Wnt3a induces myofibroblast differentiation by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent manner

Growing evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events. However, the role of the Wnt pathway in such settings remains unclear. In the current study, we treated mouse fibroblasts with 250 ng/mL of recombinant Wnt3a for 72 hours and examined its affect on cell morphology and function. Wnt3a induced a spindle-like morphology in fibroblasts characterized by the increased formation of stress fibres. Wnt3a decreased the proliferation of fibroblasts, but significantly increased cell migration as well as fibroblast-mediated contraction of a collagen lattice. Wnt3a significantly increased the expression of TGF-β and its associated signaling through SMAD2. Consistent with this, we observed significantly increased smooth muscle α-actin expression and incorporation of this contractile protein into stress fibres following Wnt3a treatment. Knockdown of β-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through β-catenin. Neutralization of TGF-β with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-β signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent mechanism. As myofibroblasts are critical regulators of wound healing responses, these findings may have important implications for our understanding of normal and aberrant injury and repair events.     

Targeting pro-inflammatory cytokines following joint injury: acute intra-articular inhibition of interleukin-1 following knee injury prevents post-traumatic arthritis

Introduction

Post-traumatic arthritis (PTA) is a progressive, degenerative response to joint injury, such as articular fracture. The pro-inflammatory cytokines, interleukin 1(IL-1) and tumor necrosis factor alpha (TNF-α), are acutely elevated following joint injury and remain elevated for prolonged periods post-injury. To investigate the role of local and systemic inflammation in the development of post-traumatic arthritis, we targeted both the initial acute local inflammatory response and a prolonged 4 week systemic inflammatory response by inhibiting IL-1 or TNF-α following articular fracture in the mouse knee.

Methods

Anti-cytokine agents, IL-1 receptor antagonist (IL-1Ra) or soluble TNF receptor II (sTNFRII), were administered either locally via an acute intra-articular injection or systemically for a prolonged 4 week period following articular fracture of the knee in C57BL/6 mice. The severity of arthritis was then assessed at 8 weeks post-injury in joint tissues via histology and micro computed tomography, and systemic and local biomarkers were assessed in serum and synovial fluid.

Results

Intra-articular inhibition of IL-1 significantly reduced cartilage degeneration, synovial inflammation, and did not alter bone morphology following articular fracture. However, systemic inhibition of IL-1, and local or systemic inhibition of TNF provided no benefit or conversely led to increased arthritic changes in the joint tissues.

Conclusion

These results show that intra-articular IL-1, rather than TNF-α, plays a critical role in the acute inflammatory phase of joint injury and can be inhibited locally to reduce post-traumatic arthritis following a closed articular fracture. Targeted local inhibition of IL-1 following joint injury may represent a novel treatment option for PTA.     

Study on Correlation of Maedi-Visna Virus (MVV) with Ovine Subclinical Mastitis in Iran

Maedi-Visna is an important slow viral disease of sheep leading to progressive pneumonia, encephalitis and mastitis. Udder is one of the organs affected by MVV. Despite the fact that in Iran Maedi-Visna is known since 2000, to the authors’ knowledge correlation of subclinical mastitis and infection with MVV has not been assayed. In this study 50 milk samples from 10 flocks in East Azerbaijan Province of Iran were tested. None of the animals exhibited any clinical signs of the disease. Forty samples were collected from CMT positive ewes and ten were taken from CMT negative ewes. Milk samples were analyzed using PCR targeting gag sequence. Presence of provirus DNA was detected in one sample from CMT negative and seven samples from CMT positive ewes. These data demonstrate that 16.5 % of sheep with subclinical mastitis were infected to MVV. Thus this virus can be considered one of the main pathogenic agents of mastitis and can be dramatically transmitted to lambs by milk.     

Diffusional anisotropy in collagenous tissues: fluorescence imaging of continuous point photobleaching

Molecular transport in avascular collagenous tissues such as articular cartilage occurs primarily via diffusion. The presence of ordered structures in the extracellular matrix may influence the local transport of macromolecules, leading to anisotropic diffusion depending on the relative size of the molecule and that of extracellular matrix structures. Here we present what we believe is a novel photobleaching technique for measuring the anisotropic diffusivity of macromolecules in collagenous tissues. We hypothesized that macromolecular diffusion is anisotropic in collagenous tissues, depending on molecular size and the local organization of the collagen structure. A theoretical model and experimental protocol for fluorescence imaging of continuous point photobleaching was developed to measure diffusional anisotropy. Significant anisotropy was observed in highly ordered collagenous tissues such as ligament, with diffusivity ratios >2 along the fiber direction compared to the perpendicular direction. In less-ordered tissues such as articular cartilage, diffusional anisotropy was dependent on site in the tissue and size of the diffusing molecule. Anisotropic diffusion was also dependent on the size of the diffusing molecule, with greatest anisotropy observed for larger molecules. These findings suggest that diffusional transport of macromolecules is anisotropic in collagenous tissues, with higher rates of diffusion along primary orientation of collagen fibers.     

Dose-dependent therapeutic effects of topical 1,25 OH-vitamin D3 on corneal wound healing

Molecular Biology Reports - Conflicting results have been reported regarding the effects of 1,25 OH-vitamin D3 on corneal wound healing. Therefore, we undertook this study to determine whether the...     

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.     

Compressive properties of mouse articular cartilage determined in a novel micro-indentation test method and biphasic finite element model

The mechanical properties of articular cartilage serve as important measures of tissue function or degeneration, and are known to change significantly with osteoarthritis. Interest in small animal and mouse models of osteoarthritis has increased as studies reveal the importance of genetic background in determining predisposition to osteoarthritis. While indentation testing provides a method of determining cartilage mechanical properties in situ, it has been of limited value in studying mouse joints due to the relatively small size of the joint and thickness of the cartilage layer. In this study, we developed a micro-indentation testing system to determine the compressive and biphasic mechanical properties of cartilage in the small joints of the mouse. A nonlinear optimization program employing a genetic algorithm for parameter estimation, combined with a biphasic finite element model of the micro-indentation test, was developed to obtain the biphasic, compressive material properties of articular cartilage. The creep response and material properties of lateral tibial plateau cartilage were obtained for wild-type mouse knee joints, by the micro-indentation testing and optimization algorithm. The newly developed genetic algorithm was found to be efficient and accurate when used with the finite element simulations for nonlinear optimization to the experimental creep data. The biphasic mechanical properties of mouse cartilage in compression (average values: Young's modulus, 2.0 MPa; Poisson's ratio, 0.20; and hydraulic permeability, 1.1 x 10(-16) m4/N-s) were found to be of similar orders of magnitude as previous findings for other animal cartilages, including human, bovine, rat, and rabbit and demonstrate the utility of the new test methods. This study provides the first available data for biphasic compressive properties in mouse cartilage and suggests a promising method for detecting altered cartilage mechanics in small animal models of osteoarthritis.     

A Mechano-chemical Model for the Passive Swelling Response of an Isolated Chondron under Osmotic Loading

The chondron is a distinct structure in articular cartilage that consists of the chondrocyte and its pericellular matrix (PCM), a narrow tissue region surrounding the cell that is distinguished by type VI collagen and a high glycosaminoglycan concentration relative to the extracellular matrix. We present a theoretical mechano-chemical model for the passive volumetric response of an isolated chondron under osmotic loading in a simple salt solution at equilibrium. The chondrocyte is modeled as an ideal osmometer and the PCM model is formulated using triphasic mixture theory. A mechano-chemical chondron model is obtained assuming that the chondron boundary is permeable to both water and ions, while the chondrocyte membrane is selectively permeable to only water. For the case of a neo-Hookean PCM constitutive law, the model is used to conduct a parametric analysis of cell and chondron deformation under hyper- and hypo-osmotic loading. In combination with osmotic loading experiments on isolated chondrons, model predictions will aid in determination of pericellular fixed charge density and its relative contribution to PCM mechanical properties.