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(Circulation Research. 2003;92:1162.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Peroxisome Proliferator-Activated Receptor Gamma (PPAR) Expression Is Decreased in Pulmonary Hypertension and Affects Endothelial Cell Growth

Shingo Ameshima, Heiko Golpon, Carlyne D. Cool, Daniel Chan, R. William Vandivier, Shyra J. Gardai, Marilee Wick, Raphael A. Nemenoff, Mark W. Geraci, Norbert F. Voelkel

From the Pulmonary Hypertension Center (S.A., N.F.V., R.W.V., S.J.G., M.W.G.), Division of Renal Medicine (R.A.N., M.W.), Cancer Center (D.C., R.A.N., M.W.G.), and Department of Pathology (C.D.C.), University of Colorado Health Sciences Center, Denver, Colo; National Jewish Research Center (R.W.V., S.J.G.), Denver, Colo; Pulmonary Division (H.G.), Universitätsklinik Magdeburg, Germany.

Correspondence to Norbert F. Voelkel, MD, Division of Pulmonary Sciences and Critical Care Medicine, 4200 East Ninth Ave, C272, Denver, CO 80262. E-mail Norbert.Voelkel{at}uchsc.edu

	Abstract

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PPAR is a member of a family of nuclear receptors/ligand–dependent transcription factors, which bind to hormone response elements on target gene promoters. An antiproliferative and proapoptotic action profile of PPAR has been described and PPAR may function as a tumor suppressor gene, but little is known about the role of PPAR in vascular remodeling. One group of human diseases that shows impressive vascular remodeling exclusively in the lungs is the group of severe pulmonary hypertensive disorders, which is characterized by complex, endothelial cell–proliferative lesions of lung precapillary arterioles composed of clusters of phenotypically altered endothelial cells that occlude the vessel lumen and contribute to the elevation of the pulmonary arterial pressure and reduce local lung tissue blood flow. In the present study, we report the ubiquitous PPAR expression in normal lungs, and in contrast, a reduced lung tissue PPAR gene and protein expression in the lungs from patients with severe PH and loss of PPAR expression in their complex vascular lesions. We show that fluid shear stress reduces PPAR expression in ECV304 endothelial cells, that ECV304 cells that stably express dominant-negative PPAR (DN-PPAR ECV304) form sprouts when placed in matrigel and that DN-PPAR ECV304 cells, after tail vein injection in nude mice, form lumen-obliterating lung vascular lesions. We conclude that fluid shear stress decreases the expression of PPAR in endothelial cells and that loss of PPAR expression characterizes an abnormal, proliferating, apoptosis-resistant endothelial cell phenotype.

Key Words: severe pulmonary hypertension • peroxisome proliferator-activated receptor &ggr • endothelial cell growth • apoptosis • shear stress

	Introduction

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Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear hormone receptor/transcription factor superfamily.^1,2 The transcription factor PPAR forms a heterodimeric complex with the retinoid X receptor, and the complex of PPAR and RXR binds to specific DNA recognition sites and regulates transcriptional events. PPAR controls expression of genes that are involved in metabolism and cellular differentiation and is highly expressed in adipose tissue.³ Differentiation of preadipocytes into adipocytes is regulated by PPARs, which in turn are responsive to glucocorticoids, xanthines, retinoids, and prostanoids.⁴ PPAR expression occurs in the lung in alveolar type II cells, coincidentally with surfactant protein A expression during type II cell differentiation.⁵ Recently, antiinflammatory properties of PPAR have been demonstrated; in particular, the attenuation of the oxidative burst in activated macrophages⁶ and decreased cytokine production by monocytes⁷ via inhibition of the AP-1 and NF-B transcription factors. Although it has been demonstrated that oxidized alkyl phospholipids are high-affinity PPAR ligands,⁸ which may induce PPAR-dependent gene expression in atherosclerotic lesions,⁹ and that PPAR can regulate vascular smooth muscle cell migration and proliferation, little information is available regarding the potential role of PPARs in pulmonary vascular remodeling. Because PPAR can act as a tumor suppressor protein,^10–13 induce apoptosis,^14–18 inhibit angiogenesis,^19,20 and because prostacyclin is decreased in severe pulmonary hypertension,^21,22 we wondered whether PPAR expression is reduced in the lung vascular lesions found in severe pulmonary hypertension (PH).

Severe pulmonary hypertension is characterized by complex precapillary arteriolar plexiform lesions,^22,23–25 which contain phenotypically altered smooth muscle and endothelial cells,²³ which express 5-lipoxygenase²⁶ but not p27²³ or caveolin-1 and 2 (R. Achcar, N.F. Voelkel, L. Taraseviciene-Stewart, M. Kasper, C.D. Cool, unpublished data, 2003). By microarray gene expression screening, we found a decrease in PPAR transcripts in random lung tissue samples from patients with primary PH.²⁷

In the present study, we assessed the PPAR gene and protein expression in normal human lungs and in lungs from patients with severe primary and secondary pulmonary hypertension, and we used immunohistochemistry to localize PPAR in normal and pulmonary hypertensive lungs. Because in severe PH the plexiform lesions form at sites of bifurcations²¹ where shear stress is likely high,²⁸ we examined whether fluid shear stress affects PPAR expression. Indeed, increased fluid shear stress reduced the expression of PPAR in a human cell (ECV304 cell) line which has endothelial cell features.^29–33 We found that in normal human lung tissue, PPAR is abundantly expressed especially in endothelial cells, but that the PPAR expression is reduced or lacking in all of the angiogenic plexiform lesions of the pulmonary hypertensive lungs and in the vascular lesions of a rat model of severe PH.³⁴ We also found that ECV304 cells that stably express dominant-negative PPAR are hyperproliferative, relatively apoptosis-resistant, and rapidly form tubes when embedded in matrigel, whereas ECV304 cells that overexpress PPAR do not. We conclude that lack of endothelial cell PPAR expression is a marker of an abnormal endothelial cell phenotype and lack of PPAR expression inhibits apoptosis and facilitates endothelial cell growth and angiogenesis.

	Materials and Methods

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Patients With Severe Pulmonary Hypertension and COPD, Tissue Samples, Immunohistochemistry
Lung tissue was obtained from 6 patients with primary pulmonary hypertension and from 3 patients with collagen vascular disease associated pulmonary hypertension and 1 patient with PH and Eisenmenger physiology. All of these patients had severely elevated pulmonary artery pressures as documented by right heart catheterization (online Table, in the online data supplement available at http://www.circresaha.org). We also obtained lungs from 6 patients with severe emphysema who were undergoing lung transplantation, lung volume reduction surgery, or lobectomy. All 6 patients were chronic cigarette smokers with pulmonary obstructive changes documented by lung function studies. Histologically normal lung tissue was obtained from 6 patients (3 males, 3 females; 62.5±6.2 [SEM] years;) undergoing lung biopsy for diagnostic purposes (localized inflammation [n=1] or primary or metastatic malignancies [n=5]).

Rat Model of Severe Pulmonary Hypertension
Adult Sprague-Dawley rats (Harlan, Indianapolis, Ind; UCHSC-approved animal protocol was used.) (n=5) received a single sc injection of the VEGF receptor inhibitor SU5416 (Sugen) and then the animals were housed in a hypobaric pressure chamber at a simulated altitude of 15 000 feet for 3 weeks. The combination of SU5416 treatment and chronic hypoxia causes severe pulmonary hypertension and lumen-obliterating pulmonary vascular lesions as described previously.²⁰

Quantitative PCR
Quantitative RT-PCR was performed using the SYBR Green PCR Core reagents (Perkin- Elmer). Primers were designed to meet specific criteria by using the Primer Express software (Perkin-Elmer). The primers used were for human-specific PPAR- (forward: 5'-GGGATGTCTCATAATGCCATCA-3'; reverse: 5'-CGCCAACAGCTTCTCCTTCT-3') and for PPAR₂ (forward: 5'-CCCAGAAAGCCATTCCTTCA-3'; reverse: 5'-AATGCGATCT-CTGTGTCAACCA-3'). We isolated total RNA from human lung tissue and ECV304 cells using the RNeasy Mini Kit (Qiagen). Five microliters (200 ng total RNA) was used as a template for the one-step RT-PCR. To obtain the copy numbers for the PPAR₁ gene, we subtracted the copy number of PPAR₂ from that of total PPAR.

Immunohistochemistry
Formalin-fixed, paraffin-embedded sections (5 µm) of normal, emphysematous, and pulmonary hypertensive lungs were deparaffinized and microwave-treated for 15 minutes. Endogenous peroxidase was blocked with 3% H₂O₂ for 30 minutes. Immunohistochemical staining was performed using the Vectastain Universal Quick kit (Vector Laboratories). To block nonspecific binding of Biotin/Avidin system reagents, the Avidin/Biotin blocking kit from Vector laboratories was used. The sections were incubated with mouse monoclonal anti-PPAR- (Santa Cruz) at 1:200 dilution in a humid chamber at room temperature for 30 minutes. The universal second antibody was incubated for 10 minutes, developed with DAB (Vector laboratories) and counterstained with Gill’s Hematoxylin (solution No. 1; Electron Microscopy Sciences). Negative controls were performed using a polyclonal rabbit IgG control primary antibody (Vector Laboratories) at 1:200. To stain the lung tissues for cleaved caspase 3 (the large fragment of activated caspase 3), we used a polyclonal antibody (Cell Signaling Technology).

Immunoblots
Lung tissue was homogenized with 20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl₂, 0.15 mmol/L spermine, 0.5 mmol/L spermidine, 0.25 mol/L sucrose, 100 mmol/L NaCl, 0.2 mmol/L EDTA, 200 µmol/L PMSF, 0.5 mmol/L DTT, 1 µg/µL leupeptin, and 1 µg/µL aprotinin, using a Tissuemizer (Tekmar, Cincinnati, OH) at 4°C, centrifuged at 10 000g for 10 minutes at 4°C to remove tissue fragments, and the supernatant collected. Protein (25 µg, measured with D_C protein assay kit; Bio-Rad) was loaded in each lane of a 4% to 20% Tris-HCl Ready Gel (Bio-Rad). After 1 hour-incubation in a primary antibody (either 1:1000 rabbit polyclonal IgG against the full-length (32kDa) and cleaved fragments of human caspase 3 or ß-actin; both Santa Cruz Biotechnology), the membranes were incubated with horseradish-peroxidase–conjugated second antibodies for 2 hours and visualized by chemiluminescence (ECL kit; Amersham Pharmacia Biotech). The densitometry results for PPAR- were normalized to ß-actin using the Gel Doc 2000 Gel Documentation System (Bio-Rad).

Shear Stress of ECV304 Cells
ECV304^29–33 cells were grown to 90% confluence, trypsinized, pooled, resuspended in media with 1% fetal bovine serum (FBS), and inoculated into polypropylene capillaries contained within a sterile cartridge (CELLMAX Cell Culture Systems, Spectrum Laboratories), which contain artificial capillaries lined with fibronectin. The ECV304 cells were exposed to no flow, low flow (2 dyne/cm²), or high flow 14.3 dyne/cm²) for 24 hours. The cartridge containing the capillary tubes was dismantled, and the cells within the capillary lumens were stripped with trypsin and washed in preparation for quantitative RT-PCR. Alternatively, the capillary tubes were fixed in formalin and embedded in paraffin for routine histology and immunohistochemistry.

Stable Transfection of ECV304 Cells
For stable transfections,^35–37 WT or DN PPAR1 cDNA (gift of Carl Clay, Bowman Gray School of Medicine, Winston-Salem, NC) was inserted into the pLNCS₂ retroviral expression vector (Clontech). The cDNAs for the WT and DN PPAR1 were as reported by Gurnell et al.³⁷ Recombinant virus was prepared in 293T cells by transfecting cells with Gag/Pol/Env vectors using Lipofectin (Gibco BRL). Polybrene (8 µg/mL) was added to the retroviral-containing medium from the packaging cells and filtered before two 24-hour incubations with subconfluent layers of ECV304 cells. The infected cells were replated, selected for G418 resistance, and expanded. Clones were selected for expression of PPAR by immunoblotting with specific antibodies.

ECV304 Cell Tube Formation in Matrigel
Matrigel (50 µL; Collaborative Biomedical Products) at 4°C was coated on each well of a 96-well culture plate and allowed to gel at 37°C for 1 hour. ECV304 (2x10⁴; wild-type or ECV-PPAR dominant-negative or PPAR-overexpressing ECV304 cells) were plated in each well in 200 µL RPMI 1640 supplemented with 10% FCS. The cells were then treated with saline solution (control), ciglitazone, or 15-deoxy-^12,14 prostaglandin J₂ at various concentrations indicated for 24 hours or more. Pictures were taken under an Olympus microscope (10x10 or 10x40 magnifications) equipped with a Nikon Coolpix 995 camera.

ECV304 Cell Apoptosis
Wild-type, PPAR overexpressing, and PPAR dominant-negative endothelial cells were incubated in the presence of medium alone, human TNF (1000 U/mL; R&D Systems), and cycloheximide (10 µg/mL; Calbiochem), or H₂O₂ (1 mmol/L; Sigma-Aldrich) for 6 hours at 37°C, 5% CO₂. Endothelial cells were centrifuged onto slides (Cytospin; Shandon), stained with modified Wright’s Giemsa (Fisher), and analyzed blindly for apoptosis using nuclear condensation. In some experiments, endothelial cells were processed for Western blotting as described in the immunoblot section above.

Tail Vein Injection of ECV304 Cells in Nude Mice
Athymic nude mice (nu/nu) were obtained from NIH-NCI (Rockville, Md). Experiments were performed with an approved IACUC protocol. One million ECV304 cells each were injected intravenously via the tail vein of each mouse under anesthesia. Animals were monitored closely for any changes in health or activity. Animals were euthanized 3 months later. Lungs were isolated, fixed in buffered formalin, and examined histologically.

An expanded Materials and Methods section is available in the online data supplement at http://www.circresaha.org.

	Results

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Decreased PPAR-1 Gene Expression in the Lung Tissue From Patients With Severe Pulmonary Hypertension
Quantitative PCR analysis of lung tissue samples showed that total PPAR mRNA was decreased in patients with severe pulmonary hypertension when compared with normal lung tissue or tissue from patients with emphysema. Using primer sets designed to specifically identify PPAR₂, we found that the majority of the PPAR expressed in the lung tissue was accounted for by PPAR₁, and not PPAR₂ (Figure 1A). PPAR protein expression was decreased in whole lung tissue extracts from patients with both primary and secondary PH (Figure 1B).

View larger version (26K): [in this window] [in a new window]   Figure 1. Reduction in lung tissue PPAR expression in severe pulmonary hypertension (PH) when compared with normal lung tissue or tissue from patients with chronic obstructive pulmonary disease (COPD). Decrease in total PPAR RNA is accounted for by a reduction of the expression of PPAR1 because there was no change in the expression of PPR2 (A). In comparison to normal or COPD patient lung tissue, the PPAR protein expression is reduced in lung tissue from patients with primary (PPH) or secondary (2nd) pulmonary hypertension (PH) (B).

Immunohistochemistry revealed in normal lungs the ubiquitous expression of PPAR in alveolar septal structures and in small vessel endothelium; bronchial epithelial cells did not express PPAR (Figures 2A and 2B). In PH, remodeled, muscularized precapillary arterioles showed frequently reduced endothelial cell PPAR expression. Most remarkable was the reduction or lack of PPAR staining of the lumen-obliterating cells of the plexiform lesions (Figures 2C and 2D) in the lungs from patients with primary or secondary PH (Figure 2E). In fact, all of the plexiform lesions (n=38) in the lungs from the 9 patients with severe PH were characterized by pale centers, ie, relative or total absence of the nuclear PPAR staining. Figure 2 also shows serial sections of plexiform lesions. The lack of smooth muscle cell actin staining of the cells in the lesion (Figure 2D) indicates that the PPAR-negative cells of the lesion are not smooth muscle cells. Lack of cells undergoing apoptosis in this lesion (Figure 2F) and in the lesion from a patient with Eisenmenger physiology (VSD) is also apparent (Figure 2G). Two different lesions shown in Figure 2, with diminished PPAR expression also demonstrate a lack of expression of active caspase 3 (Figures 2F and 2H). An example of a complex vascular lesion in a rat lung with severe pulmonary hypertension, documenting that most of the lesion cells do not express PPAR is shown in Figure 2I.

View larger version (127K): [in this window] [in a new window]   Figure 2. PPAR immunohistochemistry of normal lung tissue. Ubiquitous staining of alveolar septal cells and of alveolar macrophage (A), x400. Absence of PPAR staining in vascular smooth muscle (B), x100. Serial sections of 1 plexiform lesion (C through F) showing expression of FVIII-r.ag (C), -smooth muscle actin (D), PPAR (E), and active caspase 3 (F); there is complete lack of caspase 3 staining of the cells of the lesion and also of the surrounding alveolar structures (the insert, right lower corner shows positive staining for active caspase 3 in a lymph node). A vascular lesion from a patient with PH and Eisenmenger physiology is shown in G and H. Lack of PPAR staining is shown in G and lack of staining for active caspase 3 in H. A complex vascular lesion in a rat lung from an animal exposed to SU5416 and hypoxia is shown in I. Alveolar septal cells stain positive for PPAR, the vascular lesion cells lack PPAR staining.

Decreased Expression of PPAR Protein in the Lung Vascular Lesions of Rats With Severe PH
A single sc injection of 25 mg/kg of the VEGF receptor blocker SU5416 causes severe pulmonary hypertension and intense vascular remodeling in the lungs of rats exposed to chronic hypobaric hypoxia.³⁴ Immunohistochemistry was performed on such lungs using the antibody directed against PPAR and lung sections from 5 different rats. Figure 2I shows that the lung vascular lesion, which is composed of proliferating endothelial cells³⁴ lacks PPAR protein expression. Thus, absence of PPAR staining serves as a marker for easy recognition of abnormal, proliferating pulmonary vascular endothelial cells also in this animal model of severe PH.

Decreased PPAR Expression of ECV304 Cells by Fluid Shear Stress
PPAR gene expression in cultured confluent ECV304 cells, which had been placed on a rocking platform that was tilted 15 times a minute for 8 hours in order to move the cell culture medium rhythmically back and forth over the monolayer and thus apply fluid shear stress, was decreased in the tilted cells when compared with resting cells. Similar results were also obtained with human umbilical vein endothelial cells (HUVECs) (data not shown). Subsequently, ECV304 cells were seeded into the CellMax system and exposed to no shear, low fluid shear, or high fluid shear stress for 24 hours. High shear stress for 24 hours dramatically reduced PPAR gene expression (Figure 3A) and so did chronic shear stress applied to the CellMax system for 3 weeks (Figure 3B).

View larger version (43K): [in this window] [in a new window]   Figure 3. Decreased PPAR gene expression after exposure of ECV304 cells to high shear stress (A). Decreased PPAR gene expression by reverse transcription PCR in ECV304 cells exposed to shear stress for 3 weeks (B). High shear stress for 3 weeks in the artificial capillary lumen: (1) ECV304 wild-type cells form cell clusters that partially obliterate the lumen; (2) ECV304 cells that overexpress the PPAR gene form monolayers in the artificial capillaries; (3) ECV304 cells that are dominant-negative for the PPAR gene grow to obstruct the artificial capillary lumen (C).

Increased Angiogenic Potential of Cells Expressing DN-PPAR
We stably transfected ECV304 cells with either full-length WT-PPAR or a construct encoding DN-PPAR. Cells overexpressing WT-PPAR had a marked increase in activation of a PPAR responsive promoter, whereas promoter activity was inhibited in cells expressing DN-PPAR (data not shown). In 3-dimensional collagen gels, wild-type ECV304 cells formed clumps, whereas cells DN-PPAR consistently formed reticular tube-like structures (Figure 4), suggesting that lack of PPAR results in an angiogenic potential. PPAR overexpressing ECV304 cells did not form tubes at all. Treatment of DN-PPAR ECV304 cells with the PPAR agonists 15-deoxy ^12,14 prostaglandin J₂ and ciglitazone suppressed the formation of tube-like structures at concentrations of 6 µmol/L, but not of concentrations of 3 µmol/L (Figure 4).

View larger version (143K): [in this window] [in a new window]   Figure 4. Neither wild-type (WT) ECV304 cells nor ECV304 cells overexpressing the PPAR gene (middle) form reticular tube structures at 18 hours after being embedded in matrigel. DN-PPAR cells form tube-like structures (A). Treatment of the ECV304 cells with the PPAR agonist 15-deoxy-12,14 prostaglandin J2 had little effect on ECV304 cell tube formation at a concentration of 3 µmol/L (B) or 6 µmol/L (C).

Tail vein injection into nude mice of ECV304 cells expressing DN-PPAR caused the growth of intravascular tumors exclusively in the lungs but not in other organs (Figure 5), whereas wild-type or PPAR overexpressing cells did not grow after similar injection.

View larger version (115K): [in this window] [in a new window]   Figure 5. Lung histology of a nude mouse 12 weeks after intravenous (tail vein) injection of DN- PPAR ECV304 cells showing intrapulmonary arterial ECV304 cell growth, x100 (A) and x400 (B).

Apoptosis of ECV304 Cells
ECV cells exposed to the combination of TNF- plus cycloheximide or to H₂O₂ underwent apoptosis as assessed by nuclear morphology (Figure 6A). Overexpression of PPAR significantly increased the number of apoptotic cells, ie, facilitated apoptosis. The combination of TNF- plus cycloheximide did not facilitate apoptosis in the DN-PPAR cells when compared with the unchallenged cells, indicating that DN-PPAR cells were relatively apoptosis resistant. Although H₂O₂ exposure increased the number of apoptotic cells when compared with the untreated groups, DN-PPAR ECV cells showed a trend toward apoptosis protection when compared with the PPAR-overexpressing ECV cells (Figure 6A). The effect of DN-PPAR as an inhibitor of apoptosis was confirmed in separate experiments (Figure 6B), where DN-PPAR prevented the loss of procaspase 3 compared with either wild-type or overexpressing PPAR ECV cells.

View larger version (27K): [in this window] [in a new window]   Figure 6. Dominant-negative (DN) PPAR ECV cells are relatively resistant to apoptosis. PPAR-overexpressing (OE), DN, or wild-type ECV cells received either no treatment, or were exposed for 6 hours to tumor necrosis factor- (TNF-; 1000 U/mL) plus cycloheximide (10 µg/mL) or to hydrogen peroxide (H2O2; 1 mmol/L). Blinded assessment of nuclear morphology (A) (n=5) or procaspase 3 staining (B) (n=3) was used to determine apoptosis. *Significantly different from untreated OE-PPAR (P<0.05; Dunnett’s test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The remodeling of precapillary arterioles in severe pulmonary hypertension is in part caused by the uncontrolled growth of endothelial cells contributing to the lumen obliteration of these arterioles.^22,23–26 The phenotype of these proliferating endothelial cells is abnormal as these cells express VEGF and 5-lipoxygenase,^24,26 have absent or decreased expression of the gene encoding prostacyclin synthase,²² decreased expression of the p27 protein,²³ and loss of expression of the tumor suppressor TGF-ß RII protein in the plexiform lesions in the lungs from patients with primary pulmonary hypertension.³⁸ Our present study characterizes the phenotype of the proliferating endothelial cells further. All of the plexiform lesions examined had reduced staining or lacked staining when the lung sections from the patients with severe PH were probed with the antibody directed against PPAR; a similar loss of staining (expression) was observed in the vascular lesions, which characterize our rat model of severe PH.³⁴ In addition, PPAR gene expression was reduced by fluid shear stress in cultures of human endothelial cell–like (ECV304) cells.

Activation of PPAR can induce cell growth inhibition,³⁹ even in cancer cells^11–13; therefore, our data that show a global tissue decrease in both PPAR gene and protein expression in the lungs from patients with severe primary or secondary PH—but not in the lungs from patients with chronic obstructive lung disease (Figure 1), which are characterized by apoptosis of endothelial cells⁴⁰—suggest to us the loss of a cell growth inhibitor in the vascular lesions in severely pulmonary hypertensive lungs.

We offer two pathobiological explanations: either loss of a tumor suppressor gene and protein facilitates endothelial cell proliferation in PH, or the loss of PPAR expression is another marker of angiogenic endothelial cell growth²² and impaired apoptosis. We could not find any information regarding control mechanisms of PPAR expression in the lung or in endothelial cells; for example, it is not known whether hypoxia or shear stress regulate PPAR expression. What is known, however, is that PPAR ligands are potent inhibitors of angiogenesis.^19,20 Although pulmonary blood flow is low in many patients with severe pulmonary hypertension, we postulate that regional shear stress is high, in particular at sites of precapillary arteriolar bifurcations, where plexiform lesions form.²³ We thus examined whether fluid shear stress applied for 24 hours or 3 weeks affects endothelial PPAR gene expression. Indeed in the CellMax endothelial cell model, shear stress reduced PPAR expression (Figure 3). The mechanism whereby shear stress decreases PPAR expression is unclear because the promoter region of the PPAR gene does not contain the known shear stress response motif (GAGACC).⁴¹ Because shear stress inhibits endothelial cell apoptosis,^42–44 it is an intriguing hypothesis that endothelial cell apoptosis resistance might be related to decreased or lost PPAR expression.

Because it had been shown previously that 15-deoxy-^12,14 prostaglandin J₂ induces endothelial cell,¹⁵ synoviocyte,¹⁶ and T-lymphocyte¹⁸ apoptosis, we conducted experiments to assess the effect of PPAR activation on the growth of stably expressing DN-PPAR, overexpressing PPAR, and wild-type ECV304 cells. In comparison to the wild-type cells, the ECV304 cells expressing DN-PPAR sprouted tubes; ECV304 cells overexpressing PPAR did not grow any sprouts. Both ciglitazone (data not shown) and 15-deoxy-^12,14 prostaglandin J₂ (Figure 4) decreased sprout formation in ECV304 cells expressing DN-PPAR at higher concentrations (6 µmol/L), but had little effect at the 3 µmol/L concentration; this is consistent with the concept that diminished or impaired expression of the nuclear receptor PPAR permits angiogenesis and that ligand activation of PPAR induces endothelial cell apoptosis.¹⁵ In fact, when ECV304 cells overexpressing PPAR were subjected to TNF- plus cycloheximide, apoptosis was facilitated when compared with the DN-PPAR cells (Figure 6).

Finally, when we injected ECV304 cells into the tail-veins of nude mice, only the ECV304 cells expressing DN-PPAR formed tumors in the lungs (Figure 5). No tumor formation was observed in liver, spleen, or kidney, perhaps because the lung vessels provided a filter for the injected cells. As we observed tumor formation after injection of ECV304 cells carrying dominant-negative mutations of PPAR, anti-tumor growth effects of PPAR ligands have been observed in mice injected with breast cancer cells.¹³

In conclusion, we propose that lack of PPAR expression is an important aspect of an abnormal, apoptosis-resistant, angiogenesis-promoting endothelial cell phenotype, and that in severe forms of pulmonary hypertension, lumen-obliterating endothelial cell growth may be facilitated by the loss of the tumor suppressor function of PPAR. Although our data indicate that PPAR plays a role in endothelial cell sprout formation, in rapid growth of endothelial cells in an artificial tube system and also in endothelial cell apoptosis resistance, it remains unclear what the initiating events leading to severe pulmonary hypertensive remodeling are. In the SU5416/chronic hypoxia model of severe PH,³⁴ it could be shown that initial endothelial cell apoptosis is critical for the subsequent endothelial cell growth, which results eventually in lumen obliteration. Whether serotonin excess,⁴⁵ potassium channel activity,⁴⁶ protease/anti-protease imbalances,^47,48 or germline mutations of the bone morphogenic protein receptor II⁴⁹ relate to loss of pulmonary vascular endothelial growth control, the evolution of apoptosis-resistant endothelial cell phenotypes, which also have lost PPAR expression, requires further investigation. Although we can show that the cells of the plexiform lesions in severe PH lack both apoptotic events and PPAR expression, inhibition of apoptosis and lack of PPAR expression may be associated or causally linked as suggested by our experiments that used PPAR-overexpressing and DN-PPAR ECV304 cells.

	Acknowledgments

 
This work was supported by the following NIH-HLB grants, PO1 HL66254 (N.F.V.) and KO8 HL 03911 (C.D.C.), DR 39902, DK 19928, HL62824 (R.A.N.), HL 34303 (Peter Henson), and a grant by the Deutsche Forschungs Gemeinschaft, Bonn-Bad Godesberg, Germany (H.G.). The authors thank K. Wood for technical assistance.

Received September 4, 2002; revision received April 7, 2003; accepted April 14, 2003.

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