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Vol. 49, No. 5, 2012
Issue release date: August 2012
Section title: Research Paper

Free access is sponsored by an educational grant of the European Society for Microcirculation
J Vasc Res 2012;49:425–431
(DOI:10.1159/000337921)

Combined Therapy with Sonic Hedgehog Gene Transfer and Bone Marrow-Derived Endothelial Progenitor Cells Enhances Angiogenesis and Myogenesis in the Ischemic Skeletal Muscle

Palladino M.a, b · Gatto I.a · Neri V.a, c · Stigliano E.a, d · Smith R.C.f · Pola E.c · Straino S.e · Gaetani E.a · Capogrossi M.e · Leone G.b · Hlatky L.f · Pola R.a, f
aLaboratory of Vascular Biology and Genetics, Department of Medicine, and Departments of bHematology, cOrthopedics and dPathology, A. Gemelli University Hospital, Catholic University School of Medicine, and eLaboratory of Vascular Pathology, IDI IRCCS Research Institute, Rome, Italy; fCenter of Cancer Systems Biology (CCSM), Steward St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Mass., USA
email Corresponding Author

Abstract

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases.

© 2012 S. Karger AG, Basel


  

Key Words

  • Sonic hedgehog gene therapy
  • Endothelial progenitor cells
  • Peripheral ischemia

 Introduction

In the last decade, we have demonstrated that the embryonic morphogen sonic hedgehog (Shh), in addition to regulating epithelial-mesenchymal interactions during development, is active in postnatal life and regulates angiogenesis and tissue regeneration by modulating the expression of multiple growth factors [1,2,3,4,5,6]. We have also shown that Shh gene transfer is a powerful and promising therapeutic approach to improve angiogenesis and functional recovery after ischemia in experimental models of myocardial infarction, peripheral limb ischemia and diabetic peripheral neuropathy [3,5,6]. One of the mechanisms through which Shh gene therapy promotes revascularization of ischemic tissues is the mobilization and homing of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). Indeed, Shh gene therapy increases the number of circulating BM-derived EPCs [5] and enhances their incorporation into the growing neovasculature [3,5]. In addition, when Shh gene therapy is carried out in combination with pharmacological agents that mobilize BM-derived EPCs, the incorporation of endogenous EPCs in the ischemic tissue is increased, compared to when Shh gene therapy is carried out alone [7]. Taken together, these data strongly suggest that Shh is an important regulator of BM-derived EPC mobilization and homing and that its effects on vasculogenesis deserve further investigation. In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs might be more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve ischemia-induced angiogenesis in these animals. We found that combined treatment with Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than either treatment individually.

 Methods


 Mice

Old (18-month-old) C57BL/6J male mice (Charles River Laboratories) were used for the experimental model of hindlimb ischemia. Young (8–12-week-old) C57BL/6J male mice (Charles River Laboratories) were used for the isolation of BM-derived EPCs. All the experiments were conducted in accordance with the local institutional review boards and ethics committees.

 Ischemic Hindlimb Model

Unilateral hindlimb ischemia was induced in twenty 18-month-old C57BL/6J male mice, by ligation and excision of the left femoral artery, as previously described by our group [1,2,4,5]. Induction of ischemia was confirmed by laser Doppler perfusion imaging (Lisca, PeriMed AB), as established.

 Preparation of the Plasmid Encoding the Shh Human Gene

The 4,878-bp plasmid containing the 600-bp amino-terminal domain coding sequence of the human Shh gene [i.e. the plasmid encoding the Shh human gene (phShh)] was prepared as previously described [3,5].

 EPC Culture and Preparation

BM cell suspensions were prepared by crushing the femurs, tibiae, sternum, backbone and iliac crests of 8-week-old C57BL/6J mice. Mononuclear cells (MNCs) were obtained by density gradient centrifugation on Ficoll Lympholite (Cedarlane). Isolated MNCs were resuspended by 10 ml of EGM-2 BulletKit system (CC-3162, Clonetics), seeded on 2% gelatin-coated 100-mm plates (1 × 107 cells per well) and incubated in a 5% CO2 incubator at 37°C, as previously described [8]. The medium was changed every 3 days for 3 weeks. Direct fluorescent staining was used to detect dual binding of CellTracker CM-DiI (Invitrogen) and FITC-labeled Bandeiraea simplicifolia lectin I (BS-1 lectin, Vector Laboratories) on attached BM-derived MNCs after 18 days of culture. Nuclei were counterstained with 10 µg/ml Hoechst 33258 (Sigma). Cells demonstrating double-positive fluorescence were identified as differentiating EPCs, as established [9] (fig. 1). After 3 weeks, the EPCs were detached with trypsin and PBS and counted by the Trypan blue dead-cell exclusion method. After washing with PBS, EPCs were labeled with CM-DiI and then used for treatment, as described below [10].

FIG01
Fig. 1. Murine BM-derived MNCs were cultured in dishes coated with 2% gelatin and maintained in endothelial cell basal medium supplemented with 5% FBS, human VEGF-1, human fibroblast growth factor-2 (FGF-2), human epidermal growth factor (EGF), insulin-like growth factor (IGF-1) and ascorbic acid. a Morphology of adherent murine MNCs on day 10. Phase contrast. ×10. b EPCs were identified by uptake of DiI (red fluorescence) and binding to BS-1 lectin (green fluorescence); nuclei were counterstained with DAPI. ×20.

 Treatment Groups

Of the 20 mice used for unilateral hindlimb ischemia, 5 were treated with 200 µg of phShh, by direct intramuscular injection immediately after induction of ischemia, as previously described [5]. Five other mice were treated with 1 × 106 DiI-labeled EPCs, administered intramuscularly immediately after induction of ischemia, as described [11]. Five others received combined therapy with phShh and EPCs. The remaining 5 received treatment with PBS and were used as controls.

 Assessment of Capillary Density and EPC Homing and Survival

At day 28 after the induction of ischemia, mice were sacrificed and both right and left adductor muscles were harvested, fixed in PFA, embedded in paraffin and sectioned for histological evaluation. Capillaries were identified by BS-1 lectin-positive fluorescent staining and appropriate morphology and localization (adjacent to or associated with muscle fibers) and counted by 2 independent operators blinded to the treatment regimen. The number of capillaries from 10 randomly selected different fields was counted for each muscle. Capillary density was defined as the mean number of capillaries per field, as established [12]. The number of EPCs incorporated into the vasculature was determined by counting cells that were double-positive for BS-1 lectin and DiI, as established [13]. The number of apoptotic EPCs was determined by TUNEL staining, as previously described [3]. Sections were observed with an inverted Zeiss Axioplan fluorescence microscope (Jena). Images were acquired with a ×20 objective and a digital camera system and analyzed, using the I.A.S. Delta System software, by 2 blinded operators.

 Assessment of Myoblast Proliferation in vitro

Satellite cell-derived primary myoblasts were isolated from the lower hindlimb muscles of 2-month-old C57BL/10SnJ mice, as described [14,15]. EPCs were isolated from the BM of the same mice, as described above. Myoblasts were cultured in myoblast growth medium, as described [16], with or without EPCs and with or without the addition of 10 µg/ml of Shh recombinant protein (R&D Systems). Myoblast proliferation was quantified by the number of desmin-positive cells 3 days after coculture, as described [16]. At least 100 desmin-positive myoblasts were counted for each experiment.

 Assessment of Regenerating Myofibers

Hematoxilin and eosin (H&E) staining was used to identify regenerating myofibers, defined as fibers with centrally located nuclei [17]. Nuclei more than 1 nuclear diameter from the fiber border were classified as ‘central’. Fibers intersecting the right and top border of the field were not counted. Images were taken using a Zeiss Axioplan microscope (Jena). Ten fields (×20 objective) were analyzed in each mouse by 2 individuals in a blinded manner.

 Statistical Analyses

Results are presented as mean ± SD. Statistically significant differences between groups were determined by Student’s t test and ANOVA when appropriate.

 Results

The first parameter evaluated to determine the advantage of combined phShh and EPC therapy was the quantification of capillary density at day 28 after ischemia. Histological analysis of the ischemic tissues with fluorescent BS-1 lectin, an endothelial cell marker, demonstrated that, although phShh gene transfer alone and EPCs alone are able to significantly increase capillary density compared to control (PBS-treated) animals (p < 0.001 and p < 0.001, respectively), the combination of the two therapeutic strategies results in a further significant increase in capillary density, compared to phShh gene transfer (p < 0.001) or EPC administration (p < 0.001) alone (fig. 2a, b).

FIG02
Fig. 2. Histological observation of neovascularization in ischemic hindlimb. At day 28 after ischemia, adductor muscles harvested from the ischemic hindlimbs were analyzed for capillary density by staining with BS-1 lectin. a Capillary density was significantly increased in mice receiving combined phShh + EPC therapy compared to the other treatment groups (ANOVA, p < 0.001). b Representative images of capillaries in muscles of phShh-treated mice, EPC-treated mice and those treated with phShh + EPCs in combination.

We next examined whether the contribution of exogenous EPCs to vascularization may be enhanced by combined treatment with phShh gene transfer. Our analyses were performed 28 days after induction of ischemia on the adductor muscles of mice treated with either DiI-labeled EPCs alone or DiI-labeled EPCs in combination with phShh gene transfer. Double-positive staining for DiI and BS-1 lectin was used to identify EPCs that had incorporated into blood vessels. We found that the number of DiI/BS-1 lectin double-positive cells was significantly higher in the group of mice treated with phShh + EPCs than in the group that only received EPCs (p = 0.003), thus suggesting that phShh therapy increases the incorporation of transplanted EPCs into the site of ischemia (fig. 3a–c). We also performed a TUNEL staining to identify apoptotic EPCs and found a significantly reduced number of TUNEL-positive DiI-labeled EPCs in the phShh + EPC group than in the group that received EPCs alone (p < 0.01) (fig. 3d).

FIG03
Fig. 3. Treatment with phShh increases the incorporation of transplanted EPCs into the site of ischemia. a The number of transplanted DiI-labeled EPCs, 28 days after ischemia, is significantly higher in the group treated with phShh + EPCs compared to the EPC-treated group (Student’s t test, p = 0.003). b Representative images of transplanted DiI-labeled EPCs in the hindlimb of mice, 28 days after ischemia, in the EPC-treated group and in the group treated with phShh + EPCs, respectively. c DiI-labeled transplanted red fluorescent cells (EPCs) were identified in small vessels marked with green fluorescent BS-1 lectin. d The number of apoptotic (TUNEL-positive) DiI-labeled EPCs is significantly lower in mice receiving phShh + EPC combination therapy compared to those treated with EPCs alone (Student’s t test, p < 0.01).

It has been demonstrated that endothelial cells support the proliferation of myoblasts in vitro [16]. Here, we cultured satellite cell-derived primary myoblasts in the presence or absence of BM-derived EPCs and found increased myoblast proliferation in cultures also containing EPCs. Likewise, we found that treatment with Shh increases myoblast proliferation in vitro. These findings are consistent with previous data in the literature [18,19,20,21]. Interestingly, we detected a further increase of myoblast proliferation when these myogenic cells were cocultured with EPCs and concomitantly treated with Shh (fig. 4a). These in vitro data were mirrored by the in vivo evidence that the number of regenerating myofibers, identified by the presence of centrally located nuclei (as opposed to peripheral nuclei), was significantly higher in the EPC + Shh-treated muscles compared to the other treatment groups (fig. 4b). This analysis was performed on 10 nonoverlapping fields of each muscle section from mice treated with phShh, EPCs, phShh + EPCs and PBS.

FIG04
Fig. 4.a The number of desmin-positive cells is significantly higher when satellite cell-derived primary myoblasts are cocultured with EPCs and concomitantly treated with Shh (ANOVA, p < 0.01). b Histological analysis of limb muscles. Four weeks after hindlimb ischemia, tissue samples were stained with H&E to quantify regenerative areas. The number of regenerating fibers was significantly higher in mice concomitantly treated with phShh + EPCs (ANOVA, p < 0.001).

 Discussion

Several reports from our laboratory and others have demonstrated clearly that Shh is a potent angiogenic agent with important therapeutic potentials in various types of ischemic and degenerative diseases [1,2,3,4,5,6,7,22]. It has also been demonstrated that the increased mobilization and homing of endogenous EPCs are among the mechanisms through which Shh exerts its angiogenic and vasculogenic properties [3,5]. Indeed, we have previously demonstrated that Shh gene therapy increases the number of circulating EPCs after hindlimb ischemia in mice and enhances EPC homing in the myocardium and skeletal muscle in rodent and porcine models of coronary artery disease and peripheral ischemia [3,5]. On the other hand, many experimental studies and clinical trials have demonstrated that the transplantation of exogenous EPCs restores blood flow and improves organ function in ischemic cardiovascular diseases [23,24,25,26,27,28,29,30,31]. In this study, we tested the hypothesis that combined treatment with Shh gene transfer and exogenous BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than either treatment individually. Our results demonstrate that the number of transplanted EPCs that are recruited in the site of ischemia is higher when these cells are injected in combination with phShh, compared to cell therapy alone. Our data also show that capillary density in the ischemic hindlimb is higher in animals treated with combination therapy than in the other treatment groups. Because Shh regulates the expression of numerous angiogenic factors, the beneficial effects of Shh + EPC combination therapy likely evolve through several mechanisms. Indeed, we have previously demonstrated that phShh gene therapy increases the expression levels of vascular endothelial growth factor (VEGF) and SDF-1α in the ischemic muscle [5]. The higher EPC recruitment detected in animals treated with combination therapy compared to that observed in mice treated with EPCs alone may depend on the ability of phShh gene therapy to upregulate the expression of SDF-1α, which is required for EPC recruitment and homing upon ischemia [32]. However, it has been shown that SDF-1α alone may not sufficiently enhance cell incorporation [33]. Thus, the Shh-induced upregulation of other growth factors, such as VEGF, may contribute to EPC incorporation in our model. The same mechanism may be responsible for the increased survival of EPCs observed in animals treated with combination therapy. The fact that phShh gene therapy is able to increase the recruitment, homing and survival of exogenous EPCs may be particularly important from a clinical and therapeutic standpoint. Indeed, although the administration of exogenous EPCs has been proposed as a treatment for ischemic cardiovascular diseases [23,24,25,26,27,28,29,30,31], a major limiting factor of this therapeutic approach is the relatively small number of cells that incorporate into the existing and growing neovasculature in an efficient and stable manner [34]. Our findings demonstrate that we may overcome such limitation by administering phShh in combination with EPCs. This approach also has advantages over the administration of phShh alone. Indeed, although Shh gene therapy is able to increase the contribution of endogenous EPCs to the process of neovascularization [3,5,7], one should consider that, in both humans and animal models, the availability and function of endogenous EPCs is significantly impaired by those pathological conditions that are responsible for the development and progression of ischemic cardiovascular diseases, such as age, diabetes, hypercholesterolemia, hypertension and smoking [35,36]. Thus, growth factors may act on endogenous EPCs in only a limited manner. For this reason, it is possible to hypothesize that a more effective promotion of angiogenesis and vasculogenesis may be achieved by transplanting exogenous EPCs in combination with Shh gene therapy, in order to provide Shh with the possibility of acting on healthy EPCs.

Another interesting finding of our study is the demonstration that treatment with phShh + EPCs increases myogenesis in vitro and muscle regeneration in vivo. We know that in response to skeletal muscle injury the quiescent myogenic stem cells (satellite cells) become activated, proliferate and fuse with each other to form myotubes, which eventually mature into myofibers [37,38]. We have already demonstrated that Shh is able to stimulate the process of myogenesis after injury of the skeletal muscle in vivo [4], and other studies in the literature report that Shh increases myoblast proliferation in vitro [18,19,20,21]. The fact that such myogenic effects are enhanced by phShh + EPC combination therapy is consistent with the concept that angiogenesis and myogenesis are interdependent mechanisms and that the vascular niche promotes the proliferation of myoblasts [16,39]. The increased number of proliferating myoblasts and regenerating fibers observed upon combined treatment with phShh and EPCs might be the result of increased myoblast proliferation, promoted directly by the endothelial cells or indirectly by factors produced by them [16,39].

In conclusion, this study demonstrates that in the setting of peripheral limb ischemia combined therapy with phShh and EPCs is more effective, in terms of neovascularization and muscle repair, than single phShh or EPC therapy. These findings may establish the foundation for novel therapeutic strategies for ischemic diseases.

 Acknowledgements

This study was supported by grant RCSNE3 from the ‘Fondazione Roma’ and grant RBID08-MAFS (FIRB-IDEAS) from the Italian Department of University and Research (MIUR).


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Author Contacts

Dr. Roberto Pola
Center of Cancer Systems Biology, CBR4
Steward St. Elizabeth’s Medical Center, Tufts University School of Medicine
736 Cambridge Street, Boston, MA 02135 (USA)
Tel. +1 617 562 7275, E-Mail roberto.pola@tufts.edu

  

Article Information

Received: August 30, 2011
Accepted after revision: February 23, 2012
Published online: June 22, 2012
Number of Print Pages : 7
Number of Figures : 4, Number of Tables : 0, Number of References : 39

  

Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 49, No. 5, Year 2012 (Cover Date: August 2012)

Journal Editor: Pohl U. (Munich), Meininger G.A. (Columbia, Mo.)
ISSN: 1018-1172 (Print), eISSN: 1423-0135 (Online)

For additional information: http://www.karger.com/JVR


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Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases.

© 2012 S. Karger AG, Basel


  

Author Contacts

Dr. Roberto Pola
Center of Cancer Systems Biology, CBR4
Steward St. Elizabeth’s Medical Center, Tufts University School of Medicine
736 Cambridge Street, Boston, MA 02135 (USA)
Tel. +1 617 562 7275, E-Mail roberto.pola@tufts.edu

  

Article Information

Received: August 30, 2011
Accepted after revision: February 23, 2012
Published online: June 22, 2012
Number of Print Pages : 7
Number of Figures : 4, Number of Tables : 0, Number of References : 39

  

Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 49, No. 5, Year 2012 (Cover Date: August 2012)

Journal Editor: Pohl U. (Munich), Meininger G.A. (Columbia, Mo.)
ISSN: 1018-1172 (Print), eISSN: 1423-0135 (Online)

For additional information: http://www.karger.com/JVR


Article / Publication Details

First-Page Preview
Abstract of Research Paper

Received: 8/30/2011
Accepted: 3/9/2012
Published online: 6/22/2012
Issue release date: August 2012

Number of Print Pages: 7
Number of Figures: 4
Number of Tables: 0

ISSN: 1018-1172 (Print)
eISSN: 1423-0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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