Essential Role of Vascular Endothelial Growth Factor and Flt-1 Signals in Neointimal Formation After Periadventitial Injury
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《动脉硬化血栓血管生物学》
From the Department of Cardiovascular Medicine (Q.Z., K.E., K.H., M.I., S.I., K.O., C.T., A.T., K.S.), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and the Department of Genetics (M.S.), Institute of Medical Science, University of Tokyo, Japan.
ABSTRACT
Objective— Vascular endothelial growth factor (VEGF) is upregulated after arterial injury. Its role in the pathogenesis of neointimal formation after periadventitial injury, however, has not been addressed.
Methods and Results— Expression of VEGF and its receptors but not that of placental growth factor markedly increased with the development of neointimal formation in hypercholesterolemic mice after cuff-induced periarterial injury. Transfection with the murine soluble Flt-1 (sFlt-1) gene to block VEGF in vivo in mice inhibited early inflammation and later neointimal formation. The sFlt-1 gene transfer did not affect plasma lipid levels but attenuated increased expression of VEGF, Flt-1, Flk-1, monocyte chemoattractant protein-1, and other inflammation-promoting factors. Mice with Flt-1 kinase deficiency also displayed reduced neointimal formation.
Conclusions— Inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury. VEGF might promote neointimal formation by acting as a proinflammatory cytokine.
We examined the role of vascular endothelial growth factor (VEGF) in the pathogenesis of neointimal formation after cuff-induced periadventitial injury in mice. Blockade of VEGF by transfecting mice with the sFlt-1 gene inhibited neointimal formation associated with reduced expression of various inflammation-promoting factors. Therefore, VEGF might promote neointimal formation by acting as a proinflammatory cytokine after cuff-induced periadventitial injury.
Key Words: remodeling ? growth substances ? inflammation ? arteriosclerosis ? gene therapy
Introduction
Neointimal formation is a major cause of restenosis after coronary intervention.1,2 Vascular endothelial growth factor (VEGF) and its receptors (VRGFR-1: Flt-1, VEGFR-2: Flk-1) are upregulated in vascular inflammatory and proliferative disorders such as atherosclerosis and restenosis.3–6 VEGF is thought to protect the artery from such disorders by inducing endothelial regeneration and improving endothelial function.7 VEGF gene transfer or administration of its protein induces endothelial regeneration and attenuates neointimal formation after endothelial injury.7–9 VEGF is reported to inhibit leukocyte infiltration through hemeoxygenase-1.10 There is still considerable debate, however, over the role of VEGF in the development of neointimal formation after injury.11,12 Emerging evidence suggests that VEGF causes or promotes the development of atherosclerosis or neointimal formation after injury. VEGF induces migration and activation of monocytes,13 adhesion molecules,14 or monocyte chemoattractant protein-1 (MCP-1)15 through its receptor Flt-1. Moreover, administration of VEGF protein to hypercholesterolemic animals enhances atherogenesis by inducing monocyte infiltration and activation.16 In addition, VEGF might promote migration of vascular smooth muscle cells though Flt-1.17,18 Angiogenesis inhibitors are shown to reduce intimal neovascularization and plaque growth in hyperlipidemic mice.19
One major reason for the inconsistent reports regarding the role of VEGF might be because there are no selective VEGF inhibitors tested. The only known endogenous VEGF inhibitor is a soluble form of the VEGF receptor-1, Flt-1 (sFlt-1).20 This isoform is mainly expressed by vascular endothelial cells and can inhibit VEGF activity by directly sequestering VEGF and by functioning as a dominant-negative inhibitor.20 We and others previously demonstrated that intramuscular transfection of the sFlt-1 gene blocks VEGF signaling and thus quenches VEGF activity in vivo.21,22 Therefore, sFlt-1 gene transfer can be used as an inhibitor against VEGF and its receptors (Flt-1, Flk-1). In addition, Flt-1 tyrosine kinase–deficient mice can be used to determine the role of Flt-1 signals.23
In this study, we investigated the role of VEGF and Flt-1 signals in the pathogenesis of neointimal formation after cuff-induced periadventitial injury in hypercholesterolemic mice. Several animal models for evaluation of neointimal formation after injury have been reported, including balloon injury, wire injury, chemical injury, and cuff injury, among others. The ideal animal model for human neointimal formation is uncertain. The cuff model was chosen because cuff placement in the presence of hypercholesterolemia offers the advantage of inducing reproducible site-controlled neointimal formation and stenosis.24,25 In addition, the cuff-induced injury triggers vascular inflammation and induces neointimal lesions that are partly similar to the restenotic and atherosclerotic lesions observed in humans.24,25 Our present data provide direct evidence suggesting that inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury.
Methods
Expression Vector
The 3.3-kb mouse sFlt-1 gene was obtained from a mouse lung cDNA library26 and cloned into the BamHI(5') and NotI(3') sites of the eukaryotic expression vector plasmid cDNA3 (Invitrogen).
Experimental Animals
The study protocol was reviewed and approved by the Committee on Ethics for Animal Experiments, Kyushu University Faculty of Medicine, and the experiments were conducted according to the Guidelines of the American Physiological Society. A part of this study was performed at the Kyushu University Station for Collaborative Research.
Apolipoprotein E–deficient (apoE-KO) and wild-type mice (8 to 10 weeks old, n=5 to 9 each group) with a genetic background of C57BL/6J were purchased from The Jackson Laboratory (Bar Harbor, Me) and fed with commercial standard chow. Placement of cuff and gene transfer were performed as previously described.21,27 A nonconstrictive polyethylene cuff (1.5 mm long; PE20, 0.38-mm inner diameter, 1.09-mm outer diameter) was placed loosely around the left femoral artery. Either empty plasmid or sFlt-1 plasmid (300 μg/100 μL PBS) was injected into the right femoral muscle using a 27-gauge needle immediately and 10 days after cuff placement. To enhance transgene expression, these animals received electroporation at the injected site immediately after injection.21,27–29 It has been shown that electroporation-mediated gene transfer is useful to introduce genes into muscle tissues in vivo with no serious tissue injury.30 To determine the role of flt-1 signals, Flt-1 tyrosine kinase–deficient mice with a genetic background of C57BL/6J were used.23
In Vivo Matrigel Plug Assay
An in vivo matrigel plug assay was used to determine the effect of sFlt-1 gene transfer on VEGF activity.21,27 Matrigel matrix alone (300 μL) or mixed with recombinant VEGF protein (100 ng/mL) was injected subcutaneously into the flanks of C57BL/6J mice. The matrigels were then removed 7 or 14 days after injection, and angiogenesis and inflammation were examined by histopathologic analysis.
Histopathology, Immunohistochemistry, and Morphometry
Mice were anesthetized with pentobarbital, and the femoral artery was harvested, fixed overnight in 3.7% formaldehyde in PBS, and paraffin-embedded.27 Serial cross sections (5 μm thick) throughout the entire length of the cuffed femoral artery were used for histological analysis. Cryosections were made from 2 mice in each condition. All sections were routinely stained with hematoxylin-eosin or van Gieson. Mac-3 (PharMingen) staining was used to detect monocytes/macrophages, and CD3 (Santa Cruz Biotechnology) was used for T cells. Proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) was used to detect vascular proliferation. An antibody against von Willebrand factor (vWF; Sigma Chemical Co) was used to mark endothelial cells. Antibodies against VEGF (Santa Cruz Biotechnology) and placental growth factor (PlGF; R&D Systems Inc) were also used. Indirect immunofluorescence double-staining with matched primary and fluorescein-conjugated secondary antibodies was used to stain for colocalization with VEGF receptors in smooth muscle cells or monocytes as follows: Rabbit anti-mouse Flt-1 (Santa Cruz Biotechnology), rabbit anti-mouse Flk-1 (Santa Cruz Biotechnology), rat anti-mouse Mac-3, anti–-smooth muscle actin (-SMA; Boehringer Mannheim Corp), anti-rabbit IgG conjugated with fluorescein isothiocyanate or rhodamine, and anti-rat IgG conjugated with fluorescein isothiocyanate or rhodamine (Santa Cruz Biotechnology).
Ten equally-spaced cross sections were examined in all mice to quantify intimal lesions. Using image analysis software, the total cross-sectional medial area was measured between the external and internal elastic lamina; the total cross-sectional intimal area was measured between the endothelial cell monolayer and the internal elastic lamina.
Plasma Measurements
Plasma total cholesterol and triacylglycerol levels were determined with commercially available kits (Wako Pure Chemicals). Plasma concentrations of sFlt-1, VEGF, and PlGF were measured by the use of ELISA kit (R&D Systems Inc).
RT-PCR and RNAse Protection Assay
RNA was prepared from the pooled samples (5 to 7 arteries for 1 sample).21 First-strand DNA was synthesized using reverse transcriptase with random hexamers from 1 μg total RNA in a 20-μL reaction volume according to the manufacturer’s protocol (GeneAmp RNA polymerase chain reaction Kit; Perkin–Elmer). Primers used for amplification of VEGF were 5'-GGA TCC ATG AAC TTT CTG CT-3' and 5'-GAA TTC ACC GCC TCG GCT TGT C-3' with expected sizes of 654, 582, and 450 bp for the 3 VEGF isoforms (VEGF 188, 164, and 120, respectively). Primers for PlGF were 5'-CCC ACA CCC AGC TCA CGT ATT TA-3' and 5'-TCC CCT CTA CAT GCC TTC AAT GC-3'. Primers for Flk-1 were 5'-ACT GCA GTG ATT GCC ATG TTC T-3' and 5'-GCT CAT CCA AGG GCA ATT CAT-3'. Primers for the internal control, ?-actin, were 5'-ATG GAT GAC GAT ATC GCT-3' and 5'-ATG AGG TAG TCT GCT AGG T-3' with an expected product of 550 bp.
RNAse protection assays were performed using 5 μg total RNA with 2 custom template sets according to the manufacturer’s protocol (PharMingen).27
Statistical Analysis
Data are expressed as the mean±SE. Statistical analysis of differences was compared by ANOVA. Post hoc analyses were performed using Bonferroni correction for multiple comparison tests. P<0.05 was considered to be statistically significant.
Results
In Vivo Matrigel Plug Assay in ApoE-KO Mice
Seven days after injection of matrigel, there were significant angiogenic (number of CD31-positive cells per mm2) and inflammatory (number of Mac3-positive cells per mm2) reactions in the matrigel plugs containing recombinant VEGF protein compared with matrigel alone. Soluble Flt-1 gene transfer but not injection of an empty plasmid suppressed both the angiogenic and inflammatory reactions to VEGF to a level similar to that of matrigel plugs without VEGF (Figure I, available online at http://atvb.ahajournals.org). This suppression of angiogenic and inflammatory reactions to VEGF was noted on day 14 but not on day 21 of sFlt-1 gene transfer (data not shown).
Gene Expression and Immunoreactivity in ApoE-KO Mice
The mRNA levels of 2 VEGF isoforms (188 and 164) markedly increased after cuff placement, whereas they were undetectably low in control intact artery (Figure 1A and 1B). Peak expression was observed on day 7. VEGF 121 mRNA was undetectable before and after cuff placement. Gene expression of Flt-1, Flk-1, VEGF, and PlGF was also increased on day 7 (Figure 1C). Plasma concentrations of sFlt-1, VEGF, and PlGF were measured on day 7 in several groups of animals (Figure 1D). Plasma sFlt-1 was increased in sFlt-1 transfection group. There was no significant change in plasma VEGF nor in PlGF among groups (Figure 1D).
Figure 1. Gene expression VEGF, Flt-1, Flk-1, and PlGF in cuffed femoral artery. A, Time course of VEGF mRNA levels (RT-RCR) and expression of arterial VEGF and ?-actin mRNA after cuff placement. mRNA levels were assessed at the indicated times. This is a representative assay from 5 separate experiments. B, Densitometric analysis of data in A. Expression of VEGF mRNA in each sample was normalized by ?-actin mRNA expression in the same sample. N=5 for each bar. *P<0.01 vs control intact artery. C, Gene expressions of Flt-1, Flk-1, PlGF, and VEGF (RT-PCR) in femoral arteries before or 7 days after cuff placement/sFlt-1 gene transfer. *P<0.01 vs intact control. D, Plasma levels of sFlt-1, PlGF, and VEGF 7 days after cuff placement in 4 animal groups: (1) untreated control, (2) mice with cuff alone, (3) cuff+empty plasmid, and (4) cuff+sFlt-1 plasmid. N=6 for each. *P<0.01 vs control group.
Immunohistochemical staining indicated that compared with faint staining in the control artery, VEGF increased in the vicinity of inflammatory lesions (mononuclear cell infiltration) in the intima and adventitia on day 7 and in cells of 3 layers of cuffed artery on day 21 (Figure 2A). The endothelial layer, as detected by vWF staining, was preserved before and after cuff placement (Figure 2A). No detectable increase in PlGF staining was observed before and after cuff placement (Figure 2B).
Figure 2. Immunostaining of VEGF, Flt-1, Flk-1, and PlGF in cuffed femoral artery. A, Cross sections of intact or cuffed femoral arteries were stained immunohistochemically against VEGF, VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flk-1), or vWF 7 and 21 days after cuff placement in empty plasmid group. Immunohistochemical sections of cuffed arteries on day 21 in sFlt-1 group are also shown. Black lines indicate internal and external elastin laminas. L indicates lumen. Scale bar=50 μm. B, Immunohistochemical staining of PlGF in femoral arteries before or after cuff placement/sFlt-1 gene transfer. Black lines indicate internal or external elastin laminas. L indicates lumen. Scale bar=50 μm.
Both Flk-1 and Flt-1 were undetectable, except in endothelial layers in control intact arteries, but both were increased in the intima, media, and adventitia 7 and 21 days after cuff placement (Figure 2A). Compared with Flt-1 staining, Flk-1 staining was less impressive on day 7 but was apparently noted on day 21. To localize VEGF receptors, immunofluorescent double-staining was performed (Figure 3). On day 7, -SMA–positive cells in the media and neointima expressed little Flk-1, whereas they did express Flt-1 (Figure 3A). Also, some -SMA–positive cells in the adventitia (possibly adventitial myofibroblasts) expressed Flt-1. Mac-3 positive cells recruited to the neointima, media, and adventitia expressed VEGF and Flt-1. On day 21, most -SMA–positive cells in the neointima and media expressed VEGF and its receptors (Figure 3B).
Figure 3. Immunofluorescence double-staining of VEGF receptors, monocytes, and -SMA in cuffed femoral artery. A, Micrographs of cuffed femoral arteries doubly stained with Flt-1 (VEGF-R1, green) and -SMA (red), with Flk-1 (VEGF-R2, green) and -SMA (red), and with Mac-3 (green) and Flt-1 (VEGF-R1, red) 7 days after cuff placement. Scale bar=50 μm. B, Micrographs of cuffed femoral arteries doubly stained with Flt-1 (VEGF-R1) and -SMA, Flk-1 (VEGF-R2) and -SMA, and VEGF and -SMA in the cuffed femoral arteries 21 days after cuff placement. Single fluorescence–positive cells were stained green or red, whereas double-positive cells were stained yellow. White lines indicate external elastin laminas. L indicates lumen. Scale bar=50 μm.
Time Course of Development of Neointimal Hyperplasia in ApoE-KO Mice
As published,24,27,31,32 within 7 days of cuff placement, mononuclear leukocytes, most of which were Mac3-positive monocytes, were recruited into the adventitia, media, and intima (Figure 3). After 7 days, neointimal lesions developed and became thick over time (Figure 4A). Monocyte infiltration declined spontaneously and -SMA–positive cells appeared predominantly in the neointima. On day 21, significant neointimal formation with luminal stenosis developed (Figure 4A). Endothelial staining with vWF antibody showed that no significant neointimal neovascularization was observed during the course of experiments (Figure 2A).
Figure 4. Histopathology of cuffed femoral artery. A, Time course of cuff injury–induced neointimal formation and effect of sFlt-1 gene transfer. Micrographs of cross sections of control (intact) and cuffed arteries stained with van Gieson Elastica on days 3, 7, and 21 are shown. Scale bar=100 μm. B through D, Effects of sFlt-1 gene transfer on neointimal thickening (B), intima/media ratio (C), and % stenosis (D) 21 days after cuff placement. *P<0.01 vs cuff only and cuff+empty plasmid group.
Effects of Soluble Flt-1 Gene Transfer on Cuff-Induced Neointimal Hyperplasia in ApoE-KO Mice
As we previously reported,27 Mac3-positive monocytes/macrophages and PCNA-positive cells were detected mainly in the adventitia and intima. There was markedly less inflammation (Mac3-positive cells) and proliferation (the PCNA index) in sFlt-1–transfected mice than in empty plasmid-transfected mice on day 7 (Figure II, available online at http://atvb.ahajournals.org). There was no detectable change in the number of CD3-positive T cells in empty plasmid or sFlt-1–transferred mice (Figure II). The sFlt-1 gene transfer significantly reduced neointimal formation (increases in neointimal area, intima/media ratio, and luminal stenosis) 21 days after cuff placement (Figure 4A through 4D).
Because sFlt-1 gene transfer markedly reduced monocyte-mediated inflammation, gene expression of a battery of inflammatory cytokines, chemokines, and chemokine receptors was examined by RNAse protection assays (Figure III, available online at http://atvb.ahajournals.org) or by RT-PCR (Figure 1C) 7 days after cuff placement. The sFlt-1 gene transfer did not affect gene expression of RANTES, macrophage inflammatory protein-1, transforming growth factor-?, macrophage inflammatory protein-2, and PlGF, but prevented or attenuated the increased gene expression of CCR1, interleukin-6, CCR2, MCP-1, Flt-1, CXCR2, eotaxin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, Flk-1, and VEGF. The sFlt-1 gene transfer reduced increased immunostainings against VEGF, Flt-1, and Flk-1, but did not affect staining against vWF (Figure 2A).
Time course of plasma concentrations of sFlt-1 after sFlt-1 gene transfer was determined. Plasma sFlt-1 levels before and 3, 7, and 14 days after sFlt-1 transfection were 467±37, 1037±132 (P<0.01 versus baseline), 927±215 (P<0.01), and 649±83 pg/mL (P<0.05, n=6 each), indicating that sFlt-1 was released from the transfected muscle.
Plasma Lipid Levels in ApoE-KO Mice
Total cholesterol and triacylglycerol levels were 503±11 and 38±6 mg/dL in the control group, 512±16 and 40±5 mg/dL in the empty plasmid group, and 497±10 and 39±3 mg/dL in the sFlt-1 group, indicating that the observed effects of sFlt-1 gene transfer were not caused by changes in serum lipid levels.
Effects of Flt-1 Tyrosine Kinase Deficiency on Neointimal Hyperplasia
Wild-type and Flt-1 tyrosine kinase–deficient mice were fed a high-fat diet for 2 weeks, and cuff was placed as mentioned above. Mice received a high-fat diet for an additional 3 weeks. Neointimal formation was noted 21 days after cuff placement in wild-type mice. Compared with wild-type mice, Flt-1 tyrosine kinase–deficient mice displayed reduced neointimal formation (Figure IV, available online at http://atvb.ahajournals.org). Total cholesterol levels at 5 weeks of high-fat diet were 520±21 and 511±18 mg/dL in wild-type and Flt-1 tyrosine kinase–deficient mice, respectively.
Discussion
This study is the first to demonstrate the essential role of VEGF and Flt-1 signals in the development of neointimal formation after cuff-induced periadventitial injury in hypercholesterolemic mice. VEGF is conventionally thought to be an endothelial cell–specific growth factor and to attenuate vascular disease by inducing endothelial proliferation and regeneration mainly through the endothelial type 2 receptor Flk-1.7 Recent evidence, however, suggests that functional Flt-1 and Flk-1 are expressed in injured arterial wall cells other than endothelial cells. In this study, Flt-1, Flk-1, and VEGF were increased in lesional monocytes and medial smooth muscle cells at early stages and in neointimal and medial smooth muscle cells at later stages. Flt-1 is demonstrated to act as an important mediator of chemotaxis through vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and MCP-1.13–15 We demonstrated that sFlt-1 gene transfer reduced the early inflammatory and proliferative changes and thus attenuated the development of neointimal formation. It is speculated, therefore, that VEGF might cause inflammation (monocyte recruitment and activation) and proliferation through Flt-1–mediated signals and thus cause neointimal formation after cuff-induced periadventitial injury. In addition, Flt-1 in smooth muscle cells is reported to mediate migration and proliferation in vitro.18,33 An alternative interpretation is that VEGF directly caused migration and proliferation of smooth muscle cells resulting in neointimal formation. In any way, our present finding of Flt-1 tyrosine kinase–deficient mice suggests the involvement of Flt-1–related signals in the pathogenesis of neointimal formation after periadventitial injury.
Periadventitial inflammation has a major role in the pathogenesis of cuff-induced neointimal formation.27,32 To gain insight into the mechanism of VEGF-mediated inflammation and neointimal formation, we assessed gene expression of various inflammatory genes. The sFlt-1 gene transfer attenuated increased gene expression of inflammatory cytokines, adhesion molecules, chemokines, and chemokine receptors. Yamada et al34 showed that MCP-1 is essential in VEGF-induced angiogenesis, vascular leakage, and inflammation. An essential role of MCP-1 in the development of neointimal formation after arterial injury has also been reported.27–29,35,36 The sFlt-1 gene transfer attenuated increased VEGF, Flk-1, and Flt-1 gene expression, indicating that VEGF regulates its activity by an autocrine loop mechanism within diseased arterial wall cells, such as smooth muscle cells, endothelial cells, and lesional monocytes. A positive feedback effect of VEGF is supported by previous studies that demonstrated enhanced VEGF production by monocytes through Flt-1 stimulation.37 Therefore, sFlt-1 gene transfer might attenuate cuff-induced neointimal formation mainly by suppressing inflammation (monocyte recruitment and activation).
This study has potentially significant clinical implications. Blockade of VEGF by sFlt-1 gene transfer can be an attractive anti-VEGF therapy for inflammatory vascular disease and other inflammatory disorders. The efficacy of this strategy for experimental tumor angiogenesis has already been tested.22 Luttun et al38 recently reported that treatment with anti–Flt-1 antibody attenuated the development of experimental tumor angiogenesis, arthritis, and atherosclerosis. One limitation of the present finding is that the pathogenesis of neointimal formation after periadventitial injury differs from that after endothelial injury/denudation and from that in humans. The endothelial integrity is preserved in cuff-induced periadventitial injury. For clinical application of our findings to human vascular disease, future studies are needed to examine the efficacy and safety of anti-VEGF strategy with sFlt-1 in experimental atherosclerosis and restenosis.
In conclusion, inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury. These data support the notion that VEGF promote neointimal formation by acting as a proinflammatory cytokine after cuff-induced periadventitial injury.
Acknowledgments
This study was supported by Grants-in-Aid for Scientific Research (14657172, 14207036) from the Ministry of Education, Science, and Culture by Health Science Research Grants (Comprehensive Research on Aging and Health and Research on Translational Research), from the Ministry of Health, Labor, and Welfare, and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, Tokyo, Japan.
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Ohtani K, Usui M, Nakano K, Kohjimoto Y, Kitajima S, Hirouchi Y, Li XH, Kitamoto S, Takeshita A, Egashira K. Antimonocyte chemoattractant protein-1 gene therapy reduces experimental in-stent restenosis in hypercholesterolemic rabbits and monkeys. Gene Ther. 2004; 11: 1273–1282.
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ABSTRACT
Objective— Vascular endothelial growth factor (VEGF) is upregulated after arterial injury. Its role in the pathogenesis of neointimal formation after periadventitial injury, however, has not been addressed.
Methods and Results— Expression of VEGF and its receptors but not that of placental growth factor markedly increased with the development of neointimal formation in hypercholesterolemic mice after cuff-induced periarterial injury. Transfection with the murine soluble Flt-1 (sFlt-1) gene to block VEGF in vivo in mice inhibited early inflammation and later neointimal formation. The sFlt-1 gene transfer did not affect plasma lipid levels but attenuated increased expression of VEGF, Flt-1, Flk-1, monocyte chemoattractant protein-1, and other inflammation-promoting factors. Mice with Flt-1 kinase deficiency also displayed reduced neointimal formation.
Conclusions— Inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury. VEGF might promote neointimal formation by acting as a proinflammatory cytokine.
We examined the role of vascular endothelial growth factor (VEGF) in the pathogenesis of neointimal formation after cuff-induced periadventitial injury in mice. Blockade of VEGF by transfecting mice with the sFlt-1 gene inhibited neointimal formation associated with reduced expression of various inflammation-promoting factors. Therefore, VEGF might promote neointimal formation by acting as a proinflammatory cytokine after cuff-induced periadventitial injury.
Key Words: remodeling ? growth substances ? inflammation ? arteriosclerosis ? gene therapy
Introduction
Neointimal formation is a major cause of restenosis after coronary intervention.1,2 Vascular endothelial growth factor (VEGF) and its receptors (VRGFR-1: Flt-1, VEGFR-2: Flk-1) are upregulated in vascular inflammatory and proliferative disorders such as atherosclerosis and restenosis.3–6 VEGF is thought to protect the artery from such disorders by inducing endothelial regeneration and improving endothelial function.7 VEGF gene transfer or administration of its protein induces endothelial regeneration and attenuates neointimal formation after endothelial injury.7–9 VEGF is reported to inhibit leukocyte infiltration through hemeoxygenase-1.10 There is still considerable debate, however, over the role of VEGF in the development of neointimal formation after injury.11,12 Emerging evidence suggests that VEGF causes or promotes the development of atherosclerosis or neointimal formation after injury. VEGF induces migration and activation of monocytes,13 adhesion molecules,14 or monocyte chemoattractant protein-1 (MCP-1)15 through its receptor Flt-1. Moreover, administration of VEGF protein to hypercholesterolemic animals enhances atherogenesis by inducing monocyte infiltration and activation.16 In addition, VEGF might promote migration of vascular smooth muscle cells though Flt-1.17,18 Angiogenesis inhibitors are shown to reduce intimal neovascularization and plaque growth in hyperlipidemic mice.19
One major reason for the inconsistent reports regarding the role of VEGF might be because there are no selective VEGF inhibitors tested. The only known endogenous VEGF inhibitor is a soluble form of the VEGF receptor-1, Flt-1 (sFlt-1).20 This isoform is mainly expressed by vascular endothelial cells and can inhibit VEGF activity by directly sequestering VEGF and by functioning as a dominant-negative inhibitor.20 We and others previously demonstrated that intramuscular transfection of the sFlt-1 gene blocks VEGF signaling and thus quenches VEGF activity in vivo.21,22 Therefore, sFlt-1 gene transfer can be used as an inhibitor against VEGF and its receptors (Flt-1, Flk-1). In addition, Flt-1 tyrosine kinase–deficient mice can be used to determine the role of Flt-1 signals.23
In this study, we investigated the role of VEGF and Flt-1 signals in the pathogenesis of neointimal formation after cuff-induced periadventitial injury in hypercholesterolemic mice. Several animal models for evaluation of neointimal formation after injury have been reported, including balloon injury, wire injury, chemical injury, and cuff injury, among others. The ideal animal model for human neointimal formation is uncertain. The cuff model was chosen because cuff placement in the presence of hypercholesterolemia offers the advantage of inducing reproducible site-controlled neointimal formation and stenosis.24,25 In addition, the cuff-induced injury triggers vascular inflammation and induces neointimal lesions that are partly similar to the restenotic and atherosclerotic lesions observed in humans.24,25 Our present data provide direct evidence suggesting that inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury.
Methods
Expression Vector
The 3.3-kb mouse sFlt-1 gene was obtained from a mouse lung cDNA library26 and cloned into the BamHI(5') and NotI(3') sites of the eukaryotic expression vector plasmid cDNA3 (Invitrogen).
Experimental Animals
The study protocol was reviewed and approved by the Committee on Ethics for Animal Experiments, Kyushu University Faculty of Medicine, and the experiments were conducted according to the Guidelines of the American Physiological Society. A part of this study was performed at the Kyushu University Station for Collaborative Research.
Apolipoprotein E–deficient (apoE-KO) and wild-type mice (8 to 10 weeks old, n=5 to 9 each group) with a genetic background of C57BL/6J were purchased from The Jackson Laboratory (Bar Harbor, Me) and fed with commercial standard chow. Placement of cuff and gene transfer were performed as previously described.21,27 A nonconstrictive polyethylene cuff (1.5 mm long; PE20, 0.38-mm inner diameter, 1.09-mm outer diameter) was placed loosely around the left femoral artery. Either empty plasmid or sFlt-1 plasmid (300 μg/100 μL PBS) was injected into the right femoral muscle using a 27-gauge needle immediately and 10 days after cuff placement. To enhance transgene expression, these animals received electroporation at the injected site immediately after injection.21,27–29 It has been shown that electroporation-mediated gene transfer is useful to introduce genes into muscle tissues in vivo with no serious tissue injury.30 To determine the role of flt-1 signals, Flt-1 tyrosine kinase–deficient mice with a genetic background of C57BL/6J were used.23
In Vivo Matrigel Plug Assay
An in vivo matrigel plug assay was used to determine the effect of sFlt-1 gene transfer on VEGF activity.21,27 Matrigel matrix alone (300 μL) or mixed with recombinant VEGF protein (100 ng/mL) was injected subcutaneously into the flanks of C57BL/6J mice. The matrigels were then removed 7 or 14 days after injection, and angiogenesis and inflammation were examined by histopathologic analysis.
Histopathology, Immunohistochemistry, and Morphometry
Mice were anesthetized with pentobarbital, and the femoral artery was harvested, fixed overnight in 3.7% formaldehyde in PBS, and paraffin-embedded.27 Serial cross sections (5 μm thick) throughout the entire length of the cuffed femoral artery were used for histological analysis. Cryosections were made from 2 mice in each condition. All sections were routinely stained with hematoxylin-eosin or van Gieson. Mac-3 (PharMingen) staining was used to detect monocytes/macrophages, and CD3 (Santa Cruz Biotechnology) was used for T cells. Proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) was used to detect vascular proliferation. An antibody against von Willebrand factor (vWF; Sigma Chemical Co) was used to mark endothelial cells. Antibodies against VEGF (Santa Cruz Biotechnology) and placental growth factor (PlGF; R&D Systems Inc) were also used. Indirect immunofluorescence double-staining with matched primary and fluorescein-conjugated secondary antibodies was used to stain for colocalization with VEGF receptors in smooth muscle cells or monocytes as follows: Rabbit anti-mouse Flt-1 (Santa Cruz Biotechnology), rabbit anti-mouse Flk-1 (Santa Cruz Biotechnology), rat anti-mouse Mac-3, anti–-smooth muscle actin (-SMA; Boehringer Mannheim Corp), anti-rabbit IgG conjugated with fluorescein isothiocyanate or rhodamine, and anti-rat IgG conjugated with fluorescein isothiocyanate or rhodamine (Santa Cruz Biotechnology).
Ten equally-spaced cross sections were examined in all mice to quantify intimal lesions. Using image analysis software, the total cross-sectional medial area was measured between the external and internal elastic lamina; the total cross-sectional intimal area was measured between the endothelial cell monolayer and the internal elastic lamina.
Plasma Measurements
Plasma total cholesterol and triacylglycerol levels were determined with commercially available kits (Wako Pure Chemicals). Plasma concentrations of sFlt-1, VEGF, and PlGF were measured by the use of ELISA kit (R&D Systems Inc).
RT-PCR and RNAse Protection Assay
RNA was prepared from the pooled samples (5 to 7 arteries for 1 sample).21 First-strand DNA was synthesized using reverse transcriptase with random hexamers from 1 μg total RNA in a 20-μL reaction volume according to the manufacturer’s protocol (GeneAmp RNA polymerase chain reaction Kit; Perkin–Elmer). Primers used for amplification of VEGF were 5'-GGA TCC ATG AAC TTT CTG CT-3' and 5'-GAA TTC ACC GCC TCG GCT TGT C-3' with expected sizes of 654, 582, and 450 bp for the 3 VEGF isoforms (VEGF 188, 164, and 120, respectively). Primers for PlGF were 5'-CCC ACA CCC AGC TCA CGT ATT TA-3' and 5'-TCC CCT CTA CAT GCC TTC AAT GC-3'. Primers for Flk-1 were 5'-ACT GCA GTG ATT GCC ATG TTC T-3' and 5'-GCT CAT CCA AGG GCA ATT CAT-3'. Primers for the internal control, ?-actin, were 5'-ATG GAT GAC GAT ATC GCT-3' and 5'-ATG AGG TAG TCT GCT AGG T-3' with an expected product of 550 bp.
RNAse protection assays were performed using 5 μg total RNA with 2 custom template sets according to the manufacturer’s protocol (PharMingen).27
Statistical Analysis
Data are expressed as the mean±SE. Statistical analysis of differences was compared by ANOVA. Post hoc analyses were performed using Bonferroni correction for multiple comparison tests. P<0.05 was considered to be statistically significant.
Results
In Vivo Matrigel Plug Assay in ApoE-KO Mice
Seven days after injection of matrigel, there were significant angiogenic (number of CD31-positive cells per mm2) and inflammatory (number of Mac3-positive cells per mm2) reactions in the matrigel plugs containing recombinant VEGF protein compared with matrigel alone. Soluble Flt-1 gene transfer but not injection of an empty plasmid suppressed both the angiogenic and inflammatory reactions to VEGF to a level similar to that of matrigel plugs without VEGF (Figure I, available online at http://atvb.ahajournals.org). This suppression of angiogenic and inflammatory reactions to VEGF was noted on day 14 but not on day 21 of sFlt-1 gene transfer (data not shown).
Gene Expression and Immunoreactivity in ApoE-KO Mice
The mRNA levels of 2 VEGF isoforms (188 and 164) markedly increased after cuff placement, whereas they were undetectably low in control intact artery (Figure 1A and 1B). Peak expression was observed on day 7. VEGF 121 mRNA was undetectable before and after cuff placement. Gene expression of Flt-1, Flk-1, VEGF, and PlGF was also increased on day 7 (Figure 1C). Plasma concentrations of sFlt-1, VEGF, and PlGF were measured on day 7 in several groups of animals (Figure 1D). Plasma sFlt-1 was increased in sFlt-1 transfection group. There was no significant change in plasma VEGF nor in PlGF among groups (Figure 1D).
Figure 1. Gene expression VEGF, Flt-1, Flk-1, and PlGF in cuffed femoral artery. A, Time course of VEGF mRNA levels (RT-RCR) and expression of arterial VEGF and ?-actin mRNA after cuff placement. mRNA levels were assessed at the indicated times. This is a representative assay from 5 separate experiments. B, Densitometric analysis of data in A. Expression of VEGF mRNA in each sample was normalized by ?-actin mRNA expression in the same sample. N=5 for each bar. *P<0.01 vs control intact artery. C, Gene expressions of Flt-1, Flk-1, PlGF, and VEGF (RT-PCR) in femoral arteries before or 7 days after cuff placement/sFlt-1 gene transfer. *P<0.01 vs intact control. D, Plasma levels of sFlt-1, PlGF, and VEGF 7 days after cuff placement in 4 animal groups: (1) untreated control, (2) mice with cuff alone, (3) cuff+empty plasmid, and (4) cuff+sFlt-1 plasmid. N=6 for each. *P<0.01 vs control group.
Immunohistochemical staining indicated that compared with faint staining in the control artery, VEGF increased in the vicinity of inflammatory lesions (mononuclear cell infiltration) in the intima and adventitia on day 7 and in cells of 3 layers of cuffed artery on day 21 (Figure 2A). The endothelial layer, as detected by vWF staining, was preserved before and after cuff placement (Figure 2A). No detectable increase in PlGF staining was observed before and after cuff placement (Figure 2B).
Figure 2. Immunostaining of VEGF, Flt-1, Flk-1, and PlGF in cuffed femoral artery. A, Cross sections of intact or cuffed femoral arteries were stained immunohistochemically against VEGF, VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flk-1), or vWF 7 and 21 days after cuff placement in empty plasmid group. Immunohistochemical sections of cuffed arteries on day 21 in sFlt-1 group are also shown. Black lines indicate internal and external elastin laminas. L indicates lumen. Scale bar=50 μm. B, Immunohistochemical staining of PlGF in femoral arteries before or after cuff placement/sFlt-1 gene transfer. Black lines indicate internal or external elastin laminas. L indicates lumen. Scale bar=50 μm.
Both Flk-1 and Flt-1 were undetectable, except in endothelial layers in control intact arteries, but both were increased in the intima, media, and adventitia 7 and 21 days after cuff placement (Figure 2A). Compared with Flt-1 staining, Flk-1 staining was less impressive on day 7 but was apparently noted on day 21. To localize VEGF receptors, immunofluorescent double-staining was performed (Figure 3). On day 7, -SMA–positive cells in the media and neointima expressed little Flk-1, whereas they did express Flt-1 (Figure 3A). Also, some -SMA–positive cells in the adventitia (possibly adventitial myofibroblasts) expressed Flt-1. Mac-3 positive cells recruited to the neointima, media, and adventitia expressed VEGF and Flt-1. On day 21, most -SMA–positive cells in the neointima and media expressed VEGF and its receptors (Figure 3B).
Figure 3. Immunofluorescence double-staining of VEGF receptors, monocytes, and -SMA in cuffed femoral artery. A, Micrographs of cuffed femoral arteries doubly stained with Flt-1 (VEGF-R1, green) and -SMA (red), with Flk-1 (VEGF-R2, green) and -SMA (red), and with Mac-3 (green) and Flt-1 (VEGF-R1, red) 7 days after cuff placement. Scale bar=50 μm. B, Micrographs of cuffed femoral arteries doubly stained with Flt-1 (VEGF-R1) and -SMA, Flk-1 (VEGF-R2) and -SMA, and VEGF and -SMA in the cuffed femoral arteries 21 days after cuff placement. Single fluorescence–positive cells were stained green or red, whereas double-positive cells were stained yellow. White lines indicate external elastin laminas. L indicates lumen. Scale bar=50 μm.
Time Course of Development of Neointimal Hyperplasia in ApoE-KO Mice
As published,24,27,31,32 within 7 days of cuff placement, mononuclear leukocytes, most of which were Mac3-positive monocytes, were recruited into the adventitia, media, and intima (Figure 3). After 7 days, neointimal lesions developed and became thick over time (Figure 4A). Monocyte infiltration declined spontaneously and -SMA–positive cells appeared predominantly in the neointima. On day 21, significant neointimal formation with luminal stenosis developed (Figure 4A). Endothelial staining with vWF antibody showed that no significant neointimal neovascularization was observed during the course of experiments (Figure 2A).
Figure 4. Histopathology of cuffed femoral artery. A, Time course of cuff injury–induced neointimal formation and effect of sFlt-1 gene transfer. Micrographs of cross sections of control (intact) and cuffed arteries stained with van Gieson Elastica on days 3, 7, and 21 are shown. Scale bar=100 μm. B through D, Effects of sFlt-1 gene transfer on neointimal thickening (B), intima/media ratio (C), and % stenosis (D) 21 days after cuff placement. *P<0.01 vs cuff only and cuff+empty plasmid group.
Effects of Soluble Flt-1 Gene Transfer on Cuff-Induced Neointimal Hyperplasia in ApoE-KO Mice
As we previously reported,27 Mac3-positive monocytes/macrophages and PCNA-positive cells were detected mainly in the adventitia and intima. There was markedly less inflammation (Mac3-positive cells) and proliferation (the PCNA index) in sFlt-1–transfected mice than in empty plasmid-transfected mice on day 7 (Figure II, available online at http://atvb.ahajournals.org). There was no detectable change in the number of CD3-positive T cells in empty plasmid or sFlt-1–transferred mice (Figure II). The sFlt-1 gene transfer significantly reduced neointimal formation (increases in neointimal area, intima/media ratio, and luminal stenosis) 21 days after cuff placement (Figure 4A through 4D).
Because sFlt-1 gene transfer markedly reduced monocyte-mediated inflammation, gene expression of a battery of inflammatory cytokines, chemokines, and chemokine receptors was examined by RNAse protection assays (Figure III, available online at http://atvb.ahajournals.org) or by RT-PCR (Figure 1C) 7 days after cuff placement. The sFlt-1 gene transfer did not affect gene expression of RANTES, macrophage inflammatory protein-1, transforming growth factor-?, macrophage inflammatory protein-2, and PlGF, but prevented or attenuated the increased gene expression of CCR1, interleukin-6, CCR2, MCP-1, Flt-1, CXCR2, eotaxin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, Flk-1, and VEGF. The sFlt-1 gene transfer reduced increased immunostainings against VEGF, Flt-1, and Flk-1, but did not affect staining against vWF (Figure 2A).
Time course of plasma concentrations of sFlt-1 after sFlt-1 gene transfer was determined. Plasma sFlt-1 levels before and 3, 7, and 14 days after sFlt-1 transfection were 467±37, 1037±132 (P<0.01 versus baseline), 927±215 (P<0.01), and 649±83 pg/mL (P<0.05, n=6 each), indicating that sFlt-1 was released from the transfected muscle.
Plasma Lipid Levels in ApoE-KO Mice
Total cholesterol and triacylglycerol levels were 503±11 and 38±6 mg/dL in the control group, 512±16 and 40±5 mg/dL in the empty plasmid group, and 497±10 and 39±3 mg/dL in the sFlt-1 group, indicating that the observed effects of sFlt-1 gene transfer were not caused by changes in serum lipid levels.
Effects of Flt-1 Tyrosine Kinase Deficiency on Neointimal Hyperplasia
Wild-type and Flt-1 tyrosine kinase–deficient mice were fed a high-fat diet for 2 weeks, and cuff was placed as mentioned above. Mice received a high-fat diet for an additional 3 weeks. Neointimal formation was noted 21 days after cuff placement in wild-type mice. Compared with wild-type mice, Flt-1 tyrosine kinase–deficient mice displayed reduced neointimal formation (Figure IV, available online at http://atvb.ahajournals.org). Total cholesterol levels at 5 weeks of high-fat diet were 520±21 and 511±18 mg/dL in wild-type and Flt-1 tyrosine kinase–deficient mice, respectively.
Discussion
This study is the first to demonstrate the essential role of VEGF and Flt-1 signals in the development of neointimal formation after cuff-induced periadventitial injury in hypercholesterolemic mice. VEGF is conventionally thought to be an endothelial cell–specific growth factor and to attenuate vascular disease by inducing endothelial proliferation and regeneration mainly through the endothelial type 2 receptor Flk-1.7 Recent evidence, however, suggests that functional Flt-1 and Flk-1 are expressed in injured arterial wall cells other than endothelial cells. In this study, Flt-1, Flk-1, and VEGF were increased in lesional monocytes and medial smooth muscle cells at early stages and in neointimal and medial smooth muscle cells at later stages. Flt-1 is demonstrated to act as an important mediator of chemotaxis through vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and MCP-1.13–15 We demonstrated that sFlt-1 gene transfer reduced the early inflammatory and proliferative changes and thus attenuated the development of neointimal formation. It is speculated, therefore, that VEGF might cause inflammation (monocyte recruitment and activation) and proliferation through Flt-1–mediated signals and thus cause neointimal formation after cuff-induced periadventitial injury. In addition, Flt-1 in smooth muscle cells is reported to mediate migration and proliferation in vitro.18,33 An alternative interpretation is that VEGF directly caused migration and proliferation of smooth muscle cells resulting in neointimal formation. In any way, our present finding of Flt-1 tyrosine kinase–deficient mice suggests the involvement of Flt-1–related signals in the pathogenesis of neointimal formation after periadventitial injury.
Periadventitial inflammation has a major role in the pathogenesis of cuff-induced neointimal formation.27,32 To gain insight into the mechanism of VEGF-mediated inflammation and neointimal formation, we assessed gene expression of various inflammatory genes. The sFlt-1 gene transfer attenuated increased gene expression of inflammatory cytokines, adhesion molecules, chemokines, and chemokine receptors. Yamada et al34 showed that MCP-1 is essential in VEGF-induced angiogenesis, vascular leakage, and inflammation. An essential role of MCP-1 in the development of neointimal formation after arterial injury has also been reported.27–29,35,36 The sFlt-1 gene transfer attenuated increased VEGF, Flk-1, and Flt-1 gene expression, indicating that VEGF regulates its activity by an autocrine loop mechanism within diseased arterial wall cells, such as smooth muscle cells, endothelial cells, and lesional monocytes. A positive feedback effect of VEGF is supported by previous studies that demonstrated enhanced VEGF production by monocytes through Flt-1 stimulation.37 Therefore, sFlt-1 gene transfer might attenuate cuff-induced neointimal formation mainly by suppressing inflammation (monocyte recruitment and activation).
This study has potentially significant clinical implications. Blockade of VEGF by sFlt-1 gene transfer can be an attractive anti-VEGF therapy for inflammatory vascular disease and other inflammatory disorders. The efficacy of this strategy for experimental tumor angiogenesis has already been tested.22 Luttun et al38 recently reported that treatment with anti–Flt-1 antibody attenuated the development of experimental tumor angiogenesis, arthritis, and atherosclerosis. One limitation of the present finding is that the pathogenesis of neointimal formation after periadventitial injury differs from that after endothelial injury/denudation and from that in humans. The endothelial integrity is preserved in cuff-induced periadventitial injury. For clinical application of our findings to human vascular disease, future studies are needed to examine the efficacy and safety of anti-VEGF strategy with sFlt-1 in experimental atherosclerosis and restenosis.
In conclusion, inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury. These data support the notion that VEGF promote neointimal formation by acting as a proinflammatory cytokine after cuff-induced periadventitial injury.
Acknowledgments
This study was supported by Grants-in-Aid for Scientific Research (14657172, 14207036) from the Ministry of Education, Science, and Culture by Health Science Research Grants (Comprehensive Research on Aging and Health and Research on Translational Research), from the Ministry of Health, Labor, and Welfare, and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, Tokyo, Japan.
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