Potentiation of Collagen Deposition by the Combination of Substance P with Transforming Growth Factor Beta in Rat Skin Fibroblasts.

A role for substance P has been proposed in musculoskeletal fibrosis, with effects mediated through transforming growth factor beta (TGFß). We examined the in vitro effects of substance P on proliferation, collagen secretion, and collagen deposition in rat primary dermal fibroblasts cultured in medium containing 10% fetal bovine serum, with or without TGFß. In six-day cultures, substance P increased cell proliferation at concentrations from 0.0002 to 100 nM. TGFß increased proliferation at concentrations from 0.0002 to 2 pg/mL, although higher concentrations inhibited proliferation. Substance P treatment alone at concentrations of 100, 0.2, and 0.00002 nM did not increase collagen deposition per cell, yet when combined with TGFß (5 ng/mL), increased collagen deposition compared to TGFß treatment alone. Substance P treatment (100 nM) also increased smooth muscle actin (SMA) expression at 72 h of culture at a level similar to 5 ng/mL of TGFß; only TGFß increased SMA at 48 h of culture. Thus, substance P may play a role in potentiating matrix deposition in vivo when combined with TGFß, although this potentiation may be dependent on the concentration of each factor. Treatments targeting substance P may be a viable strategy for treating fibrosis where both substance P and TGFß play roles.

Pharmacotherapies in Dupuytren Disease: Current and Novel Strategies.

Dupuytren disease is a benign, progressive fibroproliferative disorder of the hands. To date, only one pharmacotherapy (clostridial collagenase) has been approved for use in Dupuytren disease. There is a great need for additional nonsurgical methods that can be used to either avoid the risks of invasive treatments or help minimize recurrence rates following treatment. A number of nonsurgical modalities have been discussed in the past and continue to appear in discussions among hand surgeons, despite highly variable and often poor or no long-term clinical data. This article reviews many of the pharmacotherapies discussed in the treatment of Dupuytren disease and novel therapies used in inflammation and fibrosis that offer potential treatment options.

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-ß-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.

Manual Therapy Facilitates Homeostatic Adaptation to Bone Microstructural Declines Induced by a Rat Model of Repetitive Forceful Task.

The effectiveness of manual therapy in reducing the catabolic effects of performing repetitive intensive force tasks on bones has not been reported. We examined if manual therapy could reduce radial bone microstructural declines in adult female Sprague-Dawley rats performing a 12-week high-repetition and high-force task, with or without simultaneous manual therapy to forelimbs. Additional rats were provided 6 weeks of rest after task cessation, with or without manual therapy. The control rats were untreated or received manual therapy for 12 weeks. The untreated TASK rats showed increased catabolic indices in the radius (decreased trabecular bone volume and numbers, increased osteoclasts in these trabeculae, and mid-diaphyseal cortical bone thinning) and increased serum CTX-1, TNF-α, and muscle macrophages. In contrast, the TASK rats receiving manual therapy showed increased radial bone anabolism (increased trabecular bone volume and osteoblast numbers, decreased osteoclast numbers, and increased mid-diaphyseal total area and periosteal perimeter) and increased serum TNF-α and muscle macrophages. Rest, with or without manual therapy, improved the trabecular thickness and mid-diaphyseal cortical bone attributes but not the mineral density. Thus, preventive manual therapy reduced the net radial bone catabolism by increasing osteogenesis, while rest, with or without manual therapy, was less effective.

Blocking CTGF/CCN2 reduces established skeletal muscle fibrosis in a rat model of overuse injury.

Tissue fibrosis is a hallmark of overuse musculoskeletal injuries and contributes to functional declines. We tested whether inhibition of CCN2 (cellular communication network factor 2, previously known as connective tissue growth factor, CTGF) using a specific antibody (termed FG-3019 or pamrevlumab) reduces established overuse-induced muscle fibrosis in a clinically relevant rodent model of upper extremity overuse injury. Young adult rats performed a high repetition high force (HRHF) reaching and lever-pulling task for 18 weeks, after first being shaped for 6 weeks to learn this operant task. Rats were then euthanized (HRHF-Untreated), or rested and treated for 6 weeks with FG-3019 (HRHF-Rest/FG-3019) or a human IgG as a vehicle control (HRHF-Rest/IgG). HRHF-Untreated and HRHF-Rest/IgG rats had higher muscle levels of several fibrosis-related proteins (TGFß1, CCN2, collagen types I and III, and FGF2), and higher muscle numbers of alpha SMA and pERK immunopositive cells, compared to control rats. Each of these fibrogenic changes was restored to control levels by the blocking of CCN2 signaling in HRHF-Rest/FG-3019 rats, as were HRHF task-induced increases in serum CCN2 and pro-collagen I intact N-terminal protein. Levels of cleaved CCN3, an antifibrotic protein, were lowered in HRHF-Untreated and HRHF-Rest/IgG rats, compared to control rats, yet elevated back to control levels in HRHF-Rest/FG-3019 rats. Significant grip strength declines observed in HRHF-Untreated and HRHF-Rest/IgG rats, were restored to control levels in HRHF-Rest/FG-3019 rats. These results are highly encouraging for use of FG-3019 for therapeutic treatment of persistent skeletal muscle fibrosis, such as those induced with chronic overuse.

Force dependent effects of chronic overuse on fibrosis-related genes and proteins in skeletal muscles.

AIM: To examine the chronic effect of force on mRNA and protein expression levels of fibrosis-related genes in flexor digitorum muscles in a rat model of repetitive overuse injury that induces muscle fibrosis at high force levels. MATERIALS AND METHODS: Two groups of rats were trained to perform a voluntary repetitive lever-pulling task at either a high (HFHR) or a low force (LFHR) for 18 weeks, while a control group (FRC) performed no task. RNA and protein were prepared from forelimb flexor digitorum muscles. Fibrosis-related gene RNA transcripts were evaluated using quantitative PCR (qPCR) and analyzed using the geometric mean of three housekeeping genes or the mean of each individually as reference. Protein levels were quantified using ELISA, western blot, or immunohistofluorescence. RESULTS: Of eight fibrosis-related mRNAs examined, only FGF2 demonstrated a consistent significant increase in the HFHR group, compared to the FRC group. However, protein amounts of collagen type 1, collagen type 3, and TGFß1 were significantly higher in the HFHR, compared to the FRC and LFHR groups, while CCN2 and FGF2 were higher in both HFHR and LFHR, compared to the FRC group. CONCLUSIONS: Our results suggest that there is steady-state transcription of fibrogenic genes in muscles with established fibrosis, implying that post-transcriptional processes are responsible for the increased protein levels of fibrotic factors during muscle overuse conditions. We hypothesize that targeting such pathways represents a valid approach to treat overuse injury. Alternatively, FGF2 gene expression may represent a valid target for therapy.

Blocking CCN2 preferentially inhibits osteoclastogenesis induced by repetitive high force bone loading.

Purpose/Aim: We recently found that blocking CCN2 signaling using a monoclonal antibody (FG-3019) may be a novel therapeutic strategy for reducing overuse-induced tissue fibrosis. Since CCN2 plays roles in osteoclastogenesis, and persistent performance of a high repetition high force (HRHF) lever pulling task results in a loss in trabecular bone volume in the radius, we examined here whether blocking CCN2 signaling would reduce the early catabolic effects of performing a HRHF task for 3 weeks. Materials and Methods: Young adult, female, Sprague-Dawley rats were operantly shaped to learn to pull at high force levels, before performing the HRHF task for 3 weeks. HRHF task rats were then left untreated (HRHF Untreated), treated in task weeks 2 and 3 with a monoclonal antibody that antagonizes CCN2 (HRHF+FG-3019), or treated with an IgG (HRHF+IgG), while continuing to perform the task. Non-task control rats were left untreated. Results: In metaphyseal trabeculae of the distal radius, HRHF Untreated and HRHF-IgG rats showed increased osteoblast numbers and other indices of bone formation, compared to controls, yet decreased trabecular bone volume, increased osteoclast numbers, and increased serum CTX-1 (a serum biomarker of bone resorption). HRHF+FG-3019 rats also showed increased osteoblast numbers and bone formation, but in contrast to HRHF Untreated and HRHF-IgG rats, showed higher trabecular bone volume, and reduced osteoclast numbers and serum CTX-1 levels (and statistically similar to Control levels). Conclusions: HRHF loading increased bone formation in each task group, yet blocking CCN2 dampened trabecular bone catabolism by reducing osteoclast numbers and activity.

Occupational Activities: Factors That Tip the Balance From Bone Accrual to Bone Loss.

It is commonly assumed that beneficial adaptations in bone occur with vigorous exercise, yet any adaptive re/modeling in bone undergoing persistent overloading can be counteracted by superimposed inflammatory, compressive, and tensile loading-induced damage responses above thresholds of tissue fatigue failure and repair. This leads to a tenuous balance between achieving bone accrual and loss.

Comparing effects of rest with or without a NK1RA on fibrosis and sensorimotor declines induced by a voluntary moderate demand task.

OBJECTIVES: Fibrosis is one contributing factor in motor dysfunction and discomfort in patients with overuse musculoskeletal disorders. We pharmacologically targeted the primary receptor for Substance P, neurokinin-1, using a specific antagonist (NK1RA) in a rat model of overuse with the goal of improving tissue fibrosis and discomfort. METHODS: Female rats performed a low repetition, high force (LRHF) grasping task for 12 weeks, or performed the task for 12 weeks before being placed on a four week rest break, with or without simultaneous NK1RA treatment. Results were compared to control rats (untreated, or treated 4 weeks with NK1RA or vehicle). RESULTS: Rest improved LRHF-induced declines in grip strength, although rest plus NK1RA treatment (Rest/NK1RA) rescued it. Both treatments improved LRHF-induced increases in muscle TGFß1 and collagen type 1 levels, forepaw mechanical hypersensitivity (Rest/NK1RA more effectively), macrophage influx into median nerves, and enhanced collagen deposition in forepaw dermis. Only Rest/NK1RA reduced muscle hypercellularity. However, LRHF+4wk Rest /NK1RA rats showed hyposensitivity to noxious hot temperatures. CONCLUSIONS: While the NK1RA induced hot temperature hyposensitivity should be taken into consideration if this or related drug were used long-term, the NK1RA more effectively reduced muscle hypercellularity and improved grip strength and forepaw mechanical hypersensitivity.

Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes.

Transforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFß-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-ß1 and TGF-ß receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo.

Growth and repair factors, osteoactivin, matrix metalloproteinase and heat shock protein 72, increase with resolution of inflammation in musculotendinous tissues in a rat model of repetitive grasping.

BACKGROUND: Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA's expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats. METHODS: Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1, -2, -3, and -13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines. RESULTS: Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1, -2 and -3 progressively increased in muscles whereas MMP-1 and -3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6. CONCLUSION: The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.

The extensor carpi ulnaris pseudolesion: evaluation with microCT, histology, and MRI.

OBJECTIVE: To determine if magic angle plays a role in apparent central increased signal intensity of the distal extensor carpi ulnaris tendon (ECU) on MRI, to see if histologic findings of tendon degeneration are associated with increased T1 or T2 tendon signal on MR imaging, and to determine the prevalence of the ECU "pseudolesion". MATERIALS AND METHODS: A standard 3 Tesla protocol was utilized to scan ten cadaveric wrists. A 40 mm length of 10 ECU and four extensor carpi radialis brevis (ECRB) tendons were immersion fixed before microCT scanning. Staining with Alcian blue, Masson's trichrome and Safranin O was performed before light microscopy. Fifty clinical wrist MRIs were also reviewed for the presence of increased T1 and/or T2 signal. RESULTS: Central increased T1 and/or T2 signal was observed in 9 of 10 cadaveric ECU tendons, but not in ECRB tendons. MicroCT and histology showed inter-tendinous matrix between the two distal heads of the ECU. Increased mucoid degeneration correlated with increased MRI signal intensity. The tendon fibers were at a maximum of 8.39° to the longitudinal axis on microCT. Clinical MRIs showed increased T1 signal in 6%, increased T2 signal in 8%, increased T1 and T2 signal in 80%, and 6% showing no increased signal. CONCLUSION: Central increased T1 and/or T2 signal in the ECU tendon indicates the presence of normal inter-tendinous ground substance, with increased proteoglycan content (mucoid degeneration) responsible for increased signal intensity. None of the fibers were shown on microCT to approach the magic angle.

Connective tissue growth factor (CTGF/CCN2) negatively regulates BMP-2 induced osteoblast differentiation and signaling.

Connective tissue growth factor (CTGF/CCN2) and bone morphogenetic protein (BMP)-2 are both produced and secreted by osteoblasts. Both proteins have been shown to have independent effects in regulating osteoblast proliferation, maturation and mineralization. However, how these two proteins interact during osteoblast differentiation remains unknown. In this study, we utilized two cell culture model systems, osteoblasts derived from CTGF knockout (KO) mice and osteoblasts infected with an adenovirus which over-expresses CTGF (Ad-CTGF), to investigate the effects of CTGF and BMP-2 on osteoblast development and function in vitro. Contrary to a previously published report, osteoblast maturation and mineralization were similar in osteogenic cultures derived from KO and WT calvaria in the absence of BMP-2 stimulation. Interestingly, in KO and WT osteoblast cultures stimulated with BMP-2, the KO osteoblasts exhibited enhanced osteoblast differentiation. This increase in osteoblast differentiation was accompanied by increased protein levels of phosphorylated Smad 1/5/8 and mRNA expression levels of bone morphogenetic protein receptor Ib. We also examined osteoblast differentiation in cultures that were infected with an adenoviral-CTGF vector (Ad-CTGF) and in controls. Continuous over-expression of CTGF resulted in decreased osteoblast maturation and mineralization in both unstimulated and BMP-2 stimulated cultures. Impaired osteoblast differentiation in cultures over-expressing CTGF was accompanied by decreased protein levels of phosphorylated Smad 1/5/8. Collectively, the data from these studies demonstrate that CTGF acts to negatively regulate BMP-2 induced signaling and osteoblast differentiation, and warrant additional studies to determine the precise mechanism(s) responsible for this effect. J. Cell. Physiol. 229: 672-681, 2014. © 2013 Wiley Periodicals, Inc.

The interaction of force and repetition on musculoskeletal and neural tissue responses and sensorimotor behavior in a rat model of work-related musculoskeletal disorders.

BACKGROUND: We examined the relationship of musculoskeletal risk factors underlying force and repetition on tissue responses in an operant rat model of repetitive reaching and pulling, and if force x repetition interactions were present, indicative of a fatigue failure process. We examined exposure-dependent changes in biochemical, morphological and sensorimotor responses occurring with repeated performance of a handle-pulling task for 12 weeks at one of four repetition and force levels: 1) low repetition with low force, 2) high repetition with low force, 3) low repetition with high force, and 4) high repetition with high force (HRHF). METHODS: Rats underwent initial training for 4-6 weeks, and then performed one of the tasks for 12 weeks, 2 hours/day, 3 days/week. Reflexive grip strength and sensitivity to touch were assayed as functional outcomes. Flexor digitorum muscles and tendons, forelimb bones, and serum were assayed using ELISA for indicators of inflammation, tissue stress and repair, and bone turnover. Histomorphometry was used to assay macrophage infiltration of tissues, spinal cord substance P changes, and tissue adaptative or degradative changes. MicroCT was used to assay bones for changes in bone quality. RESULTS: Several force x repetition interactions were observed for: muscle IL-1alpha and bone IL-1beta; serum TNFalpha, IL-1alpha, and IL-1beta; muscle HSP72, a tissue stress and repair protein; histomorphological evidence of tendon and cartilage degradation; serum biomarkers of bone degradation (CTXI) and bone formation (osteocalcin); and morphological evidence of bone adaptation versus resorption. In most cases, performance of the HRHF task induced the greatest tissue degenerative changes, while performance of moderate level tasks induced bone adaptation and a suggestion of muscle adaptation. Both high force tasks induced median nerve macrophage infiltration, spinal cord sensitization (increased substance P), grip strength declines and forepaw mechanical allodynia by task week 12. CONCLUSIONS: Although not consistent in all tissues, we found several significant interactions between the critical musculoskeletal risk factors of force and repetition, consistent with a fatigue failure process in musculoskeletal tissues. Prolonged performance of HRHF tasks exhibited significantly increased risk for musculoskeletal disorders, while performance of moderate level tasks exhibited adaptation to task demands.

The skeletal site-specific role of connective tissue growth factor in prenatal osteogenesis.

BACKGROUND: Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted. RESULTS: We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-ß-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype. CONCLUSIONS: We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation.

Let's Focus on the Fibrosis in Dupuytren Disease: Cell Communication Network Factor 2 as a Novel Target.

Dupuytren disease is a progressive, benign fibroproliferative disorder of the hands that can lead to debilitating hand contractures. Once symptomatic, treatment involves either surgical intervention, specifically fasciectomy or percutaneous needle aponeurotomy, or enzymatic degradation with clostridial collagenase. Currently, collagenase is the only pharmacotherapy that has been approved for the treatment of Dupuytren contracture. There is a need for a pharmacotherapeutic that can be administered to limit disease progression and prevent recurrence after treatment. Targeting the underlying fibrotic pathophysiology is critical. We propose a novel target to be considered in Dupuytren disease-cell communication network factor 2/connective tissue growth factor-an established mediator of musculoskeletal tissue fibrosis.

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

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.

Homeodomain transcription factors regulate BMP-2-induced osteoactivin transcription in osteoblasts.

Osteoactivin (OA) is required for the differentiation of osteoblast cells. OA expression is stimulated by bone morphogenetic protein-2 (BMP-2). BMP-2 recruits homeodomain transcription factors Dlx3, Dlx5, and Msx2 to selectively activate or repress transcription of osteogenic genes and hence tightly regulate their transcription during osteoblast differentiation. Considering the key roles of Dlx3, Dlx5, and Msx2 in osteoblast differentiation, here we hypothesize that homeodomain proteins regulate BMP-2-induced OA transcription during osteoblast differentiation. Four classical homeodomain binding sites were identified in the proximal 0.96 kb region of rat OA promoter. Deletions and mutagenesis studies of the OA promoter region indicated that all four homeodomain binding sites are crucial for BMP-2-induced OA promoter activity. Simultaneous disruption of homeodomain binding sites at -852 and -843 of the transcription start site of OA gene significantly decreased the BMP-2-induced OA transcription and inhibited binding of Dlx3, Dlx5, and Msx2 proteins to the OA promoter. Dlx3 and Dlx5 proteins were found to activate the OA transcription, whereas, Msx2 suppressed BMP-2-induced OA transcription. Using chromatin immunoprecipitation assays, we demonstrated that the OA promoter is predominantly occupied by Dlx3 and Dlx5 during the proliferation and matrix maturation stages of osteoblast differentiation, respectively. During the matrix mineralization stage, BMP-2 robustly enhanced the recruitment of Dlx5 and to a lesser extent of Dlx3 and Msx2 to the OA promoter region. Collectively, our results show that the BMP-2-induced OA transcription is differentially regulated by Dlx3, Dlx5, and Msx2 during osteoblast differentiation.

The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis.

Connective tissue growth factor (CTGF) is a 38 kDa, cysteine rich, extracellular matrix protein composed of 4 domains or modules. CTGF has been shown to regulate a diverse array of cellular functions and has been implicated in more complex biological processes such as angiogenesis, chondrogenesis, and osteogenesis. A role for CTGF in the development and maintenance of skeletal tissues first came to light in studies demonstrating its expression in cartilage and bone cells, which was dramatically increased during skeletal repair or regeneration. The physiological significance of CTGF in skeletogenesis was confirmed in CTGF-null mice, which exhibited multiple skeletal dysmorphisms as a result of impaired growth plate chondrogenesis, angiogenesis, and bone formation/mineralization. Given the emerging importance of CTGF in osteogenesis and chondrogenesis, this review will focus on its expression in skeletal tissues, its effects on osteoblast and chondrocyte differentiation and function, and the skeletal implications of ablation or over-expression of CTGF in knockout or transgenic mouse models, respectively. In addition, this review will examine the role of integrin-mediated signaling and the regulation of CTGF expression as it relates to skeletogenesis. We will emphasize CTGF studies in bone or bone cells, and will identify opportunities for future investigations concerning CTGF and chondrogenesis/osteogenesis.