Macrophage-specific expression of urokinase-type plasminogen activator promotes skeletal muscle regeneration.

Macrophages (Mp) and the plasminogen system play important roles in tissue repair following injury. We hypothesized that Mp-specific expression of urokinase-type plasminogen activator (uPA) is sufficient for Mp to migrate into damaged muscle and for efficient muscle regeneration. We generated transgenic mice expressing uPA only in Mp, and we assessed the ability of these mice to repair muscle injury. Mp-only uPA expression was sufficient to induce wild-type levels of Mp accumulation, angiogenesis, and new muscle fiber formation. In mice with wild-type uPA expression, Mp-specific overexpression further increased Mp accumulation and enhanced muscle fiber regeneration. Furthermore, Mp expression of uPA regulated the level of active hepatocyte growth factor, which is required for muscle fiber regeneration, in damaged muscle. In vitro studies demonstrated that uPA promotes Mp migration through proteolytic and nonproteolytic mechanisms, including proteolytic activation of hepatocyte growth factor. In summary, Mp-derived uPA promotes muscle regeneration by inducing Mp migration, angiogenesis, and myogenesis.

Urokinase-type plasminogen activator plays essential roles in macrophage chemotaxis and skeletal muscle regeneration.

Although macrophages are thought to play important roles in tissue repair, the molecular mechanisms involved remain to be elucidated. Mice deficient in urokinase-type plasminogen activator (uPA-/-) exhibit decreased accumulation of macrophages following muscle injury and severely impaired muscle regeneration. We tested whether macrophage-derived uPA plays essential roles in macrophage chemotaxis and skeletal muscle regeneration. Macrophage uPA was required for chemotaxis, even when invasion through matrix was not necessary. The mechanism by which macrophage uPA promoted chemotaxis was independent of receptor binding but appeared to depend on proteolytic activity. Exogenous uPA restored chemotaxis to uPA-/- macrophages and rescued muscle regeneration in uPA-/- mice. Macrophage depletion in wild-type (WT) mice using clodronate liposomes resulted in impaired muscle regeneration, confirming that macrophages are required for efficient healing. Furthermore, transfer of WT bone marrow cells to uPA-/- mice restored macrophage accumulation and muscle regeneration. In this rescue, transferred WT cells appeared to contribute to IGF-1 expression but did not fuse to regenerating fibers. These data indicate that WT leukocytes, including macrophages, that express uPA were sufficient to rescue muscle regeneration in uPA-/- mice. Overall, the results indicate that uPA plays a fundamental role in macrophage chemotaxis and that macrophage-derived uPA promotes efficient muscle regeneration.

The urokinase-type plasminogen activator receptor is not required for skeletal muscle inflammation or regeneration.

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration.

Mechanical strain increases gene transfer to skeletal muscle cells.

Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport.

Mice deficient in plasminogen activator inhibitor-1 have improved skeletal muscle regeneration.

Skeletal muscle possesses a remarkable capacity for regeneration. Although the regulation of this process at the molecular level remains largely undefined, the plasminogen system appears to play a critical role. Specifically, mice deficient in either urokinase-type plasminogen activator (uPA-/- mice) or plasminogen demonstrate markedly impaired muscle regeneration after injury. In the present study, we tested the hypothesis that loss of the primary inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), would improve muscle regeneration. Repair of the extensor digitorum longus muscle was assessed after cardiotoxin injury in wild-type, uPA-/-, and PAI-1-deficient (PAI-1-/-) mice. As expected, there was no uPA activity in the injured muscles of uPA-/- mice, and muscles from these transgenic animals demonstrated impaired regeneration. On the other hand, uPA activity was increased in injured muscle from PAI-1-/- mice to a greater extent than in wild-type controls. Furthermore, PAI-1-/- mice demonstrated increased expression of MyoD and developmental myosin after injury as well as accelerated recovery of muscle morphology, protein levels, and muscle force compared with wild-type animals. The injured muscles of PAI-1-null mice also demonstrated increased macrophage accumulation, contrasting with impaired macrophage accumulation in uPA-deficient mice. The extent of macrophage accumulation correlated with both the clearance of protein after injury and the efficiency of regeneration. Taken together, these results indicate that PAI-1 deficiency promotes muscle regeneration, and this protease inhibitor represents a therapeutic target for enhancing muscle regeneration.