Published online before print November 8, 2007, doi: 10.1161/CIRCRESAHA.107.155556
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© 2008 American Heart Association, Inc.
Molecular Medicine |
15-Lipoxygenase-1 Prevents Vascular Endothelial Growth Factor A– and Placental Growth Factor–Induced Angiogenic Effects in Rabbit Skeletal Muscles via Reduction in Growth Factor mRNA Levels, NO Bioactivity, and Downregulation of VEGF Receptor 2 Expression
From the Department of Biotechnology and Molecular Medicine (H.V., J.M., E.E., M.N., K.K., M.B., T.H., S.T., S.L., T.T., S.Y.-H.), A. I. Virtanen Institute for Molecular Sciences, and Department of Medicine (S.Y.-H.), University of Kuopio; and Gene Therapy Unit (S.Y.-H.), Kuopio University Hospital, Finland.
Correspondence to Seppo Ylä-Herttuala, MD, PhD, Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, PO Box 1627, FI-70211 Kuopio, Finland. E-mail seppo.ylaherttuala{at}uku.fi
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Abstract |
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Human 15-lipoxygenase-1 (15-LO-1) is an oxidizing enzyme capable of producing reactive lipid hydroperoxides. 15-LO-1 and its products have been suggested to be involved in many pathological conditions, such as inflammation, atherogenesis, and carcinogenesis. We used adenovirus-mediated gene transfers to study the effects of 15-LO-1 on vascular endothelial growth factor (VEGF)-A165– and placental growth factor (PlGF)-induced angiogenesis in rabbit skeletal muscles. 15-LO-1 significantly decreased all angiogenic effects induced by these growth factors, including capillary perfusion, vascular permeability, vasodilatation, and an increase in capillary number. The effects are attributable to the reduction in the amount of VEGF-A165 and PlGF transcripts by 15-LO-1, resulting in reduced protein expression. The most likely mediator of the VEGF family–induced capillary vasodilatation is nitric oxide (NO), which is produced by NO synthases. Endothelial NO synthase protein expression and NO synthase activity were significantly induced by VEGF-A165, and these inductions were reduced by 15-LO-1. VEGF-A165 induces its angiogenic effects primarily via vascular endothelial growth factor receptor (VEGFR)2, and also PlGF mediates angiogenic signaling via VEGFR2, even though it binds to VEGFR1. VEGFR2 expression is induced by peroxisome proliferator-activating receptor
. We showed by quantitative RT-PCR and immunohistochemistry that expression of endogenous rabbit peroxisome proliferator-activating receptor and VEGFR2 were significantly increased in the growth factor–transduced muscles, but these inductions were efficiently prevented by 15-LO-1. In conclusion, the results suggest that expression of 15-LO-1 has an efficient antiangiogenic effect in vivo via reduction in growth factor mRNA levels, NO bioactivity, and VEGFR2 expression.
Key Words: 15-lipoxygenase • vascular endothelial growth factor • vascular endothelial growth factor receptor • peroxisome proliferator-activating receptor • angiogenesis
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Introduction |
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Human 15-lipoxygenase-1 (15-LO-1) is a lipid-peroxidizing enzyme capable of introducing molecular oxygen to polyunsaturated fatty acids either in free form or in more complex compounds, such as biological membranes, phospholipids, cholesterol esters, and plasma lipoproteins.1,2 The primary 15-LO-1 products from arachidonic acid and linoleic acid are 15-hydroperoxyeicosatetraenoic acid and 13-hydroperoxyoctadecadienoic acid, respectively.1 15-LO-1 belongs to the class of reticulocyte-type lipoxygenases, which under basal conditions are mainly expressed in reticulocytes and eosinophils.2 In reticulocytes, 15-LO-1 reacts with mitochondrial membrane lipids, initiating a controlled breakdown of mitochondria during the reticulocyte maturation process.3 The physiological role of 15-LO-1 in cells other than reticulocytes still remains unknown. Even less is known about the effects of 15-LO-1 on the expression of other genes. Because of its potential to produce reactive oxygen species and lipid hydroperoxides, 15-LO-1 may act on several different signal transduction pathways and thus affect expression of a number of different genes.4 15-LO-1 and its products have been suggested to be involved in many pathological conditions, such as in skin and respiratory symptoms in allergic diseases, inflammation, atherogenesis, and carcinogenesis, but the results are controversial.2,5,6
Vascular endothelial growth factors (VEGFs) play a pivotal role in physiological angiogenesis, but they also induce angiogenesis in many pathological conditions, such as carcinogenesis and circulatory diseases.7,8 Our group has previously used adenovirus-mediated gene transfer to study the angiogenic effects of different VEGF family members in mice,9 rabbits,10–13 and pigs.14 In this work, we studied the effects of 15-LO-1 on VEGF-A165– and placental growth factor (PlGF)-induced angiogenic effects in rabbit skeletal muscles after adenovirus-mediated intramuscular gene transfers. We show that 15-LO-1 very efficiently prevents all angiogenic effects induced by these growth factors. We also provide molecular evidence at the level of gene expression to explain potential mechanisms behind these changes.
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Materials and Methods |
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Recombinant adenoviruses containing human 15-LO-1,15 human VEGF-A165,10 mouse PlGF-2,12 and the mature, short form of human VEGF-D (VEGF-D
NC)10 were produced in 293 cells16 by homologous recombination as described.17 Recombinant adenovirus containing nuclear targeted Escherichia coli lacZ gene was used as a control.17 Functionality of the transduced 15-LO-1 protein was confirmed by measuring 15-LO-1 enzyme activity from in vitro–transduced rabbit abdominal aortic smooth muscle cells (RAASMCs) by gas chromatographic hydroxy fatty acid analysis18 as described.19
New Zealand white rabbits (n=30) were anesthetized as described.13 Intramuscular injections of 1.0x1011 virus particles of each recombinant adenovirus (AdlacZ, Adh15-LO-1, AdhVEGF-A165, AdmPlGF-2, AdhVEGF-DNC, and combinations of Adh15-LO-1 with AdhVEGF-A165, AdmPlGF-2, or AdhVEGF-DNC [combination groups]) in a total volume of 1 mL divided into 10 separate 100-µL injections were performed into the semimembranosus thigh muscles. The animals were euthanized 6 days after gene transfer, when the maximal gene transfer effects are detected.20 Perfusion fixation was performed for some animals20 to obtain tissue samples for modified Miles assay and immunohistochemistry, and another set of animals was used to get snap-frozen, fresh tissue samples for RNA, protein, and enzyme activity analyses. Capillary perfusion was measured with contrast-enhanced ultrasound (CEU)21 and vascular permeability with modified Miles assay.20 Capillary mean area (µm2) and capillary density (capillaries/myocytes) were measured from CD31-immunostained sections.20 Nitration of protein tyrosines was detected by mouse monoclonal anti-nitrotyrosine antibody, clone 1A6 (Upstate, Lake Placid, NY; dilution, 1:50)22 and NO synthase (NOS) activity was measured as NADPH diaphorase activity.23 Expression of the transduced VEGF-A and 15-LO-1 proteins and endogenous endothelial (e)NOS, peroxisome proliferator-activating receptor (PPAR)-, and VEGFR2 were detected by immunohistochemistry. A detailed description of the immunostainings is presented in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
The production of the transduced human VEGF-A165 and mouse PlGF-2 proteins was quantified by ELISA (Quantikine human VEGF and Quantikine mouse PlGF-2, R&D Systems, Minneapolis, Minn) after homogenization of the muscles in T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, Ill). Total protein concentrations of the tissue homogenates were measured with the BCA Protein Assay Kit (Pierce, Rockford, Ill). RT-PCR was used to analyze the mRNA expression of the transduced genes. The RT-PCR primers and reaction conditions are described in detail in the expanded Materials and Methods section in the online data supplement. Quantitative RT-PCR was used to quantify the mRNA expression of endogenous rabbit PPAR- and VEGFR2. A detailed description of the quantitative RT-PCR is presented in the expanded Materials and Methods section in the online data supplement.
All animal experiments were approved by the Experimental Animal Committee, University of Kuopio.
Results are presented as means±SD. Statistical significance was evaluated by GraphPad Prism 4.00 software package using 1-way ANOVA, followed by the Newman–Keuls multiple comparison test or using the Kruskal–Wallis test when necessary. A value of P<0.05 was considered statistically significant.
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Results |
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Adh15-LO-1 Recombinant Adenovirus Produces Enzymatically Active 15-LO-1
Verification of the functionality of the recombinant Adh15-LO-1 virus was confirmed by gas chromatographic enzyme activity analysis from in vitro–transduced RAASMCs. Significant increase in 15-LO-1 activity, measured as 13-hydroxyoctadecadienoic acid (13-HODE) production after incubation with linoleic acid, was detected 72 hour after transduction in the Adh15-LO-1–transduced cells compared with the untransduced or the AdlacZ-transduced cells (P<0.01) (Figure 1). The production levels of the side products 2- and 9-HODE did not differ between the groups. The proportion of 13-HODE of all the HODEs detected was 78% in the Adh15-LO-1–transduced cells.
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Adh15-LO-1 Prevents AdhVEGF-A165–, AdmPlGF-2–, and AdhVEGF-DNC–Induced Increases in Capillary Perfusion
AdhVEGF-A165 and AdmPlGF-2 gene transfers induced significant 16- and 30-fold increases, respectively, in the CEU perfusion index (P<0.001 compared with AdlacZ-transduced muscles), whereas in the combination groups in which Adh15-LO-1 was transduced together with the respective growth factor, these inductions were prevented (P<0.001 AdhVEGF-A165 and AdmPlGF-2 versus their respective combination groups) (Figure 2 and Videos 1 through 6 in the online data supplement). The same effect was also detected with AdhVEGF-DNC (supplemental Videos 7 and 8). The CEU perfusion index of the Adh15-LO-1 group did not differ from the AdlacZ group.
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Adh15-LO-1 Prevents AdhVEGF-A165–Induced Increases in Vascular Permeability, Capillary Vasodilatation, and Capillary-to-Myocyte Ratio
AdhVEGF-A165 gene transfer resulted in significant increases in vascular permeability (52-fold) (Figure 3), the mean capillary area (24-fold) (Figure 4A and 4B), and the average number of capillaries per myocyte (1.3-fold) (Figure 4A and 4C) compared with the AdlacZ control group. All of these angiogenic effects were efficiently prevented by Adh15-LO-1. Adh15-LO-1 alone had no effect on vascular permeability, mean capillary area, or capillary/myocyte ratio.
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Total area covered by capillaries and the mean capillary size were also reduced by Adh15-LO-1 in a rabbit hindlimb ischemia model compared with muscles transduced with AdlacZ (section Rabbit Hindlimb Ischemia Study in the expanded Materials and Methods section and supplemental Figure I).
Adh15-LO-1 Reduces the Expression of Transduced VEGF-A165 and PlGF-2 mRNA, Resulting in a Significant Reduction in the Production of These Growth Factors
RT-PCR showed that the expressions of the transduced VEGF-A165 and PlGF-2 mRNAs were reduced in the combination groups compared with the AdhVEGF-A165– and AdmPlGF-2–transduced samples (supplemental Figure IIA and IID). Expression of the transduced, spliced 15-LO-1 mRNA could be clearly seen as a 129-bp product both in the Adh15-LO-1 group samples and in the combination group samples (supplemental Figure IIB and IIE). The 15-LO-1 RT-PCR also showed that there is no contaminating DNA, because the amplified PCR fragment from the Adh15-LO-1 vector DNA would have been of a different size (956 bp). Rabbit 
-actin RT-PCR showed equal expression from all samples (supplemental Figure IIC and IIF). The reduced expression of the transduced VEGF-A165 and PlGF-2 transcripts was reflected also in the human VEGF-A165 (Figure 5A) and mouse PlGF-2 (Figure 5B) ELISAs. AdhVEGF-A165 and AdmPlGF-2 gene transfers resulted in significant 94- and 425-fold inductions in the production of VEGF-A165 and PlGF-2 proteins, respectively, in the rabbit semimembranosus muscles, whereas in the combination groups, the production levels did not differ from those detected in the control AdlacZ samples or in the Adh15-LO-1 transduced samples. Reduction of VEGF-A protein production by 15-LO-1 was confirmed in vitro in human umbilical vein endothelial cells (HUVECs) (section In Vitro Study With HUVECs and Figure III in the online data supplement).
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Expression of the transduced VEGF-A and 15-LO-1 proteins was detected by immunohistochemistry showing localization of the VEGF-A protein in the muscle and the 15-LO-1 protein in the capillary endothelial cells (Figure 5C).
AdhVEGF-A165–Induced NADPH Diaphorase Activity, Nitrotyrosine Formation, and eNOS Protein Expression Are Reduced by Adh15-LO-1
AdhVEGF-A165 gene transfer resulted in a significant, 1.5-fold induction (P<0.001) in the NADPH diaphorase activity compared with the AdlacZ group (Figure 6A), and this induction was prevented in the combination group, in which the activity was at the same level as in the control AdlacZ- and Adh15-LO-1–transduced samples. Immunohistochemical detection of nitrotyrosine formation showed a significant, 21-fold increase in the amount of positive cells per millimeter squared in the AdhVEGF-A165 group compared with the control AdlacZ (P<0.001) (Figure 6B). In the combination group, the amount of nitrotyrosine-positive cells was 7-fold less than in the AdhVEGF-A165 group (P<0.001). AdhVEGF-A165 gene transfer resulted also in induced eNOS protein expression in the muscle capillaries compared with the control AdlacZ- and Adh15-LO-1–transduced muscles (Figure 6C). In the combination group muscles, the induction of eNOS protein expression was prevented by 15-LO-1 (Figure 6C).
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Reduction of VEGF-A induced eNOS expression by 15-LO-1 was confirmed in vitro in HUVECs by quantitative RT-PCR (section In Vitro Study With HUVECs and Figure IVA in the online data supplement).
AdhVEGF-A165– and AdmPlGF-2–Induced mRNA and Protein Expression of Endogenous Rabbit PPAR- and VEGFR2 Are Prevented by Adh15-LO-1
Quantitative RT-PCR analysis was used to measure the expression levels of endogenous rabbit PPAR- and VEGFR2 mRNAs in the transduced semimembranosus muscles. AdhVEGF-A165 gene transfer resulted in significant 9- and 16-fold increases in PPAR- and VEGFR2 mRNA expression, respectively (Figure 7A and 7C), and AdmPlGF-2 gene transfer led to significant 3- and 6-fold inductions in the mRNA expression levels of these genes (Figure 7B and 7D). In the combination groups, these inductions were completely blocked. Reduction of VEGF-A–induced VEGFR2 expression by 15-LO-1 was confirmed in vitro in HUVECs by quantitative RT-PCR (section In Vitro Study With HUVECs and Figure IVB in the online data supplement).
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Protein expression of endogenous PPAR- and VEGFR2 were studied by immunohistochemistry from the AdhVEGF-A165 gene transfer groups. The expression of both of these proteins was induced by AdhVEGF-A165 compared with the control AdlacZ and Adh15-LO-1 samples (Figure 7E). In the combination group samples, these inductions were prevented by 15-LO-1 (Figure 7E).
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Discussion |
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We have previously used the rabbit hindlimb model to study angiogenic effects of the different members of the VEGF family10 and shown that both VEGF-A165 and PlGF induce strong angiogenic effects in skeletal muscles. 15-LO-1 and its product 13-HODE have been implicated to be antitumorigenic in some cancer studies, but controversial results also have been presented.24,25 Recently, in a transgenic mouse model overexpressing human 15-LO-1, inhibition of tumor growth with increased apoptosis was reported. The potential antitumorigenic effect of 15-LO-1 could also be related to angiogenesis.26 However, the effect of 15-LO-1 on angiogenesis induced by VEGF is not known. Therefore, we wanted to clarify the effects of 15-LO-1 on the induction of angiogenesis by VEGF family members in the well-established rabbit hindlimb model. The study was primarily focused on animal experiments, because mechanistic studies in cell cultures may not truly reflect the net effects of transduced growth factors in vivo, especially in the situation where the majority of the transduced cells (muscle cells, fibroblasts) are different from the cells in which the effects are detected (vascular wall, endothelium) and in which end products of the 15-LO-1 activity are hydro(pero)xy lipid derivatives capable of interacting with reactive compounds, such as NO, produced by several cell types in the transduced muscles.
One of the most important mediators of the VEGF-induced angiogenic effects is NO, a highly diffusible free radical formed from L-arginine by NOS enzymes.27 eNOS is an important regulator of vascular permeability.28,29 Multiple studies exploring the role of NO in endothelial cell proliferation have been performed using chemical NO donors and NOS inhibitors, but the results are very controversial. Recently, other approaches have been undertaken to study the effects of endogenously produced NO on endothelial cell proliferation. Overexpression of eNOS in an in vitro coculture model30 and in pig myocardium31 support the idea that eNOS induces endothelial cell proliferation, whereas another study using adenoviral-mediated gene transfer of eNOS32 suggests that NO is antiproliferative in endothelial cells. However, studies with eNOS knockout mice show that eNOS is required for proper endothelial cell migration, proliferation, and differentiation and plays a significant role in VEGF-induced angiogenesis and vascular permeability.28,33–35
Our results show that VEGF-A165 and PlGF-2 gene transfers resulted in increased capillary perfusion, permeability, vasodilatation, and capillary number, compared with the AdlacZ control group, and 15-LO-1 completely abolished all of the angiogenic effects induced by VEGF-A165 and PlGF in the rabbit skeletal muscle. Endothelial NO is required in arteriolar vasodilatation,36 and stimulation of NO release both endogenously and exogenously can increase also capillary diameters.37 We have shown in our previous studies10,21 that AdhVEGF-A165 gene transfer induces arteriogenesis, as nearly all enlarged capillaries in the AdhVEGF-A165–transduced muscles have a pericyte coverage positive for -smooth muscle actin. Therefore, it is likely that the capillaries, which have been shifted toward an arteriolar phenotype, can undergo NO-mediated vasodilatation.
VEGF-A induces the production of NO via VEGFR2.38–40 The AdhVEGF-A165–induced expression of both VEGF-A and VEGFR2 were prevented by 15-LO-1, resulting in the attenuation of the downstream effects mediated by NO, as VEGF-A–induced eNOS protein expression, NOS activity, and nitrotyrosine production were reduced in the combination gene transfer group (Figure 6). 15-LO-1 can also affect the bioavailability of NO by catalytically consuming it.41,42
The expressions of the transduced VEGF-A165 and PlGF-2 were reduced in the combination groups already at the mRNA level, resulting in a very low production of human VEGF-A and mouse PlGF-2 proteins. 15-LO-1 might affect the production of the transduced human VEGF-A165 and mouse PlGF-2 either by preventing their transcription or by destabilizing the transcripts, or both. VEGF-A165 mRNA production and stability are both highly regulated. VEGF-A165 transcription is induced in hypoxia by hypoxia-inducible factor 1,43 and the mRNA is stabilized by several factors binding to the 3' untranslated region (UTR).44–48 PlGF-2 transcription is induced in hypoxia as well, by metal transcription factor-149 and BF-2.50 However, our constructs lack all of the regulatory elements of the endogenous human VEGF-A165 and mouse PlGF-2, containing only the protein-coding regions under the cytomegalovirus (CMV) promoter. It is thus unlikely that the reduction in the mRNA expression of the transduced growth factors would be caused by blocking the CMV promoter, because the human 15-LO-1 cDNA in our adenovirus construct is also under the same CMV promoter, and there was no reduction in the expression of the transduced human 15-LO-1 in the combination groups. Thus, the most likely explanation is that 15-LO-1 expression leads to destabilization of the growth factor transcripts by an as yet unidentified mechanism that would affect the coding sequences of these growth factors.
To our knowledge, this is the first study reporting that VEGF family growth factors induce the mRNA and protein expression of PPAR-. The growth factor–induced expressions of PPAR- and VEGFR2 were completely blocked in the combination groups. The 15-LO-1 product 13-HODE is an endogenous activator and ligand of PPAR-.51 Interestingly, it has been shown in a rabbit eye model that PPAR- binding to VEGFR2 promoter induces VEGFR2 expression, but ligand binding to PPAR- actually results in an inhibition of VEGFR2 expression.52 We hypothesize, that 15-LO-1 product(s) binding to PPAR- in the rabbit skeletal muscle could prevent the AdhVEGF-A165– and AdmPlGF-2–induced expression of VEGFR2. It has also been shown that PPAR- ligands inhibit choroidal53 and corneal neovascularization.54–56 Thus, the effect on VEGFR2 could be an additional mechanism whereby 15-LO-1 prevents the angiogenic effects of AdhVEGF-A165 and AdmPlGF-2.
In conclusion, we have shown that Adh15-LO-1 gene transfer prevents all angiogenic effects induced by AdhVEGF-A165 and AdmPlGF-2. The mechanism for this blocking effect seems to be the reduction of the growth factor expression at the mRNA level by an as yet unidentified mechanism associated with reduction of NO bioactivity and decreased expression of VEGFR2 (Figure 8).
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Acknowledgments |
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We thank Mervi Nieminen, Anne Martikainen, Sari Järveläinen, Tiina Koponen, Seija Sahrio, and Marja Poikolainen for technical assistance.
Sources of Funding
This study was supported by grants from the University of Kuopio; the Finnish Foundation for Cardiovascular Research; the Finnish Academy; and the European Vascular Genomics Network, funded by the European Union (LSHM-CT-2003-503254). We also thank Ark Therapeutics Oy for supporting this study.
Disclosures
None.
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Footnotes |
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Original received December 11, 2006; resubmission received May 7, 2007; revised resubmission received October 9, 2007; accepted October 30, 2007.
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References |
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