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VASCULAR BIOLOGY
1Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany; 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas
Submitted 28 January 2008 ; accepted in final form 9 September 2008
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ABSTRACT |
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cytochrome P-450; matrigel plug assay
The realization that EETs, in particular 11,12- and 14,15-EET, can activate large conductance Ca2+-activated K+ channels on vascular smooth muscle cells to elicit hyperpolarization and relaxation led to their identification as a class of "endothelium-derived hyperpolarizing factor or EDHF" (2, 13). However, although CYP epoxygenase activation and EET production is generally associated with vasodilatation, the different EETs exert a variety of membrane potential-independent effects and are now recognized as intracellular signaling molecules and have been attributed with anti-inflammatory, fibrinolytic and anti-apoptotic properties (for review see Ref 31). One of the more recently explored functions of these enzymes is their ability to promote endothelial cell proliferation (4, 16, 28, 43) as well as endothelial cell migration and degradation of the extracellular matrix (29). The mechanisms underlying these actions of the EETs have not been completely resolved but are reported to involve activation of several signaling pathways, including activation of tyrosine kinases and phosphatases (16, 22), activation of mitogen-activated protein (MAP) kinase phosphatases, and inhibition of the c-Jun NH2-terminal kinase (35) as well as transactivation of the epidermal growth factor receptor (6, 30).
The endogenous expression of CYP epoxygenases can be modulated by different stimuli, in particular, hemodynamic and physiochemical forces are thought essential to maintain CYP2C expression in endothelial cells in situ (14). Hypoxia is also an effective inducer of CYP2C8/9 expression, a mechanism that contributes to hypoxia-induced angiogenesis in human endothelial cells (29). However, nothing is known about the involvement of CYP2C epoxygenases in the signaling pathways activated by other growth factors.
The vascular endothelial growth factor (VEGF) is a key regulator of physiological and pathological angiogenesis. In vitro, VEGF induces endothelial cell proliferation and migration and is a survival factor for endothelial cells (for review see Ref. 3). Since the expression of VEGF is dependent on hypoxia (27), we postulated a link between VEGF- and CYP-epoxygenase signaling. Therefore, in the present study we assessed the effects of VEGF on CYP2C epoxygenase expression and activity in human endothelial cells. In addition, we determined the effects of interfering with CYP activity or EET levels on VEGF-induced angiogenesis in vivo.
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METHODS |
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Cell Culture Human umbilical vein endothelial cells (30) and murine lung endothelial cells (15) were isolated and cultured as described. The porcine aortic endothelial cell lines overexpressing VEGFR1 and VEGFR2 were kindly provided by Dr. J. Waltenberger (Maastricht, The Netherlands). The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovasc Res 35: 2-41997). In some experiments subconfluent human endothelial cells were infected with adenoviruses to overexpress constitutively active AMP-activated protein kinase (AMPK) or dominant-negative AMPK as reported (10).
Immunoprecipitation and Western Blotting For immunoprecipitation human endothelial cells were lysed in Triton X-100, left on ice for 10 min, and centrifuged at 10,000 g for 10 min. After being precleared with protein A/G Sepharose, proteins were immunoprecipitated from the cell supernatant with phosphotyrosine antibody (Santa Cruz Biotechnology, Heidelberg, Germany). For Western blot analysis, cells were lysed in Triton X-100 lysis buffer and separated by SDS-PAGE as described (30).
Reporter Gene Assay Endothelial cells overexpressing either VEGFR1 or VEGFR2 were transiently cotransfected with the noncoding 5' region of CYP2C9 (–2,088 to +21; kindly provided by Dr. P. Maurel, Montpellier, France), subcloned into pGL3basic (Promega, Mannheim, Germany) and a plasmid containing lacZ under the control of the cytomegalovirus (CMV) promoter. After 12 h, the cells were treated with either solvent (PBS, 0.6%) or VEGF (30 ng/ml) for 6 h. Thereafter, luciferase (Promega, Mannheim, Germany) and β-galactosidase (Tropix, Bedford, MA) activity was assayed according to the manufacturers’ protocols. Promoter activity was quantified as luciferase activity relative to that of β-galactosidase and normalized to the respective nontreated controls.
Isolation of RNA and RT-qPCR Total RNA was isolated from cultured endothelial cells using phenol and guanidine isothiocyanate (TriReagenz, Sigma, Germany). Random hexanucleotide primers were used for reverse transcription of equal amounts of RNA. The cDNA was used for real-time PCR using Taqman probes for the detection of the specific amplification products. For the detection of CYP2C8 mRNA by real-time RT-PCR the oligonucleotides used were the following: 2C8 forward: 5'-GGACTTTATCGATTGCTTCCTG-3', reverse: 5'-CCATATCTCAGAGTGGTGCTTG-3'; FAM- and dabcyl-labeled taqman probe: 5'-TTGGCACTGTAGCTGATCTATTTGTTGCTGGA-3' corresponding to base pair positions 878 to 899 (forward); 1000 to 1021 (reverse) and 958 to 989 (taqman probe) in the CYP2C8 cDNA (NCBI accession no.: NM_000770). RNA polymerase2 (RNA Pol2) was amplified by qRT-PCR with the primers: forward: 5'-GCACCACGTCCAATGACAT-3', reverse: 5'-GTGCGGCTGCTTCCATAA-3', Hex and dabcyl-labeled Taqman probe: 5'-TACCACGTCATCTCCTTTGATGGCTCCTAT-3' corresponding to base pair positions 4301 to 4319 (forward), 4550 to 4567 (reverse) and 4381 to 4404 in RNA Pol2 cDNA (NCBI accession number NM_000937). The amount of cDNA in the samples was calculated on the basis of the amplification of a serial dilution of a plasmid (CYP2C8) or the serial dilution of the cDNA (RNA Pol2). The CYP2C8 levels were normalized to that of Pol2. At least two RT reactions were performed using each RNA preparation and at least two PCR reactions were performed with each cDNA sample.
Antisense Oligonucleotides In some experiments, an antisense oligonucleotide approach was used to prevent the VEGF-induced upregulation of CYP2C, as described (32). Cells were treated with CYP2C sense and antisense oligonucleotides (2 µmol/l; antisense: 5'-TCCATTGAAGCCTTCTCTTCTT-3'; sense: 5'-AAGAAGAGAAGGCTTCAATGGA-3'; in both cases the three 5' nucleotides were modified with phosphothioate; MWG-Biotech, Ebersberg, Germany) using GeneTrans II according to manufacturer's protocol (MobiTec, Göttingen, Germany). The sequence of these oligonucleotides spans the ATG and is 100% identical with human CYP2C8 and contains one mismatch to the other three human CYP2C isoforms.
Downregulation (siRNA) of the AMPK
To downregulate the AMPK 
-subunit in human and murine endothelial cells specific small interfering RNAs (siRNAs) were generated (Eurogentec, Searing, Belgium). The sequence used to target the human isoform was CCAAGUGGAUAGUAGAACU-dTdT, whereas a mixture or two siRNAs were used to target the murine isoform (GGACCCAUCUUAUAGUUCA-dTdT and GGUCAUCAGUACACCAUCU-dTdT; only the coding strand of the duplex is indicated). For siRNA transfection, Gene trans II was used according to the manufacturers protocol. For the Matrigel plug assay, equal amounts (2 µmol/l) of both murine siRNAs were added to Matrigel just before implantation (see below).
Liquid Chromatography-Mass Spectrometry Measurements
Human endothelial cells, treated as described in RESULTS and harvested by scraping, and the pellets (from 
8 x 106 cells) were suspended in 100 µl potassium phosphate buffer (0.1 mol/l, pH 7.2), hydrolyzed for 1 h in NaOH (0.5 N), and neutralized with HCl (2 mol/l) before deuterated internal standards (5-HETE-d8, 12-HETE-d8, 15-HETE-d8, 20-HETE-d6, 8,9-EET-d8, 11,12-EET-d8, and 14,15-EET-d8) were added. A liquid-liquid extraction was performed twice using ethyl acetate (0.5 ml). After evaporation of the solvent in a vacuum block under a gentle stream of nitrogen, samples were reconstituted with 50 µl of methanol-water (1:1, vol/vol) and eicosanoids were determined with a Sciex API4000 mass spectrometer operating in the multiple reaction monitoring mode. Chromatographic separation was performed on a Gemini C18 column (150 x 2 mm ID, 5-µm particle size, Phenomenex, Aschaffenburg, Germany).
In Vitro Angiogenesis Assays Tube formation. Primary cultures of endothelial cells were seeded on cell culture dishes coated with fibronectin and cultured in MCDB 133 medium containing 4% FCS and either solvent or VEGF (30 ng/ml). After 48 h, angiogenesis was quantified in three randomly chosen fields of view by measuring tube length with a computer-assisted microscope.
Endothelial cell spheroids. Spheroids containing 400 cells were generated as described (26). After 24 h in the collagen gel, angiogenesis was quantified by measuring the cumulative length of all capillary like sprouts originating from the central plain of an individual spheroid using a computer assisted microscope. At least five spheroids per experimental group and experiment were analyzed.
Matrigel plug assay.
Female C57BL/6 mice (8 wk old) were lightly anesthetized with chloralhydrate (200 µl of a 4% solution injected subcutaneously) and then injected subcutaneously with 0.5 ml of Matrigel impregnated with heparin (0.0025 U/ml), VEGF (150 ng/ml), bFGF (150 ng/ml), EEZE (100 µmol/l), or combinations thereof, along the dorsal midline on each site of the spine. Fourteen days later the mice were euthanized, the Matrigel plugs were removed, embedded in tissue tech (Sakura Finetec), and frozen. Plugs were sectioned (10 µm) by cryosection and processed for staining for CD31 (BD Biosciences, San Jose, CA) or -smooth muscle actin (Sigma). Afterwards preparations were mounted and viewed using a confocal microscope (LSM 510 META, Zeiss). Vessel formation was quantified by analyzing at least five five sections per plug. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), assurance number A5626-01. The angiogenic response was graded by three observers who were blind to the experimental conditions. The vessel formation index scores were as follows: 0, no effect, no invaded cells; 1, few invaded endothelial cells; 2, clear invasion of endothelial cells; 3, clear invasion of endothelial cells and capillary formation; and 4, endothelial and smooth muscle cell invasion, clear vessel formation. To facilitate comparison between the different groups the scores were normalized with respect to the effects observed in the control group.
Statistics Data are expressed as means ± SE. Statistical analysis of Matrigel plugs analyzed by ultrasound was performed using a paired t-test. For all other statistical comparisons evaluation was performed with Student's t-test for unpaired data or one-way ANOVA followed by a Bonferroni t-test. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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To more directly assess the impact of CYP2C on VEGF-induced endothelial cell sprouting in vitro, we performed a collagen-based spheroid assay and combined it with a previously described antisense oligonucleotide approach (13) to prevent the VEGF-induced increase in CYP2C expression. VEGF (30 ng/ml, 24 h) stimulated endothelial cell sprouting (Fig. 4); however, transfecting endothelial cells with CYP2C antisense oligonucleotides 14 h before generating the spheroids markedly attenuated VEGF-induced sprouting. CYP2C sense oligonucleotides used as a control were without effect on the response to VEGF (Fig. 4). Similarly, CYP2C antisense oligonucleotides also prevented VEGF-induced tube formation (Fig. 4B).
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VEGF induced a rapid (within 5 min) phosphorylation of the AMPK on Thr172. The response was biphasic with an initial peak at 10 min (813 ± 55% of control, n = 4, P < 0.001) followed by a return to basal levels and a secondary but sustained AMPK activation after 24 h (593 ± 120% of control, n = 4, P < 0.01; Fig. 5A). The overexpression of constitutively active AMPK increased CYP2C RNA levels in endothelial cells, whereas the overexpression of a dominant negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA (Fig. 5B). The dominant negative AMPK mutant also prevented the VEGF-induced increase in CYP2C protein (Fig. 5C) as well as endothelial cell tube formation (Fig. 5D). The CYP inducer phenobarbital (1 mmol/l) stimulated the phosphorylation of AMPK in endothelial cells (Fig. 5A) and increased endothelial CYP2C expression (Fig. 5C).
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-actin) were detected in the VEGF-impregnated plugs after 14 days. Only a few endothelial cells and no smooth muscle cells/pericytes were detected in the solvent- or 14,15-EEZE-containing plugs. However, 14,15-EEZE almost completely abolished the angiogenic effect of VEGF when both were applied in combination (Fig. 6A).
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Effect of AMPK Downregulation on VEGF-Induced Angiogenesis Because our data indicated that the VEGF-induced increase in CYP2C expression is dependent on the activation of the AMPK, we next assessed the consequences of AMPK downregulation on endothelial cell tube formation in vitro in human endothelial cells and endothelial cell invasion of Matrigel in vivo in mice.
The siRNA used almost abrogated AMPK expression in human endothelial cells (Fig. 7A) and prevented the VEGF-induced formation of tube-like structures (Fig. 7B). The same procedure was repeated using murine lung endothelial cells in which we compared the ability of two siRNAs to downregulate the AMPK (Fig. 7C). Both of the siRNA tested downregulated the AMPK with one of them (siRNA2) being more effective.
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DISCUSSION |
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Although interest in the CYP epoxygenases expressed in the vasculature was focused on the role played by the EETs in the regulation of membrane potential, these eicosanoids are now recognized as signaling molecules, able to stimulate several steps in the angiogenic process, including endothelial cell proliferation, migration, and tube formation (31). However, the majority of the studies performed to date have relied on overexpression systems, and little is known about the involvement of CYP epoxygenases/EETs in angiogenic pathways stimulated by classical growth factors. Because we recently reported that EETs are involved in hypoxia-induced angiogenesis (29), it seemed logical to hypothesize a potential link between VEGF and EET signaling, since the induction of VEGF by hypoxia and the role of the latter growth factor in hypoxia-induced cell migration is well documented (11). The results of our study demonstrate that endothelial cell stimulation with VEGF increases CYP2C expression and EET production and that preventing the induction or inhibiting the activity of CYP2C significantly attenuates the angiogenic response to VEGF. These observations fit well with a recent report indicating that another CYP-derived metabolite of arachidonic acid, namely 20-HETE, can be activated by VEGF in pulmonary arteries (23).
The CYP2C enzyme sensitive to VEGF was identified as CYP2C8, and we were able to show that both CYP2C8 mRNA and protein increased in the cells studied. One limitation of the present study was that we were only able to analyze the effects of VEGF on the activity of a CYP2C9 and not a CYP2C8 promoter construct. However, the enzymes are highly homologous and both have been implicated in angiogenesis. Moreover, we have frequently found similar effects (Ca2+ signaling and angiogenesis) in native endothelial cells, which express CYP2C8 and in cultured endothelial cells overexpressing CYP2C9 (18, 41). The stereochemistry of the EETs produced by these two enzymes is however quite different, thus it will be important to determine whether different effects in angiogenesis and cell signaling in general are observed using different EET enantiomers. Which of the EETs is responsible for the effects reported also remains to be clarified, and although we have repeatedly been able to reproduce the effects of CYP activation/overexpressing using 11,12-EET and not 14,15-EET (30, 41), this finding is not shared by others (28).
The importance of VEGF and its receptors in vascular development has been demonstrated in various knockout animals. For example, the deletion of either VEGFR1 (38) or VEGFR2 (19) has proven to be lethal due to abnormalities in vessel formation, therefore, it appears both receptors are essential for the development of the vasculature in the mouse embryo. Analysis of VEGFR signaling has led to the conclusion that although affinity for VEGF binding is approximately 10-fold higher for VEGFR1 than for VEGFR2, it is activation of the latter that is believed to convey the VEGF-mediated effects in endothelial cells (21). We observed however that VEGF was able to increase the activity of the CYP2C9 promoter in cells expressing either VEGFR1 or VEGFR2. Although EETs have been suggested to act as second messengers in the EGF-activated signaling cascade (5), not all growth factors are able to upregulate CYP epoxygenases, and we found that although the EET antagonist 14,15-EEZE almost abolished VEGF-induced endothelial tube formation in vitro and in vivo, it did not affect the angiogenesis induced by bFGF.
In the search to identify the molecular mechanism(s) underlying the VEGF-induced increase in CYP2C expression, we analyzed the role of the AMPK as VEGF has previously been demonstrated to activate the kinase in endothelial cells (36), and activation of AMPK has been linked to endothelial cell migration and angiogenesis (33, 34). Moreover, activation of the AMPK by phenobarbital is reported to increase the expression of CYP2B in chicken and human hepatocytes (1, 37). Consistent with these reports, both VEGF and phenobarbital stimulated the phosphorylation of the AMPK and increased in CYP2C expression in endothelial cells. Moreover, the overexpression of a dominant-negative AMPK mutant significantly reduced VEGF-induced CYP2C expression, and AMPK downregulation markedly attenuated VEGF-induced angiogenesis in vitro and in vivo. Although we found this involvement of AMPK in the VEGF-induced CYP2C expression, the remaining steps that involved in this process have not been elucidated, but LKB1 (serine/threonine kinase 11) is likely to be involved (1).
The VEGF-activated signaling cascade leading to angiogenesis has previously been linked to an increase in the formation of reactive oxygen species (9). Although the NADPH oxidase has been implicated as the source of these radicals (40), it should be noted that the activation of CYP2C epoxygenases also results in the generation of superoxide anions (O2
–) in sufficient amounts to alter the bioavailability of nitric oxide and the expression of adhesion molecules (17). To verify that CYP2C-derived EETs rather than reactive oxygen species were involved in mediating VEGF-induced angiogenesis, we employed the EET antagonist 14,15-EEZE. In contrast to the epoxygenase inhibitors MS-PPOH and sulfaphenazole, as well as the CYP2C antisense treatment, which attenuate the production of all the CYP2C products (EETs, 20-HETE, and O2–), 14,15-EEZE antagonizes only the effects of EETs without interfering with those of 20-HETE (20) or the production of O2– by CYP2C enzymes (U. R. Michaelis, unpublished observations). We found that 14,15-EEZE inhibited VEGF-induced tube formation in vitro and angiogenesis in vivo, indicating that EETs and not reactive oxygen species were responsible for the effects observed. Moreover, we were unable to detect an effect of VEGF on 20-HETE formation, indicating that 20-HETE does not contribute to angiogenesis under the conditions studied.
Taken together our data indicate the potential importance of 11,12-EET to (patho)physiological angiogenic processes. At this point it is important to point out that CYP enzymes are not only expressed in endothelial cells and CYP2C as well as CYP2J enzymes have been detected in different tumor tissues (24, 42) and can induce tumor growth as well as promote metastasis (25). It is therefore tempting to speculate that CYP enzymes might represent a new target for the treatment of tumor growth and therefore cancer therapy. Pharmacological inhibitors of some CYP isoforms have been identified as promising anti-cancer agents (7, 8); however, the majority of work published to date has focused on the consequences of CYP inhibition on the bioavailability of anti-cancer agents rather than determining the consequences of CYP inhibition per se.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* Both authors contributed equally to this study. 
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P < 0.05 vs. VEGF stimulation in cells treated with Se oligonucleotides. B: effect of CYP2C sense or antisense oligonucleotides on endothelial cell tube formation assessed after 48 h stimulation with VEGF (30 ng/ml). The images shown are representative of data obtained in three experiments, each performed using a different cell batch.
