Collagen gel contraction as a measure of fibroblast function in an animal model of subsynovial connective tissue fibrosis

Carpal tunnel syndrome (CTS) is a peripheral neuropathy characterized by non‐inflammatory fibrosis of the subsynovial connective tissues (SSCT). A rabbit model of CTS was developed to test the hypothesis that SSCT fibrosis causes the neuropathy. We used a cell‐seeded collagen‐gel contraction model to characterize the fibrosis in this model in terms of cellular mechanics, specifically to compare the ability of SSCT cells from the rabbit model and normal rabbits to contract the gel, and to assess the effect of transforming growth factor‐β1,which is upregulated in CTS, on these cells. SSCT fibrosis was induced in six retired breeder female rabbits which were sacrificed at 6 weeks (N = 3) and 12 weeks (n = 3). An additional two rabbits served as controls. SSCT was harvested according to a standard protocol. Gels seeded with SSCT cells from rabbits sacrificed at 6 weeks had significantly higher tensile strength (p < 0.001) and Young's modulus (p < 0.001) than gels seeded with cells from rabbits sacrificed at 12 weeks or control animals. TGF‐β1 significantly increased the decay time constant (p < 0.001), tensile strength (p < 0.001), and Young's modulus (p < 0.001) regardless of the cell source. This model may be useful in screening therapeutic agents that may block SSCT fibrosis, identifying possible candidates for CTS treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:668–674, 2015.     

Dysregulated TGF‐β signaling alters bone microstructure in a mouse model of Loeys‐Dietz syndrome

Loeys‐Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor‐β (TGF‐β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF‐β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein‐labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three‐point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF‐β1–induced Smad2 and Erk1/2 phosphorylation and upregulated TGF‐β1 ligand mRNA expression, compatible with G357W as a loss‐of‐function mutation in the TGF‐β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross‐activation of TGF‐β–related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF‐β receptor signaling in bone. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1447–1454, 2015.     

Biomarkers for the Prediction and Diagnosis of Fibrostenosing Crohn’s Disease: A Systematic Review

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NO-releasing xanthine KMUP-1 bonded by simvastatin attenuates bleomycin-induced lung inflammation and delayed fibrosis

Background and purposePulmonary fibrosis (PF) is a progressing lung injury initiated by pulmonary inflammation (PI). Bleomycin (BLM) is the most common pathogenesis of PF through early PI and extensive extracellular matrix deposition. This study is aimed to determine whether NO-releasing KMUP-1 inhibits PI and PF, and if so, the benefits of KMUP-1S resulted from simvastatin (SIM)-bonding to KMUP-1.Experiment approachC57BL/6 male mice were intra-tracheally administered BLM (4 U/kg) at day 0. KMUP-1 (1–5 mg/kg), KMUP-1S (2.5 mg/kg), SIM (5 mg/kg), Plus (KMUP-1 2.5 mg/kg + SIM 2.5 mg/kg), and clarithromycin (CAM, 10 mg/kg) were orally and daily administered for 7 and 28 days, respectively, to mice, sacrificed at day-7 and day-28 to isolate the lung tissues, for examining the inflammatory and fibrotic signaling and measuring the cell population and MMP-2/MMP-9 activity in broncholaveolar lavage fluid (BAL).Key resultsKMUP-1 and KUP-1S significantly decreased neutrophil counts in BAL fluid. Fibroblastic foci were histologically assessed by H&E and Masson's trichrome stain and treated with KMUP-1 and references. Lung tissues were determined the contents of collagen and the expressions of TGF-β, α-SMA, HMGB1, CTGF, eNOS, p-eNOS, RhoA, Smad3, p-Smad3, MMP-2 and MMP-9 by Western blotting analyses, respectively. These changes areregulated by NO/cGMP and inhibited by various treatments. KMUP-1 and KMUP-1S predominantly prevented HMGB1/MMP-2 expression at day-7 and reduced TGF-β/phosphorylated Smad3 and CTGF at day-28.Conclusions and implicationsKMUP-1 and KMUP-S restore eNOS, inhibit iNOS/ROCKII/MMP-2/MMP-9, attenuate histologic collagen disposition and reduce BALF inflammatory cells, potentially useful for the treatment of BLM-lung PF.     

TGF‐β & BMP Receptors Endoglin and ALK1: Overview of their Functional Role and Status as Antiangiogenic Targets

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF‐β superfamily, TGF‐β1, TGF‐β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell–cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1‐mediated cells signaling.