MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen

Tissue Microenvironments Define and Get Reinforced by Macrophage Phenotypes in Homeostasis or during Inflammation, Repair and Fibrosis

Current macrophage phenotype classifications are based on distinct in vitro culture conditions that do not adequately mirror complex tissue environments. In vivo monocyte progenitors populate all tissues for immune surveillance which supports the maintenance of homeostasis as well as regaining homeostasis after injury. Here we propose to classify macrophage phenotypes according to prototypical tissue environments, e.g. as they occur during homeostasis as well as during the different phases of (dermal) wound healing. In tissue necrosis and/or infection, damage- and/or pathogen-associated molecular patterns induce proinflammatory macrophages by Toll-like receptors or inflammasomes. Such classically activated macrophages contribute to further tissue inflammation and damage. Apoptotic cells and antiinflammatory cytokines dominate in postinflammatory tissues which induce macrophages to produce more antiinflammatory mediators. Similarly, tumor-associated macrophages also confer immunosuppression in tumor stroma. Insufficient parenchymal healing despite abundant growth factors pushes macrophages to gain a profibrotic phenotype and promote fibrocyte recruitment which both enforce tissue scarring. Ischemic scars are largely devoid of cytokines and growth factors so that fibrolytic macrophages that predominantly secrete proteases digest the excess extracellular matrix. Together, macrophages stabilize their surrounding tissue microenvironments by adapting different phenotypes as feed-forward mechanisms to maintain tissue homeostasis or regain it following injury. Furthermore, macrophage heterogeneity in healthy or injured tissues mirrors spatial and temporal differences in microenvironments during the various stages of tissue injury and repair. Copyright (C) 2012 S. Karger AG, Base

Biologic Treatments for Sports Injuries II Think Tank-Current Concepts, Future Research, and Barriers to Advancement, Part 2:Rotator Cuff

Rotator cuff tears are common and result in considerable morbidity. Tears within the tendon substance or at its insertion into the humeral head represent a considerable clinical challenge because of the hostile local environment that precludes healing. Tears often progress without intervention, and current surgical treatments are inadequate. Although surgical implants, instrumentation, and techniques have improved, healing rates have not improved, and a high failure rate remains for large and massive rotator cuff tears. The use of biologic adjuvants that contribute to a regenerative microenvironment have great potential for improving healing rates and function after surgery. This article presents a review of current and emerging biologic approaches to augment rotator cuff tendon and muscle regeneration focusing on the scientific rationale, preclinical, and clinical evidence for efficacy, areas for future research, and current barriers to advancement and implementation

The Effect of Sustained Compression on Oxygen Metabolic Transport in the Intervertebral Disc Decreases with Degenerative Changes

Intervertebral disc metabolic transport is essential to the functional spine and provides the cells with the nutrients necessary to tissue maintenance. Disc degenerative changes alter the tissue mechanics, but interactions between mechanical loading and disc transport are still an open issue. A poromechanical finite element model of the human disc was coupled with oxygen and lactate transport models. Deformations and fluid flow were linked to transport predictions by including strain-dependent diffusion and advection. The two solute transport models were also coupled to account for cell metabolism. With this approach, the relevance of metabolic and mechano-transport couplings were assessed in the healthy disc under loading-recovery daily compression. Disc height, cell density and material degenerative changes were parametrically simulated to study their influence on the calculated solute concentrations. The effects of load frequency and amplitude were also studied in the healthy disc by considering short periods of cyclic compression. Results indicate that external loads influence the oxygen and lactate regional distributions within the disc when large volume changes modify diffusion distances and diffusivities, especially when healthy disc properties are simulated. Advection was negligible under both sustained and cyclic compression. Simulating degeneration, mechanical changes inhibited the mechanical effect on transport while disc height, fluid content, nucleus pressure and overall cell density reductions affected significantly transport predictions. For the healthy disc, nutrient concentration patterns depended mostly on the time of sustained compression and recovery. The relevant effect of cell density on the metabolic transport indicates the disturbance of cell number as a possible onset for disc degeneration via alteration of the metabolic balance. Results also suggest that healthy disc properties have a positive effect of loading on metabolic transport. Such relation, relevant to the maintenance of the tissue functional composition, would therefore link disc function with disc nutrition

Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells

In this study, we explore the therapeutic potential of lapatinib a selective inhibitor of both the EGFR and HER2 tyrosine kinases for the treatment of endometrial cancer. The effect of lapatinib on tumour cell growth and receptor activation was studied in a panel of human endometrial cancer cell lines. Candidate molecular markers predicting sensitivity were assessed by baseline gene expression profiling, ELISA, and western blot analyses. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions between chemotherapeutic drugs and lapatinib. Concentration-dependent anti-proliferative effects of lapatinib were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC50 range: 0.052–10.9 μmol). HER2 overexpression or increased expression of EGFR was significantly associated with in vitro sensitivity (P=0.024 or 0.011, respectively). Lapatinib exerts growth inhibition in a PTEN-independent manner. Sensitive cell lines also exhibited increased expression of EGFR ligands or HER3. In contrast, lapatinib-resistant cell lines exhibited high androgen receptor (AR) levels or epithelial-to-mesenchymal transition (post-EMT) features. In endometrial cancer cells, at a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for lapatinib plus carboplatin, paclitaxel, docetaxel, and doxorubicin. These observations provide a clear biologic rational to test lapatinib as a single agent or in combination with chemotherapy in endometrial cancer with HER2 overexpression. Expression of EGFR, its ligands, HER3, AR, and post-EMT markers warrant further evaluation to help define patients with HER2-nonoverexpressing endometrial cancer most likely to benefit from lapatinib

Hyperoxic Treatment Induces Mesenchymal-to-Epithelial Transition in a Rat Adenocarcinoma Model

Tumor hypoxia is relevant for tumor growth, metabolism and epithelial-to-mesenchymal transition (EMT). We report that hyperbaric oxygen (HBO) treatment induced mesenchymal-to-epithelial transition (MET) in a dimetyl-α-benzantracene induced mammary rat adenocarcinoma model, and the MET was associated with extensive coordinated gene expression changes and less aggressive tumors. One group of tumor bearing rats was exposed to HBO (2 bar, pO2 = 2 bar, 4 exposures à 90 minutes), whereas the control group was housed under normal atmosphere (1 bar, pO2 = 0.2 bar). Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation, cell death, collagen fibrils and gene expression profile. Tumor growth was significantly reduced (∼16%) after HBO treatment compared to day 1 levels, whereas control tumors increased almost 100% in volume. Significant decreases in tumor cell proliferation, tumor blood vessels and collagen fibrils, together with an increase in cell death, are consistent with tumor growth reduction and tumor stroma influence after hyperoxic treatment. Gene expression profiling showed that HBO induced MET. In conclusion, hyperoxia induced MET with coordinated expression of gene modules involved in cell junctions and attachments together with a shift towards non-tumorigenic metabolism. This leads to more differentiated and less aggressive tumors, and indicates that oxygen per se might be an important factor in the “switches” of EMT and MET in vivo. HBO treatment also attenuated tumor growth and changed tumor stroma, by targeting the vascular system, having anti-proliferative and pro-apoptotic effects

Metacarpal trabecular bone varies with distinct hand-positions used in hominid locomotion

Trabecular bone remodels during life in response to loading and thus should, at least in part, reflect potential variation in the magnitude, frequency and direction of joint loading across different hominid species. Here we analyse the trabecular structure across all non-pollical metacarpal distal heads (Mc2-5) in extant great apes, expanding on previous volume of interest and whole-epiphysis analyses that have largely focussed on only the first or third metacarpal. Specifically, we employ both a univariate statistical mapping and a multivariate approach to test for both inter-ray and interspecific differences in relative trabecular bone volume fraction (RBV/TV) and degree of anisotropy (DA) in Mc2-5 subchondral trabecular bone. Results demonstrate that while DA values only separate Pongo from African apes (Pan troglodytes, Pan paniscus, Gorilla gorilla), RBV/TV distribution varies with the predicted loading of the metacarpophalangeal (McP) joints during locomotor behaviours in each species. Gorilla exhibits a relatively dorsal distribution of RBV/TV consistent with habitual hyper-extension of the McP joints during knuckle-walking, whereas Pongo has a palmar distribution consistent with flexed McP joints used to grasp arboreal substrates. Both Pan species possess a disto-dorsal distribution of RBV/TV, compatible with multiple hand postures associated with a more varied locomotor regime. Further inter-ray comparisons reveal RBV/TV patterns consistent with varied knuckle-walking postures in Pan species in contrast to higher RBV/TV values toward the midline of the hand in Mc2 and Mc5 of Gorilla, consistent with habitual palm-back knuckle-walking. These patterns of trabecular bone distribution and structure reflect different behavioural signals that could be useful for determining the behaviours of fossil hominins