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.

Keywords:
Sonic hedgehog gene therapy, Endothelial progenitor cells, Peripheral ischemia
1.
Pola R, Ling LE, Silver M, Corbley MJ, Kearney M, Blake Pepinsky R, Shapiro R, Taylor FR, Baker DP, Asahara T, Isner JM: The morphogen sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med 2001;7:706–711.
2.
Pola R, Ling LE, Aprahamian TR, Barban E, Bosch-Marce M, Curry C, Corbley M, Kearney M, Isner JM, Losordo DW: Postnatal recapitulation of embryonic hedgehog pathway in response to skeletal muscle ischemia. Circulation 2003;108:479–485.
3.
Kusano KF, Pola R, Murayama T, Curry C, Kawamoto A, Iwakura A, Shintani S, Ii M, Asai J, Tkebuchava T, Thorne T, Takenaka H, Aikawa R, Goukassian D, von Samson P, Hamada H, Yoon YS, Silver M, Eaton E, Ma H, Heyd L, Kearney M, Munger W, Porter JA, Kishore R, Losordo DW: Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med 2005;11:1197–1204.
4.
Straface G, Aprahamian T, Flex A, Gaetani E, Biscetti F, Smith RC, Pecorini G, Pola E, Angelini F, Stigliano E, Castellot JJ Jr, Losordo DW, Pola R: Sonic hedgehog regulates angiogenesis and myogenesis during post-natal skeletal muscle regeneration. J Cell Mol Med 2009;13:2424–2435.
5.
Palladino M, Gatto I, Neri V, Straino S, Silver M, Tritarelli A, Piccioni A, Smith RC, Gaetani E, Losordo DW, Crea F, Capogrossi M, Pola R: Pleiotropic beneficial effects of sonic hedgehog gene therapy in an experimental model of peripheral limb ischemia. Mol Ther 2011;19:658–666.
6.
Kusano KF, Allendoerfer KL, Munger W, Pola R, Bosch-Marce M, Kirchmair R, Yoon YS, Curry C, Silver M, Kearney M, Asahara T, Losordo DW: Sonic hedgehog induces arteriogenesis in diabetic vasa nervorum and restores function in diabetic neuropathy. Arterioscler Thromb Vasc Biol 2004;24:2102–2107.
7.
Roncalli J, Renault MA, Tongers J, Misener S, Thorne T, Kamide C, Jujo K, Tanaka T, Ii M, Klyachko E, Losordo DW: Sonic hedgehog-induced functional recovery after myocardial infarction is enhanced by AMD3100-mediated progenitor-cell mobilization. J Am Coll Cardiol 2011;57:2444–2452.
8.
Hur J, Yoon CH, Kim HS, Choi JH, Kang HJ, Hwang KK, Oh BH, Lee MM, Park YB: Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol 2004;24:288–293.
9.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.
10.
Yoon YS, Wecker A, Heyd L, Park JS, Tkebuchava T, Kusano K, Hanley A, Scadova H, Qin G, Cha DH, Johnson KL, Aikawa R, Asahara T, Losordo DW: Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction. J Clin Invest 2005;115:326–338.
11.
Gianella A, Guerrini U, Tilenni M, Sironi L, Milano G, Nobili E, Vaga S, Capogrossi MC, Tremoli E, Pesce M: Magnetic resonance imaging of human endothelial progenitors reveals opposite effects on vascular and muscle regeneration into ischaemic tissues. Cardiovasc Res 2010;85:503–513.
12.
Brill A, Dashevsky O, Rivo J, Gozal Y, Varon D: Platelet-derived microparticles induce angiogenesis and stimulate post-ischemic revascularization. Cardiovasc Res 2005;67:30–38.
13.
Kahler CM, Wechselberger J, Hilbe W, Gschwendtner A, Colleselli D, Niederegger H, Boneberg EM, Spizzo G, Wendel A, Gunsilius E, Patsch JR, Hamacher J: Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury. Respir Res 2007;8:50.
14.
Asakura A, Hirai H, Kablar B, Morita S, Ishibashi J, Piras BA, Christ AJ, Verma M, Vineretsky KA, Rudnicki MA: Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle. Proc Natl Acad Sci USA 2007;104:16552–16557.
15.
Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki MA: Reduced differentiation potential of primary MyoD–/– myogenic cells derived from adult skeletal muscle. J Cell Biol 1999;144:631–643.
16.
Verma M, Asakura Y, Hirai H, Watanabe S, Tastad C, Fong GH, Ema M, Call JA, Lowe DA, Asakura A: Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in MDX mice. Hum Mol Genet 2010;19:4145–4159.
17.
Spurney CF, Gordish-Dressman H, Guerron AD, Sali A, Pandey GS, Rawat R, Van Der Meulen JH, Cha HJ, Pistilli EE, Partridge TA, Hoffman EP, Nagaraju K: Preclinical drug trials in the MDX mouse: assessment of reliable and sensitive outcome measures. Muscle Nerve 2009;39:591–602.
18.
Duprez D, Fournier-Thibault C, Le Douarin N: Sonic hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development 1998;125:495–505.
19.
Koleva, M, Kappler R, Vogler M, Herwig A, Fulda S, Hahn H: Pleiotropic effects of sonic hedgehog on muscle satellite cells. Cell Mol Life Sci 2005;62:1863–1870.
20.
Elia D, Madhala D, Ardon E, Reshef R, Halevy O: Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: involvement of MAPK/ERK and PI3K/Akt pathways. Biochim Biophys Acta 2007;1773:1438–4614.
21.
Madhala-Levy D, Williams VC, Hughes SM, Reshef R, Halevy O: Cooperation between Shh and IGF-I in promoting myogenic proliferation and differentiation via the MAPK/ERK and PI3K/Akt pathways requires Smo activity. J Cell Physiol 2012;227:1455–1464.
22.
Asai J, Takenaka H, Kusano KF, Ii M, Luedemann C, Curry C, Eaton E, Iwakura A, Tsutsumi Y, Hamada H, Kishimoto S, Thorne T, Kishore R, Losordo DW: Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial progenitor cell-mediated microvascular remodeling. Circulation 2006;113:2413–2424.
23.
Edelberg JM, Tang L, Hattori K, Lyden D, Rafii S: Young adult bone marrow-derived endothelial precursor cells restore aging-impaired cardiac angiogenic function. Circ Res 2002;90:E89-E93.
24.
Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, Masaki H, Mori Y, Iba O, Tateishi E, Kosaki A, Shintani S, Murohara T, Imaizumi T, Iwasaka T: Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104:1046–1052.
25.
Chen F, Tan Z, Dong CY, Li X, Xie Y, Wu Y, Chen X, Guo S: Combination of VEGF(165)/angiopoietin-1 gene and endothelial progenitor cells for therapeutic neovascularization. Eur J Pharmacol 2007;568:222–230.
26.
Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S, Epstein SE: Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation. 2004;109:1543–1549.
27.
Zhang X, Wei M, Zhu W, Han B: Combined transplantation of endothelial progenitor cells and mesenchymal stem cells into a rat model of isoproterenol-induced myocardial injury. Arch Cardiovasc Dis 2008;101:333–342.
28.
Shibata T, Naruse K, Kamiya H, Kozakae M, Kondo M, Yasuda Y, Nakamura N, Ota K, Tosaki T, Matsuki T, Nakashima E, Hamada Y, Oiso Y, Nakamura J: Transplantation of bone marrow-derived mesenchymal stem cells improves diabetic polyneuropathy in rats. Diabetes 2008;57:3099–3107.
29.
Yang Z, von Ballmoos MW, Diehm N, Baumgartner I, Kalka C, Di Santo S: Call for a reference model of chronic hind limb ischemia to investigate therapeutic angiogenesis. Vascul Pharmacol 2009;51:268–274.
30.
Kawamoto A, Katayama M, Handa N, Kinoshita M, Takano H, Horii M, Sadamoto K, Yokoyama A, Yamanaka T, Onodera R, Kuroda A, Baba R, Kaneko Y, Tsukie T, Kurimoto Y, Okada Y, Kihara Y, Morioka S, Fukushima M, Asahara T: Intramuscular transplantation of G-CSF-mobilized CD34(+) cells in patients with critical limb ischemia: a phase I/IIa, multicenter, single-blinded, dose-escalation clinical trial. Stem Cells 2009;27:2857–2864.
31.
Lara-Hernandez R, Lozano-Vilardell P, Blanes P, Torreguitart-Mirada N, Galmés A, Besalduch J: Safety and efficacy of therapeutic angiogenesis as a novel treatment in patients with critical limb ischemia. Ann Vasc Surg 2010;24:287–294.
32.
Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, Murasawa S, Bosch-Marce M, Masuda H, Losordo DW, Isner JM, Asahara T: Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation 2003;107:1322–1328.
33.
Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ: Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation 2004;110:3300–3305.
34.
Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner JM, Asahara T: Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002;105:732–738.
35.
Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T: Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593–600.
36.
Dimmeler S, Leri A: Aging and disease as modifiers of efficacy of cell therapy. Circ Res 2008;102:1319–1330.
37.
Reimann J, Irintchev A, Wernig A: Regenerative capacity and the number of satellite cells in soleus muscles of normal and mdx mice. Neuromuscul Disord 2000;10:276–282.
38.
Barbero A, Benelli R, Minghelli S, Tosetti F, Dorcaratto A, Ponzetto C, Wernig A, Cullen MJ, Albini A, Noonan DM: Growth factor supplemented matrigel improves ectopic skeletal muscle formation – a cell therapy approach. J Cell Physiol 2001;186:183–192.
39.
Christov C, Chrétien F, Abou-Khalil R, Bassez G, Vallet G, Authier FJ, Bassaglia Y, Shinin V, Tajbakhsh S, Chazaud B, Gherardi RK: Muscle satellite cells and endothelial cells: close neighbors and privileged partners. Mol Biol Cell 2007;18:1397–1409.
© 2012 S. Karger AG, Basel
2012
Copyright / Drug Dosage / Disclaimer
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.
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 government 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.
You do not currently have access to this content.