Lysophosphatidic acid-induced RhoA signaling and prolonged macrophage infiltration worsens fibrosis and fatty infiltration following rotator cuff tears

Abstract

Previous studies have suggested that macrophage-mediated chronic inflammation is involved in the development of rotator cuff muscle atrophy and degeneration following massive tendon tears. Increased RhoA signaling has been reported in chronic muscle degeneration, such as muscular dystrophy. However, the role of RhoA signaling in macrophage infiltration and rotator muscle degeneration remains unknown. Using a previously established rat model of massive rotator cuff tears, we found RhoA signaling is upregulated in rotator cuff muscle following a massive tendon-nerve injury. This increase in RhoA expression is greatly potentiated by the administration of a potent RhoA activator, lysophosphatidic acid (LPA), and is accompanied by increased TNFα and TGF-β1 expression in rotator cuff muscle. Boosting RhoA signaling with LPA significantly worsened rotator cuff muscle atrophy, fibrosis, and fatty infiltration, accompanied with massive monocytic infiltration of rotator cuff muscles. Co-staining of RhoA and the tissue macrophage marker CD68 showed that CD68+ tissue macrophages are the dominant cell source of increased RhoA signaling in rotator cuff muscles after tendon tears. Taken together, our findings suggest that LPA-mediated RhoA signaling in injured muscle worsens the outcomes of atrophy, fibrosis, and fatty infiltration by increasing macrophage infiltraion in rotator cuff muscle. Clinically, inhibiting RhoA signaling may represent a future direction for developing new treatments to improve muscle quality following massive rotator cuff tears. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1539-1547, 2017.

Keywords: LPA; atrophy; fatty infiltration; macrophage; muscle; rotator cuff tear.

Figure 1

Representative immunohistofluorescence demonstrating increase in total RhoA expression in injured muscle (bottom row) compared to uninjured muscle (top row). White arrow indicates RhoA-positive cell. Scale bar=10 μm.

Figure 2

Real-time qRT-PCR demonstrating increase in total RhoA expression at 2 and 6 weeks after injury and initiation of treatment, TT+DN ΔC_T values normalized to mean sham ΔC_T values to calculate ΔΔC_T. Student’s two-tailed t-test was performed between ΔC_T values of injured and sham muscle, *p<0.05.

Figure 3

Real-time qRT-PCR comparing the effects of LPA vs. vehicle on total RhoA expression at 2 and 6 weeks after injury and initiation of treatment, LPA-treatment ΔC_T values normalized to those for the vehicle group for both TT+DN and sham muscle to calculate ΔΔC_T. Student’s two-tailed t-test was performed between ΔC_T values of LPA treatment and vehicle groups for both injured and sham muscle, *p<0.05, ***p<0.001.

Figure 4

Real-time qRT-PCR comparing the effects of LPA versus vehicle on inflammatory markers at 2 and 6 weeks after injury and initiation of treatment, LPA-treatment ΔCT values normalized to those for the vehicle group for both TT DN and sham muscle to calculate ΔΔC_T. Student’s two-tailed t-test was performed between ΔC_T values of LPA treatment and vehicle groups for both injured and sham muscle, *p<0.01.

Figure 5

Representative double-esterase staining at 2 weeks (A and C) and 6 weeks (B and D) after injury. Black arrows indicate α-naphthyl acetate esterase positive, monocyte-derived cells; red arrows indicate naphthol ASD-chloroacetate esterase positive, granulocyte-derived cells. Scale bar=50 μm. Cell density shown in C–D, *p<0.05, *p<0.01.

Figure 6

Representative immunohistofluorescence showing co-expression of CD68 and RhoA in tissue macrophages in rotator cuff muscles. Area within yellow box is enlarged in white box. Scale bar=20μm. White arrow (bottom row) indicates an additional CD68+/RhoA+ cell.

Figure 7

LPA treatment significantly reduced muscle wet weights (g) at 6 weeks after TT+DN injury, but not after sham surgery.

Figure 8

(A) Representative Masson’s trichrome stain at 6 weeks after injury (Scale bar=100 μm.). (A) Quantitative analysis of fibrosis index showed LPA treatment significantly increased fibrosis of rotator cuff muscles after TT DN injury (*p<0.05).

Figure 9

(A) Representative oil red-O stain at 6 weeks after injury (Scale bar=500 μm). (B) Quantitative analysis of fatty infiltration index showed LPA treatment significantly increased fatty infiltration of rotator cuff muscles after TT DN injury (*p<0.05).