Increased Serum and Musculotendinous Fibrogenic Proteins following Persistent Low-Grade Inflammation in a Rat Model of Long-Term Upper Extremity Overuse.

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

Development of Normal and Cleft Palate: A Central Role for Connective Tissue Growth Factor (CTGF)/CCN2

Development of the palate is the result of an organized series of events that require exquisite spatial and temporal regulation at the cellular level. There are a myriad of growth factors, receptors and signaling pathways that have been shown to play an important role in growth, elevation and/or fusion of the palatal shelves. Altered expression or activation of a number of these factors, receptors and signaling pathways have been shown to cause cleft palate in humans or mice with varying degrees of penetrance. This review will focus on connective tissue growth factor (CTGF) or CCN2, which was recently shown to play an essential role in formation of the secondary palate. Specifically, the absence of CCN2 in KO mice results in defective cellular processes that contribute to failure of palatal shelf growth, elevation and/or fusion. CCN2 is unique in that it has been shown to interact with a number of other factors important for palate development, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), Wnt proteins and transforming growth factor-βs (TGF-βs), thereby influencing their ability to bind to their receptors and mediate intracellular signaling. The role that these factors play in palate development and their specific interactions with CCN2 will also be reviewed. Future studies to elucidate the precise mechanisms of action for CCN2 and its interactions with other regulatory proteins during palatogenesis are expected to provide novel information with the potential for development of new pharmacologic or genetic treatment strategies for clinical intervention of cleft palate during development

Blocking CCN2 Reduces Established Palmar Neuromuscular Fibrosis and Improves Function Following Repetitive Overuse Injury

The matricellular protein cell communication factor 2/connective tissue growth factor (CCN2/CTGF) is critical to development of neuromuscular fibrosis. Here, we tested whether anti-CCN2 antibody treatment will reduce established forepaw fibro-degenerative changes and improve function in a rat model of overuse injury. Adult female rats performed a high repetition high force (HRHF) task for 18 weeks. Tissues were collected from one subset after 18 wks (HRHF-Untreated). Two subsets were provided 6 wks of rest with concurrent treatment with anti-CCN2 (HRHF-Rest/anti-CCN2) or IgG (HRHF-Rest/IgG). Results were compared to IgG-treated Controls. Forepaw muscle fibrosis, neural fibrosis and entheseal damage were increased in HRHF-Untreated rats, compared to Controls, and changes were ameliorated in HRHF-Rest/anti-CCN2 rats. Anti-CCN2 treatment also reduced phosphorylated-β-catenin (pro-fibrotic protein) in muscles and distal bone/entheses complex, and increased CCN3 (anti-fibrotic) in the same tissues, compared to HRHF-Untreated rats. Grip strength declines and mechanical sensitivity observed in HRHF-Untreated improved with rest; grip strength improved further in HRHF-Rest/anti-CCN2. Grip strength declines correlated with muscle fibrosis, entheseal damage, extraneural fibrosis, and decreased nerve conduction velocity, while enhanced mechanical sensitivity (a pain-related behavior) correlated with extraneural fibrosis. These studies demonstrate that blocking CCN2 signaling reduces established forepaw neuromuscular fibrosis and entheseal damage, which improves forepaw function, following overuse injury

Blocking CCN2 Reduces Established Bone Loss Induced by Prolonged Intense Loading by Increasing Osteoblast Activity in Rats

ABSTRACT We have an operant model of reaching and grasping in which detrimental bone remodeling is observed rather than beneficial adaptation when rats perform a high‐repetition, high‐force (HRHF) task long term. Here, adult female Sprague–Dawley rats performed an intense HRHF task for 18 weeks, which we have shown induces radial trabecular bone osteopenia. One cohort was euthanized at this point (to assay the bone changes post task; HRHF‐Untreated). Two other cohorts were placed on 6 weeks of rest while being simultaneously treated with either an anti‐CCN2 (FG‐3019, 40 mg/kg body weight, ip; twice per week; HRHF‐Rest/anti‐CCN2), or a control IgG (HRHF‐Rest/IgG), with the purpose of determining which might improve the trabecular bone decline. Results were compared with food‐restricted control rats (FRC). MicroCT analysis of distal metaphysis of radii showed decreased trabecular bone volume fraction (BV/TV) and thickness in HRHF‐Untreated rats compared with FRCs; responses improved with HRHF‐Rest/anti‐CCN2. Rest/IgG also improved trabecular thickness but not BV/TV. Histomorphometry showed that rest with either treatment improved osteoid volume and task‐induced increases in osteoclasts. Only the HRHF‐Rest/anti‐CCN2 treatment improved osteoblast numbers, osteoid width, mineralization, and bone formation rate compared with HRHF‐Untreated rats (as well as the latter three attributes compared with HRHF‐Rest/IgG rats). Serum ELISA results were in support, showing increased osteocalcin and decreased CTX‐1 in HRHF‐Rest/anti‐CCN2 rats compared with both HRHF‐Untreated and HRHF‐Rest/IgG rats. These results are highly encouraging for use of anti‐CCN2 for therapeutic treatment of bone loss, such as that induced by chronic overuse. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research

Design of experiment.

<p>All rats were handled daily for week 1. Then, all but normal control rats (NC) were food restricted to within 5% of the weights of the NC rats for the remainder of the experiments. Food restricted control rats (FRC) did not undergo training and did not perform the high repetition low force (HRLF) task. A cohort of rats were trained only for 4 weeks, and then euthanized (TR0). Another cohort was trained only for 4 weeks, and then rested for 24 weeks (TR24). Two groups of rats performed the HRLF task for either 18 weeks or 24 weeks (18-week HRLF and 24-week HRLF rats). After euthanasia, serum (S) was collected from all rats and assayed in nearly all rats. Half of the tissues were analyzed using biochemical methods (B) or histological methods (H).</p

Weight and grip strength changes across weeks of task performance.

<p>(A) All rats gained weight across the 29 weeks. There were no significant differences in body weights between trained only (TR0 and TR24) or high repetition low force (HRLF) rats, compared to normal controls (NC) rats. (B) Maximum reflexive grip strength is shown for control rats (normal and food-restricted controls were combined as there were no significant differences between these groups), TR24 (TR+Rest; trained rats that rested after training for 24 weeks), and HRLF rats. The preferred reach limbs and the contralateral support limbs were analyzed separately in HRLF rats. The week 0 time point of each group is after the training period and before the HRLF task performance or the rest period. Symbols: *:p<0.05 and ***:p<0.001, compared to age-matched control rats; ^aa: p<0.01 and ^aaa:p<0.001, compared to age-matched TR24 rats; ^b: p<0.05, compared to the support limb of HRLF rats.</p

Transforming growth factor beta 1 (TGFB1) in serum and flexor digitorum tissues of the reach limbs.

<p>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.</p

Pro- and anti-inflammatory cytokines in serum.

<p>Groups are as defined in figure legend 2. Data is shown for (A) TNF-alpha, (B) IL-6, (C) IL-10, (D), IL-1alpha, (E) IL-12 (only TR24 and 18 wk HRLF rat serum were tested for IL-12), and (F) MIP2. Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats. ^a: p<0.05, compared to age-matched trained rats that rested for 24 weeks after the initial training period.</p

Pro- and anti-inflammatory cytokines in forelimb flexor digitorum tissues.

<p>Data shown for cytokines assayed in muscles and tendons after collection from control rats (indicated as C; normal and food restricted control data was combined as there was no significant difference), trained only rats euthanized immediately after training ( indicated as TR0 on the x-axes), TR+Rest (TR24; trained rats that rested for 24 weeks; blue dashed lines), and high repetition low force (HRLF) rats that worked for 18 or 24 weeks of task performance before tissue collection. The preferred reach limbs (red line) and the contralateral support limbs (green lines) were examined separately in HRLF rats. (A & B) Muscle and Tendon IL-1beta. (C & D) Muscle and Tendon TNF-alpha. (E & F) Muscle and Tendon IL-6. (G & H) Muscle and Tendon IL-10. Each analyte was assayed in duplicate using single-plex ELISA kits. Symbols: *:p<0.05, **:p<0.01 and ***:p<0.001, compared to age-matched control rats; ^a: p<0.05 and ^aa:p<0.01, compared to age-matched TR+Rest rats; ^b: p<0.05, compared to the support limb of HRLF rats.</p