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Atherosclerosis 233 (2014) 641e647
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Local inhibition of hypoxia-inducible factor reduces neointima formation after arterial injury in ApoE/ mice Marian Christoph 1, Karim Ibrahim 1, Kathleen Hesse, Antje Augstein, Alexander Schmeisser 2, Ruediger C. Braun-Dullaeus 2, Gregor Simonis, Carsten Wunderlich, Silvio Quick, Ruth H. Strasser, David M. Poitz* University of Dresden, Heart Center, University Hospital, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 July 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 2 February 2014
Objective: Hypoxia plays a pivotal role in development and progression of restenosis after vascular injury. Under hypoxic conditions the hypoxia-inducible factors (HIFs) are the most important transcription factors for the adaption to reduced oxygen supply. Therefore the aim of the study was to investigate the effect of a local HIF-inhibition and overexpression on atherosclerotic plaque development in a murine vascular injury model. Methods and results: After wire-induced vascular injury in ApoE/ mice a transient, local inhibition of HIF as well as an overexpression approach of the different HIF-subunits (HIF-1a, HIF-2a) by adenoviral infection was performed. The local inhibition of the HIF-pathway using a dominant-negative mutant dramatically reduced the extent of neointima formation. The diminished plaque size was associated with decreased expression of the well-known HIF-target genes vascular endothelial growth factor-A (VEGF-A) and its receptors Flt-1 and Flk-1. In contrast, the local overexpression of HIF-1a and HIF-2a further increased the plaque size after wire-induced vascular injury. Conclusions: Local HIF-inhibition decreases and HIF-a overexpression increases the injury induced neointima formation. These findings provide new insight into the pathogenesis of atherosclerosis and may lead to new therapeutic options for the treatment of in stent restenosis. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Atherosclerosis Restenosis Hypoxia-inducible factor Hypoxia
1. Introduction Coronary restenosis still represents one of the major complications after coronary stent implantation even in the era of drug eluting stents [1]. The main mechanism for the developing of restenosis after vascular injury is an exceeding neointimal hyperplasia [2]. This pathomechanism is characterized by a massive invasion of mononuclear cells as well as chronic inflammation with excessive local release of cytokines and chemokines, which finally results in a pathological increased proliferation of smooth muscle cells and endothelial cells [3]. Despite the histological composition of neointimal hyperplasia is well characterized, the molecular
* Corresponding author. University of Technology Dresden, Heart Center Dresden University hospital, Fetscherstr. 76, 01307 Dresden, Germany. Tel.: þ49 351 458 6627; fax: þ49 351 458 6329. E-mail address: [email protected] (D.M. Poitz). 1 Authors contributed equally to this work. 2 Present address: Internal Medicine, Department of Cardiology, Angiology and Pneumology, Magdeburg University, Germany. 0021-9150/$ e see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.048
regulation remains poorly understood even after numerous of different in vitro and in vivo studies. The hypoxia-inducible factor (HIF) pathway is thought to be associated with atherosclerotic lesion progression, suggesting its involvement in the response to hypoxia and regulation of human intra-plaque angiogenesis. This hypothesis is supported by the in vivo detection of hypoxia in plaque-macrophages and the expression of several hypoxia-induced genes in atherosclerotic lesions [4]. The hypoxia-inducible factors are key transcriptional regulators of physiological as well as pathophysiological hypoxic response in both embryonic and adult organisms and are activated in many different human diseases including cancer, rheumatoid arthritis, stroke, and chronic lung disease [5e8]. The hypoxia-inducible factors are heterodimeric transcription factors consisting of an oxygen-sensitive HIF-a subunit and a constitutively expressed HIFb subunit [9,10]. The HIF-a/b-dimer binds to a core DNA-motif, the so called hypoxia-response element (HRE) and induces a variety of hypoxia-responsive genes that are involved in a variety cellular processes like angiogenesis, erythropoiesis, glucose metabolism and vasomotor regulation [11].
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The HIF-family consists of the three closely related subunits HIF1a, HIF-2a and HIF-3a. Whereas HIF-1a is ubiquitously expressed, analysis of cell-type-specific expression patterns of HIF-2a showed predominantly expression in endothelial, epithelial cells as well as fibroblasts and macrophages [8,12e14]. Under normoxic conditions the HIF-1a and HIF-2a subunits are continuously hydroxylated by prolyl hydroxylase domain-containing enzymes (PHDs) [15]. The hydroxylated HIF-a subunits are bound by the von Hippel-Lindau protein complex (pVHL), leading to ubiquitinilation and subsequent proteosomal degradation [16]. However, in the absence of oxygen the HIF-a-subunits are stabilized, dimerize with the HIF-1bsubunit and induce transcription of numerous genes [17]. With consideration of the above-mentioned findings of hypoxic areas within atherosclerotic plaques, it can be assumed that the HIFpathway is strongly associated with atherosclerotic plaque progression. The reactive or causative role of HIF in atherogenesis remains unclear to date [18]. Therefore, this in vivo study investigates the role of a transient and local HIF inhibition as well as a local overexpression of HIF-1a and -2a subunits in neointima formation after vascular injury.
femoral bifurcation), as described previously [20]. These sections were stained with Elastica van Gieson stain, and digital microscopic images were taken (Carl Zeiss). The area of lumen, neointima (area between lumen and internal elastic lamina) and media (area between internal and external elastic lamina) were determined by planimetry (Image J, National Institutes of Health) (see Supplemental Fig. 1A). From the obtained areas of lumen, neointima and media the intima/media ratio (area of neointima divided to area of media, IMR) and lumen/vessel-wall ratio (area of lumen divided to area of media and neointima, LVR) were calculated. The mean values of IMR and LVR of multiple sections within the same vessel were obtained in order to consider the biological longitudinal variability of neointimal plaque morphology. 2.6. X-Gal-staining and immunohistochemistry Details are described in the data supplement. 2.7. Fluorescence-lifetime imaging microscopy Details are described in the data supplement.
2. Materials and methods 2.8. Statistical analysis 2.1. Cell culture, adenovirus construction and adenoviral infection Details are described in the data supplement. 2.2. Real-time RT-PCR Details are described in the data supplement. 2.3. Mouse model The details are described in the data supplement. 2.4. Atherogenic vascular injury model All procedures were approved by the local ethical committee (24D-9168.11-1/2008-17). The unilateral femoral artery injury was performed as published previously [19]. In brief male mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). First, a groin incision was made and the common and deep femoral artery were dissected and mobilized. After temporarily clamping of the femoral artery a small transverse incision of the deep femoral artery was made and an angioplasty guide wire (0.014 inch diameter) was introduced and advanced to the level of aortic bifurcation. The wire was left inside the vessel for 1 min in order to achieve endothelial injury. Afterwards the wire was removed and the deep femoral artery was ligated. The contralateral femoral artery was not injured and served as negative control. For transient local adenoviral infection of the femoral artery, immediately after the vascular injury the treated artery was locally incubated with 40 ml sodium chloride solution containing adenoviral vectors at a dose of 1 109 infectious units (ifu) for 30 min. Subsequently the virus solution was carefully aspirated and the skin was closed with interrupted sutures. 2.5. Histological and immunohistological analysis To examine the development of the atherosclerotic lesions, the treated arteries were harvested at specific time points (between 2 and 5 weeks). For the quantification of neointimal and medial areas, 8 sections from the paraformaldehyde-fixed and paraffinembedded femoral arteries were taken (each 4 mm in thickness, 50 mm distance between the sections, started 50 mm from the
Results were expressed as means standard deviation. Statistical analysis was done using the 2-tailed, unpaired Student’s t-test. Level of significance was set to p < 0.05. p values below 0.05(0.01/ 0.001) are indicated by *(**/***). 3. Results 3.1. In vivo local inhibition of the HIF pathway by Ad-HIFadn significantly reduces neointima formation after wire-induced vascular injury To investigate the impact of local inhibition of the HIF pathway on neointima formation, a wire-induced endovascular injury of the femoral artery in ApoE/ mice was performed and a local transduction with an adenovirus was established. To show the biological functionality of the dominant-negative HIF-a mutant in murine cells, vascular smooth muscle cells (VSMC) and macrophages were infected in vitro with an adenovirus encoding either for b-galactosidase (Ad-LacZ) as a control or for a dominant-negative mutant based on HIF-2a (Ad-HIFadn), which leads to a competitive inhibition of both HIF-1a and HIF-2a [21e 24]. Subsequently, the mRNA expression of the HIF target genes VEGF-A and PGK1 (Phosphoglycerate-kinase) were analyzed by real-time RT-PCR. The infection with Ad-HIFadn decreased the hypoxia-triggered mRNA induction significantly compared to the corresponding Ad-LacZ-control (Supplemental Fig. 3). To test the in vivo local infection efficiency of the recombinant replication-deficient adenoviruses, the femoral arteries of ApoE/ mice were in vivo incubated in a solution with Ad-LacZ for 30 min. Subsequently the skin was closed. X-Gal staining 72 h after the local infection showed sufficient local transduction efficiency. 2 weeks after infection no positive X-Gal staining was observed. To exclude that infection with Ad-LacZ itself has an influence on the neointima formation as a consequence of increased local inflammation, the femoral artery was incubated in vivo with either Ad-LacZ or saline as control for 30 min after wire induced vascular injury in ApoE/ mice. After 5 weeks there was no difference in IMR after local infection with Ad-LacZ compared to saline control (Supplemental Fig. 1C). To study the effect of local HIF inhibition on neointima formation, immediately after the vascular injury, the treated femoral
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artery was locally infected either with Ad-HIFadn or with Ad-LacZ as control for 30 min. After 5 weeks the femoral arteries were harvested and the IMR (intima/media ratio) and LVR (lumen/vesselwall ratio) as values of the atherosclerotic plaque size were measured by planimetry. After 5 weeks the intima/media ratio was dramatically reduced by 65.4% in Ad-HIFadn infected arteries compared to Ad-LacZ controls (Fig. 1). Additionally, a significant increase by 113.8% of the lumen/vessel-wall ratio as sign of a preserved vessel lumen after Ad-HIFadn infection in comparison to Ad-LacZ was observed. To evaluate the effect of Ad-HIFadn infection on neointima formation in a time course after transluminal wire-injury, the femoral arteries of ApoE/ mice were directly infected after injury either with Ad-HIFadn or with Ad-LacZ as control. Afterwards the treated vessels were harvested at time points of 2 h (0 weeks), 2 weeks and 5 weeks after vascular injury and the IMR and LVR were calculated (Fig. 1). As expected, 2 h after vascular injury, no relevant neointimal area and no relevant change in LMR could be measured in both groups. 2 weeks after vascular injury and local infection with Ad-HIFadn, a significant reduction by 42.3% of IMR and a trend of a LMR reduction of 22.1% is already evident in comparison to AdLacZ treated vessels. In contrast to Ad-LacZ control which show an advanced plaque progression after 5 weeks, no further relevant plaque progression could be detected in Ad-HIFadn infected femoral arteries between 2 and 5 weeks.
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Next, it should be investigated if this inhibition of neointima formation could be further enhanced by local Ad-HIFadn infection 72 h before wire-induced vascular injury in order to ensure the biological functionality of HIFadn directly at the time point of vascular injury. To address the role of HIF inhibition directly to vascular injury the femoral arteries of ApoE/ mice were isolated, mobilized and infected with Ad-HIFadn 72 h before the injury and the IMR and LVR were measured after 5 weeks. No difference of IMR and LVR could be found between infections with Ad-HIFadn 72 h before or directly to vascular injury (Supplemental Fig. 2). 3.2. Effects of local suppression of the HIF pathway by Ad-HIFadn on the content of VSMC and MC of neointimal lesions In the next step the role of the HIF pathway on neointimal content of vascular smooth muscle cells (VSMC) and monocytes/ macrophages (MC) of wire-induced atherosclerotic plaque was examined by quantitative immunohistochemistry. For this purpose, a-actin staining for detection of VSMC (Fig. 2A) and galectin-3 staining for determination of MC (Fig. 2B) was performed 2 and 5 weeks after transluminal artery injury. After 2 weeks, a significant decrease by 57.5% of VSMC positive neointimal area and a decrease by 62.8% monocyte/macrophage positive neointimal area could be detected in HIF-adn treated arteries compared to LacZ controls. In
Fig. 1. Local HIF-inhibition reduces the atherosclerotic plaque development. Local Ad-HIFadn infection directly after wire induced vascular injury of femoral artery in ApoE/ mice dramatically reduces the restenosis compared to Ad-LacZ infection 5 weeks after vascular injury. Time course of atherosclerotic plaque development after local Ad-HIFadn infection 2 h (0 weeks), 2 weeks and 5 weeks after wire induced injury of femoral artery in comparison to Ad-LacZ control. Representative images of Elastica van Gieson stained sections are shown (scale bar ¼ 100 mm) (A). Planimetrical analysis revealed a significantly reduced plaque development already 2 weeks after injury in Ad-HIFadn treated animals compared Ad-LacZ controls. This results in a significant decrease of IMR (B) and a significant increase of LVR (C) in Ad-HIFadn treated arteries compared Ad-LacZ infected vessels after 5 weeks. (n ¼ 10).
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Fig. 2. Reduction in plaque size is accompanied by lowered neointimal VSMC and MC content. Effect of HIFadn on the neointimal cellular atherosclerotic plaque composition 2 and 5 weeks after transluminal vascular injury revealed significant lowered a-actin (A) and galactin-3 expression (B) in Ad-HIFadn infected arteries. (A) Representative images of a-actin staining (VSMC) 2 and 5 weeks after vascular injury and subsequent infection with either Ad-HIFadn or Ad-LacZ control are shown (scale bar ¼ 100 mm). Statistical analysis revealed a significant decrease of total neointimal VSMC positive area in Ad-HIFadn treated arteries compared to Ad-LacZ controls 2 and 5 weeks after vessel injury (n ¼ 6). (B) Representative images of galectin-3 staining 2 and 5 weeks after vascular injury and local infection with either Ad-HIFadn or Ad-LacZ control are illustrated (scale bar ¼ 100 mm). Statistical analysis revealed a significantly diminished total neointimal monocyte/macrophage (MC) positive area in Ad-HIFadn treated arteries in comparison to Ad-LacZ controls 2 and 5 weeks after vessel injury. The neointimal VSMC/MC ratio remained unchanged in HIF-adn and LacZ group 2 and 5 weeks after vascular injury (n ¼ 6).
addition, no further changes in the cellular composition of the atherosclerotic plaque were observed 5 weeks after vascular injury (VSMC: decrease by 69.5%; MC: decrease by 50.6% in HIF-adn treated arteries) (Fig. 2). Because of the unchanged positive area of VSMC and MCs despite of the further increased neointimal area after 5 weeks, we additionally performed fluorescence-lifetime imaging microscopy of the arteries 5 weeks after vascular injury to clarify further content of the neointimal lesions. Using this approach clearly shows that the lacZ-treated arteries has a much higher content of collagen within the neointima, which might be an explanation for the increase in neointima but not in smooth muscle cells (Supplemental Fig. 4). 3.3. Expression of the HIF target proteins VEGF-A, Flt-1 and Flk-1 in neointimal lesions after local suppression of the HIF pathway by AdHIFadn To further study the regulation of HIF target proteins within atherosclerotic lesions, quantitative immunohistochemical analysis of neointimal expression of the vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 1 (Flt-1, VEGFR-1) and vascular endothelial growth factor receptor 2 (Flk-1, VEGFR-2) were conducted 2 and 5 weeks after transluminal vascular injury. 2 weeks after vascular injury, a significant reduction of the absolute neointimal positive area of VEGF-A (60.0%), Flt1 (77.8%) and Flk-1 (49.7%) was detected in the HIF-adn treated animals compared to LacZ controls (Fig. 3). This diminished neointimal expression of HIF target proteins was unchanged up to 5
weeks after vascular injury. Looking on the relative expression within the neointima (ratio positive staining area to neointima area) revealed a difference between lacZ and Hifadn arteries after 2 weeks but not after 5 weeks, suggesting an important role of the HIF signaling especially in the beginning of neointima formation (Supplemental Fig. 5).
3.4. Local overexpression of HIF-1a and -2a increase restenosis after vascular injury It should be clarified whether an overexpression of the HIF-a subunits, Hif-1a and -2a, also modulates the neointimal formation after vascular injury. To address this issue, femoral arteries of ApoE/ mice were locally in vivo infected with adenoviruses encoding for HIF-1a(Ad-HIF-1a), -2a(Ad-HIF-2a) or b-galactosidase (Ad-LacZ, as negative control) immediately after vascular injury. The treated arteries were harvested 5 weeks after vascular injury and the IMR and LVR were determined. The results of this experiment are summarized in Fig. 4. The overexpression of HIF-1a caused a significant increase by 64.7% of IMR and a decrease by 55.6% of LVR compared to LacZ controls 5 weeks after transluminal vessel injury. The local overexpression of the HIF-2a subunit also resulted in an increased plaque size 5 weeks after the injury in comparison to the LacZ controls (increase of IMR by 41.8%, decrease of LVR by 59.4%). In addition, an increased expression of HIF-target genes VEGF-A, Flt-1 and Flk-1 was detected in HIF-a overexpressing arteries (Supplemental Fig. 6).
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Fig. 3. Inhibition of the HIF-pathway reduces the expression of HIF-target genes within the plaque. Local suppression of the HIF pathway by Ad-HIFadn after vascular injury dramatically reduces neointimal VEGF-A, Flt-1 and Flk-1 expression. Representative images of VEGF-A (A), Flt-1 (B) and Flk-1 (C) staining 2 and 5 weeks after vascular injury and local infection with either Ad-HIFadn or Ad-LacZ control are shown (scale bar ¼ 100 mm). Statistical analysis revealed a significant decrease of total neointimal positive area of VEGFA, Flt-1 and Flk-1 in Ad-HIFadn treated arteries compared to Ad-LacZ controls 2 and 5 weeks after vessel injury (n ¼ 5).
4. Discussion Restenosis after coronary stent implantation still keeps one of the major complications caused by massive infiltration of mononuclear cells, chronic inflammation, and proliferation of smooth muscle cells and endothelial cells [25,26]. The regulatory mechanisms of this morphologically well characterized clinical problem remains poorly understood. In the development and the progression of atherosclerotic lesions, local hypoxic areas within the arterial wall seem to play a central role [4]. As the HIF-system plays
a pivotal role for hypoxic tissue response, the present animal study investigated the effect of a local suppression of the HIF pathway on neointima formation in a model of transluminal wire-injured arteries of ApoE/ mice. Secondary, the impact of overexpression of the HIF-a subunits, HIF-1a and HIF-2a, on neointima formation was studied. The salient finding of the study is the significant reduction of injury-induced neointima formation through local inhibition of the HIF pathway. In addition, the work also shows an increased neointima formation after local overexpression of the HIF-1a and -2a
Fig. 4. Local overexpression of HIF-1a and -2a increases the plaque size. Local overexpression of the HIF subunits HIF-1a and HIF-2a cause an advanced plaque progression compared to LacZ treatment 5 weeks after transluminal artery injury. Representative images of Elastica van Gieson stained sections are shown (scale bar ¼ 100 mm). Statistical analysis (n ¼ 5) revealed a similar increase in IMR and a decreased LVR in Ad-HIF-1a and Ad-HIF-2a infected arteries compared to LacZ control.
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subunit, demonstrating the importance of the HIF-system in the pathogenesis of atherosclerosis. Within human atherosclerotic plaques, obtained by carotid endarterectomy both hypoxic areas within arterial walls and a colocalization of hypoxic areas and HIF expression could be detected [4,27,28]. Moreover, histological analysis of atheromatous carotid arteries revealed an increased expression of inflammatory as well as apoptotic molecules in plaque areas in which HIF-1a was upregulated [29]. However, whether the induction of the HIF pathway promotes plaque development or hypoxia-induced, intraplaque HIF stabilization protects against further plaque progression remained unclear so far. In the present study, five weeks after transluminal vascular injury the restenosis was dramatically reduced in arteries with locally inhibited HIF pathway compared to controls (Fig. 1). In contrast, Ben-Shoshan et al. studied the effect of systemic overexpression of HIF-1a in lymphocytes of ApoE/ mice and found an inhibition of atherosclerotic plaque progression [30]. But, our findings are consistent to a previous work of Karshovska et al. demonstrating a reduction of neointimal area after vascular injury by local HIF-1a silencing using siRNA [31]. These data suggest a discrepancy between the systemic, cell-specific and local function of HIF in atherosclerosis. In the next step we investigated the cellular composition of neointimal lesions after vascular injury and local HIF suppression. Karshovska et al. described an isolated reduced VSMC proliferation as a result of decreased expression of the stromal cell-derived factor (SDF-1a) due to inhibition of HIF-1a as the major cause of the suppressed neointimal formation [31]. We could confirm the reduction of the absolute amount of VSMC due to inhibition of HIF, but not in the relative neointimal content of VSMC and MC. Moreover, using FLIM, we could detect an increase in collagen content in control arteries compared to HIF-inhibited arteries, which might be an additional explanation for the neointima decrease. These results suggest, that the anti-atherosclerotic effect of local HIF inhibition cannot exclusively explained by isolated suppression of VSMC proliferation. Instead, a modulation of numerous HIF target genes in at least VSMC, MC and probably different cell types is a more likely explanation in the present model. In contrast to the study of Kashovska we used an adenoviral approach instead of siRNA-mediated silencing of HIF-1a, which probably not only affects the VSMC within the arterial wall but also other cell types like fibroblasts and macrophages. Furthermore the use of HIF-adn leads to a competitive inhibition of the HIF-pathway as it is directed against HIF-1 and HIF-2 signaling. The HIF-system increases the expression of several hundred genes and affects indirectly the mRNA of an equal number of genes that mediate cellular adaptive responses to hypoxic conditions [32]. Various studies had shown the importance of this mechanism: Deguchi et al. recently reported about a hypoxia-triggered and potential HIF dependent activation of the Akt and b-catenin axis and expression of downstream gene products, which amplify inflammatory milieu in permanent atherosclerotic plaque macrophages [33]. Another study demonstrated a hypoxia-triggered upregulation of LRP1 (low-density lipoprotein receptor-related protein 1) and aggregated LDL-derived intracellular cholesterol ester accumulation in human VSMCs. These effects of intravascular LDL metabolism were also induced through HIF-1a in atherosclerotic plaques [34]. Another in vitro study showed that hypoxia stimulates the autocrine regulation of VSMCs migration via HIF-1a dependent expression of Thrombospondin-1 as cause of enhanced atherogenesis [35]. Furthermore, elevated levels of VEGF-C and its receptor Flt-4 could be measured in hypoxic cell cultures [36]. As described previously, we also could detect VEGF-C and Flt-4 expression in permanent atherosclerotic monocytes/macrophages, which was associated with an increased apoptosis of these mononuclear cells [37]. The implication of all these findings is that
the reduction of atherosclerotic neointima formation through inhibition of the HIF pathway is likely caused by a multifactorial regulation of downstream genes in VSMC, MC and probably further plaque-associated cell types. Another observation of the present study is, that already in the early stage, two weeks after plaque initiation, a significant reduction of atherosclerotic lesion progression was observed after inhibition of HIF signaling using Ad-HIFadn (Fig. 1). This cannot exclusively be explained by local suppression of hypoxia-triggered HIF accumulation after transluminal vascular injury, because neointimal hypoxia doesn’t seem to play a relevant role in the early stage of atherosclerotic lesion initiation. More likely is the early local inhibition of oxygen-independent HIF accumulation in the injured arterial walls. In this context we recently demonstrated that oxLDL, which is predominantly located in atherosclerotic plaques causes an oxygen-independent accumulation of HIF-1a in macrophages. Furthermore, we could prove that HIF-1a protects macrophages against oxLDL induced apoptosis [24]. In addition, other non-hypoxic stimuli like angiotensin-II increases HIF-1a translation by a reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase pathway, which acts on the 50 untranslated region of HIF-1a mRNA was described [38]. Another finding of the present study is the decreased neointimal expression of both VEGF-A and its receptors Flt-1 (VEGFR-1) and Flk-1 (VEGFR2) after HIF-pathway inhibition (Fig. 3), as possible cause of the reduced atherosclerotic plaque progression. So, previous immunohistological findings of human endarterectomy specimens revealed that in atherosclerotic plaques, the transcription factor HIF-1a is associated with VEGF-A expression and with an atheromatous inflammatory plaque phenotype [39,40]. Additionally, in vitro data demonstrated a hypoxia induced proliferation of coronary artery VSMCs which was mediated through the expression of VEGF-A and Flt-1 [41]. Furthermore, recent works demonstrated that VEGF-A accelerates neointima formation through Flk-1 by regulating MCP-1 (monocyte chemoattractant protein-1) expression in VSMCs and macrophage-mediated inflammation in murine wire-injured arteries [42,43]. Moreover, the findings of Khurana et al. indicate that VEGF induced adventitial angiogenesis stimulates neointimal thickening but does not initiate it [44]. In contrast to the local inhibition of the HIF pathway, which causes a reduced restenosis, we also performed local adenoviral overexpression of the HIF-1a and -2a after vascular injury in ApoE/ mice (Fig. 4). In both cases the overexpression resulted in an increased restenosis compared to control animals. These results together with the data the HIF inhibition-experiments validated our hypothesis, that the HIF pathway plays an important role in wire-induced restenosis. The limitation of the present work is the unspecific inhibition of different cell types only inside the adventitia and the media. But several works agree that especially the adventitial angiogenesis and HIF induced expression of proproliferative and proinflammatory genes may significantly impact neointimal formation [45]. Further investigations are necessary to examine the role of cell-specific local HIF inhibition in atherosclerotic restenosis. In conclusion, the present study demonstrated the pivotal role of the hypoxia-inducible factor HIF in atherosclerotic plaque development after wire-induced injury. The reduction of neointimal hyperplasia after inhibition of HIF is accompanied by a reduction of the HIF target genes VEGF-A and its receptors. The current findings demonstrate the important effect of the key transcription factor HIF rather than modification of putative single downstream target genes of HIF. Therefore, an intravascular gene transfer for local HIF inhibition may provide an alternative option for treatment of atherosclerosis and in stent restenosis in future.
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Disclosures None. Conflict of interest
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None. [22]
Acknowledgments [23]
This project was supported by grants to M.C. from the MeDDrive program, University of Technology Dresden. The authors thank Peggy Barthel, Janet Lehmann and Anita Männel for excellent technical support. We thank Martin Hammer, PhD, Department of Ophthalmology, University of Jena, Germany for performing of the fluorescence-lifetime imaging microscopy.
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[25] [26]
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.01.048.
[27] [28] [29]
References [30] [1] Dangas GD, Claessen BE, Caixeta A, Sanidas EA, Mintz GS, Mehran R. In-stent restenosis in the drug-eluting stent era. J Am Coll Cardiol 2010;56:1897e907. [2] Gaspardone A, Versaci F. Coronary stenting and inflammation. Am J Cardiol 2005;96:65Le70L. [3] Davies MG, Hagen PO. Pathobiology of intimal hyperplasia. Br J Surg 1994;81: 1254e69. [4] Sluimer JC, Gasc JM, van Wanroij JL, et al. Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. J Am Coll Cardiol 2008;51:1258e65. [5] Szekanecz Z, Besenyei T, Szentpetery A, Koch AE. Angiogenesis and vasculogenesis in rheumatoid arthritis. Curr Opin Rheumatol 2010;22:299e306. [6] Li Y, Ye D. Cancer therapy by targeting hypoxia-inducible factor-1. Curr Cancer Drug Targets 2010;10:782e96. [7] Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438: 932e6. [8] Bracken CP, Whitelaw ML, Peet DJ. The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses. Cell Mol Life Sci 2003;60:1376e 93. [9] Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basichelix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92:5510e4. [10] Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 1995;270:1230e7. [11] Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 2004;5:343e54. [12] Wiesener MS, Turley H, Allen WE, et al. Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxiainducible factor-1alpha. Blood 1998;92:2260e8. [13] Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, Fujii-Kuriyama Y. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A 1997;94:4273e8. [14] Gu YZ, Moran SM, Hogenesch JB, Wartman L, Bradfield CA. Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha. Gene Expr 1998;7:205e13. [15] Kaelin WG, Ratcliffe PJ. Oxygen sensing by Metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 2008;30:393e402. [16] Miyata T, Takizawa S, van Ypersele de Strihou C. Hypoxia. 1. Intracellular sensors for oxygen and oxidative stress: novel therapeutic targets. Am J Physiol Cell Physiol 2011;300:C226e31. [17] Rey S, Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovasc Res 2010;86:236e42. [18] Sluimer JC, Daemen MJ. Novel concepts in atherogenesis: angiogenesis and hypoxia in atherosclerosis. J Pathol 2009;218:7e29. [19] Roque M, Fallon JT, Badimon JJ, Zhang WX, Taubman MB, Reis ED. Mouse model of femoral artery denudation injury associated with the rapid
[31]
[32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44] [45]
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accumulation of adhesion molecules on the luminal surface and recruitment of neutrophils. Arterioscler Thromb Vasc Biol 2000;20:335e42. Shagdarsuren E, Bidzhekov K, Mause SF, et al. C5a receptor targeting in neointima formation after arterial injury in atherosclerosis-prone mice. Circulation 2010;122:1026e36. Augstein A, Poitz DM, Braun-Dullaeus RC, Strasser RH, Schmeisser A. Cellspecific and hypoxia-dependent regulation of human HIF-3alpha: inhibition of the expression of HIF target genes in vascular cells. Cell Mol Life Sci 2011;68:2627e42. Jing D, Wobus M, Poitz DM, Bornhauser M, Ehninger G, Ordemann R. Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro. Haematologica 2012;97:331e9. Maemura K, Hsieh CM, Jain MK, et al. Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain. J Biol Chem 1999;274:31565e70. Poitz DM, Augstein A, Weinert S, Braun-Dullaeus RC, Strasser RH, Schmeisser A. OxLDL and macrophage survival: essential and oxygenindependent involvement of the Hif-pathway. Basic Res Cardiol 2011;106: 761e72. Hoffmann R, Mintz GS. Coronary in-stent restenosis e predictors, treatment and prevention. Eur Heart J 2000;21:1739e49. Stone GW, Ellis SG, Cox DA, et al. A polymer-based, paclitaxel-eluting stent in patients with coronary artery disease. N Engl J Med 2004;350:221e31. Bjornheden T, Levin M, Evaldsson M, Wiklund O. Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol 1999;19:870e6. Luque A, Turu M, Juan-Babot O, et al. Overexpression of hypoxia/inflammatory markers in atherosclerotic carotid plaques. Front Biosci 2008;13:6483e90. Bitto A, De Caridi G, Polito F, et al. Evidence for markers of hypoxia and apoptosis in explanted human carotid atherosclerotic plaques. J Vasc Surg 2010;52:1015e21. Ben-Shoshan J, Afek A, Maysel-Auslender S, et al. HIF-1alpha overexpression and experimental murine atherosclerosis. Arterioscler Thromb Vasc Biol 2009;29:665e70. Karshovska E, Zernecke A, Sevilmis G, et al. Expression of HIF-1 in injured arteries controls SDF-1 mediated neointima formation in apolipoprotein E deficient mice. Arterioscler Thromb Vasc Biol 2007;27:2540e7. Semenza GL. Oxygen sensing, homeostasis, and disease. N Engl J Med 2011;365:537e47. Deguchi Jo, Yamazaki H, Aikawa E, Aikawa M. Chronic hypoxia Activates the Akt and -Catenin pathways in human macrophages. Arterioscler Thromb Vasc Biol 2009;29:1664e70. Castellano J, Aledo R, Sendra J, et al. Hypoxia stimulates low-density lipoprotein receptor-related protein-1 expression through hypoxia-inducible factor-1alpha in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2011;31:1411e20. Osada-Oka M, Ikeda T, Akiba S, Sato T. Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alphadependent expression of thrombospondin-1. J Cell Biochem 2008;104: 1918e26. Simiantonaki N, Jayasinghe C, Michel-Schmidt R, Peters K, Hermanns MI, Kirkpatrick CJ. Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines. Int J Oncol 2008;32:585e92. Schmeisser A, Christoph M, Augstein A, et al. Apoptosis of human macrophages by Flt-4 signaling: implications for atherosclerotic plaque pathology. Cardiovasc Res 2006;71:774e84. Page EL, Robitaille GA, Pouyssegur J, Richard DE. Induction of hypoxiainducible factor-1alpha by transcriptional and translational mechanisms. J Biol Chem 2002;277:48403e9. Vink A, Schoneveld AH, Lamers D, et al. HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages. Atherosclerosis 2007;195:e69e75. Chen F, Eriksson P, Kimura T, Herzfeld I, Valen G. Apoptosis and angiogenesis are induced in the unstable coronary atherosclerotic plaque. Coron Artery Dis 2005;16:191e7. Osada-Oka M, Ikeda T, Imaoka S, Akiba S, Sato T. VEGF-enhanced proliferation under hypoxia by an autocrine mechanism in human vascular smooth muscle cells. J Atheroscler Thromb 2008;15:26e33. Bhardwaj S, Roy H, Heikura T, Yla-Herttuala S. VEGF-A, VEGF-D and VEGFD(DeltaNDeltaC) induced intimal hyperplasia in carotid arteries. Eur J Clin Invest 2005;35:669e76. Koga J, Matoba T, Egashira K, et al. Soluble Flt-1 gene transfer ameliorates neointima formation after wire injury in flt-1 tyrosine kinase-deficient mice. Arterioscler Thromb Vasc Biol 2009;29:458e64. Khurana R, Zhuang Z, Bhardwaj S, et al. Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation 2004;110:2436e43. Simons M. VEGF and restenosis: the rest of the story. Arterioscler Thromb Vasc Biol 2009;29:439e40.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Local inhibition of hypoxia-inducible factor reduces neointima formation after arterial injury in ApoE/ mice Marian Christoph 1, Karim Ibrahim 1, Kathleen Hesse, Antje Augstein, Alexander Schmeisser 2, Ruediger C. Braun-Dullaeus 2, Gregor Simonis, Carsten Wunderlich, Silvio Quick, Ruth H. Strasser, David M. Poitz* University of Dresden, Heart Center, University Hospital, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 July 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 2 February 2014
Objective: Hypoxia plays a pivotal role in development and progression of restenosis after vascular injury. Under hypoxic conditions the hypoxia-inducible factors (HIFs) are the most important transcription factors for the adaption to reduced oxygen supply. Therefore the aim of the study was to investigate the effect of a local HIF-inhibition and overexpression on atherosclerotic plaque development in a murine vascular injury model. Methods and results: After wire-induced vascular injury in ApoE/ mice a transient, local inhibition of HIF as well as an overexpression approach of the different HIF-subunits (HIF-1a, HIF-2a) by adenoviral infection was performed. The local inhibition of the HIF-pathway using a dominant-negative mutant dramatically reduced the extent of neointima formation. The diminished plaque size was associated with decreased expression of the well-known HIF-target genes vascular endothelial growth factor-A (VEGF-A) and its receptors Flt-1 and Flk-1. In contrast, the local overexpression of HIF-1a and HIF-2a further increased the plaque size after wire-induced vascular injury. Conclusions: Local HIF-inhibition decreases and HIF-a overexpression increases the injury induced neointima formation. These findings provide new insight into the pathogenesis of atherosclerosis and may lead to new therapeutic options for the treatment of in stent restenosis. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Atherosclerosis Restenosis Hypoxia-inducible factor Hypoxia
1. Introduction Coronary restenosis still represents one of the major complications after coronary stent implantation even in the era of drug eluting stents [1]. The main mechanism for the developing of restenosis after vascular injury is an exceeding neointimal hyperplasia [2]. This pathomechanism is characterized by a massive invasion of mononuclear cells as well as chronic inflammation with excessive local release of cytokines and chemokines, which finally results in a pathological increased proliferation of smooth muscle cells and endothelial cells [3]. Despite the histological composition of neointimal hyperplasia is well characterized, the molecular
* Corresponding author. University of Technology Dresden, Heart Center Dresden University hospital, Fetscherstr. 76, 01307 Dresden, Germany. Tel.: þ49 351 458 6627; fax: þ49 351 458 6329. E-mail address: [email protected] (D.M. Poitz). 1 Authors contributed equally to this work. 2 Present address: Internal Medicine, Department of Cardiology, Angiology and Pneumology, Magdeburg University, Germany. 0021-9150/$ e see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.048
regulation remains poorly understood even after numerous of different in vitro and in vivo studies. The hypoxia-inducible factor (HIF) pathway is thought to be associated with atherosclerotic lesion progression, suggesting its involvement in the response to hypoxia and regulation of human intra-plaque angiogenesis. This hypothesis is supported by the in vivo detection of hypoxia in plaque-macrophages and the expression of several hypoxia-induced genes in atherosclerotic lesions [4]. The hypoxia-inducible factors are key transcriptional regulators of physiological as well as pathophysiological hypoxic response in both embryonic and adult organisms and are activated in many different human diseases including cancer, rheumatoid arthritis, stroke, and chronic lung disease [5e8]. The hypoxia-inducible factors are heterodimeric transcription factors consisting of an oxygen-sensitive HIF-a subunit and a constitutively expressed HIFb subunit [9,10]. The HIF-a/b-dimer binds to a core DNA-motif, the so called hypoxia-response element (HRE) and induces a variety of hypoxia-responsive genes that are involved in a variety cellular processes like angiogenesis, erythropoiesis, glucose metabolism and vasomotor regulation [11].
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The HIF-family consists of the three closely related subunits HIF1a, HIF-2a and HIF-3a. Whereas HIF-1a is ubiquitously expressed, analysis of cell-type-specific expression patterns of HIF-2a showed predominantly expression in endothelial, epithelial cells as well as fibroblasts and macrophages [8,12e14]. Under normoxic conditions the HIF-1a and HIF-2a subunits are continuously hydroxylated by prolyl hydroxylase domain-containing enzymes (PHDs) [15]. The hydroxylated HIF-a subunits are bound by the von Hippel-Lindau protein complex (pVHL), leading to ubiquitinilation and subsequent proteosomal degradation [16]. However, in the absence of oxygen the HIF-a-subunits are stabilized, dimerize with the HIF-1bsubunit and induce transcription of numerous genes [17]. With consideration of the above-mentioned findings of hypoxic areas within atherosclerotic plaques, it can be assumed that the HIFpathway is strongly associated with atherosclerotic plaque progression. The reactive or causative role of HIF in atherogenesis remains unclear to date [18]. Therefore, this in vivo study investigates the role of a transient and local HIF inhibition as well as a local overexpression of HIF-1a and -2a subunits in neointima formation after vascular injury.
femoral bifurcation), as described previously [20]. These sections were stained with Elastica van Gieson stain, and digital microscopic images were taken (Carl Zeiss). The area of lumen, neointima (area between lumen and internal elastic lamina) and media (area between internal and external elastic lamina) were determined by planimetry (Image J, National Institutes of Health) (see Supplemental Fig. 1A). From the obtained areas of lumen, neointima and media the intima/media ratio (area of neointima divided to area of media, IMR) and lumen/vessel-wall ratio (area of lumen divided to area of media and neointima, LVR) were calculated. The mean values of IMR and LVR of multiple sections within the same vessel were obtained in order to consider the biological longitudinal variability of neointimal plaque morphology. 2.6. X-Gal-staining and immunohistochemistry Details are described in the data supplement. 2.7. Fluorescence-lifetime imaging microscopy Details are described in the data supplement.
2. Materials and methods 2.8. Statistical analysis 2.1. Cell culture, adenovirus construction and adenoviral infection Details are described in the data supplement. 2.2. Real-time RT-PCR Details are described in the data supplement. 2.3. Mouse model The details are described in the data supplement. 2.4. Atherogenic vascular injury model All procedures were approved by the local ethical committee (24D-9168.11-1/2008-17). The unilateral femoral artery injury was performed as published previously [19]. In brief male mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). First, a groin incision was made and the common and deep femoral artery were dissected and mobilized. After temporarily clamping of the femoral artery a small transverse incision of the deep femoral artery was made and an angioplasty guide wire (0.014 inch diameter) was introduced and advanced to the level of aortic bifurcation. The wire was left inside the vessel for 1 min in order to achieve endothelial injury. Afterwards the wire was removed and the deep femoral artery was ligated. The contralateral femoral artery was not injured and served as negative control. For transient local adenoviral infection of the femoral artery, immediately after the vascular injury the treated artery was locally incubated with 40 ml sodium chloride solution containing adenoviral vectors at a dose of 1 109 infectious units (ifu) for 30 min. Subsequently the virus solution was carefully aspirated and the skin was closed with interrupted sutures. 2.5. Histological and immunohistological analysis To examine the development of the atherosclerotic lesions, the treated arteries were harvested at specific time points (between 2 and 5 weeks). For the quantification of neointimal and medial areas, 8 sections from the paraformaldehyde-fixed and paraffinembedded femoral arteries were taken (each 4 mm in thickness, 50 mm distance between the sections, started 50 mm from the
Results were expressed as means standard deviation. Statistical analysis was done using the 2-tailed, unpaired Student’s t-test. Level of significance was set to p < 0.05. p values below 0.05(0.01/ 0.001) are indicated by *(**/***). 3. Results 3.1. In vivo local inhibition of the HIF pathway by Ad-HIFadn significantly reduces neointima formation after wire-induced vascular injury To investigate the impact of local inhibition of the HIF pathway on neointima formation, a wire-induced endovascular injury of the femoral artery in ApoE/ mice was performed and a local transduction with an adenovirus was established. To show the biological functionality of the dominant-negative HIF-a mutant in murine cells, vascular smooth muscle cells (VSMC) and macrophages were infected in vitro with an adenovirus encoding either for b-galactosidase (Ad-LacZ) as a control or for a dominant-negative mutant based on HIF-2a (Ad-HIFadn), which leads to a competitive inhibition of both HIF-1a and HIF-2a [21e 24]. Subsequently, the mRNA expression of the HIF target genes VEGF-A and PGK1 (Phosphoglycerate-kinase) were analyzed by real-time RT-PCR. The infection with Ad-HIFadn decreased the hypoxia-triggered mRNA induction significantly compared to the corresponding Ad-LacZ-control (Supplemental Fig. 3). To test the in vivo local infection efficiency of the recombinant replication-deficient adenoviruses, the femoral arteries of ApoE/ mice were in vivo incubated in a solution with Ad-LacZ for 30 min. Subsequently the skin was closed. X-Gal staining 72 h after the local infection showed sufficient local transduction efficiency. 2 weeks after infection no positive X-Gal staining was observed. To exclude that infection with Ad-LacZ itself has an influence on the neointima formation as a consequence of increased local inflammation, the femoral artery was incubated in vivo with either Ad-LacZ or saline as control for 30 min after wire induced vascular injury in ApoE/ mice. After 5 weeks there was no difference in IMR after local infection with Ad-LacZ compared to saline control (Supplemental Fig. 1C). To study the effect of local HIF inhibition on neointima formation, immediately after the vascular injury, the treated femoral
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artery was locally infected either with Ad-HIFadn or with Ad-LacZ as control for 30 min. After 5 weeks the femoral arteries were harvested and the IMR (intima/media ratio) and LVR (lumen/vesselwall ratio) as values of the atherosclerotic plaque size were measured by planimetry. After 5 weeks the intima/media ratio was dramatically reduced by 65.4% in Ad-HIFadn infected arteries compared to Ad-LacZ controls (Fig. 1). Additionally, a significant increase by 113.8% of the lumen/vessel-wall ratio as sign of a preserved vessel lumen after Ad-HIFadn infection in comparison to Ad-LacZ was observed. To evaluate the effect of Ad-HIFadn infection on neointima formation in a time course after transluminal wire-injury, the femoral arteries of ApoE/ mice were directly infected after injury either with Ad-HIFadn or with Ad-LacZ as control. Afterwards the treated vessels were harvested at time points of 2 h (0 weeks), 2 weeks and 5 weeks after vascular injury and the IMR and LVR were calculated (Fig. 1). As expected, 2 h after vascular injury, no relevant neointimal area and no relevant change in LMR could be measured in both groups. 2 weeks after vascular injury and local infection with Ad-HIFadn, a significant reduction by 42.3% of IMR and a trend of a LMR reduction of 22.1% is already evident in comparison to AdLacZ treated vessels. In contrast to Ad-LacZ control which show an advanced plaque progression after 5 weeks, no further relevant plaque progression could be detected in Ad-HIFadn infected femoral arteries between 2 and 5 weeks.
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Next, it should be investigated if this inhibition of neointima formation could be further enhanced by local Ad-HIFadn infection 72 h before wire-induced vascular injury in order to ensure the biological functionality of HIFadn directly at the time point of vascular injury. To address the role of HIF inhibition directly to vascular injury the femoral arteries of ApoE/ mice were isolated, mobilized and infected with Ad-HIFadn 72 h before the injury and the IMR and LVR were measured after 5 weeks. No difference of IMR and LVR could be found between infections with Ad-HIFadn 72 h before or directly to vascular injury (Supplemental Fig. 2). 3.2. Effects of local suppression of the HIF pathway by Ad-HIFadn on the content of VSMC and MC of neointimal lesions In the next step the role of the HIF pathway on neointimal content of vascular smooth muscle cells (VSMC) and monocytes/ macrophages (MC) of wire-induced atherosclerotic plaque was examined by quantitative immunohistochemistry. For this purpose, a-actin staining for detection of VSMC (Fig. 2A) and galectin-3 staining for determination of MC (Fig. 2B) was performed 2 and 5 weeks after transluminal artery injury. After 2 weeks, a significant decrease by 57.5% of VSMC positive neointimal area and a decrease by 62.8% monocyte/macrophage positive neointimal area could be detected in HIF-adn treated arteries compared to LacZ controls. In
Fig. 1. Local HIF-inhibition reduces the atherosclerotic plaque development. Local Ad-HIFadn infection directly after wire induced vascular injury of femoral artery in ApoE/ mice dramatically reduces the restenosis compared to Ad-LacZ infection 5 weeks after vascular injury. Time course of atherosclerotic plaque development after local Ad-HIFadn infection 2 h (0 weeks), 2 weeks and 5 weeks after wire induced injury of femoral artery in comparison to Ad-LacZ control. Representative images of Elastica van Gieson stained sections are shown (scale bar ¼ 100 mm) (A). Planimetrical analysis revealed a significantly reduced plaque development already 2 weeks after injury in Ad-HIFadn treated animals compared Ad-LacZ controls. This results in a significant decrease of IMR (B) and a significant increase of LVR (C) in Ad-HIFadn treated arteries compared Ad-LacZ infected vessels after 5 weeks. (n ¼ 10).
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Fig. 2. Reduction in plaque size is accompanied by lowered neointimal VSMC and MC content. Effect of HIFadn on the neointimal cellular atherosclerotic plaque composition 2 and 5 weeks after transluminal vascular injury revealed significant lowered a-actin (A) and galactin-3 expression (B) in Ad-HIFadn infected arteries. (A) Representative images of a-actin staining (VSMC) 2 and 5 weeks after vascular injury and subsequent infection with either Ad-HIFadn or Ad-LacZ control are shown (scale bar ¼ 100 mm). Statistical analysis revealed a significant decrease of total neointimal VSMC positive area in Ad-HIFadn treated arteries compared to Ad-LacZ controls 2 and 5 weeks after vessel injury (n ¼ 6). (B) Representative images of galectin-3 staining 2 and 5 weeks after vascular injury and local infection with either Ad-HIFadn or Ad-LacZ control are illustrated (scale bar ¼ 100 mm). Statistical analysis revealed a significantly diminished total neointimal monocyte/macrophage (MC) positive area in Ad-HIFadn treated arteries in comparison to Ad-LacZ controls 2 and 5 weeks after vessel injury. The neointimal VSMC/MC ratio remained unchanged in HIF-adn and LacZ group 2 and 5 weeks after vascular injury (n ¼ 6).
addition, no further changes in the cellular composition of the atherosclerotic plaque were observed 5 weeks after vascular injury (VSMC: decrease by 69.5%; MC: decrease by 50.6% in HIF-adn treated arteries) (Fig. 2). Because of the unchanged positive area of VSMC and MCs despite of the further increased neointimal area after 5 weeks, we additionally performed fluorescence-lifetime imaging microscopy of the arteries 5 weeks after vascular injury to clarify further content of the neointimal lesions. Using this approach clearly shows that the lacZ-treated arteries has a much higher content of collagen within the neointima, which might be an explanation for the increase in neointima but not in smooth muscle cells (Supplemental Fig. 4). 3.3. Expression of the HIF target proteins VEGF-A, Flt-1 and Flk-1 in neointimal lesions after local suppression of the HIF pathway by AdHIFadn To further study the regulation of HIF target proteins within atherosclerotic lesions, quantitative immunohistochemical analysis of neointimal expression of the vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 1 (Flt-1, VEGFR-1) and vascular endothelial growth factor receptor 2 (Flk-1, VEGFR-2) were conducted 2 and 5 weeks after transluminal vascular injury. 2 weeks after vascular injury, a significant reduction of the absolute neointimal positive area of VEGF-A (60.0%), Flt1 (77.8%) and Flk-1 (49.7%) was detected in the HIF-adn treated animals compared to LacZ controls (Fig. 3). This diminished neointimal expression of HIF target proteins was unchanged up to 5
weeks after vascular injury. Looking on the relative expression within the neointima (ratio positive staining area to neointima area) revealed a difference between lacZ and Hifadn arteries after 2 weeks but not after 5 weeks, suggesting an important role of the HIF signaling especially in the beginning of neointima formation (Supplemental Fig. 5).
3.4. Local overexpression of HIF-1a and -2a increase restenosis after vascular injury It should be clarified whether an overexpression of the HIF-a subunits, Hif-1a and -2a, also modulates the neointimal formation after vascular injury. To address this issue, femoral arteries of ApoE/ mice were locally in vivo infected with adenoviruses encoding for HIF-1a(Ad-HIF-1a), -2a(Ad-HIF-2a) or b-galactosidase (Ad-LacZ, as negative control) immediately after vascular injury. The treated arteries were harvested 5 weeks after vascular injury and the IMR and LVR were determined. The results of this experiment are summarized in Fig. 4. The overexpression of HIF-1a caused a significant increase by 64.7% of IMR and a decrease by 55.6% of LVR compared to LacZ controls 5 weeks after transluminal vessel injury. The local overexpression of the HIF-2a subunit also resulted in an increased plaque size 5 weeks after the injury in comparison to the LacZ controls (increase of IMR by 41.8%, decrease of LVR by 59.4%). In addition, an increased expression of HIF-target genes VEGF-A, Flt-1 and Flk-1 was detected in HIF-a overexpressing arteries (Supplemental Fig. 6).
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Fig. 3. Inhibition of the HIF-pathway reduces the expression of HIF-target genes within the plaque. Local suppression of the HIF pathway by Ad-HIFadn after vascular injury dramatically reduces neointimal VEGF-A, Flt-1 and Flk-1 expression. Representative images of VEGF-A (A), Flt-1 (B) and Flk-1 (C) staining 2 and 5 weeks after vascular injury and local infection with either Ad-HIFadn or Ad-LacZ control are shown (scale bar ¼ 100 mm). Statistical analysis revealed a significant decrease of total neointimal positive area of VEGFA, Flt-1 and Flk-1 in Ad-HIFadn treated arteries compared to Ad-LacZ controls 2 and 5 weeks after vessel injury (n ¼ 5).
4. Discussion Restenosis after coronary stent implantation still keeps one of the major complications caused by massive infiltration of mononuclear cells, chronic inflammation, and proliferation of smooth muscle cells and endothelial cells [25,26]. The regulatory mechanisms of this morphologically well characterized clinical problem remains poorly understood. In the development and the progression of atherosclerotic lesions, local hypoxic areas within the arterial wall seem to play a central role [4]. As the HIF-system plays
a pivotal role for hypoxic tissue response, the present animal study investigated the effect of a local suppression of the HIF pathway on neointima formation in a model of transluminal wire-injured arteries of ApoE/ mice. Secondary, the impact of overexpression of the HIF-a subunits, HIF-1a and HIF-2a, on neointima formation was studied. The salient finding of the study is the significant reduction of injury-induced neointima formation through local inhibition of the HIF pathway. In addition, the work also shows an increased neointima formation after local overexpression of the HIF-1a and -2a
Fig. 4. Local overexpression of HIF-1a and -2a increases the plaque size. Local overexpression of the HIF subunits HIF-1a and HIF-2a cause an advanced plaque progression compared to LacZ treatment 5 weeks after transluminal artery injury. Representative images of Elastica van Gieson stained sections are shown (scale bar ¼ 100 mm). Statistical analysis (n ¼ 5) revealed a similar increase in IMR and a decreased LVR in Ad-HIF-1a and Ad-HIF-2a infected arteries compared to LacZ control.
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subunit, demonstrating the importance of the HIF-system in the pathogenesis of atherosclerosis. Within human atherosclerotic plaques, obtained by carotid endarterectomy both hypoxic areas within arterial walls and a colocalization of hypoxic areas and HIF expression could be detected [4,27,28]. Moreover, histological analysis of atheromatous carotid arteries revealed an increased expression of inflammatory as well as apoptotic molecules in plaque areas in which HIF-1a was upregulated [29]. However, whether the induction of the HIF pathway promotes plaque development or hypoxia-induced, intraplaque HIF stabilization protects against further plaque progression remained unclear so far. In the present study, five weeks after transluminal vascular injury the restenosis was dramatically reduced in arteries with locally inhibited HIF pathway compared to controls (Fig. 1). In contrast, Ben-Shoshan et al. studied the effect of systemic overexpression of HIF-1a in lymphocytes of ApoE/ mice and found an inhibition of atherosclerotic plaque progression [30]. But, our findings are consistent to a previous work of Karshovska et al. demonstrating a reduction of neointimal area after vascular injury by local HIF-1a silencing using siRNA [31]. These data suggest a discrepancy between the systemic, cell-specific and local function of HIF in atherosclerosis. In the next step we investigated the cellular composition of neointimal lesions after vascular injury and local HIF suppression. Karshovska et al. described an isolated reduced VSMC proliferation as a result of decreased expression of the stromal cell-derived factor (SDF-1a) due to inhibition of HIF-1a as the major cause of the suppressed neointimal formation [31]. We could confirm the reduction of the absolute amount of VSMC due to inhibition of HIF, but not in the relative neointimal content of VSMC and MC. Moreover, using FLIM, we could detect an increase in collagen content in control arteries compared to HIF-inhibited arteries, which might be an additional explanation for the neointima decrease. These results suggest, that the anti-atherosclerotic effect of local HIF inhibition cannot exclusively explained by isolated suppression of VSMC proliferation. Instead, a modulation of numerous HIF target genes in at least VSMC, MC and probably different cell types is a more likely explanation in the present model. In contrast to the study of Kashovska we used an adenoviral approach instead of siRNA-mediated silencing of HIF-1a, which probably not only affects the VSMC within the arterial wall but also other cell types like fibroblasts and macrophages. Furthermore the use of HIF-adn leads to a competitive inhibition of the HIF-pathway as it is directed against HIF-1 and HIF-2 signaling. The HIF-system increases the expression of several hundred genes and affects indirectly the mRNA of an equal number of genes that mediate cellular adaptive responses to hypoxic conditions [32]. Various studies had shown the importance of this mechanism: Deguchi et al. recently reported about a hypoxia-triggered and potential HIF dependent activation of the Akt and b-catenin axis and expression of downstream gene products, which amplify inflammatory milieu in permanent atherosclerotic plaque macrophages [33]. Another study demonstrated a hypoxia-triggered upregulation of LRP1 (low-density lipoprotein receptor-related protein 1) and aggregated LDL-derived intracellular cholesterol ester accumulation in human VSMCs. These effects of intravascular LDL metabolism were also induced through HIF-1a in atherosclerotic plaques [34]. Another in vitro study showed that hypoxia stimulates the autocrine regulation of VSMCs migration via HIF-1a dependent expression of Thrombospondin-1 as cause of enhanced atherogenesis [35]. Furthermore, elevated levels of VEGF-C and its receptor Flt-4 could be measured in hypoxic cell cultures [36]. As described previously, we also could detect VEGF-C and Flt-4 expression in permanent atherosclerotic monocytes/macrophages, which was associated with an increased apoptosis of these mononuclear cells [37]. The implication of all these findings is that
the reduction of atherosclerotic neointima formation through inhibition of the HIF pathway is likely caused by a multifactorial regulation of downstream genes in VSMC, MC and probably further plaque-associated cell types. Another observation of the present study is, that already in the early stage, two weeks after plaque initiation, a significant reduction of atherosclerotic lesion progression was observed after inhibition of HIF signaling using Ad-HIFadn (Fig. 1). This cannot exclusively be explained by local suppression of hypoxia-triggered HIF accumulation after transluminal vascular injury, because neointimal hypoxia doesn’t seem to play a relevant role in the early stage of atherosclerotic lesion initiation. More likely is the early local inhibition of oxygen-independent HIF accumulation in the injured arterial walls. In this context we recently demonstrated that oxLDL, which is predominantly located in atherosclerotic plaques causes an oxygen-independent accumulation of HIF-1a in macrophages. Furthermore, we could prove that HIF-1a protects macrophages against oxLDL induced apoptosis [24]. In addition, other non-hypoxic stimuli like angiotensin-II increases HIF-1a translation by a reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase pathway, which acts on the 50 untranslated region of HIF-1a mRNA was described [38]. Another finding of the present study is the decreased neointimal expression of both VEGF-A and its receptors Flt-1 (VEGFR-1) and Flk-1 (VEGFR2) after HIF-pathway inhibition (Fig. 3), as possible cause of the reduced atherosclerotic plaque progression. So, previous immunohistological findings of human endarterectomy specimens revealed that in atherosclerotic plaques, the transcription factor HIF-1a is associated with VEGF-A expression and with an atheromatous inflammatory plaque phenotype [39,40]. Additionally, in vitro data demonstrated a hypoxia induced proliferation of coronary artery VSMCs which was mediated through the expression of VEGF-A and Flt-1 [41]. Furthermore, recent works demonstrated that VEGF-A accelerates neointima formation through Flk-1 by regulating MCP-1 (monocyte chemoattractant protein-1) expression in VSMCs and macrophage-mediated inflammation in murine wire-injured arteries [42,43]. Moreover, the findings of Khurana et al. indicate that VEGF induced adventitial angiogenesis stimulates neointimal thickening but does not initiate it [44]. In contrast to the local inhibition of the HIF pathway, which causes a reduced restenosis, we also performed local adenoviral overexpression of the HIF-1a and -2a after vascular injury in ApoE/ mice (Fig. 4). In both cases the overexpression resulted in an increased restenosis compared to control animals. These results together with the data the HIF inhibition-experiments validated our hypothesis, that the HIF pathway plays an important role in wire-induced restenosis. The limitation of the present work is the unspecific inhibition of different cell types only inside the adventitia and the media. But several works agree that especially the adventitial angiogenesis and HIF induced expression of proproliferative and proinflammatory genes may significantly impact neointimal formation [45]. Further investigations are necessary to examine the role of cell-specific local HIF inhibition in atherosclerotic restenosis. In conclusion, the present study demonstrated the pivotal role of the hypoxia-inducible factor HIF in atherosclerotic plaque development after wire-induced injury. The reduction of neointimal hyperplasia after inhibition of HIF is accompanied by a reduction of the HIF target genes VEGF-A and its receptors. The current findings demonstrate the important effect of the key transcription factor HIF rather than modification of putative single downstream target genes of HIF. Therefore, an intravascular gene transfer for local HIF inhibition may provide an alternative option for treatment of atherosclerosis and in stent restenosis in future.
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Disclosures None. Conflict of interest
[20]
[21]
None. [22]
Acknowledgments [23]
This project was supported by grants to M.C. from the MeDDrive program, University of Technology Dresden. The authors thank Peggy Barthel, Janet Lehmann and Anita Männel for excellent technical support. We thank Martin Hammer, PhD, Department of Ophthalmology, University of Jena, Germany for performing of the fluorescence-lifetime imaging microscopy.
[24]
[25] [26]
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.01.048.
[27] [28] [29]
References [30] [1] Dangas GD, Claessen BE, Caixeta A, Sanidas EA, Mintz GS, Mehran R. In-stent restenosis in the drug-eluting stent era. J Am Coll Cardiol 2010;56:1897e907. [2] Gaspardone A, Versaci F. Coronary stenting and inflammation. Am J Cardiol 2005;96:65Le70L. [3] Davies MG, Hagen PO. Pathobiology of intimal hyperplasia. Br J Surg 1994;81: 1254e69. [4] Sluimer JC, Gasc JM, van Wanroij JL, et al. Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. J Am Coll Cardiol 2008;51:1258e65. [5] Szekanecz Z, Besenyei T, Szentpetery A, Koch AE. Angiogenesis and vasculogenesis in rheumatoid arthritis. Curr Opin Rheumatol 2010;22:299e306. [6] Li Y, Ye D. Cancer therapy by targeting hypoxia-inducible factor-1. Curr Cancer Drug Targets 2010;10:782e96. [7] Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438: 932e6. [8] Bracken CP, Whitelaw ML, Peet DJ. The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses. Cell Mol Life Sci 2003;60:1376e 93. [9] Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basichelix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92:5510e4. [10] Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 1995;270:1230e7. [11] Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 2004;5:343e54. [12] Wiesener MS, Turley H, Allen WE, et al. Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxiainducible factor-1alpha. Blood 1998;92:2260e8. [13] Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, Fujii-Kuriyama Y. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A 1997;94:4273e8. [14] Gu YZ, Moran SM, Hogenesch JB, Wartman L, Bradfield CA. Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha. Gene Expr 1998;7:205e13. [15] Kaelin WG, Ratcliffe PJ. Oxygen sensing by Metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 2008;30:393e402. [16] Miyata T, Takizawa S, van Ypersele de Strihou C. Hypoxia. 1. Intracellular sensors for oxygen and oxidative stress: novel therapeutic targets. Am J Physiol Cell Physiol 2011;300:C226e31. [17] Rey S, Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovasc Res 2010;86:236e42. [18] Sluimer JC, Daemen MJ. Novel concepts in atherogenesis: angiogenesis and hypoxia in atherosclerosis. J Pathol 2009;218:7e29. [19] Roque M, Fallon JT, Badimon JJ, Zhang WX, Taubman MB, Reis ED. Mouse model of femoral artery denudation injury associated with the rapid
[31]
[32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44] [45]
647
accumulation of adhesion molecules on the luminal surface and recruitment of neutrophils. Arterioscler Thromb Vasc Biol 2000;20:335e42. Shagdarsuren E, Bidzhekov K, Mause SF, et al. C5a receptor targeting in neointima formation after arterial injury in atherosclerosis-prone mice. Circulation 2010;122:1026e36. Augstein A, Poitz DM, Braun-Dullaeus RC, Strasser RH, Schmeisser A. Cellspecific and hypoxia-dependent regulation of human HIF-3alpha: inhibition of the expression of HIF target genes in vascular cells. Cell Mol Life Sci 2011;68:2627e42. Jing D, Wobus M, Poitz DM, Bornhauser M, Ehninger G, Ordemann R. Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro. Haematologica 2012;97:331e9. Maemura K, Hsieh CM, Jain MK, et al. Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain. J Biol Chem 1999;274:31565e70. Poitz DM, Augstein A, Weinert S, Braun-Dullaeus RC, Strasser RH, Schmeisser A. OxLDL and macrophage survival: essential and oxygenindependent involvement of the Hif-pathway. Basic Res Cardiol 2011;106: 761e72. Hoffmann R, Mintz GS. Coronary in-stent restenosis e predictors, treatment and prevention. Eur Heart J 2000;21:1739e49. Stone GW, Ellis SG, Cox DA, et al. A polymer-based, paclitaxel-eluting stent in patients with coronary artery disease. N Engl J Med 2004;350:221e31. Bjornheden T, Levin M, Evaldsson M, Wiklund O. Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol 1999;19:870e6. Luque A, Turu M, Juan-Babot O, et al. Overexpression of hypoxia/inflammatory markers in atherosclerotic carotid plaques. Front Biosci 2008;13:6483e90. Bitto A, De Caridi G, Polito F, et al. Evidence for markers of hypoxia and apoptosis in explanted human carotid atherosclerotic plaques. J Vasc Surg 2010;52:1015e21. Ben-Shoshan J, Afek A, Maysel-Auslender S, et al. HIF-1alpha overexpression and experimental murine atherosclerosis. Arterioscler Thromb Vasc Biol 2009;29:665e70. Karshovska E, Zernecke A, Sevilmis G, et al. Expression of HIF-1 in injured arteries controls SDF-1 mediated neointima formation in apolipoprotein E deficient mice. Arterioscler Thromb Vasc Biol 2007;27:2540e7. Semenza GL. Oxygen sensing, homeostasis, and disease. N Engl J Med 2011;365:537e47. Deguchi Jo, Yamazaki H, Aikawa E, Aikawa M. Chronic hypoxia Activates the Akt and -Catenin pathways in human macrophages. Arterioscler Thromb Vasc Biol 2009;29:1664e70. Castellano J, Aledo R, Sendra J, et al. Hypoxia stimulates low-density lipoprotein receptor-related protein-1 expression through hypoxia-inducible factor-1alpha in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2011;31:1411e20. Osada-Oka M, Ikeda T, Akiba S, Sato T. Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alphadependent expression of thrombospondin-1. J Cell Biochem 2008;104: 1918e26. Simiantonaki N, Jayasinghe C, Michel-Schmidt R, Peters K, Hermanns MI, Kirkpatrick CJ. Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines. Int J Oncol 2008;32:585e92. Schmeisser A, Christoph M, Augstein A, et al. Apoptosis of human macrophages by Flt-4 signaling: implications for atherosclerotic plaque pathology. Cardiovasc Res 2006;71:774e84. Page EL, Robitaille GA, Pouyssegur J, Richard DE. Induction of hypoxiainducible factor-1alpha by transcriptional and translational mechanisms. J Biol Chem 2002;277:48403e9. Vink A, Schoneveld AH, Lamers D, et al. HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages. Atherosclerosis 2007;195:e69e75. Chen F, Eriksson P, Kimura T, Herzfeld I, Valen G. Apoptosis and angiogenesis are induced in the unstable coronary atherosclerotic plaque. Coron Artery Dis 2005;16:191e7. Osada-Oka M, Ikeda T, Imaoka S, Akiba S, Sato T. VEGF-enhanced proliferation under hypoxia by an autocrine mechanism in human vascular smooth muscle cells. J Atheroscler Thromb 2008;15:26e33. Bhardwaj S, Roy H, Heikura T, Yla-Herttuala S. VEGF-A, VEGF-D and VEGFD(DeltaNDeltaC) induced intimal hyperplasia in carotid arteries. Eur J Clin Invest 2005;35:669e76. Koga J, Matoba T, Egashira K, et al. Soluble Flt-1 gene transfer ameliorates neointima formation after wire injury in flt-1 tyrosine kinase-deficient mice. Arterioscler Thromb Vasc Biol 2009;29:458e64. Khurana R, Zhuang Z, Bhardwaj S, et al. Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation 2004;110:2436e43. Simons M. VEGF and restenosis: the rest of the story. Arterioscler Thromb Vasc Biol 2009;29:439e40.