Volume 19, Issue 4, Pages 658-666 (April 2011) Pleiotropic Beneficial Effects of Sonic Hedgehog Gene Therapy in an Experimental Model of Peripheral Limb Ischemia  Mariangela Palladino, Ilaria Gatto, Valentina Neri, Stefania Straino, Marcy Silver, Alessandra Tritarelli, Andrea Piccioni, Roy C Smith, Eleonora Gaetani, Douglas W Losordo, Filippo Crea, Maurizio Capogrossi, Roberto Pola  Molecular Therapy  Volume 19, Issue 4, Pages 658-666 (April 2011) DOI: 10.1038/mt.2010.292 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Impaired activation of the Sonic hedgehog (Shh) signaling pathway is in ischemic muscles of middle-aged mice. Gli1 real-time PCR was performed at day 0, 3, 7, 14, and 21 after hindlimb ischemia. The ratio between Gli1 mRNA levels in the ischemic limb and the contralateral limb increases significantly in young (8-week-old) mice. In middle-aged (1-year-old) mice, Gli1 upregulation after ischemia is significantly impaired (P < 0.01). Molecular Therapy 2011 19, 658-666DOI: (10.1038/mt.2010.292) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Treatment with plasmid encoding the Sonic hedgehog human gene (phShh) induces activation of the Sonic hedgehog (Shh) pathway in the skeletal muscle of middle-aged mice. (a) phShh (100–200 µg) or empty plasmid was administered to 1-year-old mice. Gli1 expression was measured in hindlimb muscles 7 days after treatment. Significant increment of Gli1 expression was detected in phShh-treated compared to empty plasmid-treated muscles (P < 0.001). Increment was stronger in muscles treated with 200-µg phShh. (b) phShh (100–200 µg) or empty plasmid was administered to 1-year-old nls-Ptc1-LacZ mice. β-Gal activity was measured 7 days after treatment. It was significantly increased in muscles treated with 100 (P < 0.01) or 200 µg phShh (P < 0.001), compared to muscles treated with empty plasmid. (c) Real-time PCR (RT-PCR) showing strong expression of human Shh mRNA in the adductor muscle of mice treated with 200 µg of phShh. No signal is detectable in the muscles of mice treated with empty plasmid. (d) Whole mount X-gal staining showing strong and diffuse positive signal (blue staining) in large areas of nls-Ptc1-LacZ muscle treated with 200-µg phShh, whereas muscle treated with empty plasmid only shows negligible staining. (e) Presence of X-gal-positive cells (blue nuclei) in muscles of nls-Ptc1-LacZ mice treated with 200 µg phShh. (f) X-gal-expressing cells (blue nuclei) are positive for vimentin (brown cytoplasm). Molecular Therapy 2011 19, 658-666DOI: (10.1038/mt.2010.292) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Treatment with plasmid encoding the Sonic hedgehog human gene (phShh) increases blood flow and vascular density in middle-aged mice after hindlimb ischemia. Laser Doppler perfusion imaging was performed in 1-year-old mice immediately after induction of hindlimb ischemia (day 0), and at day 7, 14, 21, and 28 after surgery. In color-coded images (a), normal perfusion is depicted in red. Immediately after surgical excision of the left femoral artery (day 0), marked reduction of blood flow is depicted in blue, both in control- and phShh-treated mice. Perfusion remained severely impaired for 14 days in animals treated with control plasmid, while only for 7 days in phShh-treated mice. At day 21, perfusion is depicted in yellow and red in phShh-treated mice and in light blue/green in control mice, indicating better blood flow in the group of animals treated with Shh. Finally, perfusion returned to normal levels by day 28 in phShh-treated mice (depicted in red), whereas it was still partially impaired in control animals (depicted in yellow). (b) Quantification of laser Doppler perfusion studies demonstrated that, at day 28 after surgery, blood flow ratio between the ischemic leg and the contralateral leg was about 1 in mice treated with 200 µg phShh (black line), whereas it was still significantly compromised in mice treated with empty plasmid (gray line) (P < 0.01). (c) Representative images of CD31 immunostaining in the adductor muscle of 1-year-old mice treated with 200 µg phShh or empty plasmid, 28 days after induction of ischemia. (d) At the end of follow-up, capillary density is significantly higher in phShh-treated mice compared to control mice (P = 0.01). (e) Representative images of immunostaining for α-smooth muscle (SM)-actin in the adductor muscle of 1-year-old mice treated with 200 µg phShh or empty plasmid, 28 days after ischemia. (f) At the end of follow-up, arteriole density is significantly higher in ischemic limbs of mice treated with phShh than in those of mice treated with empty plasmid (P < 0.01). (g) Representative image of a CD31+ large-diameter vessel in muscles of phShh-treated mice. (h) Representative image of α-SM-actin-positive large-diameter vessels in muscles of phShh-treated mice. Molecular Therapy 2011 19, 658-666DOI: (10.1038/mt.2010.292) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Treatment with plasmid encoding the Sonic hedgehog human gene (phShh) increases the number of circulating progenitor cells after ischemia and increases their number in the site of neovascularization. (a) Fluorescence-activated cell sorting (FACS) analysis for expression of endothelial progenitor cell markers was performed on freshly isolated peripheral blood cells from 1-year-old mice at baseline, and at 4 and 7 days after ischemia. Mice were treated with phShh (200 µg) or control plasmid immediately after induction of ischemia. Compared to empty plasmid, treatment with phShh induced significant increase of CD45−/Sca-1+/Flk-1+ circulating cells both at day 4 (P < 0.01) and day 7 (P < 0.005). Each histogram represents the result of five independent experiments. (b) Representative image of X-gal positive cells in the hindlimb of mice transplanted with BM cells derived from nls-Ptc1-LacZ mice, 7 days after induction of ischemia. (c) The number of X-gal positive cells, 7 days after induction of ischemia, is significantly higher in the hindlimb muscles of mice transplanted with BM cells derived from nls-Ptc1-LacZ mice and treated with phShh than control (P < 0.01). (d) Representative image of β-gal (red) and CD31 (green) double fluorescent (yellow) cells in the ischemic hindlimb of mice transplanted with BM cells derived from nls-Ptc1-LacZ mice and treated with 200-µg phShh. Molecular Therapy 2011 19, 658-666DOI: (10.1038/mt.2010.292) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Treatment with plasmid encoding the Sonic hedgehog human gene (phShh) increases expression of vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), and stromal cell-derived factor-1α (SDF-1α) after hindlimb ischemia in middle-aged mice. VEGF, Ang-1, and SDF-1α expression ratio (ischemic/contralateral) is significantly increased in the ischemic muscle of 1-year-old mice treated with phShh compared to empty plasmid (P < 0.01). Molecular Therapy 2011 19, 658-666DOI: (10.1038/mt.2010.292) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions