β-Catenin Overexpression Augments Angiogenesis and Skeletal Muscle Regeneration Through Dual Mechanism of Vascular Endothelial Growth Factor–Mediated Endothelial Cell Proliferation and Progenitor Cell Mobilization
Objective— β-Catenin plays a critical role in directing cell fate during embryogenesis, and uncontrollable activation leads to cancers, suggesting its importance in cell survival and proliferation. However, little is known regarding its role in endothelial cell (EC) and skeletal muscle proliferation and progenitor cell mobilization.
Methods and Results— β-Catenin enhanced ECs proliferation, protected ECs from apoptosis, and increased the capillary forming capabilities, which was completely blocked by inhibition of its nuclear translocation. In addition, the increased proliferation by β-catenin was associated with increased expression of cyclin E2. In skeletal myocytes, β-catenin overexpression increased proliferation with cyclin D1 expression, decreased apoptosis, and induced hypertrophy. Furthermore, β-catenin induced the expression of vascular endothelial growth factor (VEGF) in skeletal myocytes, resulting in EC proliferation. In a mouse hindlimb ischemia model, β-catenin significantly increased recovery of blood perfusion, capillary density along with enhanced VEGF expression, and the number of proliferating ECs and myocytes. Local delivery of β-catenin also promoted angiogenic progenitor cell mobilization and increased the number of satellite cells.
Conclusions— β-Catenin may be an important modulator of angiogenesis and myocyte regeneration not only by directly enhancing proliferation and survival of ECs and skeletal myocytes but also by inducing VEGF expression and promoting angiogenic progenitor cell mobilization and muscle progenitor cell activation.
β-Catenin is an intracellular protein known to play dual roles in cells. In addition to its structural role in maintaining tissue architecture and cell polarity at adherens junctions, cytoplasmic β-catenin also translocates into the nucleus where it forms a complex with transcription factors of the Tcf/Lef family and activates the expression of specific genes involved in cell proliferation and survival.1,2 Although the critical role of β-catenin on the proliferative and migratory responses of cells during embryogenesis and in neoplastic disease have been well described previously, relatively little is known about the role of β-catenin on the endothelial cell (EC) in normal, controlled cell proliferation and migration.3,4 Recent data suggest that Wnt/β-catenin signaling may play a key role in vascular biology. For example, transfection of Wnt-1–expressing vector was shown to stimulate EC proliferation with β-catenin accumulation, which implies that Wnt proteins may regulate signal transduction in ECs via β-catenin.5 Furthermore, β-catenin was also identified in the cytoplasm of ECs of newly formed vessels around the area of infarction.6 In a recent study,7 the human vascular endothelial growth factor (VEGF) gene promoter has been reported to contain binding sites for β-catenin/Tcf, and the transfection of β-catenin to normal colon epithelial cells significantly increased VEGF expression. In addition, the Wnt pathway is also reported to play an important role in muscle regeneration.8
However, the downstream target genes and the exact mechanisms of the Wnt/β-catenin signaling pathway in ECs and skeletal muscle cells have not been clarified. Therefore, the aim of the present study was to evaluate the role of β-catenin in cell biological behaviors of ECs and skeletal myocytes and to elucidate the key signaling pathway of β-catenin in these cells and crosstalk between the 2 types of cells. Furthermore, we investigated the role of β-catenin as a modulator of angiogenesis and myocyte regeneration in a murine hindlimb ischemia model.
Materials and Methods
Detailed material and methods are described in the Expanded Materials and Methods section (available online at http://atvb.ahajournals.org).
In Vitro Studies
Construction of Adenoviral Vectors Expressing Wild-Type β-Catenin
Adenoviruses expressing β-catenin constructs were produced using AdEasy kits (Q Biogene), and transfected cells were determined by the coexpression of green fluorescent protein (GFP).
Cell Culture and Adenoviral Transfection
Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial growth medium (Clonetics) as described previously.9 Four to six passage cells were used. To examine the effect of β-catenin, HUVECs were serum-starved for 15 hours, then treated with the indicated agents for 1 hour and stimulated with 2% FBS. For control studies, an adenoviral vector expressing only the GFP gene was used. Under these conditions, the transfection efficiency was >95%. C2C12 myoblasts (American Type Culture Collection) were cultured as described previously.10 Cells were maintained in growth medium (DMEM supplemented with 10% FBS, GIBCO) and shifted to differentiation medium (DMEM supplemented with 5% heat-inactivated horse serum). For viral transfection, C2C12 cells were incubated with adenovirus (250 multiplicity of infection) in differentiation medium for 12 hours. Under these conditions, the transfection efficiency was >90%.
Inhibition of β-Catenin–Mediated Transactivation by Cadherin Derivatives
Dominant negative N-cadherin (NCadΔC), which lacks the extracellular domain, was used to suppress β-catenin–mediated transcriptional activity as described previously.11
Immunoblot assays were performed as described previously.9 The primary antibodies used were antitotal β-catenin antibody (1:500 dilution, Cell Signaling), anti–α-tubulin antibody (1:500 dilution, Oncogene), VEGF (1:500 dilution, Santa Cruz), cyclin D1 (1:500 dilution, Santa Cruz), and cyclin E2 (1:500 dilution, Santa Cruz). The secondary antibodies were antirabbit IgG/horseradish peroxidase (HRP) conjugate or antimouse IgG/HRP conjugate and antigoat IgG/HRP conjugate (1:2500 dilution, ECL, Amersham).
In Vivo Studies
Murine Hindlimb Ischemia Model
Male C57BL/6 (6 weeks old) mice were purchased from KBT Oriental Co Ltd (Charles River Grade, Tosu). All of the procedures were performed in accordance with the Institutional Animal Care and Use Committee of Seoul National University Hospital. To impair angiogenesis in response to hindlimb ischemia, mice were fed a 2% high-cholesterol diet.12 After 2 weeks of the high-cholesterol diet, unilateral limb ischemia was surgically induced in all of the animals. Under sufficient anesthesia with IP injection of a combination anesthetics (ketamine 50 mg/kg and xylazine 20 mg/kg, Bayer Korea), the entire left superficial femoral artery was ligated, cut, and excised.13,14 For gene therapy, 40 μL of vector solution (109 plaque forming units) was injected into 4 injection sites in the adductor and thigh muscles soon after the surgical procedure.
Progenitor Cell Mobilization: Fluorescence Activated Cell Sorter Analysis, ELISA, and Endothelial Progenitor Cell Culture
Peripheral blood was obtained from mice at 3 and 7 days after hindlimb ischemia. The VEGF concentration was measured using ELISA (R&D Systems). Fluorescence activated cell sorter (FACS) analysis was performed as described previously using CD34 (BD PharMingen) and Sca-1 (Biosource) antibodies. Endothelial progenitor cells (EPCs) were identified and calculated as described previously.15
All of the statistical analyses were performed using SPSS for Windows 10.0 (SPSS Inc). Continuous variables are expressed as mean ± SE and were analyzed by ANOVA test, using the Student-Newman-Keuls and Bonferroni post-hoc tests. All of the statistical analyses were 2-tailed, and P<0.05 was considered statistically significant.
Effect of β-Catenin on EC Proliferation, Apoptosis, and Tube Formation
Fluorescent microscopy of HUVECs at 24 hours after transfection with adenovirus (25 multiplicities of infection) showed higher proliferative activity in cells transduced with the β-catenin gene. The proproliferative effect of β-catenin on ECs was more than twice that of GFP as quantified by WST-1 assay [absorption: 249.6±18.6 vs 100.0±3.74% for β-catenin wild-type (WT) vs GFP; P<0.05], which was completely inhibited by NCadΔC. In addition, under serum-deprived conditions, the transfection of β-catenin WT to ECs significantly reduced the subdiploid apoptotic fraction of DNA as measured by FACS analysis compared with GFP-transduced cells, which was reversed by NCadΔC, suggesting that the antiapoptotic effects were mediated by the transcriptional activity of β-catenin. Furthermore, EC function as measured by Matrigel tube formation was significantly better in β-catenin WT-transduced cells compared with GFP controls (tube length relative to GFP control [%]: 258.2±31.8% in β-catenin WT; P<0.05; Figure I, available online at http://atvb.ahajournals.org).
Effect of β-Catenin on Skeletal Myocyte Proliferation, Apoptosis, and Hypertrophy
Adenovirus-mediated β-catenin overexpression in skeletal myocytes after differentiation induced hypertrophy, that is, an increase in myocyte size (mean area and width). In addition, β-catenin transfection resulted in enhanced proliferation and resistance to serum-deprived apoptosis (Figure II, available online at http://atvb.ahajournals.org). All of these effects in the skeletal myocyte were inhibited by adding NCadΔC, suggesting the importance of the transcriptional activation of β-catenin in myocyte hypertrophy, proliferation, and resistance to apoptosis.
Dual Mechanism of EC Regulation by β-Catenin
To investigate downstream target signals of β-catenin in EC, major cell-cycle regulators, cyclin E2 and cyclin D1, were examined. Cyclin E2 expression was consistently increased after β-catenin transduction, whereas no significant change was observed in cyclin D1 expression (Figure 1A). To confirm the cell biological significance of cyclin E2, cell cycle analysis was performed using flow cytometry. As expected, β-catenin WT decreased the percentage of cells in the G1 phase and increased the number of cells in the S phase, a profile that is typically associated with acceleration of G1 (Figure 1B). In addition, the increased expression of cyclin E2 with β-catenin was inhibited by NCadΔC, which suggests that β-catenin enhances cyclin E2 expression after its nuclear translocation (Figure 1C).
Because VEGF is a major cytokine involved in EC proliferation, survival, and angiogenesis and was recently discovered as a downstream molecule controlled by β-catenin in colon cancer, we hypothesized that β-catenin, in addition to the direct prosurvival and antiapoptotic effects, may have indirect effects on EC through VEGF secretion by other surrounding cells. Therefore, we targeted skeletal myocytes and examined the effects of β-catenin transduction in myocytes with regard to VEGF expression. After β-catenin transfection, VEGF expression was markedly increased in myocytes and reversed by NCadΔC (Figure 1D). To validate the hypothesis that β-catenin promotes EC proliferation by way of VEGF expression, an EC survival assay was performed using the supernatant from β-catenin–stimulated myocyte culture and blocking the antibody for VEGF. The WST-1 assay showed a significant increase of EC proliferation after adding the supernatant of C2C12 cell culture to HUVECs, which was reversed by adding anti-VEGF neutralizing antibody (Figure 1E), suggesting that the increased survival from the addition of supernatant from β-catenin–stimulated myocyte culture was through VEGF.
β-Catenin Promotes Angiogenesis and Myocyte Regeneration in a Mouse Hindlimb Ischemia Model
To investigate the in vivo effects of β-catenin on angiogenesis and skeletal muscle regeneration, we used a mouse hindlimb ischemia model. First, to confirm that ischemia induces β-catenin, we examined the expression of β-catenin in ischemic hindlimb in a baseline experiment, which showed that the expression of β-catenin was significantly increased in the ischemic limb compared with the nonischemic limb (Figure 2A). Tissue ischemia induced the expression of β-catenin and VEGF, but this phenomenon was only transient and decreased after day 3. However, when the β-catenin gene was delivered by adenoviral vector, we found that β-catenin and VEGF expression was stronger with prolonged expression up to day 5 (Figure 2B). Serial laser Doppler perfusion imaging of the ischemic left hindlimb showed that recovery of blood flow was faster and more intense in the β-catenin gene–delivered group than in the control group (Figure 2C). By day 14, the ratio of ischemic:nonischemic blood flow was significantly greater in the β-catenin WT group (0.93±0.05 versus 0.70±0.03 for β-catenin WT versus GFP group; P<0.01; Figure 2D).
Immunohistochemical staining for the EC marker PECAM-1 was performed on skeletal muscle sections retrieved from the ischemic hindlimbs of mice at day 14 to quantify capillary density (Figure 3A). There were significantly more capillary ECs in the ischemic limb of the β-catenin gene transfer group (Figure 3B). Double immunostaining showed that the β-catenin gene delivery was effective in hindlimb ischemic tissue, and β-catenin was mainly expressed in both skeletal myocytes and ECs (Figure 3C).
VEGF expressions in skeletal muscle were also significantly increased in the β-catenin group, suggesting an additional paracrine effect of β-catenin to enhance angiogenesis (Figure 4A). Augmentation of β-catenin expression in the ischemic muscle by β-catenin gene transfer resulted in significantly increased expression of downstream molecules VEGF and cyclin D1, which mainly originated from skeletal muscle in limb (Figure 4B). Double-immunohistochemical staining showed that the VEGF was highly expressed in β-catenin–transduced cells (Figure 4C).
In addition, immunohistochemical staining for proliferating cell nuclear antigen (PCNA) to measure cell proliferation showed significantly more proliferating cells in the β-catenin group compared with the GFP control group at day 5 (Figure 5A and 5B). We also observed increased skeletal muscle regeneration, as shown by increased regenerating myocytes (small cells with central nuclei), in the β-catenin group (Figure 5C).
Local β-Catenin Gene Transfer Promotes Mobilization of Angiogenic Progenitor Cells and Activates Skeletal Progenitor Cells in a Mouse Hindlimb Ischemia Model
Furthermore, to investigate the role of β-catenin in progenitor cell mobilization, peripheral blood mononuclear cells were isolated for FACS analysis. We found a greater fraction of CD34-positive and Sca1-positive cells in the β-catenin group (Figure 6A). To characterize the mobilized progenitor cell after local β-catenin gene transfer, the number of cells uptaking DiI-AcLDL among the double-positive (CD34 and Sca1) cells was counted in peripheral blood mononuclear cells after flow cytometry. More EPCs uptaking DiI-acetylated LDL (DiI-AcLDL) were observed in the β-catenin group (Figure 6B). To find out the underlying mechanism of progenitor cell mobilization, we measured the concentration of VEGF in plasma, which showed an increased level of VEGF in the β-catenin group at day 3 (Figure 6C). We also evaluated the potential effect of β-catenin gene transfer to muscle on the skeletal progenitor cell. Gene transfer of β-catenin to muscle increased both CD34/Sca1 double-positive cells (Figure 6C) and the number of satellite cells in muscle analyzed by skeletal myocyte single-fiber culture technique (Figure 6E; 4′,6-diamidino-2-phenylindole–positive satellite cells per muscle fiber; 4.4±2.9 in GFP versus 10.1±2.7 in β-catenin; P<0.05), suggesting that local gene transfer of β-catenin may have also increased skeletal progenitor cells in muscle.
In this study, we showed that overexpression of β-catenin led to enhanced EC survival, function, and proliferation. Furthermore, these prosurvival effects were mediated through cyclin E2, which is a novel downstream target molecule of β-catenin in ECs that was not reported previously in other studies. In addition, we found that β-catenin overexpression not only enhances proliferation and inhibits apoptosis of skeletal myocytes, but also increases VEGF expression in skeletal myocytes, suggesting a paracrine effect of β-catenin on ECs via surrounding myocytes. All of these effects were inhibited by NCadΔC, suggesting that these effects are, indeed, mediated by the transcriptional activity of β-catenin.
In vivo, critical ischemia led to a transiently increased expression of β-catenin, which suggests that β-catenin may be the actual modulator of angiogenesis in ischemic tissue. Accordingly, adenovirus-mediated β-catenin gene transfer resulted in a sustained increase in β-catenin, as well as VEGF expression, leading to a significant augmentation of angiogenesis and myocyte regeneration in a mouse hindlimb ischemia model. In addition, β-catenin increased the mobilization of hematopoietic progenitor cells from the bone marrow and the number of satellite cells. These are all novel findings, which have not been reported previously.
The Wnt signaling pathway is involved in the control of multiple cellular processes. Recent studies have demonstrated the expression of Wnt ligands, Wnt receptors, and Wnt inhibitors in vascular cells.16–18 However, the specific effect of β-catenin overexpression on EC survival and function and, furthermore, on new vessel formation have not been studied previously. Our results provide new insight into a possible role of the Wnt/β-catenin signaling pathway in angiogenesis.
In the present study, we showed that in ECs, in contrast to other cancer cells, the expression of cyclin E2 rather than cyclin D1 is significantly increased, which leads to the propagation of the cell cycle from the G1 phase to the S phase. Previously, it was reported that cyclin D1 is a direct target of the Tcf/ LEF-1 pathway through a binding site in the cyclin D1 promoter region in the colon cancer cell.19,20 The same finding was observed when we transferred the β-catenin gene in skeletal muscle cells in vitro and limb muscle in vivo. In ECs, however, we observed increased expression of cyclin E2 rather than cyclin D1. The finding that β-catenin increases cyclin E2 expression in ECs is a novel one, which needs to be additionally studied to understand how β-catenin controls the EC growth at a molecular level.
The modulations of β-catenin on ECs were reversed by NCadΔC. This suggests that the binding of NCadΔC to β-catenin may compete with other transcription factors that interact with β-catenin. It may be deduced, therefore, that the binding of β-catenin to dominant-negative cadherin, which has a truncated extracellular domain with an intact cytoplasmic tail, may have inhibited the binding of β-catenin to other transcription factors, such as the Tcf/Lef family, and blocked its transcriptional activity.
Another key finding of the present study is that β-catenin, in addition to the direct prosurvival effects, has an indirect effect on ECs via myocytes, which plays a critical role in angiogenesis. We found that β-catenin gene transfer on skeletal myocytes leads to a significant increase in VEGF expression. Furthermore, the importance of this so-called “paracrine” effect on EC proliferation and angiogenesis was confirmed in experiments where an antibody against VEGF resulted in partially decreased proliferation in vitro and decreased new vessel formation in vivo. These findings suggest the Wnt/β-catenin pathway may play a key role in angiogenesis both directly, by enhancing EC proliferation and function, and indirectly, by inducing the expression of proangiogenic molecules in surrounding cells.
In addition to the paracrine effects, β-catenin enhances proliferation and reduces serum deprivation–induced apoptosis in myocytes. β-Catenin overexpression also increased the mean area and size of the myocyte. These findings are compatible with previous reports showing that stabilization of β-catenin in cardiomyocytes is necessary for the hypertrophic response.21
In a hindlimb ischemia model, we observed a significantly increased but transient expression of β-catenin after the induction of ischemia, suggesting that β-catenin is a gene that responds to ischemic insult and may be involved in angiogenesis. This hypothesis was confirmed by showing that the overexpression β-catenin in the ischemic tissue by gene transfer resulted in sustained increased expression of β-catenin, leading to significantly augmented angiogenesis and recovery of blood flow. Our results also suggest that the impact of β-catenin on angiogenesis in ischemic tissue may be greater than other angiogenic molecules because of its dual effect on both EC and myocytes. On top of its direct proproliferative role, β-catenin also enhances VEGF expression in gene-transfected skeletal muscle, which leads to the increased plasma level of VEGF. The increased concentration of VEGF at local muscle may help ECs proliferate and survive, leading to angiogenesis, and the increased VEGF in circulation may lead to progenitor cell mobilization, mainly hematopoietic stem cells from bone marrow, which might augment the angiogenic effect of local β-catenin gene transfer to ischemic hind limb.
Furthermore, there have been several reports regarding the roles of β-catenin in skeletal myogenesis and regeneration.8,22 In this study, we observed that β-catenin increased CD34 and Sca1 double-positive muscular stem cells, which were reported to have an important role in skeletal regeneration.23,24 Although it is not confirmed whether the muscular stem cells are derived from the ones in muscle or from mobilized hematopoietic progenitor cells, it is possible that these progenitor cells have an important role in tissue repair of the ischemic hindlimb. Considering that the number of skeletal satellite cells significantly increased after β-catenin gene transfer, we can think that stimulatory effect of β-catenin on skeletal progenitor cells contributed to the accelerated regeneration of skeletal muscle.
There are previous reports showing induction of inflammation by adenovirus mediated gene transfection,25,26 which may have led to the increased VEGF and augmented angiogenesis. However, our study showed the clear benefit of β-catenin over GFP, both using the adenoviral transfection technique. Thus, we believe that the enhanced angiogenic potential we observed in the present study was because of β-catenin and not inflammation from adenoviral transfection.
In conclusion, we show for the first time that β-catenin directly increases proliferation of ECs through cell cycle propagation and indirectly enhances EC survival by inducing VEGF expression from surrounding myocytes. Furthermore, β-catenin gene transfer significantly induced the mobilization of angiogenic progenitor cells in the circulating blood and increased the number of skeletal muscle progenitor cells in transfected muscle, leading to enhanced angiogenesis and muscle regeneration in a mouse hindlimb ischemia model. These data suggest that β-catenin may be an important regulator of angiogenesis and skeletal muscle regeneration in ischemic tissue.
This study was supported by a grant from the Korea Health 21 Research and Development Project, Ministry of Health and Welfare (02-PJ10-PG8-EC01-0026 and A050082), and from Stem Cell Research Center (SC13122), Republic of Korea.
K-i.K. and H-J.C. contributed equally to this work.
- Received February 21, 2005.
- Accepted August 5, 2005.
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