Soluble Flt-1 Gene Transfer Ameliorates Neointima Formation After Wire Injury in flt-1 Tyrosine Kinase–Deficient Mice
Objective— We have demonstrated that vascular endothelial growth factor (VEGF) expression is upregulated in injured vascular wall, and blockade of VEGF inhibited monocyte infiltration and neointima formation in several animal models. In the present study, we aimed to clarify relative role of two VEGF receptors, flt-1 versus flk-1/KDR, in neointima formation after injury using flt-1 tyrosine kinase-deficient (Flt-1 TK−/−) mice and soluble Flt-1(sFlt-1) gene transfer.
Methods and Results— Neointima formation was comparable between wild-type and Flt-1 TK−/− mice 28 days after intraluminal wire injury in femoral arteries. By contrast, neointima formation was significantly suppressed by sFlt-1 gene transfer into Flt-1 TK−/− mice that blocks VEGF action on flk-1 (intima/media ratio: 2.8±0.4 versus 1.4±0.4, P<0.05). The inhibition of neointima formation was preceded by significant reduction of monocyte chemoattractant protein (MCP-1) expression in vascular smooth muscle cells (VSMCs) and monocyte infiltration 7 days after injury. Gene transfer of sFlt-1 or treatment of flk-1–specific antibody significantly inhibited VEGF-induced MCP-1 expression determined by RT-PCR in cultured aortic tissue and VSMCs. MCP-1–induced chemotaxis was equivalent between wild-type and Flt-1 TK−/− mice.
Conclusions— These results suggest that endogenous VEGF accelerates neointima formation through flk-1 by regulating MCP-1 expression in VSMCs and macrophage-mediated inflammation in injured vascular wall in murine model of wire injury.
Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic and vascular permeability factors playing essential roles in neonatal and postnatal vascular formation. VEGF has gathered growing attention because of its possible contribution to cardiovascular pathophysiology including therapeutic angiogenesis, endothelial regeneration, and inflammation in the vascular wall. VEGF expression is upregulated in human coronary arterial wall after stent implantation, suggesting its role in reendothelialization, perivascular angiogenesis, and neointima formation leading to clinical restenosis.1 VEGF induction is reproduced in various animal vascular injury models including wire or cuff injury in mice, and balloon injury in rats and rabbits, porcine coronary stent model.2–4 From these prior studies that supplement or inhibit VEGF pathway in animal models, two conflicting mechanisms have been demonstrated in which VEGF may contribute to neointima formation. One is that VEGF inhibits neointima formation by promoting reendothelialization and inhibiting vascular smooth muscle cell (VSMC) proliferation.5 The other is that VEGF accelerates neointima formation by promoting inflammation2,4 and adventitial angiogenesis in the vascular wall.2,3,6
In this controversy, clinical studies have been carried out to examine the effect of VEGF gene delivery after percutaneous coronary angioplasty without significant reduction in restenosis.7–9 Thus, it is crucial to understand the mechanisms underlying differential effects of VEGF during neointima formation to optimize vasculoprotective effects and minimize adverse effects of endogenous VEGF that may depend on receptor, cell type, and the mode of vascular injury including species studied. We have demonstrated that flt-1 (VEGF receptor 1) is upregulated during neointima formation after vascular injury, especially in the neointima, media, and adventitia, and flk-1/KDR (VEGF receptor 2) in the neointima and media, and that blockade of VEGF by soluble Flt-1 (sFlt-1) gene transfer inhibited monocyte infiltration and neointima formation in several animal models.2,4 However, contribution of each VEGF receptor was not fully clarified in previous studies including ours, because sFlt-1 as well as other VEGF traps sequesters VEGF from its receptors nonspecifically.2,10 It is reported that flk-1/KDR mainly mediates endothelial cell proliferation during angiogenesis,11 so that flk-1 may promote reendothelialization and suppress neointima formation after injury. By contrast, flt-1 is reported to regulate nitric oxide production in endothelial cells,12 and to regulate monocyte chemotaxis that may promote inflammation and influence neointima formation.13 Therefore, the present study aimed to determine the relative role of each VEGF receptor, flt-1 versus flk-1, in neointima formation after vascular injury, using flt-1 tyrosine kinase-deficient (Flt-1 TK−/−) mice14 in the presence or absence of sFlt-1 gene transfer2,15 in wire injury model.
All study protocols were reviewed and approved by the Committee on the Ethics of Animal Experiments in Kyushu University Graduate School of Medical Sciences. To examine the role of flt-1, we used Flt-1 TK−/− mice on C57Bl/6J background because flt-1 deficiency is known to be embryonic lethal.14 Age-matched C57Bl/6J mice (CLEA Japan, Tokyo, Japan) were used as wild-type (WT) control. All mice were fed with normal diet and water at libitum.
The 3.3-kb mouse sFLT-1 gene, originally cloned from mouse lung cDNA library, was subcloned into the BamH I (5′) and Not I (3′) sites of the eukaryotic expression vector cDNA3 plasmid (Invitrogen, Carlsbad, Calif).2,16
Femoral Arterial Wire Injury
To examine the role of flt-1 in neointima formation, femoral arterial wire injury was performed in male 12- to 20-week-old WT or Flt-1 TK−/− mice. After exposure of left femoral artery, straight spring wire (0.38 mm in diameter, COOK) was inserted into femoral artery from the muscular branch. The wire was placed for 1 minute to denude and dilate the artery. After removal of wire, branch artery was ligated and restoration of blood flow was verified by pulsation of peripheral arteries.2,17 Twenty-eight days after injury, femoral artery was excised after injection of 10% buffered formalin and evaluated histopathologically. Heart rate and systolic blood pressure were measured by tail cuff method before sacrifice.
Soluble Flt-1 Gene Transfer
To examine the role of flk-1, sFlt-1 gene transfer was performed in Flt-1 TK−/− mice as described elsewhere.2,4,15 Briefly, plasmid vector encoding sFlt-1 cDNA (100 μg) was injected in the gastrocnemial muscle followed by electroporation to facilitate gene transfer. sFlt-1 gene transfer was performed once every 2 weeks from 14 days before until 28 days after injury, based on the data that serum sFlt-1 concentration was elevated over 28 days after single injection of sFlt-1 plasmid with the peak at 14 day after injection (serum sFlt-1concentration at baseline and 3, 14, and 28 days after injection was 222±24, 396±59,* 970±55,* and 477±54* pg/mL, *P<0.05 versus baseline).
Histopathology and Immunohistochemistry
For histopathologic and immunohistochemical analysis, serial paraffin sections of the femoral artery were prepared. Briefly, femoral artery was excised after perfusion of 10% buffered formalin and fixed overnight in formalin. After fixation, the tissue was embedded in paraffin and cross sections (6 μm thick) were stained with Masson trichrome or elastica van Gieson stains. Neointima area was defined as the area surrounded by internal elastic lamina except lumen area. Extent of neointima formation was evaluated by the areal ratio of intima to media (I/M ratio) and neointima area. Other sections were subjected to immunostaining using rat anti-mouse macrophage monoclonal antibodies (Mac-3; BD Pharmingen), goat antimouse monocyte chemoattractant protein-1 (MCP-1) antibodies (Santa Cruz Biotechnology Inc). Proliferating cells were evaluated by the immunostaining with antiproliferating cell nuclear antigen (PCNA) antibody (DAKO). Alpha-smooth muscle actin (α-SMA, DAKO) and CD31 antibody (Santa Cruz) were also used as smooth muscle and endothelium marker. The respective nonimmune IgGs were used as negative controls. After incubation with biotinylated goat antirat IgG (Santa Cruz Biotechnology Inc) or rabbit anti-goat IgG (Nichirei), the sections were incubated with diaminobenzidine (DAB). The sections were then counterstained with Mayer hematoxylin. Analysis was performed using a microscope with a computerized, digital image analysis system and Scion Image Software (Scion Corporation). Fluorescent immunostaining was performed with secondary antibodies which are labeled by AlexaFluor 488 or 555 (Invitrogen).
Ex Vivo Culture of Mouse Aorta
Male 12- to 20-week-old WT Flt-1 TK−/− and sFlt-1 plasmid-injected Flt-1 TK−/− mice were used in this experiment. In the first experiment, sFlt-1 plasmid was injected 7 days before excision of aorta. Aorta was excised from ascending aorta to the bifurcation of iliac arteries and incubated with Dulbecco modified Eagle medium (DMEM) containing 1% fetal bovine serum (FBS). After overnight starvation, aorta was incubated with 50 ng/mL VEGF for 3 hours. Then, mRNA was extracted and quantitative real-time RT-PCR was performed by ABI PRISM 7000 Sequence Detection System (Applied Biosystems). MCP-1 and GAPDH primer, which is mixed with probes as TaqMan Gene Expression Assays, were commercially available and purchased from Applied Biosystems. The second experiment was performed using WT mice and blocking antibody of flt-1(R&D Systems Inc) and flk-1 (R&D Systems Inc). After overnight incubation with these antibodies, mRNA was collected in a similar fashion.
VEGF-Induced MCP-1 Expression in VSMCs
Mouse aortic VSMCs (P53LMACO1)18 was purchased from Health Science Research Resource Banks and cultured in DMEM containing 10% FBS and phorbol-12myristate-13acetate (PMA, 100 nmol/L). VSMCs were used after 9 passages in the experiment. VSMCs were starved overnight and incubated with blocking antibody for flt-1 (10 μg/mL, R&D Systems) or flk-1 (1 μg/mL, R&D Systems). MCP-1 gene expression was analyzed by real-time PCR after 4-hour stimulation with 200 ng/mL human VEGF.
Peritoneal Macrophage Chemotaxis
Peritoneal fluid containing macrophage was harvested 4 days after intraperitoneal injection of thioglycolate. Macrophage migration was measured in 96-well chemotaxis chambers (Neuro Probe Inc). MCP-1 or VEGF in RPMI 1640 was added to the lower wells and the isolated macrophages (1×107 cells per mL) were placed in the upper wells. The concentration of MCP-1 and VEGF was 5, 15, and 50 ng/mL. After incubation for 90 minutes at 37°C, the upper surface of the membrane was washed with PBS and migrated cells on the lower surface were counted after staining with trypan blue. The number of cells per field was counted. All assays were performed in triplicate.
All data are reported as the mean±SE. Statistical analysis of differences was performed by Student t test and 1-way ANOVA with Bonferroni post test. Statistical analysis of chemotaxis assay was performed by 2-way ANOVA with Bonferroni post test. Probability values less than 0.05 were considered to be statistically significant.
Distinct Role of flt-1 and flk-1 in Neointima Formation After Wire Injury
To examine the role of flt-1 in neointima formation, we examined the degree of neointima formation after wire injury in femoral arteries of Flt-1 TK−/− mice. Flt-1 TK−/− mice lack intracellular tyrosine kinase domain of flt-1 and thus downstream signaling.14 Histological analysis revealed that there was no significant difference in I/M ratio and neointimal area 28 days after wire injury between WT and Flt-1 TK−/− mice (Figure 1A), suggesting that the role of flt-1 is minor in neointima formation in this model. Deficiency of flt-1 tyrosine kinase unaffected heart rate (673±11 versus 662±19 bpm) or blood pressure (110±3 versus 104±3 mm Hg) in mice, also unaffected macrophage infiltration evaluated as mac-3 staining, perivascular fibrosis and vessel diameter (data not shown).
To examine the role of flk-1, sFlt-1 gene transfer was performed into Flt-1 TK−/− mice. The sFlt-1 sequesters VEGF from both VEGF receptors and thus we could evaluate the role of flk-1 when applied to Flt-1 TK−/− mice. The sFlt-1 gene transfer markedly inhibited neointima formation with significant reduction of I/M ratio compared with control empty plasmid in Flt-1 TK−/− mice (Figure 1B). The sFlt-1 gene transfer did not affect heart rate (662±19 versus 660±11 bpm) and blood pressure (95±5 versus 107±4 mm Hg) in Flt-1 TK−/− mice.
sFlt-1 Gene Transfer Suppressed Proliferation, Monocyte Infiltration, and MCP-1 Expression in Flt-1 TK−/− Mice
We have repeatedly shown the importance of MCP-1 and monocyte-mediated inflammation in the vascular wall during vascular remodeling in various vascular disease models.19–22 Thus, we analyzed the effect of sFlt-1 gene transfer on proliferation of vascular wall cells, MCP-1 expression, and monocyte/macrophage infiltration at an early stage of neointima formation. Histology at 7 days after injury showed a decrease in PCNA-positive cells in the neointima of Flt-1 TK−/− mice transfected with sFlt-1 gene that underpins reduction in neointima formation (Figure 2A). Prominent MCP-1 induction was found in the intimal and the medial cells in Flt-1 TK−/− mice, which was markedly suppressed by sFlt-1 gene transfer (Figure 2B). Infiltration of Mac-3–positive monocytes was found in the media and adventitia, which was suppressed by sFlt-1 gene transfer as well (Figure 2C). These results suggest that endogenous VEGF upregulates MCP-1 and macrophage recruitment via flk-1 in Flt-1 TK−/− mice during neointima formation after wire injury.
Distinct Role of flt-1 and flk-1 in VEGF-Mediated MCP-1 Induction
To elucidate detailed mechanisms of VEGF-mediated MCP-1 induction in injured arteries, we first performed double immunostaining of MCP-1 and α-SMA 3 days after injury when the endothelium has not regenerated yet. We found equivalent MCP-1 induction in the medial VSMCs in WT and Flt-1 TK−/− mice. In contrast, sFlt-1 gene transfer into Flt-1 TK−/− mice remarkably inhibited MCP-1 induction (Figure 3A). These results suggest that (1) wire injury induces MCP-1 expression primarily in the VSMCs and (2) flk-1, but not flt-1, mediates MCP-1 induction in the VSMCs immediately after vascular injury. Next, we examined the role of each VEGF receptor in MCP-1 induction in ex vivo culture model. Mouse aorta was harvested from WT, Flt-1 TK−/−, and sFlt-1 plasmid-administrated Flt-1 TK−/− mice. The aortas were stimulated with VEGF (50 ng/mL) after 24-hour starvation, and induction of MCP-1 was quantified by real-time PCR. Real-time PCR showed that VEGF-induced expression of MCP-1, which is partially but not significantly inhibited by Flt-1 TK deletion and is completely inhibited by sFlt-1 gene transfer, suggesting that VEGF-induced MCP-1 mRNA transcription is primarily mediated by flk-1 (Figure 3B). Blockade of each VEGF receptor by neutralizing antibodies showed that anti–flt-1 antibody had no effect; by contrast, anti–flk-1 antibody completely inhibited VEGF-induced MCP-1 expression (Figure 3C). Finally we examined the effect of each VEGF receptor blockade on VEGF-induced MCP-1 expression in mouse aortic VSMCs. Blocking antibody of flt-1 did not inhibit VEGF-induced MCP-1 expression. In contrast, blocking antibody of flk-1 significantly inhibited VEGF-induced MCP-1 expression (supplemental Figure I, please see http://atvb.ahajournals.org). These results suggest that VEGF induces MCP-1 expression by flk-1–mediated mechanisms in mouse VSMCs. We also examined whether blockade of VEGF influences PDGF signaling that may mediate MCP-1 induction in injured arterial wall.23 In vitro study using cultured VSMCs revealed that blockade of VEGF by sFlt-1 gene transfer or sFlt-1 protein does not inhibit PDGF-induced phosphorylation of PDGF receptors, suggesting that blockade of flk-1 inhibit MCP-1 induction via PDGF-independent mechanisms (supplemental Figure II).
Macrophage Chemotaxis in WT and Flt-1 TK−/− Mice
It is reported that human monocytes exclusively express flt-1 and that VEGF induces chemotaxis by flt-1–mediated mechanism.13 We performed Boyden chamber experiment to examine macrophage chemotactic function in response to VEGF or MCP-1 in WT and Flt-1 TK−/− mice. VEGF induced significant chemotaxis of peritoneal macrophage from WT mice, which was abolished by Flt-1 TK deficiency (Figure 4A), suggesting that flt-1 essentially mediates VEGF-induced chemotaxis. By contrast, MCP-1 caused more prominent chemotaxis in WT and Flt-1 TK−/− mice equivalently (Figure 4B). These results suggest that MCP-1–induced chemotaxis was preserved in Flt-1 TK−/− mice, and MCP-1 is a primary mediator of flk-1–dependent macrophage recruitment in injured vascular wall even in the Flt-1 TK deficiency.
Effect of sFlt-1 Gene Transfer on Adventitial Angiogenesis
Adventitial angiogenesis was evaluated by counting CD31-positive endothelial cells in the adventitia of injured femoral arteries. Adventitial angiogenesis was equivalent in WT and Flt-1 TK−/− mice, however, was significantly decreased in Flt-1 TK−/− mice after sFlt-1 gene transfer compared with WT mice (Figure 5).
In this study, we aimed to clarify the relative importance of 2 VEGF receptors, flt-1 and flk-1/KDR in neointima formation after intraluminal wire injury. Major findings were: (1) Flt-1 TK deficiency unaffected neointima formation, (2) sFlt-1 gene transfer into Flt-1 TK−/− mice remarkably suppressed neointima formation, and (3) VEGF induced MCP-1 expression in VSMCs which was blocked by flk-1–specific antibody. The inhibition of neointima formation by flk-1 blockade was preceded by significant reduction of MCP-1 expression in the medial VSMCs 3 days after injury, and monocyte infiltration, VSMC proliferation, and perivascular neovascularization 7 days after injury. These in vivo results suggest that flk-1 plays a primary role in the development of neointima by regulating macrophage-mediated inflammation in this model.
Inflammation in the vascular wall, mainly mediated by monocyte and macrophage, is a hallmark of vascular remodeling after injury as evident in our previous studies using cuff injury in mice and balloon injury in rats, rabbits, and monkeys, in which MCP-1 blockade effectively suppresses vascular inflammation and remodeling.24–26 It is reported that VEGF induces MCP-1 expression in endothelial cells27; in turn, MCP-1 induces VEGF in VSMCs.28 Macrophage-mediated inflammation activates VSMC migration and proliferation by cytokines, or by redox-dependent signaling.29,30 Although flk-1 is expressed mainly in endothelial cells, VSMCs also express flk-1,31 and the present study revealed a new mechanism that flk-1 mediates MCP-1 expression in VSMCs and monocyte recruitment after injury. This VEGF/MCP-1–positive feedback and downstream signaling is considered to be a major underlying mechanism in neointima formation after injury, as shown in our previous studies using sFlt-1 and MCP-1 mutant19,24–26 (Figure 6).
It has been reported that VEGF induces direct macrophage chemotaxis by flt-1–mediated mechanisms, whereas flk-1 is not expressed on monocytes/macrophages.13 Indeed, Flt-1 TK deficiency abrogated VEGF-induced chemotaxis in peritoneal macrophages in the present study; however, Flt-1 TK deficiency had no effect on macrophage infiltration into injured arterial wall and neointima formation in in vivo setting. In the present study, blockade of flk-1 inhibited MCP-1 expression in the medial VSMCs and abrogated monocyte accumulation in the vascular wall after injury. Thus, we considered that MCP-1, which is regulated by VEGF/flk-1 pathway, mainly mediates macrophage chemotaxis rather than VEGF/flt-1 expressed on monocyte itself. This mechanism well explains the in vivo effect of flk-1 blockade on monocyte accumulation after injury.
In the present study, sFlt-1 gene transfer also inhibited adventitial angiogenesis after injury. Inhibition of adventitial angiogenesis may be another mechanism by which flk-1 blockade inhibits neointima formation, because a positive correlation was observed between adventitial blood vessel formation and neointima formation in various injury model including rabbit collar placement model.32
The role of VEGF in neointima formation may be different depending on the mode of injury or the species studied. For example, Isner et al33 reported that local delivery of VEGF accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Hutter et al5 reported in mouse wire injury model that intravenous injection of VEGF adenovirus promoted endothelial repair and inhibited neointima formation. By contrast, Khurana et al32 reported that adenovirus-mediated VEGF gene transfer exacerbates adventitial neovascularization and neointima formation in rabbit periadventitial collar replacement model, which was abrogated by administration of sFlt-1. Thus, there remain controversies in the role of VEGF gene transfer per se in neointima formation in previous studies. In the present study, we administered sFlt-1 plasmid intramuscularly, and sFlt-1, which was detected in the serum, blocked VEGF signaling at the site of vascular injury. In our previous study, we have reported that reendothelialization is complete 14 days after wire injury in mice irrespective of VEGF blockade by sFlt-1 gene transfer.2 Thus, it is suggested that on complete reendothelialization, excess VEGF may accelerate neointima formation by promoting monocyte-mediated inflammation through flk-1–dependent MCP-1 expression in the injured vascular wall at least in the murine wire injury model studied. In this situation, Flk-1–specific VEGF blockade may be another potential approach to control vascular inflammation and subsequent remodeling after injury.
In conclusion, the present study demonstrated that soluble sFlt-1 gene transfer ameliorates neointima formation after wire injury in flt-1 tyrosine kinase–deficient mice by inhibiting MCP-1 expression in the medial VSMCs and resulting monocyte-mediated inflammation. The present findings suggest that endogenous VEGF accelerates neointima formation after injury through flk-1–dependent mechanisms, and provide new insights into complex VEGF-mediated signaling in vascular remodeling.
Sources of Funding
This study was supported by Grants-in-Aid for Scientific Research (19390216, 19650134) from the Ministry of Education, Science, and Culture, Tokyo, Japan and by Health Science Research Grants (Research on Translational Research and Nano-medicine) from the Ministry of Health Labor and Welfare, Tokyo, Japan.
Received December 2, 2007; revision accepted January 7, 2009.
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