horseradish peroxidase–conjugated secondary antibody in blocking buffer for 1 h at room temperature. Blots were developed using enhanced chemiluminescence reagent (Amersham Biosciences, Little Chalfont, UK) and rainbow markers were used for molecular weight determinations. In further experiments, the effect of inhibition of activated p38MAPK on COX-2 induction was studied using the water-soluble selective inhibitor SB203580 hydrochloride (0.1–5 µM; Calbiochem, Nottingham, UK). Cells were pretreated for 1 h with SB203580 then exposed to hypoxia for 24 h. Cell viability in the presence of increasing concentrations of inhibitor was assessed by trypan blue exclusion.

To determine whether hypoxia activated p38MAPK in PASMCs, cells were exposed to pre-gassed hypoxic medium for up to 2 h. Protein was harvested by washing cells in cold phosphate-buffered saline and immediately freezing in an ethanol/dry ice bath. Cells were scraped into 200 µl of protein loading buffer containing protease inhibitors and subjected to SDS-PAGE electrophoresis, as previously described (6, 14). After washing, blots were incubated with anti-phospho-p38MAPK (1:1,000) (Cell Signaling Technology, Inc., Beverly, MA) overnight at 4°C. Blots were then washed and incubated with an anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:2,500; DAKO, Ltd., Ely, UK) for 1 h at room temperature. Blots were the stripped and re-probed using an antibody to total p38MAPK (Cell Signaling Technology, Inc.).

COX Protein Expression in Pulmonary Arteries
Lung tissue sections (~ 5 x 5 x 3 mm) were obtained from the same surgical resection specimens used for PASMC isolation. Again, results were only included from those sections which were judged by the pathologist to show no evidence for significant pulmonary vascular disease. Sections were cut using a razor blade and incubated in DMEM at 37°C under hypoxic or normoxic conditions for 24 and 48 h. Sections were then fixed in 10% phosphate-buffered formalin, embedded, and sectioned (3 µm). Sections were immunostained using antibodies to COX-1 and COX-2 using a labeled streptavidin biotin peroxidase technique described previously (15).

Measurement of Prostaglandin Release by Gas Chromatography–Mass Spectrometry
Cells were plated in 6-well plates and grown to subconfluence, then quiesced for 48 h in serum-deprived medium. Cells were then incubated with normoxic or hypoxic 0.1%FBS/DMEM (2 ml/well) for 3, 6, 12, 24, 48, or 72 h, in the presence or absence of COX inhibitors. The conditioned medium was collected at the end of each time point and immediately frozen at -70°C.

Prostaglandins were analyzed as their methoxime-O trimethylsilyl ether-3,5-bis (trifluormethyl)benzyl ester derivatives by gas chromatography electron capture mass spectrometry as previously described (16, 17). Following extraction on C-18 Sep-Pak columns (Waters Corporation, Milford, MA), samples were derivatised and chromatographed on a 30 m DB-5 capillary gas chromatography column (J&W Scientific, Folson, CA), using a thermal gradient from 200–325°C at 20°C/min. The gas chromatograpy products were routed into the electron capture source of a Trio 1000 mass spectrometer (Thermoquest, San Jose, CA) operated in selected ion mode with ammonia as reagent gas. Deuterated standards for PGF2{alpha}, 6-keto PGF1{alpha}, PGE2, and PGD2 (Cayman Chemical Ltd, Ann Arbor, MI) were added to samples as internal standards.

Statistics
Data were expressed as mean ± SEM and analyzed with GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA). Comparisons were made by Student's t test (two-tailed) or one-way ANOVA with the Tukey post hoc test, as appropriate. A value of P < 0.05 indicated statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Hypoxia on Growth of Distal PASMCs
In initial experiments, cells were allowed to grow from explants under standard normoxic conditions. Growth of these cells was then assessed under normoxic or hypoxic conditions. The respiratory gas tensions and pH of the media were: normoxia, pH 7.32 ± 0.07, PCO2 5.3 ± 0.2 kPa, PO2 21.3 ± 0.1 kPa; hypoxia, pH 7.35 ± 0.08, PCO2 5.2 ± 0.1 kPa, PO2 3.4 ± 0.4 kPa. Proliferation assays and [3H]thymidine assays demonstrated that cell proliferation was markedly inhibited under hypoxic conditions (Figures 1A and 1B). Flow cytometry revealed that hypoxia inhibited the serum-stimulated progression of PASMCs from G0/G1 to the S phase of the cell cycle. Under normoxic versus hypoxic conditions the distribution of cells was G0/G1 88.2% versus 91.1%, S phase 4.3% versus 3.2%, and G2/M phase 7.4 versus 5.6%.



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  		Figure 1. In PASMCs derived from explants under normoxic conditions [3H]thymidine incorporation studies (A) demonstrated that DNA synthesis was inhibited by hypoxia (open bars, normoxia; closed bars, hypoxia). Cell counts (B) confirmed that hypoxia inhibited PASMC proliferation. In contrast, in hypoxia-selected cells, [3H]thymidine incorporation (C) and cell counts (D) were increased under hypoxic conditions (*P < 0.05, **P < 0.01 compared with corresponding normoxic time point).

 
The majority of freshly dispersed or first passage distal PASMCs (~ 95%) failed to proliferate under hypoxic conditions. However, in ~ 5% of wells cell proliferation was observed and when sufficient cells were available for plating (passages 6–8), growth studies revealed that "hypoxia-selected" cells proliferated faster and demonstrated increased [3H]thymidine incorporation under hypoxic compared with normoxic conditions (Figures 1C and 1D).

Induction of COX-2 by Hypoxia is Partly Dependent on Activation of p38MAPK
In normoxia-derived cells, immunoblotting for COX isoforms demonstrated constitutive expression of COX-1 under normoxic and hypoxic conditions. In contrast, COX-2 expression was minimal in normoxic cells, but was induced in a time-dependent manner under hypoxic conditions (Figure 2) and remained elevated for up to 72 h. Exposure of normoxia-derived PASMCs to hypoxia did not induce expression of iNOS or HO-1. Incubation with IL-1ß also induced COX-2 in these cells and also iNOS, though not HO-1. Induction of HO-1 was seen when cells were incubated with SNAP (Figure 2).



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  		Figure 2. Representative Western blots demonstrating the induction of COX-2 in explant-derived hypoxia-inhibited PASMCs incubated with IL-1ß (10 µM), and exposed to 72 h of hypoxia or normoxia (A). COX-2 protein expression was increased by 1 h hypoxic exposure, and persisted for up to 72 h, whereas COX-1 protein expression remained unchanged. Hypoxia failed to induce expression of iNOS (B) or HO-1 (C), though induction of these enzymes was demonstrated with IL-1ß and SNAP (1 mM), respectively. Recombinant rat Hsp32 (HO-1; Stressgen Biotechnologies Corp.) was used as a positive control for HO-1. Membranes were stripped and re-probed for ß-actin to confirm equal protein loading.

 
Because hypoxia has been shown to activate p38MAPK in pulmonary artery fibroblasts (6, 14) we first confirmed that hypoxia activated P38MAPK in human PASMCs (Figure 3). In addition, incubation of hypoxic PASMCs with SB203580 concentration-dependently inhibited the induction of COX-2 (Figure 3). Cell viability studies using trypan blue exclusion demonstrated that concentrations of SB203580 from 1–20 µM had no effect on cell viability. Although there was a small effect of SB203580 on COX-2 expression under normoxic conditions, this effect was minimal compared with the inhibition of hypoxia-inducible COX-2 expression (Figure 3).



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  		Figure 3. Time course of hypoxic induction of phospho-p38MAPK in PASMCs (A). Equal loading of lanes was confirmed by stripping and re-probing for total p38MAPK. Incubation of hypoxic cells with the p38MAPK inhibitor, SB203580, concentration-dependently inhibited induction of COX-2 (B). Results are representative of three separate experiments in distinct cell isolates.

 
Effect of Hypoxia on Release of Prostaglandins
In normoxia-derived cells, release of prostaglandins 6-keto-PGF1{alpha}, PGF2{alpha}, PGD2, and PGE2 was increased by culture under hypoxic conditions (Figure 4). Increased production of prostaglandins peaked at 12 h but persisted up to 72 h, paralleling the time course of induction of COX-2 protein expression. The increase in prostaglandin production in response to hypoxia was markedly inhibited in cells incubated with indomethacin or NS398 for 12 h, but not VS (Figure 5). There was no evidence for isoprostane formation in any of the incubations. As expected, IL-1ß also increased the release of prostaglandins from PASMCs (data not shown).



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  		Figure 4. Time course of prostaglandin release from explant-derived hypoxia-inhibited PASMCs cultured under normoxic (open bars) and hypoxic (solid bars) conditions. Hypoxia increased production of all prostaglandins measured, for up to 72 h. PGE2 and PGD2 were released in greater quantities than the other prostaglandins measured (note different axis scales; bars represent the mean of five samples from different cell isolates; *P < 0.05, **P < 0.01, ***P < 0.001 compared with corresponding normoxic time point).

 


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  		Figure 5. The hypoxia-stimulated increase in prostaglandin release from hypoxia-inhibited PASMCs at 12 h was inhibited by incubation with indomethacin (Indo; 10 µM) and NS398 (10 µM), but not by valeryl salicylate (VS; 0.5 mM). Bars represent the mean of five samples; (*P < 0.05, **P < 0.01, ***P < 0.001 compared with the hypoxic cells in the absence of inhibitors).

 
Effect of COX Inhibition on PASMC Growth
In normoxia-derived cells the inhibition of DNA synthesis and cell proliferation induced by hypoxia was reversed in the presence of indomethacin or NS398, but not by VS (Figure 6). Similar results were obtained with IL-1ß, in that COX-2, but not COX-1 inhibition partly reversed the IL-1ß–induced inhibition of DNA synthesis and cell proliferation (data not shown).



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  		Figure 6. [3H]thymidine incorporation (A) studies in hypoxia-inhibited PASMCs under hypoxic conditions. Indomethacin and NS398, but not valeryl salicylate (VS) increased serum- stimulated DNA synthesis. (B) Cell counts confirmed that PASMC proliferation under hypoxic conditions was increased in the presence of indomethacin (solid triangles) and NS398 (open triangles), but not valeryl salicylate (open circles). Closed circles indicate 10% FBS. Data points represent the mean of five samples derived from separate isolates (*P < 0.05, **P < 0.01 compared with hypoxic cells in the absence of inhibitors).

 
COX-2 in "Hypoxia-Selected" PASMCs
We questioned whether the hypoxia-induced proliferation of "hypoxia-selected" PASMCs could be due to failure of COX-2 induction in these cells. However, "hypoxia-selected" cells demonstrated induction of COX-2 (Figure 7) and increased release of prostaglandins under hypoxic conditions (Figure 7). Furthermore, hypoxia-stimulated DNA synthesis in these cells was further augmented by co-incubation with selective or nonselective COX-2 inhibition, but not by COX-1 inhibition (Figure 7).



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  		Figure 7. Hypoxia-selected cells also demonstrated induction of COX-2 protein expression under hypoxic conditions (A), and increases in prostaglandin production (B). In addition, [3H]thymidine incorporation was increased under hypoxic conditions in the presence of indomethacin or NS398, but not valeryl salicylate (VS) (C). Bars represent mean of three samples from different isolates (*P < 0.05, **P < 0.01 compared with corresponding normoxic time point).

 
Demonstration of COX-2 Induction in Intact Arteries
Immunohistochemistry of normoxic lung tissue sections demonstrated COX-1, and to a lesser extent COX-2, predominantly in the endothelium of small pulmonary arteries (Figure 8). The expression of COX-1 was similar under normoxic and hypoxic conditions. In contrast, COX-2 expression was increased in the endothelium of hypoxic arteries, and was induced in underlying smooth muscle (Figure 8).



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  		Figure 8. Photomicrographs of peripheral pulmonary arteries from lung tissue incubated under normoxic (A and C) and hypoxic (B and D) conditions for 24 h and immunostained for COX-1 (C and D) or COX-2 (A and B). Increased COX-2 immunostaining was observed under hypoxic conditions in endothelium (closed arrows) and media (open arrows) under hypoxic conditions. Control sections incubated with COX-2 antibody that was pre-absorbed with the peptide to which the antibody was raised (E), or incubated without the addition of primary antibody (F), showed no specific signal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated that at least two cell types can be derived from the human peripheral pulmonary circulation that differ in their proliferative response to hypoxia. The growth of explant-derived cells was inhibited under hypoxic conditions. However, plating cells at low density under hypoxic conditions allowed proliferation of "hypoxia-selected" cells. Exposure of either cell type to hypoxia did not alter expression of COX-1, but induced expression of COX-2, and this was associated with increased production of prostaglandins 6-keto-PGF1{alpha}, PGF2{alpha}, PGD2, and PGE2. The induction of COX-2 by hypoxia was at least partly dependent on the activation of p38MAPK. Inhibition of COX-2 activity, but not COX-1, attenuated the hypoxia-induced increase in prostaglandin release and increased DNA synthesis and proliferation of hypoxic cells. Taken together, these results suggest that the peripheral pulmonary circulation contains smooth muscle cells that are distinct with regard to their growth under hypoxic conditions. Furthermore, hypoxic induction of COX-2 in peripheral PASMCs may play a role in the inhibition of pulmonary vascular remodeling during the development of hypoxia-induced pulmonary hypertension.

Several studies have examined the effect of hypoxia on growth of bovine and rat vascular smooth muscle cells (9, 18–21) and fibroblasts (22–24). These studies suggest that the fibroblast is readily stimulated to proliferate in response to cellular hypoxia, whereas the smooth muscle cell may require a process of priming, such as serum stimulation (18) or activation of protein kinase C (25). The response of human PASMCs to hypoxia has been less extensively studied. One study demonstrated that PASMCs isolated from the main pulmonary artery were stimulated to proliferate by 5% O2 environment, but not 1% O2 (20). However, because the time course of equilibration of environmental O2 with that in the media, and the PO2 in the media bathing the cells, is usually not reported in these studies, it is difficult to draw direct comparisons with our observations.

Previous reports have focused on the endothelial cell as the main site of increased vascular prostaglandin production under hypoxic conditions (26). Marked increases in the release of PGE2, PGD2, PGF2{alpha}, and PGI2 occur in endothelial cells exposed to hypoxia (27). Although the induction of COX-2 by inflammatory cytokines (28) and growth factors (29) is well described in vascular smooth muscle cells, this is the first demonstration of the induction of COX-2 and increased release of prostaglandins by hypoxia in these cells. Hypoxia has been shown to induce COX-2 mRNA and protein expression in human umbilical vein endothelial cells (10). However, in macrophages, hypoxia inhibits lipopolysaccharide-induced COX-2 expression (30), demonstrating the cell-specific nature of hypoxic responses. The hypoxic induction of COX-2, at least in endothelial cells, appears to involve the cooperation of the transcription factors nuclear factor-{kappa}B (10), Sp1, and the high-mobility-group protein I(Y) (11) binding to the COX-2 promoter. In bovine (6) and rat (14) pulmonary artery adventitial fibroblasts, hypoxic signaling involves the activation of p38MAPK, though activation of this stress-associated protein kinase is associated with cell proliferation in these cells. Our data would indicate that activation of p38MAPK in human PASMCs is coupled to growth inhibitory pathways via induction of COX-2.

Our experiments demonstrate the selectivity of hypoxia for COX-2 induction in human peripheral PASMCs, in that hypoxia failed to induce expression of iNOS and HO-1. Indeed, additional selectivity of specific stimuli to enzyme induction was demonstrated by the induction of COX-2 and iNOS, but not HO-1, by IL-1ß, and the induction of HO-1, but not iNOS or COX-2, by the NO donor, SNAP. In rat vascular smooth muscle cells, HO-1(31) is induced by hypoxia. However, species differences have been observed between human and rat smooth muscle cells with regard to the induction of these enzymes (32).

COX-2 induction by cytokines and growth factors has been shown to regulate the growth of vascular smooth muscle cells (29). However, the effect of COX-2 induction on cell proliferation depends on several factors, including the pattern of prostanoid production and the coupling of cAMP to growth inhibitory/growth promoting pathways. Thus, in rat vascular smooth muscle cells COX-2 antagonists and thromboxane A2 antagonists inhibited tumor necrosis factor-{alpha}- and angiotensin II–mediated proliferation (29), suggesting that thromboxane A2 production by COX-2 predominates over the production of antiproliferative prostaglandins in these cells. In bovine fetal smooth muscle cells, by contrast, cAMP production in response to prostaglandin-induced stimulation of adenylyl cyclase may be coupled to mitogenic pathways (33). We have previously shown that prostacyclin analogs are potent inhibitors of human PASMC proliferation (13). In the present study, we demonstrated the simultaneous induction of COX-2 and enhanced release of prostaglandins under hypoxic conditions. Inhibition of COX-2 activity was associated with a marked reduction in prostaglandin release and enhanced proliferation of cells under hypoxic conditions.

The functional role of hypoxic induction of COX-2 in vivo remains to be determined, though we have demonstrated the ex vivo induction of COX-2 in organ-cultured lung tissue. Clearly, this requires confirmation in an in vivo model. Repeated administration of the nonselective COX inhibitor, indomethacin, over a 3-wk period caused sustained pulmonary hypertension in sheep (34). In addition, infusion of angiotensin II in the chronically hypoxic rat model of pulmonary hypertension prevents acute and chronic increases in pulmonary artery pressure, a protective effect probably due to increased prostaglandin release (35). These studies support the concept that COX products could inhibit increases in pulmonary vascular tone and remodeling during the development of pulmonary hypertension.

The concept of cellular heterogeneity within the pulmonary vascular wall is well established (36), with distinct subpopulations of smooth muscle cells in the bovine pulmonary artery responding differently in vivo and in vitro. Our observations provide further support for the hypothesis that multiple cell phenotypes exist in the human pulmonary artery that may respond differently to the same stimulus (13). Cells that proliferate preferentially under hypoxic conditions would have a growth disadvantage under normoxic culture conditions, especially if they existed in a minority in vivo. However, it remains to be established whether the "hypoxia-selected" cells isolated in this study are represented in the pulmonary vascular wall in vivo. A detailed study comparing the phenotype of these cells, looking for specific cell markers, or examination of differential gene expression, may help to establish this.

In summary, this study has demonstrated that PASMCs derived from the human peripheral pulmonary circulation differ with regard to their growth under hypoxic conditions, suggesting that the process of hypoxia-induced vascular remodeling in vivo may depend on the net result of growth stimulation and growth suppression. Hypoxic induction of COX-2 in both cell types increased production of prostaglandins and exerts an antiproliferative effect in PASMCs. Inhibition of prostanoid production releases cells from this growth inhibitory effect.


   Acknowledgments
 
This study was supported by funding from the British Heart Foundation and Medical Research Council (UK). K.S. is supported by a British Heart Foundation Ph.D. Fellowship.

Received in original form May 16, 2002

Received in final form July 16, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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