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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03300

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Ex vivo treatment with nitric oxide increases mesoangioblast therapeutic efficacy in muscular dystrophy

Clara Sciorati^1,*, Beatriz G. Galvez^1,*, Silvia Brunelli^1,2,*, Enrico Tagliafico³, Stefano Ferrari³, Giulio Cossu^1,4, and Emilio Clementi^1,5,6,

¹ CellStem Cell Research Institute, H. San Raffaele Scientific Institute, 20132, Milan, Italy
² Department of Experimental, Environmental Medicine and Medical Biotechnology, University of Milano-Bicocca, 20052 Monza, Italy
³ Department of Biomedical Science, University of Modena and Reggio Emilia, 41100 Modena, Italy
⁴ Department of Biology, University of Milano, 20130 Milan, Italy
⁵ E. Medea Scientific Institute, 23842 Bosisio Parini, Italy
⁶ Department of Preclinical Sciences LITA-Vialba, University of Milano, 20157 Milan, Italy

View larger version (60K): [in this window] [in a new window]   Fig. 1. NO/cGMP increase sarcoglycan production by intra-arterially delivered mesoangioblasts. Mesoangioblasts (5x105) pretreated for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM) were injected into the right femoral artery of 4-month-old SG-null mice. Mice were sacrificed after 21 days and quadriceps (Qd), gastrocnemius (Gs) and soleus (So) muscles collected. (A) SG mRNA expression, an index of mesoangioblast homing, was measured by real-time PCR. Values ± s.e.m. are expressed as the percentage of the total injected cells (n=3). (B,C) SG expression, evaluated by western blot analysis (B, showing both a representative western blot image and densitometric values, n=3) and immunohistological detection (C, representative of three consistent experiments). The graph in B reports the ratio of densitometric values ± s.e.m. of SG vs. those of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) used as an internal loading control. Triple asterisks and crosses, P<0.001 vs NT and DETA-NO-treated cells, respectively. Bar in C, 400 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 2. NO/cGMP increase mesoangioblast migration and engrafting in vivo. (A,B) GFP-expressing mesoangioblasts (5x105) pretreated for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM) were injected into the right femoral artery of 4-month-old SG-null mice. After 6 hours quadriceps (Qd), tibialis anterior (TA) and gastrocnemius (Gs) muscles, as well as liver, spleen and lung were collected and the number of mesoangioblasts migrated into them calculated by quantitative real-time PCR for GFP. Values are expressed as the percentage of the total injected cells (n=3). (C) GFP-expressing mesoangioblasts (5x105) pretreated as indicated above were injected into the tibialis anterior muscles of 4-month-old SG-null mice. After 12 hours muscles were recovered. Apoptosis of GFP-expressing mesoangioblasts was assessed by the TUNEL technique (images from one out of three reproducible experiments). Also shown is the DAPI staining of the nuclei and its overlay with the GFP and TUNEL staining. Arrows in the merge panels show selected GFP, TUNEL and DAPI-positive cells. The red arrow indicates the specific cell for which the GFP, TUNEL, DAPI and merge stainings are magnified in the insets. (D,E) Same conditions as in C, except that mice were sacrificed three weeks later, tibialis muscles were recovered and SG expression was analysed by real-time PCR (D, n=3) and western blot analysis (E, showing both a representative western blot image and densitometric values, n=3). The graph in E reports the ratio of densitometric values of SG vs. those of GAPDH used as an internal loading control. Triple asterisks and crosses, P<0.001 vs. NT and DETA-NO-treated cells, respectively; error bars in A, B, D and E, s.e.m.). Bar in C, 400 µm. The inset in the panel is at 6x magnification.

View larger version (44K): [in this window] [in a new window]   Fig. 3. NO/cGMP increase mesoangioblast migration and myogenic differentiation in vitro. (A-C) Mesoangioblast migration on gelatin-coated membranes. (A) Comparison of the chemoattractant properties of VEGF (10 ng/ml), TGFß (100 ng/ml), TNF (10 ng/ml), HGF (10 ng/ml) or bFGF (10 ng/ml) measured in a 6-hour migration assay (n=5). (B) Effect of pretreatment of mesoangioblasts for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM). Migration is expressed as the percentage of that observed in untreated controls (NT) (n=5). (C) Representative images of the transmigrated mesoangioblasts after staining with crystal violet. (D,E) Mesoangioblast transmigration through an H5V endothelial cell monolayer. In these experiments, LacZ-expressing mesoangioblasts, pretreated with or without DETA-NO, IMN, 8-Br-cGMP and ODQ as above were used. Transmigration induced by TNF, VEGF, HGF (D) or L6E9 myoblasts and myotubes (E) was measured after 6 hours. The migration index was calculated as described in the Materials and Methods (n=4). (F,G) Mesoangioblasts pretreated for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM) were co-cultured for 5 days in differentiating conditions together with rat L6E9 myoblasts (co-fusion conditions, F) or preformed L6E9 myotubes (post-fusion conditions, G). The graphs show the fusion index, i.e. the number of murine mesoangioblast-derived nuclei in myosin-expressing cells with more than two nuclei vs. the total number of rat and murine nuclei measured (n=5). Double and triple asterisks and crosses, P<0.01 and P<0.001, respectively vs. NT (asterisks) and DETA-NO-treated cells (crosses). Error bars, s.e.m. Bar in C, 200 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 4. NO/cGMP protect mesoangioblasts from cell death-inducing stimuli. (A,B) Mesoangioblasts were pretreated for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM) and incubated for 24 hours in the absence (NT) or presence of TNF (100 ng/ml), H2O2 (1 µM) or As2O3 (20 µM). (A) Cell death was determined by flow cytometry measuring Annexin V staining of phosphatidylserine exposed on the outer leaflet of the plasma membrane and PI incorporation, and expressed as the percentage of that observed in controls (i.e. mesoangioblasts pretreated without drugs and incubated in the absence of apoptogens) (n=4). (B) Representative dot-plot analyses showing the results obtained in one of four consistent experiments using As2O3 as the cell death-triggering stimulus. (C) Protection by NO/cGMP of mesoangioblasts (target) from cell death (specific lysis) induced by incubation for 16 hours with cytotoxic T lymphocytes (effector) at the indicated effector/target ratios. Single, double and triple asterisks and crosses, P<0.05, P<0.01 and P<0.001, respectively vs. NT (asterisks) or DETA-NO-treated cells (crosses). Error bars, s.e.m.