Inhibition of the immunoproteasome modulates innate immunity to ameliorate muscle pathology of dysferlin-deficient BlAJ mice.

Muscle repair in dysferlinopathies is defective. Although macrophage (Mø)-rich infiltrates are prominent in damaged skeletal muscles of patients with dysferlinopathy, the contribution of the immune system to the disease pathology remains to be fully explored. Numbers of both pro-inflammatory M1 Mø and effector T cells are increased in muscle of dysferlin-deficient BlAJ mice. In addition, symptomatic BlAJ mice have increased muscle production of immunoproteasome. In vitro analyses using bone marrow-derived Mø of BlAJ mice show that immunoproteasome inhibition results in C3aR1 and C5aR1 downregulation and upregulation of M2-associated signaling. Administration of immunoproteasome inhibitor ONX-0914 to BlAJ mice rescues muscle function by reducing muscle infiltrates and fibro-adipogenesis. These findings reveal an important role of immunoproteasome in the progression of muscular dystrophy in BlAJ mouse and suggest that inhibition of immunoproteasome may produce therapeutic benefit in dysferlinopathy.

Metal Nanoclusters with Synergistically Engineered Optical and Buffering Activity of Intracellular Reactive Oxygen Species by Compositional and Supramolecular Design.

Metal nanoclusters featuring tunable luminescence and high biocompatibility are receiving attention as fluorescent markers for cellular imaging. The recently discovered ability of gold clusters to scavenge cytotoxic reactive oxygen species (ROS) from the intracellular environment extends their applicability to biomedical theranostics and provides a novel platform for realizing multifunctional luminescent probes with engineered anti-cytotoxic activity for applications in bio-diagnostics and conceivably cellular therapy. This goal could be achieved by using clusters of strongly reactive metals such as silver, provided that strategies are found to enhance their luminescence while simultaneously enabling direct interaction between the metal atoms and the chemical surroundings. In this work, we demonstrate a synergic approach for realizing multifunctional metal clusters combining enhanced luminescence with strong and lasting ROS scavenging activity, based on the fabrication and in situ protection of Ag nanoclusters with a supramolecular mantle of thiolated-Au atoms (Ag/Au-t). Confocal imaging and viability measurements highlight the biocompatibility of Ag/Au-t and their suitability as fluorescent bio-markers. ROS concentration tests reveal the remarkable scavenging activity of Ag-based clusters. Proliferation tests of cells in artificially stressed culture conditions point out their prolonged anti-cytotoxic effect with respect to gold systems, ensuring positive cell proliferation rates even for long incubation time.

Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from nonfunctioning pituitary tumors.

The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 ± 17% and 35 ± 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.

Functional changes in Duchenne muscular dystrophy: a 12-month longitudinal cohort study.

OBJECTIVE: The aim of the study was to assess different outcome measures in a cohort of ambulant boys with Duchenne muscular dystrophy (DMD) over 12 months in order to establish the spectrum of possible changes in relation to age and steroid treatment. METHODS: The study is a longitudinal multicentric cohort study. A total of 106 ambulant patients with DMD were assessed using the 6-minute walk test (6MWT) and North Star Ambulatory Assessment (NSAA) at baseline and 12 months. Clinical data including age and steroid treatment were collected. RESULTS: During the 12 months of the study, we observed a mean decline of 25.8 meters in the 6MWT with a SD of 74.3 meters. On NSAA, the mean decline was 2.2 points with a SD of 3.7. Not all the boys with DMD in our cohort showed a decline over the 12 months, with young boys showing some improvement in their 6MWT and NSAA scores up to the age of 7. NSAA and the 6MWT had the highest correlation (r = 0.52, p < 0.001). CONCLUSIONS: This study provides longitudinal data of NSAA and 6MWT over a 12-month period. These data can be useful when designing a clinical trial.

Partial dysferlin reconstitution by adult murine mesoangioblasts is sufficient for full functional recovery in a murine model of dysferlinopathy.

Dysferlin deficiency leads to a peculiar form of muscular dystrophy due to a defect in sarcolemma repair and currently lacks a therapy. We developed a cell therapy protocol with wild-type adult murine mesoangioblasts. These cells differentiate with high efficiency into skeletal muscle in vitro but differ from satellite cells because they do not express Pax7. After intramuscular or intra-arterial administration to SCID/BlAJ mice, a novel model of dysferlinopathy, wild-type mesoangioblasts efficiently colonized dystrophic muscles and partially restored dysferlin expression. Nevertheless, functional assays performed on isolated single fibers from transplanted muscles showed a normal repairing ability of the membrane after laser-induced lesions; this result, which reflects gene correction of an enzymatic rather than a structural deficit, suggests that this myopathy may be easier to treat with cell or gene therapy than other forms of muscular dystrophies.

CD20-related signaling pathway is differently activated in normal and dystrophic circulating CD133(+) stem cells.

Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133(+) cells displaying myogenic properties. Unexpectedly, we observed the expression of the B-cell marker CD20 in blood-derived CD133(+) stem cells. The CD20 antigen plays a role in the modulation of intracellular calcium homeostasis through signaling pathways activation. Several observations suggest that an increase in intracellular calcium concentration ([Ca(2+)](i)) could be involved in the etiology of the Duchenne muscular dystrophy (DMD). Here, we show that a CD20-related signaling pathway able to induce an increase in [Ca(2+)](i) is differently activated after brain derived neurotrophic factor (BDNF) stimulation of normal and dystrophic blood-derived CD133(+) stem cells, supporting the assumption of a "CD20-related calcium impairment" affecting dystrophic cells. Presented findings represent the starting point toward the expansion of knowledge on pathways involved in the pathology of DMD and in the behavior of dystrophic blood-derived CD133(+) stem cells.

The new challenge of stem cell: brain tumour therapy.

The surprising similarity of much brain tumour behavior to the intrinsic properties of the neural stem/progenitor cell has triggered a recent interest in both arming stem cells to track and help eradicate tumours and in viewing stem cell biology as somehow integral to the emergence and/or production of the neoplasm itself. Moreover, based on the unique capacity of neural stem cells (NSCs) to migrate throughout the brain and to target invading tumour cells, the transplantation of NSCs offers a new potential therapeutic approach as a cell-based delivery system for gene therapy in brain tumours. On the one hand, both stem cells and cancer cells are thought to be capable of unlimited proliferation. While on the other, many tumours and cancer cell lines express stem cell markers, suggesting either that cancer cells resemble stem cells or that cancers contain stem-like cells. In this review we highlight the close relationship between normal neural stem cells and brain tumour stem cells and also suggest the possible clinical implications that these similarities could offer.

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse.

Abnormal connective tissue proliferation following muscle degeneration is a major pathological feature of Duchenne muscular dystrophy (DMD), a genetic myopathy due to lack of the sarcolemmal dystrophin protein. Since this fibrotic proliferation is likely to be a major obstacle to the efficacy of future therapies, research is needed to understand and prevent the fibrotic process in order to develop an effective treatment. Murine muscular dystrophy (mdx) is genetically homologous to DMD, and histopatological alterations are comparable to those of the muscles of patients with DMD. To investigate the development of fibrosis, we bred the mdx mouse with the scid immunodepressed mouse and analysed fibrosis histologically; we used ELISA analysis to determine TGF-beta1 expression. Significant reduction of fibrosis and TGF-beta1 expression was found in the muscles of the scid/mdx mice. However, we observed similar centrally located nuclei, necrosis, muscle degeneration and muscle force compared to the mdx animals. These data demonstrate a correlation between the absence of B and T lymphocytes and loss of fibrosis accompanied by reduction of TGF-beta1, suggesting the importance of modulation of the immune system in DMD.

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection.

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.

High-resolution X-ray microtomography for three-dimensional visualization of human stem cell muscle homing.

In the perspective of clinical translation of stem cell research, it would be advantageous to develop new techniques to detect donor cells after transplantation to track their fate and thus better understand their role in regeneration of damaged and diseased tissues. In this study we use X-ray computed microtomography for three-dimensional visualization of stem cells that were labeled with magnetic nanoparticles and transplanted via intra-arterial infusion. We show that X-ray computed microtomography offers the possibility to detect with high definition and resolution human cells after transplantation, and opens new possibilities for both experimental stem cell research.

Fate of autologous dermal stem cells transplanted into the spinal cord after traumatic injury (TSCI).

Rat dermis is a source of cells capable of growing in vitro and, in appropriate conditions, forming floating spheres constituted by nestin-positive cells. We have clonally grown these spheres up to the 15th generation. These spheres can be dissociated into cells that differentiate in vitro under appropriate conditions, these cells are labeled by antibodies to immature neuron markers such as nestin and beta-tubulin III and, later, to mature neuron markers such as microtubule-associated protein 2 and neurofilaments. However, most cells are positive to the astroglial marker glia fibrillary acidic protein (GFAP). When sphere-derived cells are transplanted into the spinal cord after traumatic injury, their migration into the lesion cavity is optimal but their differentiation is dependent upon the time interval between lesioning and cell transplantation. Injection of skin-derived stem cell within 30 min from injury yields mainly membrane activated complex-1 (MAC-1), cluster of differentiation-4 (CD-4) and CD-8 positive cells, that 60-90 days later undergo apoptosis. However, when transplantation is performed 7 days after injury, most cells (65% of total) are positive to staining with antibodies to GFAP, others (16%) to neurofilaments, and a smaller amount (2%) to the endothelial marker, platelet endothelial cell adhesion molecule. Thus our study shows that delayed transplantations of dermis-derived stem cells yield healthy cells that do not die, migrate to the lesion site, and there differentiate mainly in cells expressing glia and neuronal markers. On the other hand there is the possibility of dye transfer from labeled cells to endogenous cells, and this might influence the data.

Tumor necrosis factor-alpha (TNF-alpha) stimulates chemotactic response in mouse myogenic cells.

Migration of transplanted myogenic cells occurs during both embryogenesis and regeneration of skeletal muscles and is important for successful myoblast transplantation, but little is known about factors that promote chemotaxis of these cells. Tumor necrosis factor-alpha (TNF-alpha) is known to induce chemotactic effect on several cell types. In this study, we investigated its influence on the in vitro and in vivo motility of C2C12 and primary myoblasts. In the in vitro test performed in the blind-well Boyden chambers, we showed that TNF-alpha (50-400 U/ml) significantly enhanced the ability of myogenic cells to migrate. The dose-response curve for this factor was bell shaped, with maximum activity in the 200 U/ml range. In the in vivo test, intramuscular administration of TNF-alpha was performed by an Alzet pump connected to a perforated polyethylene microtube inserted in the tibialis anterior (TA) of CD1 mice. In these experiments, myoblasts were injected under the muscle epimysium. The recipient mice were immunosuppressed with FK506. Our results showed that, 5 days after myoblast transplantation, cells migrated further in the muscles infused with TNF-alpha than in the muscles not exposed to TNF-alpha. TNF-alpha not only has a chemotactic activity but may also modify cell migration via its action on matrix metalloproteinase (MMP) expression. The proteolytic activities of the MMPs secreted in the muscles were thus also assessed by gelatin zymography. The results showed an increased of MMP-2 and MMP-9 transcripts in the TNF-alpha-infused muscles injected with myogenic cells. Myoblast migration during transplantation may be enhanced by overlapping gradients of several effector molecules such as TNF-alpha, interferon-gamma (INF-gamma), and interleukins, released at the site of muscle injury. We propose that TNF-alpha may promote myoblast migration directly through chemotactic activity and indirectly by enhancing MMP activity at the site of muscle injury.

The urokinase plasminogen activator: an interesting way to improve myoblast migration following their transplantation.

Muscle cell migration plays an important role in the incorporation of transplanted myoblasts in muscle fibers. Understanding the mechanisms underlying the high migration capacity of the C(2)C(12) myoblast cell line may help to develop approaches to improve the migration of normal myoblasts and consequently to increase their participation to the host myofiber regeneration. We have previously shown that matrix metalloproteinases are implicated in the in vivo migration of C(2)C(12). Here, we studied the role of urokinase plasminogen activator (uPA) in this process. The expression of uPA mRNA and the enzymatic activity of uPA were studied in both normal myoblasts and the C(2)C(12) myoblast cell line. Reverse transcriptase polymerase chain reaction analysis showed that uPA mRNA was more strongly expressed in C(2)C(12) cells than in normal myoblasts. The enzymatic activity of secreted uPA analyzed by casein zymography is higher in medium conditioned by C(2)C(12) cells than in medium conditioned by normal myoblasts. Using our previously described microtube technique to assess in vivo cell migration, we showed that uPA is implicated in the in vivo migration of C(2)C(12) cells since this migration was abrogated in the presence of aprotinin (a general serine protease inhibitor) or amiloride (a uPA-specific inhibitor). We, therefore, hypothesized that increasing endogenous uPA expression by normal myoblasts may improve their migration capacity. Since an accumulating body of evidence has shown that growth factors regulate expression of uPA in a wide range of cells, we treated normal myoblasts with several growth factors alone or in combination with components of the extracellular matrix (ECM). All stimulants tested showed a minimal to strong effect on uPA enzymatic activity as assayed by zymography analysis. The positive effect of basic fibroblast growth factor (bFGF) on uPA enzymatic activity was slightly potentiated in the presence of fibronectin. Moreover, the pretreatment and coinjection of mouse myoblasts with bFGF alone or in combination with fibronectin improved significantly their in vivo migration throughout the tibialis anterior muscle of mdx mice. These results suggest that increasing uPA expression by an appropriate combination of growth factors and ECM components constitutes a possible approach to improving the migration of myogenic cells after transplantation.

Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy.

The purpose of this study was twofold: first, to evaluate the myoblast labeling of various 99mTc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding 99mTc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [99mTc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

Beta-enolase deficiency, a new metabolic myopathy of distal glycolysis.

A severe muscle enolase deficiency, with 5% of residual activity, was detected in a 47-year-old man affected with exercise intolerance and myalgias. No rise of serum lactate was observed with the ischemic forearm exercise. Ultrastructural analysis showed focal sarcoplasmic accumulation of glycogen beta particles. The enzyme enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the beta-enolase subunit, the protein product of the ENO3 gene. The beta-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while alpha-enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues; a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant beta-enolase. Muscle beta-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

In vitro and in vivo tetracycline-controlled myogenic conversion of NIH-3T3 cells: evidence of programmed cell death after muscle cell transplantation.

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to beta-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of beta-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive "blue" fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cell-mediated gene therapy of inherited muscle disorders.

Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice.

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.

T-antigen regulated expression reduces apoptosis of tag-transformed human myoblasts.

The generation of human myogenic cell lines could potentially provide a valuable source for cell transplantation in myopathies. The dysregulation of proliferative-differentiative signals by viral oncogenes can result in the induction of apoptosis. Whether apoptosis occurred in myogenic cells expressing large T antigen (Tag) from SV40 upon differentiation was unknown. Human muscle satellite cells were transfected with two different constructs, containing either an origin-defective SV40 genome or Tag under vimentin promoter control. When differentiation was triggered, Tag expression reduced the formation of myotubes and dead cells showing apoptotic features were present. However, the cells expressing SV40 Tag under vimentin promoter control retained their capacity to form myotubes and expressed the myofibrillar proteins as myosin heavy chain and dystrophin when Tag expression was silent. Their apoptotic rate was similar to that of untransfected cells. The observation that apoptosis can be prevented by the down-regulation of Tag suggests that the programmed cell death induced in transformed cells can be reversed, and confirms the regulatory efficiency of the human vimentin promoter.

Intracellular delivery of a Tat-eGFP fusion protein into muscle cells.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse biological membranes in a process called "protein transduction," delivering its cargo into cells. A Tat-eGFP fusion protein was purified from bacteria to study the transduction kinetics of Tat fusion proteins into cultured myoblasts and in the muscle tissue. Correctly folded Tat-eGFP reaches a maximum intracellular level in nearly 30 min, while its endogenous fluorescence is first detected only after 14 h. The nuclear localization signal from the basic domain of Tat was not sufficient to confer nuclear localization to Tat-eGFP, suggesting that the nuclear import pathway used by the exogenously added Tat-eGFP might be sensitive to the folding state of eGFP. In mice, the direct delivery to the muscle tissue using subcutaneous injections or the intra-arterial pathway led to few positive fibers in the muscle periphery or surrounding the blood vessels. Muscles injected with Tat-eGFP showed intense labeling of the extracellular matrix (ECM), suggesting that, although Tat fusion proteins can transduce muscle fibers, their binding by components of the ECM surrounding myofibers could interfere with the intracellular transduction process.