Flightless, secreted through a late endosome/lysosome pathway, binds LPS and dampens cytokine secretion.

Flightless (Flii) is upregulated in response to wounding and has been shown to function in wound closure and scarring. In macrophages intracellular Flii negatively modulates TLR signalling and dampens cytokine production. We now show that Flii is constitutively secreted from macrophages and fibroblasts and is present in human plasma. Secretion from fibroblasts is upregulated in response to scratch wounding and LPS-activated macrophages also temporally upregulate their secretion of Flii. Using siRNA, wild-type and mutant proteins we show that Flii is secreted via a late endosomal/lysosomal pathway that is regulated by Rab7 and Stx11. Flii contains 11 leucine rich repeat (LRR) domains in its N-terminus that have nearly 50% similarity to those in the extracellular pathogen binding portion of Toll-like receptor 4 (TLR4). We show secreted Flii can also bind LPS and has the ability to alter macrophage activation. LPS activation of macrophages in Flii depleted conditioned media leads to enhanced macrophage activation and increased TNF secretion compared to cells activated in the presence of Flii. These results show secreted Flii binds to LPS and in doing so alters macrophage activation and cytokine secretion, suggesting that like the intracellular pool of Flii, secreted Flii also has the ability to alter inflammation.

Stratum basale keratinocyte expression of the cell surface glycoprotein CDCP1 during epidermogenesis and its role in keratinocyte migration

Background: Epidermogenesis and epidermal wound healing are tightly regulated processes during which keratinocytes must migrate, proliferate and differentiate. Cell to cell adhesion is crucial to the initiation and regulation of these processes. CUB domain containing protein 1 (CDCP1) is a transmembrane glycoprotein that is differentially tyrosine phosphorylated during changes in cell adhesion and survival signalling and is expressed by keratinocytes in native human skin, as well as in primary cultures. Objectives: To investigate the expression of CDCP1 during epidermogenesis and its role in keratinocyte migration. Methods: We examined both human skin tissue and an in vitro three-dimensional human skin equivalent model to examine the expression of CDCP1 during epidermogenesis. To examine the role of CDCP1 in keratinocyte migration we used a function blocking anti-CDCP1 antibody and a real-time Transwell™ cell migration assay. Results: Immunohistochemical analysis indicated that in native human skin CDCP1 is expressed in the stratum basale and stratum spinosum. In contrast, during epidermogenesis in a 3-dimensional human skin equivalent model CDCP1 was expressed only in the stratum basale, with localization restricted to the cell-cell membrane. No expression was detected in basal keratinocytes that were in contact with the basement membrane. Further, an anti-CDCP1 function blocking antibody was shown to disrupt keratinocyte chemotactic migration in vitro. Conclusions: These findings delineate the expression of CDCP1 in human epidermal keratinocytes during epidermogenesis and demonstrate that CDCP1 is involved in keratinocyte migration.

Vitronectin – master controller or micromanager?

The concept of the cellular glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knock-out mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that vitronectin occupies a role in the earliest events of thrombogenesis and tissue repair. That role is as a foundation upon which the thrombus grows in an organised structure. In addition to closing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators and provides a provisional scaffold for the repairing tissue. In the absence of vitronectin (e.g. VN-KO animal) this cascade is disrupted before it begins. Our data demonstrates that a wide variety of biologically active species associate with VN. While initial studies were focused on mitogens, other classes of bioactives (e.g. glycosaminoglycans, metalloproteinases) are now also known to specifically interact with VN. Many of these interactions are long-lived, often resulting in multi-protein complexes, while others are stable for prolonged periods. Multiprotein complexes provide several advantages: prolonging molecular interaction; sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular / biological responses. We contend that these, or equivalent, multi-protein complexes mediate vitronectin functionality in vivo. It is also likely that many of the species demonstrated to associate with vitronectin in vitro, also associate with vitronectin in vivo in similar multi-protein complexes. Thus the predominant biological function of vitronectin is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave, and ‘how’ to behave (i.e. appropriately for the current circumstance).

Lysosomal secretion of Flightless I upon injury has the potential to alter inflammation

Intracellular Flightless I (Flii), a gelsolin family member, has been found to have roles modulating actin regulation, transcriptional regulation and inflammation. In vivo Flii can regulate wound healing responses. We have recently shown that a pool of Flii is secreted by fibroblasts and macrophages, cells typically found in wounds, and its secretion can be upregulated upon wounding. We show that secreted Flii can bind to the bacterial cell wall component lipopolysaccharide and has the potential to regulate inflammation. We now show that secreted Flii is present in both acute and chronic wound fluid.

Quantifying the roles of cell motility and cell proliferation in a circular barrier assay

Moving fronts of cells are essential features of embryonic development, wound repair and cancer metastasis. This paper describes a set of experiments to investigate the roles of random motility and proliferation in driving the spread of an initially confined cell population. The experiments include an analysis of cell spreading when proliferation was inhibited. Our data have been analysed using two mathematical models: a lattice-based discrete model and a related continuum partial differential equation model. We obtain independent estimates of the random motility parameter, D, and the intrinsic proliferation rate, λ, and we confirm that these estimates lead to accurate modelling predictions of the position of the leading edge of the moving front as well as the evolution of the cell density profiles. Previous work suggests that systems with a high λ/D ratio will be characterized by steep fronts, whereas systems with a low λ/D ratio will lead to shallow diffuse fronts and this is confirmed in the present study. Our results provide evidence that continuum models, based on the Fisher–Kolmogorov equation, are a reliable platform upon which we can interpret and predict such experimental observations.

Continuum mechanics modelling of microfilament networks with different architectures based on molecular investigation of single F-actin

The actin microfilament plays a critical role in many cellular processes including embryonic development, wound healing, immune response, and tissue development. It is commonly organized in the form of networks whose mechanical properties change with changes in their architecture due to cell evolution processes. This paper presents a new nonlinear continuum mechanics model of single filamentous actin (F-actin) that is based on nanoscale molecular simulations. Following this continuum model of the single F-actin, mechanical properties of differently architected lamellipodia are studied. The results provide insight that can contribute to the understanding of the cell edge motions of living cells.

Travelling waves for a velocity–jump model of cell migration and proliferation

Cell invasion, characterised by moving fronts of cells, is an essential aspect of development, repair and disease. Typically, mathematical models of cell invasion are based on the Fisher–Kolmogorov equation. These traditional parabolic models can not be used to represent experimental measurements of individual cell velocities within the invading population since they imply that information propagates with infinite speed. To overcome this limitation we study combined cell motility and proliferation based on a velocity–jump process where information propagates with finite speed. The model treats the total population of cells as two interacting subpopulations: a subpopulation of left–moving cells, $L(x,t)$, and a subpopulation of right–moving cells, $R(x,t)$. This leads to a system of hyperbolic partial differential equations that includes a turning rate, $\Lambda \ge 0$, describing the rate at which individuals in the population change direction of movement. We present exact travelling wave solutions of the system of partial differential equations for the special case where $\Lambda = 0$ and in the limit that $\Lambda \to \infty$. For intermediate turning rates, $0 < \Lambda < \infty$, we analyse the travelling waves using the phase plane and we demonstrate a transition from smooth monotone travelling waves to smooth nonmonotone travelling waves as $\Lambda$ decreases through a critical value $\Lambda_{crit}$. We conclude by providing a qualitative comparison between the travelling wave solutions of our model and experimental observations of cell invasion. This comparison indicates that the small $\Lambda$ limit produces results that are consistent with experimental observations.

Sensitivity of edge detection methods for quantifying cell migration assays

Quantitative imaging methods to analyze cell migration assays are not standardized. Here we present a suite of two–dimensional barrier assays describing the collective spreading of an initially–confined population of 3T3 fibroblast cells. To quantify the motility rate we apply two different automatic image detection methods to locate the position of the leading edge of the spreading population after 24, 48 and 72 hours. These results are compared with a manual edge detection method where we systematically vary the detection threshold. Our results indicate that the observed spreading rates are very sensitive to the choice of image analysis tools and we show that a standard measure of cell migration can vary by as much as 25% for the same experimental images depending on the details of the image analysis tools. Our results imply that it is very difficult, if not impossible, to meaningfully compare previously published measures of cell migration since previous results have been obtained using different image analysis techniques and the details of these techniques are not always reported. Using a mathematical model, we provide a physical interpretation of our edge detection results. The physical interpretation is important since edge detection algorithms alone do not specify any physical measure, or physical definition, of the leading edge of the spreading population. Our modeling indicates that variations in the image threshold parameter correspond to a consistent variation in the local cell density. This means that varying the threshold parameter is equivalent to varying the location of the leading edge in the range of approximately 1–5% of the maximum cell density.

Simplified method for including spatial correlations in mean-field approximations

Biological systems involving proliferation, migration and death are observed across all scales. For example, they govern cellular processes such as wound-healing, as well as the population dynamics of groups of organisms. In this paper, we provide a simplified method for correcting mean-field approximations of volume-excluding birth-death-movement processes on a regular lattice. An initially uniform distribution of agents on the lattice may give rise to spatial heterogeneity, depending on the relative rates of proliferation, migration and death. Many frameworks chosen to model these systems neglect spatial correlations, which can lead to inaccurate predictions of their behaviour. For example, the logistic model is frequently chosen, which is the mean-field approximation in this case. This mean-field description can be corrected by including a system of ordinary differential equations for pair-wise correlations between lattice site occupancies at various lattice distances. In this work we discuss difficulties with this method and provide a simplication, in the form of a partial differential equation description for the evolution of pair-wise spatial correlations over time. We test our simplified model against the more complex corrected mean-field model, finding excellent agreement. We show how our model successfully predicts system behaviour in regions where the mean-field approximation shows large discrepancies. Additionally, we investigate regions of parameter space where migration is reduced relative to proliferation, which has not been examined in detail before, and our method is successful at correcting the deviations observed in the mean-field model in these parameter regimes.

Identification of symptom clusters in patients with chronic venous leg ulcers

Context Patients with venous leg ulcers experience multiple symptoms, including pain, depression, and discomfort from lower leg inflammation and wound exudate. Some of these symptoms impair wound healing and decrease quality of life (QOL). The presence of co-occurring symptoms may have a negative effect on these outcomes. The identification of symptom clusters could potentially lead to improvements in symptom management and QOL. Objectives To identify the prevalence and severity of common symptoms and the occurrence of symptom clusters in patients with venous leg ulcers. Methods For this secondary analysis, data on sociodemographic characteristics, medical history, venous history, ulcer and lower limb clinical characteristics, symptoms, treatments, healing, and QOL were analyzed from a sample of 318 patients with venous leg ulcers who were recruited from hospital outpatient and community nursing clinics for leg ulcers. Exploratory factor analysis was used to identify symptom clusters. Results Almost two-thirds (64%) of the patients experienced four or more concurrent symptoms. The most frequent symptoms were sleep disturbance (80%), pain (74%), and lower limb swelling (67%). Sixty percent of patients reported three or more symptoms at a moderate-to-severe level of intensity (e.g., 78% reported disturbed sleep frequently or always; the mean pain severity score was 49 of 100, SD 26.5). Exploratory factor analysis identified two symptom clusters: pain, depression, sleep disturbance, and fatigue; and swelling, inflammation, exudate, and fatigue. Conclusion Two symptom clusters were identified in this sample of patients with venous leg ulcers. Further research is needed to verify these symptom clusters and to evaluate their effect on patient outcomes.

Investigating epidermogenesis in a human skin equivalent model

Skin is the largest, and arguably, the most important organ of the body. It is a complex and multi-dimensional tissue, thus making it essentially impossible to fully model in vitro in conventional 2-dimensional culture systems. In view of this, rodents or pigs are utilised to study wound healing therapeutics or to investigate the biological effects of treatments on skin. However, there are many differences between the wound healing processes in rodents compared to humans (contraction vs. re-epithelialisation) and there are also ethical issues associated with animal testing for scientific research. Therefore, the development of skin equivalent (HSE) models from surgical discard human skin has become an important area of research. The studies in this thesis compare, for the first time, native human skin and the epidermogenesis process in a HSE model. The HSE was reported to be a comparable model for human skin in terms of expression and localisation of key epidermal cell markers. This validated HSE model was utilised to study the potential wound healing therapeutic, hyperbaric oxygen (HBO) therapy. There is a significant body of evidence suggesting that lack of cutaneous oxygen results in and potentiates the chronic, non-healing wound environment. Although the evidence is anecdotal, HBO therapy has displayed positive effects on re-oxygenation of chronic wounds and the clinical outcomes suggest that HBO treatment may be beneficial. Therefore, the HSE was subjected to a daily clinical HBO regime and assessed in terms of keratinocyte migration, proliferation, differentiation and epidermal thickening. HBO treatment was observed to increase epidermal thickness, in particular stratum corneum thickening, but it did not alter the expression or localisation of standard epidermal cell markers. In order to elucidate the mechanistic changes occurring in response to HBO treatment in the HSE model, gene microarrays were performed, followed by qRT-PCR of select genes which were differentially regulated in response to HBO treatment. The biological diversity of the HSEs created from individual skin donors, however, overrode the differences in gene expression between treatment groups. Network analysis of functional changes in the HSE model revealed general trends consistent with normal skin growth and maturation. As a more robust and longer term study of these molecular changes, protein localisation and expression was investigated in sections from the HSEs undergoing epidermogenesis in response to HBO treatment. These proteins were CDCP1, Metallothionein, Kallikrein (KLK) 1 and KLK7 and early growth response 1. While the protein expression within the HSE models exposed to HBO treatment were not consistent in all HSEs derived from all skin donors, this is the first study to detect and compare both KLK1 and CDCP1 protein expression in both a HSE model and native human skin. Furthermore, this is the first study to provide such an in depth analysis of the effect of HBO treatment on a HSE model. The data presented in this thesis, demonstrates high levels of variation between individuals and their response to HBO treatment, consistent with the clinical variation that is currently observed.

Cell migration and proliferation on homogeneous and non-homogeneous domains : modelling on the scale of individuals and populations

Cell migration is a behaviour critical to many key biological effects, including wound healing, cancerous cell invasion and morphogenesis, the development of an organism from an embryo. However, given that each of these situations is distinctly different and cells are extremely complicated biological objects, interest lies in more basic experiments which seek to remove conflating factors and present a less complex environment within which cell migration can be experimentally examined. These include in vitro studies like the scratch assay or circle migration assay, and ex vivo studies like the colonisation of the hindgut by neural crest cells. The reduced complexity of these experiments also makes them much more enticing as problems to mathematically model, like done here. The primary goal of the mathematical models used in this thesis is to shed light on which cellular behaviours work to generate the travelling waves of invasion observed in these experiments, and to explore how variations in these behaviours can potentially predict differences in this invasive pattern which are experimentally observed when cell types or chemical environment are changed. Relevant literature has already identified the difficulty of distinguishing between these behaviours when using traditional mathematical biology techniques operating on a macroscopic scale, and so here a sophisticated individual-cell-level model, an extension of the Cellular Potts Model (CPM), is been constructed and used to model a scratch assay experiment. This model includes a novel mechanism for dealing with cell proliferations that allowed for the differing properties of quiescent and proliferative cells to be implemented into their behaviour. This model is considered both for its predictive power and used to make comparisons with the travelling waves which result in more traditional macroscopic simulations. These comparisons demonstrate a surprising amount of agreement between the two modelling frameworks, and suggest further novel modifications to the CPM that would allow it to better model cell migration. Considerations of the model’s behaviour are used to argue that the dominant effect governing cell migration (random motility or signal-driven taxis) likely depends on the sort of invasion demonstrated by cells, as easily seen by microscopic photography. Additionally, a scratch assay simulated on a non-homogeneous domain consisting of a ’fast’ and ’slow’ region is also used to further differentiate between these different potential cell motility behaviours. A heterogeneous domain is a novel situation which has not been considered mathematically in this context, nor has it been constructed experimentally to the best of the candidate’s knowledge. Thus this problem serves as a thought experiment used to test the conclusions arising from the simulations on homogeneous domains, and to suggest what might be observed should this non-homogeneous assay situation be experimentally realised. Non-intuitive cell invasion patterns are predicted for diffusely-invading cells which respond to a cell-consumed signal or nutrient, contrasted with rather expected behaviour in the case of random-motility-driven invasion. The potential experimental observation of these behaviours is demonstrated by the individual-cell-level model used in this thesis, which does agree with the PDE model in predicting these unexpected invasion patterns. In the interest of examining such a case of a non-homogeneous domain experimentally, some brief suggestion is made as to how this could be achieved.

Matrix metalloproteinase biosensor based on a porous silicon reflector

Matrix metalloproteinases (MMPs) are proteolytic enzymes important to wound healing. In non-healing wounds, it has been suggested that MMP levels become dysfunctional, hence it is of great interest to develop sensors to detect MMP biomarkers. This study presents the development of a label-free optical MMP biosensor based on a functionalised porous silicon (pSi) thin film. The biosensor is fabricated by immobilising a peptidomimetic MMP inhibitor in the porous layer using hydrosilylation followed by amide coupling. The binding of MMP to the immobilised inhibitor translates into a change of effective optical thickness (EOT) over the time. We investigate the effect of surface functionalisation on the stability of pSi surface and evaluate the sensing performance. We successfully demonstrate MMP detection in buffer solution and human wound fluid at physiologically relevant concentrations. This biosensor may find application as a point-of-care device that is prognostic of the healing trajectory of chronic wounds.

Quantifying alterations in cell migration : tracking fluorescently-tagged migrating cells by FACs and live-imaging

Cell migration is fundamental to many different physiological processes including embryonic development, inflammation and wound healing. Given the range and importance cell migration plays a number of assays have been developed to measure different aspects of cell migration. Here we describe two different methods to analyze cell migration. The first method analyzes the migration of fluorescently tagged cells using Boyden chambers and FACs and the second looks at migration properties using time-lapse microscopy.