Defining the E-Cadherin repressor interactome in epithelial-mesenchymal transition : the PMC42 model as a case study

Epithelial-mesenchymal transition (EMT) is a feature of migratory cellular processes in all stages of life, including embryonic development and wound healing. Importantly, EMT features cluster with disease states such as chronic fibrosis and cancer. The dissolution of the E-cadherin-mediated adherens junction (AJ) is a key preliminary step in EMT and may occur early or late in the growing epithelial tumour. This is a first step for tumour cells towards stromal invasion, intravasation, extravasation and distant metastasis. The AJ may be inactivated in EMT by directed E-cadherin cleavage; however, it is increasingly evident that the majority of AJ changes are transcriptional and mediated by an expanding group of transcription factors acting directly or indirectly to repress E-cadherin expression. A review of the current literature has revealed that these factors may regulate each other in a hierarchical pattern where Snail1 (formerly Snail) and Snail2 (formerly Slug) are initially induced, leading to the activation of Zeb family members, TCF3, TCF4, Twist, Goosecoid and FOXC2. Within this general pathway, many inter-regulatory relationships have been defined which may be important in maintaining the EMT phenotype. This may be important given the short half-life of Snail1 protein. We have investigated these inter-regulatory relationships in the mesenchymal breast carcinoma cell line PMC42 (also known as PMC42ET) and its epithelial derivative, PMC42LA. This review also discusses several newly described regulators of E-cadherin repressors including oestrogen receptor-α and new discoveries in hypoxia- and growth factor-induced EMT. Finally, we evaluated how these findings may influence approaches to current cancer treatment.

A collagen prolyl 4-hydroxylase inhibitor reduces adhesions after tendon injury

Collagen synthesis inhibition potentially can reduce adhesion formation after tendon injury but also may affect cutaneous wound healing. We hypothesized that a novel orally administered collagen synthesis inhibitor (CPHI-I) would substantially reduce flexor tendon adhesions after injury, without any clinically important effect on cutaneous wound healing. The experiments were performed in a rat model with an in-continuity crush injury model in the rat hindfoot flexor tendon to provoke adhesion formation. Assays of dermal collagen production and the rate of healing of an excised wound were performed to assess cutaneous wound healing. Animals in the treatment groups received CPHI-I for 1, 2, or 6 weeks and were assessed at either 2 or 6 weeks. The work of flexion in the injured digit was reduced in the CPHI-I-treated animals compared with control animals, (0.188 J versus 0.0307 J at 2 weeks, and 0.0231 J versus 0.0331 J at 6 weeks) The cutaneous wound healing rate was similar in all animals, but dermal collagen synthesis was reduced in the treated animals. The CPHI-I seems to reduce tendon adhesion, and although collagen synthesis was reduced in cutaneous wounds, CPHI-I did not retard wound healing.

Models for studying cellular invasion of basement membranes

Invasion of extracellular matrices is crucial to a number of physiological and pathophysiological states, including tumor cell metastasis, arthritis, embryo implantation, wound healing, and early development. To isolate invasion from the additional complexities of these scenarios a number of in vitro invasion assays have been developed over the years. Early studies employed intact tissues, like denuded amniotic membrane (1) or embryonic chick heart fragments (2), however recently, purified matrix components or complex matrix extracts have been used to provide more uniform and often more rapid analyses (for examples, see the following integrin studies). Of course, the more holistic view of invasion offered in the earlier assays is valuable and cannot be fully reproduced in these more rapid assays, but advantages of reproducibility among replicates, ease of preparation and analysis, and overall high throughput favor the newer assays. In this chapter, we will focus on providing detailed protocols for Matrigel-based assays (Matrigel=reconstituted basement membrane; reviewed in ref. (3)). Matrigel is an extract from the transplantable Engelbreth-Holm-Swarm murine sarcoma that deposits a multilammelar basement membrane. Matrigel is available commercially (Becton Dickinson, Bedford, MA), and can be manipulated as a liquid at 4°C into a variety of different formats. Alternatively, cell culture inserts precoated with Matrigel can be purchased for even greater simplicity.

Osteoimmunomodulatory properties of magnesium scaffolds coated with β-tricalcium phosphate

The osteoimmunomodulatory property of bone biomaterials is a vital property determining the in vivo fate of the implants. Endowing bone biomaterials with favorable osteoimmunomodulatory properties is of great importance in triggering desired immune response and thus supports the bone healing process. Magnesium (Mg) has been recognized as a revolutionary metal for applications in orthopedics due to it being biodegradable, biocompatible, and having osteoconductive properties. However, Mg's high rate of degradation leads to an excessive inflammatory response and this has restricted its application in bone tissue engineering. In this study, β-tricalcium phosphate (β-TCP) was used to coat Mg scaffolds in an effort to modulate the detrimental osteoimmunomodulatory properties of Mg scaffolds, due to the reported favorable osteoimmunomodulatory properties of β-TCP. It was noted that macrophages switched to the M2 extreme phenotype in response to the Mg-β-TCP scaffolds, which could be due to the inhibition of the toll like receptor (TLR) signaling pathway. VEGF and BMP2 were significantly upregulated in the macrophages exposed to Mg-β-TCP scaffolds, indicating pro-osteogenic properties of macrophages in β-TCP modified Mg scaffolds. This was further demonstrated by the macrophage-mediated osteogenic differentiation of bone marrow stromal cells (BMSCs). When BMSCs were stimulated by conditioned medium from macrophages cultured on Mg-β-TCP scaffolds, osteogenic differentiation of BMSCs was significantly enhanced; whereas osteoclastogenesis was inhibited, as indicated by the downregualtion of MCSF, TRAP and inhibition of the RANKL/RANK system. These findings suggest that β-TCP coating of Mg scaffolds can modulate the scaffold's osteoimmunomodulatory properties, shift the immune microenvironment towards one that favors osteogenesis over osteoclastogenesis. Endowing bone biomaterials with favorable osteoimmunomodulatory properties can be a highly valuable strategy for the development or modification of advanced bone biomaterials.

Atmospheric pressure plasmas : infection control and bacterial responses

Cold atmospheric pressure plasma (APP) is a recent, cutting-edge antimicrobial treatment. It has the potential to be used as an alternative to traditional treatments such as antibiotics and as a promoter of wound healing, making it a promising tool in a range of biomedical applications with particular importance for combating infections. A number of studies show very promising results for APP-mediated killing of bacteria, including removal of biofilms of pathogenic bacteria such as Pseudomonas aeruginosa. However, the mode of action of APP and the resulting bacterial response are not fully understood. Use of a variety of different plasma-generating devices, different types of plasma gases and different treatment modes makes it challenging to show reproducibility and transferability of results. This review considers some important studies in which APP was used as an antibacterial agent, and specifically those that elucidate its mode of action, with the aim of identifying common bacterial responses to APP exposure. The review has a particular emphasis on mechanisms of interactions of bacterial biofilms with APP.

Assessing the role of spatial correlations during collective cell spreading

Spreading cell fronts are essential features of development, repair and disease processes. Many mathematical models used to describe the motion of cell fronts, such as Fisher’s equation, invoke a mean–field assumption which implies that there is no spatial structure, such as cell clustering, present. Here, we examine the presence of spatial structure using a combination of in vitro circular barrier assays, discrete random walk simulations and pair correlation functions. In particular, we analyse discrete simulation data using pair correlation functions to show that spatial structure can form in a spreading population of cells either through sufficiently strong cell–to–cell adhesion or sufficiently rapid cell proliferation. We analyse images from a circular barrier assay describing the spreading of a population of MM127 melanoma cells using the same pair correlation functions. Our results indicate that the spreading melanoma cell populations remain very close to spatially uniform, suggesting that the strength of cell–to–cell adhesion and the rate of cell proliferation are both sufficiently small so as not to induce any spatial patterning in the spreading populations.

A comparative study of Sr-incorporated mesoporous bioactive glass scaffolds for regeneration of osteopenic bone defects

SUMMARY: Recently, the use of the pharmacological agent strontium ranelate has come to prominence for the treatment of osteoporosis. While much investigation is focused on preventing disease progression, here we fabricate strontium-containing scaffolds and show that they enhance bone defect healing in the femurs of rats induced by ovariectomy. INTRODUCTION: Recently, the use of the pharmacological agent strontium ranelate has come to prominence for the treatment of osteoporosis due to its ability to prevent bone loss in osteoporotic patients. Although much emphasis has been placed on using pharmacological agents for the prevention of disease, much less attention has been placed on the construction of biomaterials following osteoporotic-related fracture. The aim of the present study was to incorporate bioactive strontium (Sr) trace element into mesoporous bioactive glass (MBG) scaffolds and to investigate their in vivo efficacy for bone defect healing in the femurs of rats induced by ovariectomy. METHODS: In total, 30 animals were divided into five groups as follows: (1) empty defect (control), (2) empty defects with estrogen replacement therapy, (3) defects filled with MBG scaffolds alone, (4) defects filled with MBG + estrogen replacement therapy, and (5) defects filled with strontium-incorporated mesopore-bioglass (Sr-MBG) scaffolds. RESULTS: The two groups demonstrating the highest levels of new bone formation were the defects treated with MBG + estrogen replacement therapy and the defects receiving Sr-MBG scaffolds as assessed by μ-CT and histological analysis. Furthermore, Sr scaffolds had a reduced number of tartrate-resistant acid phosphatase-positive cells when compared to other modalities. CONCLUSION: The results from the present study demonstrate that the local release of Sr from bone scaffolds may improve fracture repair. Future large animal models are necessary to investigate the future relationship of Sr incorporation into biomaterials.

The effect of cryotherapy on the vascular regeneration following closed soft tissue trauma

INTRODUCTION Icing (cryotherapy) is being widely used for the treatment of closed soft tissue trauma (CSTT), such as those resulting from sport injuries. It is believed that cryotherapy induces vasoconstriction and through this mechanism reduces inflammation [1]. However, the impact of this technique on the healing of impaired vasculature and muscle injuries following trauma remains controversial. Recent evidence suggests that the muscle regeneration is delayed after cryotherapy [2]. Consequently, we aimed to investigate the effect of cryotherapy on the vascular morphology following CSTT using an experimental model in rats by contrast-enhanced micro-CT imaging. METHODS Fifty four rats were divided into three main groups: control (no injury, n=6), sham (CSTT but no icing treatment, n=24) and icing (CSTT, treated with one session of ice block massaged directly on the injured muscle for 20 minutes, n=24). The CSTT was induced to the left thigh (Biceps Femoris) of anaesthetised rats (Male, Wistar) to create a standardized and reproducible vascular and muscle injury using an impact device [3]. Following trauma, animals were euthanized after 1, 3, 7, and 28 days healing time (n=6 for each time point). For a three-dimensional vascular morphological assessment, the blood vessels of euthanised rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil, MV 122, Flowtech, USA) using an infusion pump. Both hind-limbs were dissected, and then the injured and non-injured limbs were imaged using a micro-CT scanner (µCT 40, Scanco Medical, Switzerland) and total volume of the perfused blood vessels (TVV) was calculated. More detailed morphological parameters such as vessel volume (VV), diameter (VD), spacing (VSp), number (VN) and connectivity (VConn) were quantified through high resolution (6 µm), micro-CT-scanned biopsy samples (diameter: 8mm) taken directly from the region of the injured muscles. The biopsies were then analysed histologically to confirm the results derived from contrast-enhanced micro-CT imaging. RESULTS AND DISCUSSION The TVV was significantly higher in the injured legs compared to the non-injured legs at day 1 and 7 in the sham group and at day 28 in both sham and icing groups. The biopsies from the injured legs of the icing group showed a significant reduction in VV, VN, VD, VConn and an increase in VSp compared to those in the sham and control groups at days 1, 3 and 7, post injury. While the injured legs of the sham group exhibited a decrease in VN and VConn 28 days post trauma, indicating a return to the original values prior to trauma, these parameters had increased in the icing group (Figure 1). Also, at day 1 post injury, VV and VD of the injured legs were significantly higher in the sham group compared to the icing group, which may be attributed to the effect of vasoconstriction induced by icing. Further histomorphological evaluation of day 1 post injury, indicated that although cryotherapy significantly reduced the injury size and influx of inflammatory cells, including macrophages and neutrophils, a delay in vascular and muscle fiber regeneration was found at later time points confirming other reports from the literature [2]. CONCLUSIONS We have demonstrated using micro-CT imaging that the vascular morphology changes after CSTT, and that its recovery is affected by therapeutic modalities such as icing. This may be useful for the development of future clinical monitoring, diagnosis and treatment of CSTT. While icing reduces the swelling after trauma, our results suggest that it may delay the recovery of the vasculature in the injured tissue.

Interpreting scratch assays using pair density dynamics and approximate Bayesian computation

Quantifying the impact of biochemical compounds on collective cell spreading is an essential element of drug design, with various applications including developing treatments for chronic wounds and cancer. Scratch assays are a technically simple and inexpensive method used to study collective cell spreading; however, most previous interpretations of scratch assays are qualitative and do not provide estimates of the cell diffusivity, D, or the cell proliferation rate,l. Estimating D and l is important for investigating the efficacy of a potential treatment and provides insight into the mechanism through which the potential treatment acts. While a few methods for estimating D and l have been proposed, these previous methods lead to point estimates of D and l, and provide no insight into the uncertainty in these estimates. Here, we compare various types of information that can be extracted from images of a scratch assay, and quantify D and l using discrete computational simulations and approximate Bayesian computation. We show that it is possible to robustly recover estimates of D and l from synthetic data, as well as a new set of experimental data. For the first time, our approach also provides a method to estimate the uncertainty in our estimates of D and l. We anticipate that our approach can be generalized to deal with more realistic experimental scenarios in which we are interested in estimating D and l, as well as additional relevant parameters such as the strength of cell-to-cell adhesion or the strength of cell-to-substrate adhesion.

Perspectives about exercise from adults with venous leg ulcers : an exploratory study

Objective Exercise has the potential to offer a range of health benefits in addition to improving healing outcomes for people with venous leg ulcers. However despite evidence based recommendations, most of these individuals do not engage in regular exercise. The aim of this study was to gain an understanding of the perspectives of adults with venous leg ulcers, in relation to exercise. Method This was a qualitative design using semi-structured interviews and discussions. Ten participants with venous leg ulceration volunteered to participate. Recruitment was through a specialist wound clinic. Verbatim data were collected by an experienced moderator using a semi-structured guide. Data saturation was reached after three group discussions and two interviews. A random selection of transcripts was sent back to the participants for verification. Thematic content analysis was used to determine major themes and categories. Two transcripts were independently analysed, categories and themes independently developed, cross checked and found comparable. Remaining transcripts were analysed using developed categories and codes. Results Regardless of their current exercise routine, participants reported exercising prior to venous leg ulceration and expressed an interest in either becoming active or maintaining an active lifestyle. Overall four themes emerged from the findings: i) participant understanding of the relationship between chronic venous insufficiency and exercise patterns, ii) fear of harm impacts upon positive beliefs and attitudes to exercise, iii) perceived factors limit exercise and iv) structured management facilitates exercise. Conclusion The value of exercise in improving outcomes in venous leg ulcers lies in its capacity to promote venous return and reduce the risk of secondary conditions in this population. Despite motivation and interest in being exercise active, people with venous leg ulcers report many obstacles. Further exploration of mechanisms that assist this patient population and promote understanding about management of barriers, coupled with promotion of enabling factors is vital for improving their exercise participation.

Deletions and other pleasures : Episode 1: The pleasures of science fiction

"Christy Dena’s online-remix-narrative takes iconic images of popular culture and builds with them a strange world where the human fallibility is programmatically deleted. Both dystopic and playful, Dena’s work is an ironic reimagining of pleasure as a state of robotic flatlining, using tropes of science fiction to critique processes of social normalisation and increasing alienation from emotionality." This creative response began as a completely different story and form. What excited me in the end was the concept of deletion and how it could be an interesting mechanic: where the only thing you can do in the world is delete. I thought about deleting parts of robots to make them better. Healing comes from taking away, from removing things. Memories of Joseph Weizenbaum’s chatbot ELIZA came flooding back: where the (human) player is a patient talking to a Rogerian psychotherapist. But in this work I’m switching the roles and making the player the doctor, a doctor to robots…a doctor that can only prescribe deletions. I conceived of the work as a branching narrative, and started writing it in Twine. With every robot patient, the player chose one of many deletions. But when I realised I wouldn’t be able to arrange an artist and sound designer I looked for another option. I played with Zeega and felt that I could get the mood I was after with that platform. So the piece transformed into a work where the player/viewer is imprisoned in the decisions of the deleting protagonist…which has its own effect on the experience and meaning.

Can bone tissue engineering contribute to therapy concepts after resection of musculoskeletal sarcoma?

Resection of musculoskeletal sarcoma can result in large bone defects where regeneration is needed in a quantity far beyond the normal potential of self-healing. In many cases, these defects exhibit a limited intrinsic regenerative potential due to an adjuvant therapeutic regimen, seroma, or infection. Therefore, reconstruction of these defects is still one of the most demanding procedures in orthopaedic surgery. The constraints of common treatment strategies have triggered a need for new therapeutic concepts to design and engineer unparalleled structural and functioning bone grafts. To satisfy the need for long-term repair and good clinical outcome, a paradigm shift is needed from methods to replace tissues with inert medical devices to more biological approaches that focus on the repair and reconstruction of tissue structure and function. It is within this context that the field of bone tissue engineering can offer solutions to be implemented into surgical therapy concepts after resection of bone and soft tissue sarcoma. In this paper we will discuss the implementation of tissue engineering concepts into the clinical field of orthopaedic oncology.

Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis

Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCc1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

Testing the effectiveness of a self-efficacy based exercise intervention for adults with venous leg ulcers : protocol of a randomised controlled trial

Background: Exercise and adequate self-management capacity may be important strategies in the management of venous leg ulcers. However, it remains unclear if exercise improves the healing rates of venous leg ulcers and if a self-management exercise program based on self-efficacy theory is well adhered to. Method/Design: This is a randomised controlled in adults with venous leg ulcers to determine the effectiveness of a self-efficacy based exercise intervention. Participants with venous leg ulcers are recruited from 3 clinical sites in Australia. After collection of baseline data, participants are randomised to either an intervention group or control group. The control group receive usual care, as recommended by evidence based guidelines. The intervention group receive an individualised program of calf muscle exercises and walking. The twelve week exercise program integrates multiple elements, including up to six telephone delivered behavioural coaching and goal setting sessions, supported by written materials, a pedometer and two follow-up booster calls if required. Participants are encouraged to seek social support among their friends, self-monitor their weekly steps and lower limb exercises. The control group are supported by a generic information sheet that the intervention group also receive encouraging lower limb exercises, a pedometer for self-management and phone calls at the same time points as the intervention group. The primary outcome is the healing rates of venous leg ulcers which are assessed at fortnightly clinic appointments. Secondary outcomes, assessed at baseline and 12 weeks: functional ability (range of ankle motion and Tinetti gait and balance score), quality of life and self-management scores. Discussion: This study seeks to address a significant gap in current wound management practice by providing evidence for the effectiveness of a home-based exercise program for adults with venous leg ulcers. Theory-driven, evidence-based strategies that can improve an individual’s exercise self-efficacy and self-management capacity could have a significant impact in improving the management of people with venous leg ulcers. Information gained from this study will provide much needed information on management of this chronic disease to promote health and independence in this population. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12612000475842 Trial status: Current follow up