Skeletal muscle healing by M1-like macrophages produced by transient expression of exogenous GM-CSF

Abstract

Background After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. Methods To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass. Results Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-β1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill. Conclusion Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

The effect of autologous repair and voluntary wheel running on force recovery in a rat model of volumetric muscle loss

“…Macrophages can be classified as M1, which are present during the inflammatory stage of skeletal muscle regeneration, or M2, which accumulate at the site of injury once necrotic tissue has been removed and participate in the regeneration and remodelling response (Martins et al., 2020). There were differential responses in our markers of macrophage polarization at the 8‐week time point.…”

Analyses of the pericyte transcriptome in ischemic skeletal muscles

“…We also observed a dramatic increase in the number of CD45 + hematopoietic cells on ischemia day 2 (Supplementary Figure 5 c). Inflammatory regulation is known to be tightly associated with muscle regeneration [ 32 , 56 ] and angiogenesis [ 32 , 34 ], and pericytes are known to exhibit an immunomodulatory ability [ 19 , 20 , 50 , 51 ]. Notably, pericytes upregulated the expression of signaling molecules that attract inflammatory cells ( Ccl2, Ccl3, Ccl4 , and Cxcl5 ) and molecules that stimulate inflammation ( Il1b and Tnf ) at the earliest time point (ischemic day 2) (Supplementary Figure 5 a).…”

“…Previous studies conducted in our laboratory [ 30 – 34 ] and by other researchers [ 35 , 36 ] have shown that monocytes, macrophages, and MSCs have the potential to regulate angiogenesis, myogenesis, and fibrogenesis in skeletal muscle; therefore, the cells and/or genes that modulate these processes may provide insight into limb ischemia treatment. The role of pericytes in the recovery process from limb ischemia, however, has not been extensively studied.…”

Analyses of the pericyte transcriptome in ischemic skeletal muscles

“…5C). In ammatory regulation is known to be tightly associated with muscle regeneration [32,55] and angiogenesis [32,34], and pericytes are known to exhibit an immunomodulatory ability [19,20,50,51]. Notably, pericytes upregulated the expression of signaling molecules that attract in ammatory cells (Ccl2, Ccl3, Ccl4, and Cxcl5) and molecules that stimulate in ammation (Il1b and Tnf) at the earliest time point (ischemic day 2) ( Supplementary Fig.…”

“…Previous studies conducted in our laboratory [30][31][32][33][34] and by other researchers [35,36] have shown that monocytes, macrophages and MSCs have the potential to regulate angiogenesis, myogenesis, and brogenesis in skeletal muscle; therefore, the cells and/or genes that modulate these processes may provide insight into limb ischemia treatment. The role of pericytes in the recovery process from limb ischemia, however, has not been extensively studied.…”