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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:63.)
© 2007 American Heart Association, Inc.

Vascular Biology

Activation of PPARß/ Induces Endothelial Cell Proliferation and Angiogenesis

Laura Piqueras; Andrew R. Reynolds; Kairbaan M. Hodivala-Dilke; Arántzazu Alfranca; Juan M. Redondo; Toshihisa Hatae; Tadashi Tanabe; Timothy D. Warner; David Bishop-Bailey

From Cardiac, Vascular & Inflammation Research (L.P., T.D.W., D.B.-B.), William Harvey Research Institute; Cell Adhesion and Disease Group (A.R.R., K.M.H.-D.), Centre for Tumour Biology, Institute of Cancer, and the CR-UK Clinical Centre, Barts, and The London, Queen Mary University London, Charterhouse Sq, London, UK; Centro de Biologia Molecular Severo Ochoa (A.A., J.M.R.), Consejo Superior de Investigaciones Cientificas (CSIC)-Universidad Autonoma de Madrid, Facultad de Ciencias, Cantoblanco, Spain; Department of Pharmacology (T.H., T.T.), National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka, Japan. Current address for J.M.R.: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernandez Almagro 3, Madrid 28029.

Correspondence to David Bishop-Bailey, From Cardiac, Vascular & Inflammation Research (L.P., T.D.W., D.B.-B.), William Harvey Research Institute, Barts and the London, Queen Mary University London, Charterhouse Sq, London EC1M 6BQ. E-mail d.bishop-bailey{at}qmul.ac.uk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-ß/ in endothelial cells remains unclear. Interestingly, the selective PPARß/ ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPARß/ in endothelial cell proliferation and angiogenesis.

Methods and Results— Western blot analysis indicated PPARß/ was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPARß/ target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPARß/. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation.

Conclusions— PPARß/ is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

PPARß/ is expressed in vascular endothelial cells; however, its roles remain unclear. The PPARß/ ligand GW501516 induced endothelial proliferation and angiogenesis. The clinical use of GW501516 in dyslipidemia may therefore need to be monitored for angiogenic processes.

Key Words: angiogenesis • endothelial cells • PPARß/ • vascular endothelial growth factor

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator activating receptors (PPARs) belong to the steroid receptor superfamily of ligand-activated transcription factors.¹ Three PPAR isotypes, PPAR, PPARß/, and PPAR have been identified.² PPAR is predominantly expressed in liver, heart, kidney, brown adipose tissue, and stomach mucosa; PPAR is found primarily in adipose tissue; PPARß/ is the most ubiquitously expressed,^3,4 although its physiological and patho-physiological roles are less clear, particularly in human tissue. The recent development of PPARß/ knockout and transgenic mice have, however, implied roles for PPARß/ in adipose tissue formation, wound healing, brain development, placental function, atherosclerosis, and colorectal carcinogenesis.^5–8

Endothelial cells play critical roles in vascular biology, being both the protective inner lining of vessels and the local site for delivery of oxygen to all tissues. Endothelial damage or dysfunction is considered a critical initiator of large vessel diseases such as atherosclerosis. In contrast excessive formation of new blood vessel capillaries in the form of angiogenesis is associated with chronic inflammatory processes, including those diseases of large vessels, and of tumor growth. Interestingly, prostacyclin, the predominant prostanoid released by vascular cells is a putative endogenous agonist for PPAR ß/.^9,10 PPAR, PPARß/, and PPAR are all expressed in endothelial cells.^11,12 PPAR and PPAR have well-characterized roles in endothelial cells, both being in general anti-inflammatory, anti-proliferative, and anti-angiogenic in a variety of in vitro and in vivo models.¹ In contrast, the role of PPARß/ in this important cell type is poorly understood. Initial reports suggested that like PPAR and PPAR, PPARß/ promotes apoptosis.¹³ While a more recent report suggested long-term culture of endothelial cells with a selective PPARß/ ligand induces proliferation.¹⁴

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
The GW501516 (2-methyl-4-(((4-methyl-2-(4-trifluoromethyphenyl)-1,3-thiazol-5-yl)-methyl) sulfanyl)phenoxy)-acetic acid) was from Axxora Biochemicals (San Diego, Calif). Cyclo-VEGFI was from Calbiochem (Beeston, UK). PPARß/ anti-sera was from Santa Cruz Biotechnology (Autogen-Bioclear, UK). Rat tail collagen type I was from BD Bioscience (Oxford, UK). Vascular endothelial growth factor (VEGF) A enzyme-linked immunosorbent assay was from R&D Systems (Oxford, UK). pcDNA/PPAR/L431/AE434A was constructed as previously described.¹⁵ Unless stated, all other reagents were from Sigma (Poole, UK).

Cell Culture and Protein Expression
The fused human endothelial/A549 cell line EAHy926¹⁶ was cultured in DMEM supplement with 10% fetal bovine serum (FBS), antibiotic/antimycotic mix, and 1% hypoxanthine, aminopterin, and thymidine. Primary human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were from PromoCell (Heidelberg, Germany), and cultured according to the manufacturer’s recommended protocols. Western blot analysis¹⁷ and immunofluorescence¹² for PPARß/ was performed as previously described.

Cell Proliferation Assays
Endothelial cell proliferation was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay¹⁸ and by cell counts. Because normal growth medium is such a strong proliferative/angiogenic stimulus, cells were seeded into 96-well plates and after 24 hours, medium was replaced with either FBS-free medium for EAHy926 or medium containing 2% FBS for primary HAECS and HUVECS before treatments added.

Endothelial Cell Differentiation Assay
Tube formation assays were performed as previously described.¹² Briefly, cells were seeded on extracellular matrix (Sigma) at 2x10⁵ cells/well in 24-well plates in absence of serum, and incubated for 24 hours in the presence or absence of GW501516. Phase contrast micrographs (Nikon, Eclipse, TE2000-S) were recorded, and the mean number of tubes in 5 low-power (x100) random fields assessed.

Aortic Ring Assay
Aortic ring assays were performed essentially as previously described¹⁹ with minor modifications. Aorta were harvested from 8- to 12-week-old mice on a C57 black 6/129Sv mixed-background and cut into 0.5-mm-thick segments. After serum starvation overnight each ring was mounted in 50 µL of pH-neutralized 1.1 mg/mL collagen type 1 in each well of a 96-well plate. After 30 minutes of collagen polymerization, each well was overlaid with 150 µL of DMEM plus 2.5% fetal calf serum supplemented with or without 30 ng/mL VEGF or 1 µmol/L GW501516. Rings were incubated for up to 8 days at 37°C in 5% CO₂ with renewal of the medium every other day. Angiogenesis was quantified by counting the number of microvessel sprouts that grew from each ring after 6 days. For staining, aortic rings were fixed for 20 minutes with 4% wt/vol formaldehyde, permeabilized with 0.2% TritonX-100, and incubated with 10 mg/mL fluorescein isothiocyanate (FITC)-conjugated Bandeiraea simplicifolia lectin for 1 hour. After washing with phosphate-buffered saline, rings were imaged using a confocal microscope (Zeiss LSM 510).

Murine Matrigel Plug Assay
Matrigel plug assay was performed as previously described.²⁰ Briefly, growth factor depleted matrigel (BD Biosciences) supplemented with 64 U/mL heparin and either vehicle (0.01% DMSO), 250 ng/mL VEGF, or 1 µmol/L GW501516, was injected into the median abdominal area of anesthetized C57BL6 mice (400 µL/mouse). Angiogenesis was determined by plug hemoglobin concentration at day 10. Hemoglobin was measured in matrigel plug supernatant after homogenization and centrifugation, using the 3,3',5 to 5'-tetramethylbenzidine liquid substrate system (TMB Sigma; 1:1), with optical density (OD) being determined at 620 nm.

Transfections
All transfections were performed using NovaFector (VennNova, Pompano Beach, Fla) according to the manufacturer’s recommended protocol. Using EAHy926 we achieved >85% transfection efficiency as determined by GFP expression using pEGFPN-1 (Clontech; data not shown). Cells were transfected with either dominant-negative (DN) PPARß/ (pcDNA/PPAR/L431/AE434A), wild-type PPARß/ (pCMV-mPPAR; a generous gift from Dr Ronald Evans, Salk Institute), or with control vector (pcDNA), and used 24 hours after transfection.

Reverse-Transcriptase Polymerase Chain Reaction
Total RNA was isolated using TRIzol reagent (Invitrogen). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using standard protocols using primers for VEGF,²¹ ß-actin,²² and human adipose differentiation-related protein (ADRP); forward 5'-ACTGGCTGGTAGGTCCCCTTT-3', reverse 5'-TGCTTCCCAATTTAGGGTTG-3' (214bp). The PCR mixtures were subjected to 4 minutes denaturation at 94°C followed by 30 cycles of amplification of 45 seconds at 94°C, 45 seconds at 58°C, and 1 minute at 72°C, and a final extension phase of 5 minutes at 72°C. The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. To test for a role of GW501516 on VEGF transcription, cells were treated with GW501516 for 6 hours in the presence of absence of 5 µg/mL actinomycin D.

Statistical Analysis
Differences between 2 groups were determined by 1-sample, paired or unpaired Student t test, as appropriate. Differences between multiple groups were determined by analysis of variance (ANOVA) followed, when necessary, by a Student-Newman-Keuls multiple comparisons test. Values were expressed as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PPARß/ Is Expressed in EAHy926, HUVECS, and HAECS
PPARß/ was detected as a band of 55 kDa in EAHy926, HUVECS, and HAECS by Western blot analysis (Figure 1a), and was localized to the nuclear and perinuclear region by indirect immunofluorescence (Figure 1a; supplemental Figure I, available online at http://atvb.ahajournals.org). Expression or localization of PPARß/ in the cell was not affected by GW501516 treatment. To show expressed PPARß/ was functional, we used the PPARß/ target gene ADRP.²⁴ GW501516 (100 nM, 24 hours) induced ADRP in EAHy926 as measured by semiquantitative RT-PCR, which was abolished in cells co-transfected with DN-PPARß/ (Figure 1b).

View larger version (31K): [in this window] [in a new window]   Figure 1. PPARß/ expression in endothelial cells. a, Left panel, Western blot of PPARß/ in EAHy926, HUVECs, and HAECs, seen as a single band of 55 kDa. Right panels, Immunoflorescence micrographs of PPARß/ in Eahy926 cells treated with vehicle (0.001% DMSO) or GW501516 (100 nM); boxes show the PPARß/ immunolocalization in individual cells. b, The effect of GW501516 on the PPARß/ target gene adipose differentiation-related protein (ADRP) and its regulation by DN-PPARß/. EAHy926 cells were transfected with either DN-PPARß/ or empty vector (pcDNA). 24 hours after transfection cells were treated with GW501516 (100 nM) or vehicle (0.001% DMSO) for 24 hours. Top panel shows representative gel for RT-PCR analysis of ADRP and ß-actin, whereas lower panel shows densitometric quantification (fold expression from vehicle control using ratio of ADRP: ß-actin in arbitrary units). *P<0.05 vs control. #P<0.05 vs GW501516-treated cells on pcDNA group. The data represents the mean±SEM from n=3 separate experiments.

PPARß/ Activation Induces Endothelial Cell Proliferation
Subconfluent (50%) endothelial cells were treated with vehicle (0.001% DMSO) or different concentrations of GW501516 (1 to 100 nM) for 72 hours. GW501516 induced a concentration-dependent increase in EAHy926, HUVECs (GW501516 100 nM;165±22% increase*; n=3), and HAECs (GW501516 100 nM; 181±29% increase* over control; n=3; *P<0.05 1-sample t test) number as determined by MTT assay (Figure 2a) and cell counts (Figure 2b). Before 72 hours, no significant induction of proliferation was observed (EAHy926 with GW501516 100 nM; 24 hours, 117±12%; 48 hours, 124±8% increases). To test for specificity of GW501516, EAHy926 were transfected with either DN-PPARß/ or with control plasmid DNA (pcDNA). Exposure of control transfected cells to GW501516 100 nM for 72 hours resulted in an increase in cell proliferation (170±9.9% of control values), similar to untransfected cells (Figure 2c). In contrast, in cells transfected with DN-PPARß/ no proliferation was observed (Figure 2c).

View larger version (15K): [in this window] [in a new window]   Figure 2. PPARß/ activation induces endothelial cell proliferation. EAHy926 were incubated with GW501516 (1 to 100 nM) for 72 hours and cell proliferation was measured. a and c, MTT assay, expressed as % of vehicle (0.001% DMSO) treated control culture conditions, whereas (b) shows cell counts determined at 72 hours. *Significant difference (P<0.05) by 1-sample t test between vehicle and GW501516 treated cells. c, Effect of DN-PPARß/ expression on GW501516-induced EAHy926 cell proliferation. * P<0.05 vs vehicle control cells. #P<0.05 vs GW501516-treated cells on pcDNA group. Data represent n=9 to 23 incubations from 4 separate experiments.

PPARß/ Activation Induces Endothelial Tube Formation
By 24 hours, GW501516 induced EAHy926 tube formation in extracellular matrix in a concentration-dependent manner (Figure 3a and 3b). Similarly, GW501516 (100 nM, 24 hours) induced HUVECs (cont 2.0±1.4; GW 7.4±2.6* tubes per field), and HAECs (cont 2.7±0.1; GW 5.8±1.2* tubes per field; n=3; *P<0.05 1-sample t test) tube formation (supplemental Figure II). In pcDNA transfected EAHy926, 100 nM GW501516 increased tube formation at 24 hours similar to untransfected cells. In contrast, in cells transfected with DN-PPARß/ (Figure 3c), no tube formation was observed. In cells transfected with wild-type PPARß/, tube formation was augmented compared with control transfections (Figure 3d); a similar result was found with wild-type PPARß/ transfected EAHy926 in proliferation assays (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 3. PPARß/ activation induces endothelial cell tube formation in 3-day extracellular matrix cultures. a, Representative micrographs of tube formation in EAHy926 incubated with vehicle (0.001% DMSO) or GW501516 (1 to 100 nM) for 24 hours, whereas (b to d) show quantitative measure of tube formation in EAHy926 treated with vehicle or GW501516 (100 nM) for 24 hours. c and d, Effect of DN-PPARß/ (c) or wild-type (wt)-PPARß/ (d) on GW501516-induced tube formation; (d) shows the expression of PPARß/ by Western blot in EAHy926 transfected with wild-type wt-PPARß/ or pcDNA. Figures show mean±SEM for the number of tube-like structures in 5 low-magnification (x100) fields per n number. This data represents n=3 separate experiments.*P<0.05 vs vehicle control cells.

This pro-angiogenic response of GW501516 was also seen in a murine aortic ring assay utilizing type 1 collagen, and in an in vivo matrigel model of angiogenesis. In vehicle-treated aortic segments no sprouting vessels were observed. In contrast, 6-day treatment with either VEGF (30 ng/mL; used as a positive control) or GW501516 (1 µmol/L) induced endothelial cell sprouting (Figure 4a). Similarly, in vivo in vehicle containing matrigel plugs a low basal angiogenesis was observed (using hemoglobin as a marker). In contrast, in plugs containing 250 ng/mL VEGF or 1 µmol/L GW501516, a significant increase in angiogenesis was observed after 10 days (Figure 4b) as determined an increase in hemoglobin content. Higher concentrations of GW501516 were not significantly different from 1 µmol/L GW501516 in either the in vitro sprouting assay (control 0.4±0.1; GW 1 µmol/L 1.6±0.1; GW 10 µmol/L 1.9±0.5 microvessels per ring; n=5) or the in vivo matrigel plug assay (control 1; GW 1 µmol/L 2.9±0.6; GW 50 µmol/L 2.4±1.2; fold Hb change; n=7 to 12).

View larger version (14K): [in this window] [in a new window]   Figure 4. GW510516 induces angiogenesis. a, The effect of GW501516 on endothelial cell out-growth in a murine aortic ring model. Mouse aortic ring segments were embedded in type 1 rat tail collagen and incubated with VEGF (30 ng/mL), GW501516 (1 µmol/L), or vehicle (0.01% DMSO) for 6 days. Top panel shows representative photomicrographs of rings incubated with vehicle, VEGF, or GW501516 after 6 days in culture (stained with FITC-BS1-lectin). Figure shows quantification of sprouting angiogenesis (mean number of microvessels per aortic rings at day 6) from n=20 aortic rings from n=4 animals. b, The effect of GW501516 on angiogenesis in a murine matrigel plug assay in vivo. C57BL6 mice were injected into the median abdominal area with growth factor depleted Matrigel containing vehicle (0.01% DMSO), VEGF (250 ng/mL), or GW501516 (1 µmol/L). Angiogenesis was determined by plug hemoglobin concentration at day 10. Figure shows fold change in hemoglobin (Hb) concentration in the matrigel of VEGF and GW501516-treated animals compared with vehicle control (given a value of 1). *P<0.05.

VEGF Mediates PPARß/-Induced Endothelial Cell Proliferation and Tube Formation/Angiogenesis
GW501516 (100 nM) significantly elevated VEGF mRNA levels in EAHy926 (Figure 5a and 5b) and VEGF peptide release into the cultured media at 24 hours (Figure 5c). Cotreatment of cells with 5 µg/mL actinomycin D strongly reduced both basal and GW501516-induced VEGF mRNA (Figure 5b). Transfection of cells with DN-PPARß/, but not control plasmid, prevented the induction of VEGF mRNA by GW501516 treatment (Figure 5d). Pretreatment of EAHy926 cells with cyclo-VEGFI, a VEGF receptor 1/2 antagonist, abolished both GW510516-induced cell proliferation (Figure 5e) and tube formation (Figure 5f).

View larger version (19K): [in this window] [in a new window]   Figure 5. VEGF mediates PPARß/ induced proliferation and morphogenesis. Expression of VEGF mRNA in EAHy926 (a) after GW501516 (3 to 100 nM) treatment for 24 hours, and (b) shows the effect of the RNA synthesis inhibitor actinomycin D (Act D; 5 µg/mL) on the VEGF mRNA levels in the presence of absence of GW501516 (GW; 100 nM) at 6 hours. c, The effect of GW501516 (100 nM; 24 hour) on VEGF peptide secretion by EAHy926. d, The effect of DN-PPARß/ on GW501516-induced VEGF mRNA in EAHy926 cells. Figures a, b, and d show densitometric quantification of VEGF mRNA expression in response to GW501516 (fold expression from vehicle using ratio of VEGF:ß-actin in arbitrary units). *P<0.05 vs control; #P<0.05 vs GW-treated cells in pcDNA group. Data represent n=3 separate experiments. e and f, The effect of the VEGF receptor1/2 antagonist cyclo-VEGFI on GW501516-induced endothelial cell proliferation (e) and tube formation (f). Cells were incubated with vehicle (0.001% DMSO) or GW501516 (100 nM) either alone or in the presence of cyclo-VEGFI (10 µmol/L). Proliferation was measured by MTT assay and expressed as % of vehicle control, whereas morphogenesis was assessed by tube formation (mean number of tube-like structures per low magnification (x100) field per n). * P<0.05 vs control; #P<0.05 vs GW-treated cells. This data represents n=3 separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In recent years there has been a great interest in the actions of PPAR and PPAR, the molecular targets for the clinically used lipid-lowering fibrates and insulin-sensitizing thiazolidinedione classes of drugs. PPARß/ similar to PPAR and PPAR has roles in metabolism, regulating adipogenesis, increasing fatty acid oxidation and energy uncoupling, decreasing insulin resistance, and improving glycemic control and elevating high-density lipoprotein.^25–27 Because of to this latter finding in particular, GW501516, the ligand used in this study (with a reported 1000-fold selectivity for PPARß/ over other subtypes²⁶) is currently in a phase II clinical trial for dyslipidemia. The concentrations of GW501516 (100 nM to 1 µmol/L) used in our study are in line with both in vitro activation of PPARß/ and the levels required in vivo to increase high-density lipoprotein cholesterol and reduce triglycerides in obese rhesus monkeys (0.3 mg/kg to 3 mg/kg dosing i.b. gives plasma concentrations of 71 ng/mL to 706 ng/mL, which equates to GW501516 concentrations of 158 to 1560 nM.²⁶ PPAR, PPARß/, and PPAR are all present in vascular and inflammatory cells and in human and experimental mouse models of atherosclerosis.¹ Little, however, is known regarding the direct vascular roles of PPARß/. In contrast, PPAR and PPAR have well-characterized effects, inhibiting endothelial cell growth, angiogenesis, and inflammatory responses.^12,28–31 Previous, contrasting reports suggested that PPARß/ activated by prostacyclin or its analogues^13,32 or selective ligands^14,32 either acutely regulate endothelial cell apoptosis,¹³ protect endothelial cells from apoptosis from oxidant stress,³² or induce HUVEC proliferation at 14 days in culture.¹⁴ In this study, we show PPARß/ expression in human endothelial cells in culture, where its activation induces proliferation and angiogenesis, through a VEGF-dependent mechanism.

PPARß/ was expressed in all the endothelial cell types tested, consistent with previous reports for EAHy926,³¹ HUVECs,^11,32 and ECV304 cells.^12,32 As an independent marker of PPARß/ activation in endothelial cells we used the induction of ADRP. ADRP has consistently been reported as a PPARß/-regulated gene, its induction being absent in PPARß/ knockout mice.²⁴ Consistent with PPARß/ activation, GW501616 induced ADRP mRNA. There are no available antagonists for PPARß/; however, the DN-PPARß/ proved a useful tool, inhibiting GW501516-induced endothelial cell ADRP, proliferation, and angiogenesis.

In vitro tube formation assays using extracellular matrix derived from Engelbreth-Holm-Swarm sarcoma allows the study of 2 key steps in the angiogenic process, the migration and the differentiation of endothelial cells.³³ They do not mimic the whole process of in vivo angiogenesis. However, our mouse aortic endothelial growth and in vivo matrigel plug assays demonstrate, similar to our in vitro observations, that activation of PPARß/ results in increased angiogenesis. In these murine angiogenesis models, 1 µmol/L GW501516 was required to induce angiogenesis, which although higher than the concentration of 100 nM required for human cells in vitro, is fully consistent with an effect on PPARß/; GW501516 has been reported to be 20-times less potent on murine than human PPARß/.²⁶

VEGF(A) is a well-characterized mediator of endothelial cell growth and angiogenesis.³⁴ In our study, similar to previously described observations,^14,23 GW501516 induced VEGF mRNA, and here we also describe protein release from endothelial cells. The VEGF promoter does contain a PPAR response element (PPRE),³⁵ and actinomycin D, an inhibitor of mRNA synthesis blocks GW501516-induced VEGF mRNA acutely. Whether, PPARß/ directly regulates VEGF in endothelial cells or indeed message stability is not known; however, in T24 bladder cancer cells, the PPARß/ ligand L-165041 induced VEGF mRNA without effecting message stability or activating a reporter promoter construct,²³ which should contain the known PPRE.³⁵ Two endothelial VEGF tyrosine kinase receptors have been identified: VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk1. VEGFR2 appears to be the most important receptor in VEGF-induced mitogenesis and permeability.³⁶ Consistent with this, treatment of endothelial cells with cyclo-VEGFI, a VEGFR1/2 antagonist,³⁷ abolished GW501516-induced proliferation and tube formation. Our findings do not, however, rule out effects on other angiogenic/proliferative processes. In addition to the induction of VEGF, we also find GW501516 induces mRNA for matrix metalloproteinase-9 in a PPARß/-dependent manner (supplemental Figure III).

Previous studies have observed that PPAR and PPAR- inhibit endothelial cell growth and angiogenesis.¹ PPAR ligands suppress HUVEC differentiation in 3-day collagen gels in vitro, and inhibit VEGF-induced angiogenesis in rat cornea in vivo.³⁸ PPARß/ has an opposite effect, protecting endothelial cells from oxidant stress mediated apoptosis, via upregulation of 14 to 3–3 protein,³² and leading to the increases in proliferation and angiogenesis we observe. Furthermore, with the recent findings of Liou et al,³² it is also tempting to speculate¹⁰ that endogenous prostacyclin may regulate endothelial cell proliferation and angiogenesis through PPARß/.

PPARß/ induces endothelial cell proliferation and angiogenesis (Figure 6). Hypercholesterolemia is associated with an increase in circulating VEGF,³⁹ but can also be associated with a reduced angiogenesis.⁴⁰ Testing GW501516 on angiogenic responses in dyslipidemia would therefore be of great interest. Nonetheless, our findings indicate regulating PPARß/ may be a novel therapeutic intervention for inflammation, wound healing, cancer, or ischemia, in which inappropriate angiogenesis occurs or therapeutic angiogenesis is required. Care and a fuller understanding of its effects may also be required for the long term use of PPARß/ ligands such as GW501516 for conditions such as dyslipidemia, especially with patients susceptible to "angiogenic" diseases, eg, diabetic individuals who are prone to retinopathy or individuals predisposed to cancer.

View larger version (16K): [in this window] [in a new window]   Figure 6. PPARß/ induces endothelial cell proliferation and angiogenesis through VEGF. Activation of endothelial cell PPARß/ (PPARß/: RXR heterodimer) with the selective PPARß/ ligand GW501516 induces mRNA for the PPARß/ target gene adipose differentiation related protein (ADRP) and VEGF. PPARß/ activation induces endothelial cell proliferation in monolayers and angiogenesis in 3-day culture. PPARß/-induced cell proliferation and angiogenesis is blocked by the VEGF receptor 1/2 antagonist cyclo-(c)VEGFI.

	Acknowledgments

 
Sources of Funding

This work was funded in part by the European Community FP6 funding (LSHM-CT-2004-0050333). Dr Laura Piqueras holds a postdoctoral grant from the Spanish Ministry of Education and Science. Dr Bishop-Bailey is the recipient of a British Heart Foundation Basic Science Lectureship (BS/02/002).

Disclosures

None.

	Footnotes

 
Original received May 16, 2006; final version accepted October 10, 2006.

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