Volume 18, Issue 12, Pages 2155-2163 (December 2010) Laminin-111: A Potential Therapeutic Agent for Duchenne Muscular Dystrophy  Sébastien Goudenege, Yann Lamarre, Nicolas Dumont, Joël Rousseau, Jérôme Frenette, Daniel Skuk, Jacques P Tremblay  Molecular Therapy  Volume 18, Issue 12, Pages 2155-2163 (December 2010) DOI: 10.1038/mt.2010.165 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Laminin-111 increases resistance, absolute (g) and specific force (N/cm2) in dystrophin-deficient muscle. (a,b) Muscle resistance was measured by performing seven consecutive eccentric contractions. The change of tetanic force between each contraction reflects the degree of muscle damage. An eccentric contraction protocol was performed on mdx injected with laminin-111 or PBS, and on normal C57BL/10J. Systemic delivery (i.p.) of laminin-111 and intramuscular injection (i.m.) are represented, respectively, in a and b. The tetanic tension developed during the first cycle was designated as 100%. The mdx laminin-111 curves were significantly lower (percentage of force drop) than the mdx PBS control following i.m. injections (P < 0.0018, b) and i.p. injections (P < 0.0164, a). Both mdx curves (laminin-111 treated and PBS-injected mice) following i.m. injections (P < 0.0001, P < 0.0001, respectively) and i.p. injections to mice (P < 0.0001, P < 0.0001, respectively) were significantly different from normal C57BL10J. The P value refers to comparison between trend of multipoint curve laminin-111, control mdx and normal mice. (c,d) Absolute maximal muscle forces of control mdx injected with PBS, mdx injected with laminin-111, and normal C57BL/10J groups were compared. No significant difference was observed on absolute maximal force between mdx groups following i.p. injection of laminin-111 (c). A significant 41% increase of the mdx absolute maximal force (d) was observed following an i.m. injection of laminin-111 relative to the mdx muscle injected with PBS. (e,f) The specific maximal force was calculated by analyzing the absolute maximal force of a muscle in function of its mass and length. The specific maximal force of the mdx muscles injected i.p. with laminin-111 or with PBS was significantly different from the wild-type control muscles. There was, however, no significant difference of the specific maximal force between the mdx muscles injected i.p. with PBS or with laminin-111 (e). However, a significant 44% increase of the mdx specific maximal force (f) was observed following an i.m. injection of laminin-111 relative to the mdx-injected i.m. with PBS. *Statistically different results (n = 5 for i.p. injection, n = 4 for i.m. injection). i.p., intraperitoneal; PBS, phosphate-buffered saline. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Immunofluorescence of laminin-111. (a) The presence of laminin-111 was detected by immunofluorescence in the extracellular space of the tibialis anterior muscles of the Rag/mdx mice injected i.m. with laminin-111. (b) Only background staining was observed in Hank's balanced salt solution–injected Rag/mdx muscle. Bar = 90 µm, it applies to both a and b. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Laminin-111 prevents muscle pathology in mdx mice after myoblast transplantation. Hematoxylin and eosin staining reveals that control mdx muscles injected with (a,c,e) myoblasts and Hank's balanced salt solution contain large clusters of regenerating myofibers (identified by their small diameter, basophilic cytoplasm, mononuclear cell infiltrate, and centrally located nuclei) compared with mdx muscles injected i.m. with (b,d,f) myoblasts and laminin-111. Pictures illustrate representative cross-sections (n = 6). a,b, Bar = 0.35 mm; c,d, bar = 160 µm; e,f, bar = 45 µm. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Laminin-111 increases sarcolemmal integrity. (a) Representative cross-sections of tibialis anterior (Rag/mdx) in optic microscopy. Bar = 150 µm. (b) Evans blue dye (EBD) staining in Rag/mdx control mice, same cross-section as in a. (c) Rag/mdx muscles injected with laminin-111 alone or co-injected i.m. with laminin-111, and myoblasts had reduced EBD uptake compared with control co-injected with Hank's balanced salt solution and myoblasts. *Results are statistically different (P < 0.05; n = 7). There is no significant difference between both laminin-111 conditions with or without myoblasts. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Laminin-111 reduced the inflammatory reaction. Macrophage immunodetection was used to follow the inflammatory reaction in tibialis anterior muscles. (a–c) Rag/mdx control mice co-injected i.m. with HBSS and myoblasts. (b–d) Mice co-injected i.m. with laminin-111 and myoblasts. a,b, Bar = 0.4 mm; c,d, bar = 130 µm. (e) Fluorescence intensity decreased by 90% with laminin-111 compared to the control indicating that macrophage infiltration was drastically reduced. *Significant difference relative to the control (n = 6; P < 0.01). HBSS, Hank's balanced salt solution. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Immunofluorescence showing dystrophin-positive myofibers. (a) Representative cross-sections of tibialis anterior muscles injected i.m. with laminin-111 and human myoblasts (Rag/mdx). Bar = 120 µm. (b) Rag/mdx muscle injected i.m. with laminin-111 contained significantly more dystrophin-positive fibers than control Rag/mdx injected i.m. with human myoblasts without laminin-111. *Statistically different results (P < 0.05; n = 8). HBSS, Hank's balanced salt solution. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Laminin-111 stimulated cell proliferation measured by the CyQUANT assay. The fluorescence intensity of the CyQUANT GR dye was measured at different times (0, 30, 72, and 96 hours) to evaluate the myoblast proliferation in a low serum medium (2% serum) (n = 3). An increased proliferation was observed at all time intervals (18 and 20%, respectively, after 30 and 72 hours, P < 0.05), and this enhancement was maximum at 96 hours (28%, P < 0.01). *Statistically different results. Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Laminin-111 improved the overall migration rate of human myoblasts in serum-free medium. 3 × 104 cells/100 µl were loaded into the upper chamber of a Transwell migration assay. The medium in the upper chamber was complemented or not with laminin-111 at 100 nmol/l, whereas the lower chamber contained only serum-free medium. The migrating cells were stained after 14 hours of incubation. Representative pictures of the colored cells are shown in (a, serum-free medium) and (b, laminin-111). (c) Histogram shows that laminin-111 enhanced considerably cell migration compared with the control without laminin-111. *Statistically different results (P < 0.01, n = 5). Molecular Therapy 2010 18, 2155-2163DOI: (10.1038/mt.2010.165) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions