Recruitment of circulating cells into skeletal muscle : occurrence and possible mechanisms

Abstract: Tissue remodeling is a central process for all life. When a tissue is exposed to environmental stress such as injury, both local cells and cells from the periphery are recruited to regenerate and remodel the tissue. During the last decades the importance of resident cells, e.g. satellite cells (SaCs), and circulating cells, e.g. endothelial progenitor cells (EPC) and monocytes, in the process of skeletal muscle remodeling has been recognized. The aims of the current thesis were to investigate 1) whether cells from the bone marrow (BM) incorporate into skeletal muscle tissue in adult humans, 2) the impact of exercise on the level of circulating cells suggested to be important for skeletal muscle remodeling, and 3) if exercise induces the expression of cell recruiting factors in the skeletal muscle tissue. Four studies are included in the thesis. Women transplanted with male hematopoietic stem cells participated in study I. Study II and IV included healthy men and women that performed 60 min of cycling exercise, and study IV included controls. Study III was a training study in which 10 men performed one-legged exercise four times per week for 5 weeks with and without blood flow restriction. In all studies, muscle biopsies were obtained, and blood samples were collected in study II. Methods used in the studies included FISH, immunohistochemistry, flow cytometry, ELISA, qPCR, laser dissection and microdialysis. Y-chromosomes were found in muscle fiber and endothelial cell (EC) nuclei in skeletal muscle of women transplanted with male bone marrow. No Y-chromosomes were detected in the SaC niche. Directly after exercise, all monocyte subsets were significantly increased with the non-classical monocytes displaying the highest fold increase. At 2 h after exercise, only the classical subset was increased. The level of EPCs tended to increase with exercise. There was a significant increase in catecholamines, known to mobilize these cells with exercise, while no change was seen in the plasma levels of VEGF-A, G-CSF and SDF-1. The levels of endothelial adhesion molecules, E-selectin, ICAM-1 and VCAM-1, were all increased in skeletal muscle with exercise. VEGF-A expression was induced in skeletal muscle with the current exercise regime, and VEGF-A stimulation increased the level of endothelial adhesion molecules in HUVECs. An increase was observed for MMP-9 with one bout of exercise and MMP-2, MMP-14 and TIMP-1 with training, indicating different regulating mechanisms. The expression pattern and laser dissection did not support a myogenic origin of MMP-9, while MMP-2 mRNA was expressed in muscle fibers. The monocyte recruiting chemoattractant CX3CL1 was increased after a single bout of exercise and was localized to the skeletal muscle endothelium. Moreover, microdialysate obtained from exercised muscle increased the expression of CX3CL1 in HUVECs. THP-1 monocytes, primary human myoblasts and myotubes stimulated with CX3CL1 increased their expression of pro-angiogenic and pro-inflammatory factors, supporting a role for CX3CL1 in the cross-talk between ECs and other cell types in the skeletal muscle. The present thesis shows that BM-derived cells incorporate into human skeletal muscle tissue both as ECs and fused with skeletal muscle fibers. Further, monocyte subsets display different temporal patterns in mobilization with exercise, and monocyte recruiting factors and endothelial adhesion molecules increase in skeletal muscle with exercise. MMPs increase with exercise with different responses to acute exercise versus a longer period of training. Data from this thesis support the idea that cross-talk between different cell types in the muscle tissue, including ECs, muscle fibers and macrophages, plays an important role in the adaptation of skeletal muscle to exercise.