Role of integrin-β3 protein in macrophage polarization and regeneration of injured muscle

Abstract

Following injury, skeletal muscle achieves repair by a highly coordinated, dynamic process resulting from interplay among numerous inflammatory, growth factors and myogenic regulators. To identify genes involved in muscle regeneration, we used a microarray analysis; there was a significant increase in the expression of a group of integrin genes. To verify these results, we used RT-PCR and Western blotting and found that 12 integrins were up-regulated from 3 h to 15 days following injury. Following muscle injury, integrin-β3 was initially expressed, mainly in macrophages. In integrin-β3 global KO mice, the expression of myogenic genes was decreased and muscle regeneration was impaired, whereas fibrosis was enhanced versus events in wild type (WT) mice. The mechanism for these responses in integrin-β3 KO mice included an infiltration of macrophages that were polarized into the M2 phenotype. These macrophages produced more TGF-β1 and increased TGF-β1/Smad signaling. In vitro, we confirmed that M2 macrophages lacking integrin-β3 produced more TGF-β1. Furthermore, transplantation of bone marrow cells from integrin-β3 KO mice into WT mice led to suppression of the infiltration and accumulation of macrophages into injured muscles. There was also impaired muscle regeneration with an increase in muscle fibrosis. Our results demonstrate that integrin-β3 plays a fundamental role in muscle regeneration through a regulation of macrophage infiltration and polarization leading to suppressed TGF-β1 production. This promotes efficient muscle regeneration. Thus, an improvement in integrin-β3 function could stimulate muscle regeneration.

FIGURE 1.

Muscle injury increases expression of integrin subunits. TA muscles of three C57/BL6 mice were collected at different times after injury. Western blotting of integrins revealed the changes that are indicated on the right side of the blots (representative blotting from muscles of three mice at each time point after injury).

FIGURE 2.

Expression of integrin-β3 in injured muscles. A, at 5 h after injury, the cryo-sections of TA muscles were immunostained with integrin-β3 (red color) and MAC2 (green, upper panel) or CD41 (green, lower panel)). The yellow cells (right panel labeled Combined) demonstrate “double positive” cells (n = muscles of 3 mice). B, at 3 days after injury, the cryo-sections of TA muscles were immunostained with integrin-β3 (red color) and MAC2 or CD41 (green). The yellow cells (right panel labeled Combined) demonstrate double positive cells (n = muscles of 3 mice).

FIGURE 3.

Muscle regeneration is impaired in integrin-β3 KO mice. A, H&E staining of cryo-cross-sections of TA muscles at 3, 5, 8, 14, and 28 days following injury revealed impairment of muscle regeneration in integrin-β3 KO mice (n = muscles of 3 mice). B, at 28 days after injury, cryo-cross-sections of TA muscles were immunostained with laminin, and the areas of newly formed myofibers (i.e. those with central nuclei) were measured to calculate the distribution of myofiber sizes (n = muscles of 5 mice; *, p ≤ 0.05, difference between values from WT and integrin-β3 KO mice).

FIGURE 4.

Myogenic gene expression is impaired in injured TA muscles of integrin-β3 KO mice. A and B, at different times after injury, TA muscles from WT (white columns) or integrin-β3 KO mice (black columns) were collected, and the mRNAs of MyoD (A) or myogenin (B) were evaluated by RT-PCR. Data are means ± S.E. (n = muscles of 4 mice, three repetitions by RT-PCR). Two-way analysis of variance revealed a significant difference in values between treatment groups (*, p ≤ 0.05, difference between WT and integrin-β3 KO mice). C, Western blots of myogenin from 5 h to 15 days were measured using GAPDH as the control. (Representative blots were from muscles of three mice at each time point after injury.)

FIGURE 5.

Fibrosis markers are up-regulated in injured TA muscles of integrin-β3 KO mice. A, cryo-cross-sections of TA muscles obtained 28 days after injury were stained with Sirius Red. The dark red staining indicates collagen I deposition (n = muscles of 3 mice). B, cryo-cross-section of TA muscles obtained at 6 days after injury were immunostained with α-SMA (red), laminin (green), and DAPI (blue) (n = muscles of 3 mice). C and D, injured TA muscles from WT (white columns) and integrin-β3 KO mice (black columns) at different times after injury were subjected to RT-PCR to evaluate the mRNAs of α-SMA (C) and collagen 1 (D) (n = muscles of 4 mice; three repeats on RT-PCR analysis, means ± S.E.; *, p ≤ 0.05, difference between values from WT and integrin-β3 KO mice). E, hydroxyproline in uninjured and injured muscle at 7 or 14 days after injury (n = muscles from 6 mice with three repeat measurements for each muscle. Means ± S.E., *, p ≤ 0.05).

FIGURE 6.

TGF-β1 and its signaling are increased in injured TA muscles of integrin-β3 KO mice. A, injured TA muscles from WT (white columns) and integrin-β3 KO mice (black columns) were collected at different times. TGF-β1 mRNA expression was evaluated by RT-PCR (n = muscles from 4 mice (means ± S.E., *, p ≤ 0.05, difference between values from WT and integrin-β3 KO mice). B and C, Western blots of phospho-Smad2 (p-Smad2) and phospho-Smad3 (p-Smad3) in injured (I) and uninjured (C) muscles from TAs of WT and integrin-β3 KO mice at hours (B) or days (C) following injury (n = muscles from 3 mice).

FIGURE 7.

Macrophages lacking integrin-β3 develop characteristics of M2 phenotype. A, CD68 mRNA was evaluated by RT-PCR using injured TA muscles at different times (n = muscles from 4 mice; means ± S.E.; *, p ≤ 0.05, difference between values from WT and integrin-β3 KO mice). B, a representative FACS analysis of cells expressing CD206 and F4/80 is shown. The cells were obtained 5 h after gastrocnemius injury; the upper right area includes M2a macrophage cells. (Data are means ± S.E.; n = muscles of 5 mice.) C, Western blot analysis of the M2 macrophage markers, CD206 and CD163, in injured TA muscles of WT and integrin-β3 KO mice. The muscles were obtained at different times after injury. D, BMDMs from WT (white column) or integrin-β3 KO mice (black column) were used to assess the expression of mRNAs of cytokines or genes (means ± S.E.; *, p ≤ 0.05; n = 3 repeated experiments). E, media from a 24-h culture of BMDMs of the M0, M1, M2a, or M2c from WT or integrin-β KO mice were analyzed for total TGF-β1 using an ELISA kit from Promega (n = 3 repeat experiments). The percentage of increase of TGF-β1 in BMDMs of integrin-β3 KO over results of WT is indicated (*, p ≤ 0.05, difference between integrin-β3 KO and WT).

FIGURE 8.

BM transplant from integrin-β3 KO to WT mice decreases muscle regeneration following muscle injury. Muscles from WT mice bearing integrin-β3 KO BM were injured 30 days after the transplant. At 3, 6, 8, or 14 days after injury, the muscles from 4–6 mice were studied. A, at 3 days, the cryo-cross-section of muscles from WT transplanted with WT BM (WT-WT) or WT mice transplanted with integrin-β3 KO BM (β3KO-WT) were immunostained with integrin-β3 (green) or MAC2 (red; a macrophage marker). B, muscle morphology was visualized in laminin- and DAPI-stained cryo-sections. Over the entire period, there was decreased muscle regeneration in the β3KO-WT transplanted mice versus the WT-WT transplanted mice. C, the distribution of regenerating fibers also confirmed that there was a decrease in muscle regeneration in mice with the β3KO-WT transplant versus the WT-WT transplant. D, the mRNAs of MyoD, CD206, collagen-1, and TGF-β1 were examined by RT-PCR. Two-way analysis of variance showed a significant difference in the average values among treatment groups (p ≤ 0.05; *, β3KO-WT transplant versus the WT-WT transplant).