Macrophage PPARγ and impaired wound healing in type 2 diabetes.

Macrophages undergo a transition from pro-inflammatory to healing-associated phenotypes that is critical for efficient wound healing. However, the regulation of this transition during normal and impaired healing remains to be elucidated. In our studies, the switch in macrophage phenotypes during skin wound healing was associated with up-regulation of the peroxisome proliferator-activated receptor (PPAR)γ and its downstream targets, along with increased mitochondrial content. In the setting of diabetes, up-regulation of PPARγ activity was impaired by sustained expression of IL-1ß in both mouse and human wounds. In addition, experiments with myeloid-specific PPARγ knockout mice indicated that loss of PPARγ in macrophages is sufficient to prolong wound inflammation and delay healing. Furthermore, PPARγ agonists promoted a healing-associated macrophage phenotype both in vitro and in vivo, even in the diabetic wound environment. Importantly, topical administration of PPARγ agonists improved healing in diabetic mice, suggesting an appealing strategy for down-regulating inflammation and improving the healing of chronic wounds.

Macrophage activation and skeletal muscle healing following traumatic injury.

Following injury to different tissues, macrophages can contribute to both regenerative and fibrotic healing. These seemingly contradictory roles of macrophages may be related to the markedly different phenotypes that macrophages can assume upon exposure to different stimuli. We hypothesized that fibrotic healing after traumatic muscle injury would be dominated by a pro-fibrotic M2a macrophage phenotype, with M1 activation limited to the very early stages of repair. We found that macrophages accumulated in lacerated mouse muscle for at least 21 days, accompanied by limited myofibre regeneration and persistent collagen deposition. However, muscle macrophages did not exhibit either of the canonical M1 or M2a phenotypes, but instead up-regulated both M1- and M2a-associated genes early after injury, followed by down-regulation of most markers examined. Particularly, IL-10 mRNA and protein were markedly elevated in macrophages from 3-day injured muscle. Additionally, though flow cytometry identified distinct subpopulations of macrophages based on high or low expression of TNFα, these subpopulations did not clearly correspond to M1 or M2a phenotypes. Importantly, cell therapy with exogenous M1 macrophages but not non-activated macrophages reduced fibrosis and enhanced muscle fibre regeneration in lacerated muscles. These data indicate that manipulation of macrophage function has potential to improve healing following traumatic injury.

Phenotypic transitions of macrophages orchestrate tissue repair.

Macrophages are essential for the efficient healing of numerous tissues, and they contribute to impaired healing and fibrosis. Tissue repair proceeds through overlapping phases of inflammation, proliferation, and remodeling, and macrophages are present throughout this progression. Macrophages exhibit transitions in phenotype and function as tissue repair progresses, although the precise factors regulating these transitions remain poorly defined. In efficiently healing injuries, macrophages present during a given stage of repair appear to orchestrate transition into the next phase and, in turn, can promote debridement of the injury site, cell proliferation and angiogenesis, collagen deposition, and matrix remodeling. However, dysregulated macrophage function can contribute to failure to heal or fibrosis in several pathological situations. This review will address current knowledge of the origins and functions of macrophages during the progression of tissue repair, with emphasis on skin and skeletal muscle. Dysregulation of macrophages in disease states and therapies targeting macrophage activation to promote tissue repair are also discussed.

The vitronectin-binding function of PAI-1 exacerbates lung fibrosis in mice.

Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.

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 increases hepatocyte growth factor activity required for skeletal muscle regeneration.

The plasminogen system plays a crucial role in the repair of a variety of tissues, including skeletal muscle. We hypothesized that urokinase-type plasminogen activator (uPA) promotes muscle regeneration by activating hepatocyte growth factor (HGF), which, in turn, stimulates proliferation of myoblasts required for regeneration. In our studies, levels of active HGF and phosphorylation of the HGF receptor c-met were increased after muscle injury in wild-type mice. Compared with wild-type animals, mice deficient in uPA (uPA(-/-)) had markedly reduced HGF levels and c-met activation after muscle damage. This reduced HGF activity in uPA(-/-) animals was associated with decreased cell proliferation, myoblast accumulation, and new muscle fiber formation. On the other hand, HGF activity was enhanced at early time points in PAI-1(-/-) mice compared with wild-type mice and the PAI-1(-/-) animals exhibited accelerated muscle fiber regeneration. Furthermore, administration of exogenous uPA rescued HGF levels and muscle regeneration in uPA(-/-) mice, and an HGF-blocking antibody reduced HGF activity and muscle regeneration in wild-type mice. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity, and this process was inhibited by an HGF-blocking antibody. Together, our findings demonstrate that uPA promotes muscle regeneration through HGF activation and subsequent myoblast proliferation.

Macrophage phenotypes during tissue repair.

Mp are crucial for tissue repair and regeneration but can also contribute to tissue damage and fibrosis. Mp can adopt a variety of functional phenotypes in response to different stimuli; two of the best-characterized in vitro phenotypes are a proinflammatory "M1" phenotype, produced by exposure to IFN-γ and TNF-α, and an anti-inflammatory "M2a" phenotype, produced by IL-4 or IL-13. M2a Mp are frequently termed "wound healing" Mp, as they express factors that are important for tissue repair. This review will summarize current knowledge of Mp phenotypes during tissue repair and will argue that these in vivo Mp populations are heterogeneous and temporally regulated and do not conform to existing, in vitro-defined M1 or M2 phenotypes. Mp during the early stages of tissue repair exhibit a more proinflammatory phenotype than their later counterparts, which in turn may exhibit some M2a-associated characteristics. However, phenotypic markers that appear to be coregulated in cultured Mp can be expressed independently of each other in vivo. Additionally, M1- and M2-associated markers may be expressed simultaneously by actual tissue-repair Mp. Improved understanding of Mp phenotypes and their regulation may assist in generation of novel therapies based on manipulating Mp function to improve healing.

Assessing macrophage phenotype during tissue repair.

Macrophages are thought to play important roles in tissue repair, from host defense to angiogenesis and new tissue formation. The role of macrophages in repair of different tissues is an active area of inquiry, particularly in settings of impaired healing. In this chapter, we describe methods for isolating monocyte/macrophage cell populations from damaged tissue and characterizing the phenotype of these cells. Cells are isolated from tissue by enzymatic digestion, and then monocyte/macrophage populations can be sorted by magnetic separation. The phenotype of these cells is assessed by real-time PCR, flow cytometry and ELISA. A complementary approach of assessing monocyte/macrophage phenotype by immunofluorescence staining of cryosections is also described. This combination of approaches to study the macrophage phenotypes expressed during tissue repair will lead to better understanding of the roles of macrophages in tissue repair and new therapeutic avenues for improving healing.

COX-2 inhibitor reduces skeletal muscle hypertrophy in mice.

Anti-inflammatory strategies are often used to reduce muscle pain and soreness that can result from high-intensity muscular activity. However, studies indicate that components of the acute inflammatory response may be required for muscle repair and growth. The hypothesis of this study was that cyclooxygenase (COX)-2 activity is required for compensatory hypertrophy of skeletal muscle. We used the synergist ablation model of skeletal muscle hypertrophy, along with the specific COX-2 inhibitor NS-398, to investigate the role of COX-2 in overload-induced muscle growth in mice. COX-2 was expressed in plantaris muscles during compensatory hypertrophy and was localized mainly in or near muscle cell nuclei. Treatment with NS-398 blunted the increases in mass and protein content in overloaded muscles compared with vehicle-treated controls. Additionally, the COX-2 inhibitor decreased activity of the urokinase type plasminogen activator, macrophage accumulation, and cell proliferation, all of which are required for hypertrophy after synergist ablation. Expression of insulin-like growth factor-1 and phosphorylation of Akt, mammalian target of rapamycin, and p70S6K were increased following synergist ablation, but were not affected by NS-398. Additionally, expression of atrogin-1 was reduced during hypertrophy, but was also not affected by NS-398. These results demonstrate that COX-2 activity is required for skeletal muscle hypertrophy, possibly through facilitation of extracellular protease activity, macrophage accumulation, and cell proliferation.