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Increased serum and musculotendinous fibrogenic proteins following persistent low-grade inflammation in a rat model of long-term upper extremity overuse.

Gao HG, Fisher PW, Lambi AG, Wade CK, Barr-Gillespie AE, Popoff SN, Barbe MF - PLoS ONE (2013)

Bottom Line: Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles.Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance.Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

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Transforming growth factor beta 1 (TGFB1) in serum and flexor digitorum tissues of the reach limbs.Groups are as defined in figure legend 2. (A&B) Serum TGFB1 and tendon TGFB1, assayed using ELISA. (C) Immunohistochemical staining for TGFB1 in tendons from NC and 18-week HRLF reach limbs shows localization of TGFB1 in fibroblast-like cells in the peritendon region of both the NC and HRLF rats, and additional stained cells in the epitendon (Epi; thickened in HRLF rats) and endotendon (T) regions of the HRLF rat tendon. (D&E) The results of Western blot analysis of muscle TGFB1 in which two bands were detected, 50 kDa and 12.5 kDa. The ratio of each band of TGFB1 normalized to GAPDH levels is shown for three replicates of the western blot. (F) A representative Western blot of reach limb muscles from normal controls (NC, n = 4 shown), 24-week HRLF rats (n = 3 shown), and 18-week HRLF rats (n = 4 shown), probed with anti-TGFB1 and GAPDH. Green bands were detected with an anti-TGFB1 antibody and a secondary antibody tagged with IRDye800CW (Li-Cor, #.926-32211). Red bands were detected with an anti-GAPDH antibody and a secondary antibody tagged IRDye680LT (Li-Cor, #926-68020). Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats; a: p<0.05, compared to TR24 rats. Scale bar = 50 micrometers.
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pone-0071875-g005: Transforming growth factor beta 1 (TGFB1) in serum and flexor digitorum tissues of the reach limbs.Groups are as defined in figure legend 2. (A&B) Serum TGFB1 and tendon TGFB1, assayed using ELISA. (C) Immunohistochemical staining for TGFB1 in tendons from NC and 18-week HRLF reach limbs shows localization of TGFB1 in fibroblast-like cells in the peritendon region of both the NC and HRLF rats, and additional stained cells in the epitendon (Epi; thickened in HRLF rats) and endotendon (T) regions of the HRLF rat tendon. (D&E) The results of Western blot analysis of muscle TGFB1 in which two bands were detected, 50 kDa and 12.5 kDa. The ratio of each band of TGFB1 normalized to GAPDH levels is shown for three replicates of the western blot. (F) A representative Western blot of reach limb muscles from normal controls (NC, n = 4 shown), 24-week HRLF rats (n = 3 shown), and 18-week HRLF rats (n = 4 shown), probed with anti-TGFB1 and GAPDH. Green bands were detected with an anti-TGFB1 antibody and a secondary antibody tagged with IRDye800CW (Li-Cor, #.926-32211). Red bands were detected with an anti-GAPDH antibody and a secondary antibody tagged IRDye680LT (Li-Cor, #926-68020). Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats; a: p<0.05, compared to TR24 rats. Scale bar = 50 micrometers.

Mentions: Since IL-6 and TNF-α are not only pro-inflammatory cytokines, but also pro-fibrogenic cytokines, we next extended our study to examine for increases of four other fibrogenic cytokines: TGFB1, CTGF, and PDGFab and PDGFbb (Figures 5–7). We focused on the reach limb tissues, since they showed consistent significant grip strength declines and inflammatory cytokine increases, from control levels.


Increased serum and musculotendinous fibrogenic proteins following persistent low-grade inflammation in a rat model of long-term upper extremity overuse.

Gao HG, Fisher PW, Lambi AG, Wade CK, Barr-Gillespie AE, Popoff SN, Barbe MF - PLoS ONE (2013)

Transforming growth factor beta 1 (TGFB1) in serum and flexor digitorum tissues of the reach limbs.Groups are as defined in figure legend 2. (A&B) Serum TGFB1 and tendon TGFB1, assayed using ELISA. (C) Immunohistochemical staining for TGFB1 in tendons from NC and 18-week HRLF reach limbs shows localization of TGFB1 in fibroblast-like cells in the peritendon region of both the NC and HRLF rats, and additional stained cells in the epitendon (Epi; thickened in HRLF rats) and endotendon (T) regions of the HRLF rat tendon. (D&E) The results of Western blot analysis of muscle TGFB1 in which two bands were detected, 50 kDa and 12.5 kDa. The ratio of each band of TGFB1 normalized to GAPDH levels is shown for three replicates of the western blot. (F) A representative Western blot of reach limb muscles from normal controls (NC, n = 4 shown), 24-week HRLF rats (n = 3 shown), and 18-week HRLF rats (n = 4 shown), probed with anti-TGFB1 and GAPDH. Green bands were detected with an anti-TGFB1 antibody and a secondary antibody tagged with IRDye800CW (Li-Cor, #.926-32211). Red bands were detected with an anti-GAPDH antibody and a secondary antibody tagged IRDye680LT (Li-Cor, #926-68020). Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats; a: p<0.05, compared to TR24 rats. Scale bar = 50 micrometers.
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Related In: Results  -  Collection

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pone-0071875-g005: Transforming growth factor beta 1 (TGFB1) in serum and flexor digitorum tissues of the reach limbs.Groups are as defined in figure legend 2. (A&B) Serum TGFB1 and tendon TGFB1, assayed using ELISA. (C) Immunohistochemical staining for TGFB1 in tendons from NC and 18-week HRLF reach limbs shows localization of TGFB1 in fibroblast-like cells in the peritendon region of both the NC and HRLF rats, and additional stained cells in the epitendon (Epi; thickened in HRLF rats) and endotendon (T) regions of the HRLF rat tendon. (D&E) The results of Western blot analysis of muscle TGFB1 in which two bands were detected, 50 kDa and 12.5 kDa. The ratio of each band of TGFB1 normalized to GAPDH levels is shown for three replicates of the western blot. (F) A representative Western blot of reach limb muscles from normal controls (NC, n = 4 shown), 24-week HRLF rats (n = 3 shown), and 18-week HRLF rats (n = 4 shown), probed with anti-TGFB1 and GAPDH. Green bands were detected with an anti-TGFB1 antibody and a secondary antibody tagged with IRDye800CW (Li-Cor, #.926-32211). Red bands were detected with an anti-GAPDH antibody and a secondary antibody tagged IRDye680LT (Li-Cor, #926-68020). Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats; a: p<0.05, compared to TR24 rats. Scale bar = 50 micrometers.
Mentions: Since IL-6 and TNF-α are not only pro-inflammatory cytokines, but also pro-fibrogenic cytokines, we next extended our study to examine for increases of four other fibrogenic cytokines: TGFB1, CTGF, and PDGFab and PDGFbb (Figures 5–7). We focused on the reach limb tissues, since they showed consistent significant grip strength declines and inflammatory cytokine increases, from control levels.

Bottom Line: Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles.Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance.Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

Show MeSH
Related in: MedlinePlus