The skin safety model: Reconceptualizing skin vulnerability in older patients

Purpose: To develop a unique skin safety model (SSM) that offers a new and unified perspective on the diverse yet interconnected antecedents that contribute to a spectrum of potential iatrogenic skin injuries in older hospitalized adults. Organizing Construct: Discussion paper. Methods: A literature search of electronic databases was conducted for published articles written in English addressing skin integrity and iatrogenic skin injury in elderly hospital patients between 1960 and 2014. Findings: There is a multiplicity of literature outlining the etiology, prevention, and management of specific iatrogenic skin injuries. Complex and interrelated factors contribute to iatrogenic skin injury in the older adult, including multiple comorbidities, factors influencing healthcare delivery, and acute situational stressors. A range of injuries can result when these factors are com- plicated by skin irritants, pressure, shear, or friction; however, despite skin injuries sharing multiple ntecedents, no unified overarching skin safety conceptual model has been published. Conclusions: The SSM presented in this article offers a new, unified framework that encompasses the spectrum of antecedents to skin vulnerability as well as the spectrum of iatrogenic skin injuries that may be sustained by older acute care patients. Current skin integrity frameworks address prevention and management of specific skin injuries. In contrast, the SSM recognizes the complex interplay of patient and system factors that may result in a range of iatrogenic skin injuries. Skin safety is reconceptualized into a single model that has the potential for application at the individual patient level, as well as health-care systems and governance levels. Clinical Relevance: Skin safety is concerned with keeping skin safe from any iatrogenic skin injury, and remains an ongoing challenge for healthcare providers. A conceptual framework that encompasses all of the factors that may contribute to a range of iatrogenic skin injuries is essential, and guides the clinician in maintaining skin integrity in the vulnerable older patient.

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.

Characterisation of a human skin equivalent model to study the effects of ultraviolet B radiation on keratinocytes

The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline both in Australia and globally. Hence, the cellular and molecular pathways associated with UVR-induced photocarcinogenesis urgently need to be elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular the highly-mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which to some extent have limited their relevance to the in vivo situation. To address this, we characterised a 3-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically-relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB-irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53 and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration following photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.

Toward the multilevel older person's transportation and road safety model: A new perspective on the role of demographic, functional, and psychosocial factors

Objectives Self-regulation refers to the practice of using self-imposed restrictions to protect oneself from situations that are, or are perceived to be, unsafe. Within the driving context, self-regulation refers the compensatory practices that some older adults adopt to restrict their driving to situations in which they feel safe. However, the way in which demographic, functional, and psychosocial factors, and the interactions between these factors, influence older adults’ driving self-regulation is not well understood. Improving this understanding could lead to new ways of considering the mobility concerns faced by older drivers. Method A systematic review of the current literature was conducted to explore this issue. Twenty-nine empirical studies investigating the factors associated with older adults’ self-regulatory driving behaviors were examined. Results The review findings were used to construct the Multilevel Older Persons Transportation and Road Safety (MOTRS) model. The MOTRS model proposes that individual and environmental factors such as age, gender, and the availability of alternative transportation predict older adults’ practice of driving-related self-regulation. However, these variables influence self-regulation through psychosocial variables such as driving confidence, affective attitude, and instrumental attitude toward driving. Discussions The MOTRS model extends previous attempts to model older adults’ driving by focusing on a novel target, driving self-regulation, and by including a wider range of predictors identified on the basis of the systematic literature review. This focus enables consideration of broader mobility issues and may inform new strategies to support the mobility of older adults.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Simulation-based cutaneous surgical-skill training on a chicken-skin bench model in a medical undergraduate program

Background: Because of ethical and medico-legal aspects involved in the training of cutaneous surgical skills on living patients, human cadavers and living animals, it is necessary the search for alternative and effective forms of training simulation. Aims: To propose and describe an alternative methodology for teaching and learning the principles of cutaneous surgery in a medical undergraduate program by using a chicken-skin bench model. Materials and Methods: One instructor for every four students, teaching materials on cutaneous surgical skills, chicken trunks, wings, or thighs, a rigid platform support, needled threads, needle holders, surgical blades with scalpel handles, rat-tooth tweezers, scissors, and marking pens were necessary for training simulation. Results: A proposal for simulation-based training on incision, suture, biopsy, and on reconstruction techniques using a chicken-skin bench model distributed in several sessions and with increasing levels of difficultywas structured. Both feedback and objective evaluations always directed to individual students were also outlined. Conclusion: The teaching of a methodology for the principles of cutaneous surgery using a chicken-skin bench model versatile, portable, easy to assemble, and inexpensive is an alternative and complementary option to the armamentarium of methods based on other bench models described. © Indian Journal of Dermatology 2013.

Ex vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.

Improving wound management for residents in Residential Aged Care Facilities: National dissemination and implementation of the evidence based Champions for Skin Integrity Program

Overview The incidence of skin tears, pressure injuries and chronic wounds increases with age [1-4] and therefore is a serious issue for staff and residents in Residential Aged Care Facilities (RACFs). A pilot project funded in Round 2 of the Encouraging Best Practice in Residential Aged Care (EBPRAC) program by the then Australian Government Department of Health and Ageing found that a substantial proportion of residents in aged care facilities experienced pressure injuries, skin tears or chronic wounds. It also found the implementation of the evidence based Champions for Skin Integrity (CSI) model of wound care was successful in significantly decreasing the prevalence and severity of wounds in residents, improving staff skills and knowledge of evidence based wound management, increasing staff confidence with wound management, increasing implementation of evidence based wound management and prevention strategies, and increasing staff awareness of their roles in evidence based wound care at all levels [5]. Importantly, during the project, the project team developed a resource kit on evidence based wound management. Two critical recommendations resulting from the project were that: - The CSI model or a similar strategic approach should be implemented in RACFs to facilitate the uptake of evidence based wound management and prevention - The resource kit on evidence based wound management should be made available to all Residential Aged Care Facilities and interested parties A proposal to disseminate or rollout the CSI model of wound care to all RACFs across Australia was submitted to the department in 2012. The department approved funding from the Aged Care Services Improvement Healthy Ageing Grant (ACSIHAG) at the same time as the Round 3 of the Encouraging Better Practice in Aged Care (EBPAC) program. The dissemination involved two crucial elements: 1. The updating, refining and distribution of a Champions for Skin Integrity Resource Kit, more commonly known as a CSI Resource Kit and 2. The presentation of intensive one day Promoting Healthy Skin “Train the Trainer” workshops in all capital cities and major regional towns across Australia Due to demand, the department agreed to fund a second round of workshops focussing on regional centres and the completion date was extended to accommodate the workshops. Later, the department also decided to host a departmental website for a number of clinical domains, including wound management, so that staff from the residential aged care sector had easy access to a central repository of helpful clinical resource material that could be used for improving the health and wellbeing of their older adults, consumers and carers. CSI Resource Kit Upgrade and Distribution: At the start of the project, a full evidence review was carried out on the material produced during the EBPRAC-CSI Stage 1 project and the relevant evidence based changes were made to the documentation. At the same time participants in the EBPRAC-CSI Stage 1 project were interviewed for advice on how to improve the resource material. Following this the documentation, included in the kit, was sent to independent experts for peer review. When this process was finalised, a learning designer and QUT’s Visual Communications Services were engaged to completely refine and update the design of the resources, and combined resource kit with the goal of keeping the overall size of the kit suitable for bookshelf mounting and the cost at reasonable levels. Both goals were achieved in that the kit is about the same size as a 25 mm A4 binder and costs between $19.00 and $28.00 per kit depending on the size of the print run. The dissemination of the updated CSI resource kit was an outstanding success. Demand for the kits was so great that a second print run of 2,000 kits was arranged on top of the initial print run of 4,000 kits. All RACFs across Australia were issued with a kit, some 2,740 in total. Since the initial distribution another 1,100 requests for kits has been fulfilled as well as 1,619 kits being distributed to participants at the Promoting Healthy Skin workshops. As the project was winding up a final request email was sent to all workshop participants asking if they required additional kits or resources to distribute the remaining kits and resources. This has resulted in requests for 200 additional kits and resources. Feedback from the residential aged care sector and other clinical providers who have interest in wound care has been very positive regarding the utility of the kit, (see Appendix 4). Promoting Healthy Skin Workshops The workshops also exceeded the project team’s initial objective. Our goal of providing workshop training for staff from one in four facilities and 450 participants was exceeded, with overwhelming demand for workshop places resulting in the need to provide a second round of workshops across Australia. At the completion of the second round, 37 workshops had been given, with 1286 participants, representing 835 facilities. A number of strategies were used to promote the workshops ranging from invitations included in the kit, to postcard mail-outs, broadcast emailing to all facilities and aged care networks and to articles and paid advertising in aged care journals. The most effective method, by far, was directly phoning the facilities. This enabled the caller to contact the relevant staff member and enlist their support for the workshop. As this is a labour intensive exercise, it was only used where numbers needed bolstering, with one venue rising from 3 registrants before the calls to 53 registrants after. The workshops were aimed at staff who had the interest and the capability of implementing evidence-based wound management within their facility or organisation. This targeting was successful in that a large proportion (68%) of participants were Registered Nurses, Nurse Managers, Educators or Consultants. Twenty percent were Endorsed Enrolled Nurses with the remaining 12% being made up of Personal Care Workers or Allied Health Professionals. To facilitate long term sustainability, the workshop employed train-the-trainer strategies. Feedback from the EBPRAC-CSI Stage 1 interviews was used in the development of workshop content. In addition, feedback from the workshop conducted at the end of the EBPRAC-CSI Stage 1 project suggested that change management and leadership training should be included in the workshops. The program was trialled in the first workshop conducted in Brisbane and then rolled out across Australia. Participants were asked to complete pre and post workshop surveys at the beginning and end of the workshop to determine how knowledge and confidence improved over the day. Results from the pre and post surveys showed significant improvements in the level of confidence in attendees’ ability to implement evidence based wound management. The results also indicated a significant increase in the level of confidence in ability to implement change within their facility or organisation. This is an important indication that the inclusion of change management/leadership training with clinical instruction can increase staff capacity and confidence in translating evidence into practice. To encourage the transfer of the evidence based content of the workshop into practice, participants were asked to prepare an Action Plan to be followed by a simple one page progress report three months after the workshop. These reports ranged from simple (e.g. skin moisturising to prevent skin tears), to complex implementation plans for introducing the CSI model across the whole organisation. Outcomes described in the project reports included decreased prevalence of skin tears, pressure injuries and chronic wounds, along with increased staff and resident knowledge and resident comfort. As stated above, some organisations prepared large, complex plans to roll out the CSI model across their organisation. These plans included a review of the organisation’s wound care system, policies and procedures, the creation of new processes, the education of staff and clients, uploading education and resource material onto internal electronic platforms and setting up formal review and evaluation processes. The CSI Resources have been enthusiastically sought and incorporated into multiple health care settings, including aged care, acute care, Medicare Local intranets (e.g. Map of Medicine e-pathways), primary health care, community and home care organisations, education providers and New Zealand aged and community health providers. Recommendations: Recommendations for RACFs, aged care and health service providers and government  Skin integrity and the evidence-practice gap in this area should be recognised as a major health issue for health service providers for older adults, with wounds experienced by up to 50% of residents in aged care settings (Edwards et al. 2010). Implementation of evidence based wound care through the Champions for Skin Integrity model in this and the pilot project has demonstrated the prevalence of wounds, wound healing times and wound infections can be halved.  A national program and Centre for Evidence Based Wound Management should be established to: - expand the reach of the model to other aged care facilities and health service providers for older adults - sustain the uptake of models such as the Champions for Skin Integrity (CSI) model - ensure current resources, expertise and training are available for consumers and health care professionals to promote skin integrity for all older adults  Evidence based resources for the CSI program and similar projects should be reviewed and updated every 3 – 4 years as per NH&MRC recommendations  Leadership and change management training is fundamental to increasing staff capacity, at all levels, to promote within-organisation dissemination of skills and knowledge gained from projects providing evidence based training Recommendations for future national dissemination projects  A formal program of opportunities for small groups of like projects to share information and resources, coordinate activities and synergise education programs interactively would benefit future national dissemination projects - Future workshop programs could explore an incentive program to optimise attendance and reduce ‘no shows’ - Future projects should build in the capacity and funding for increased follow-up with workshop attendees, to explore the reasons behind those who are unable to translate workshop learnings into the workplace and identify factors to address these barriers.

Does bench model fidelity interfere in the acquisition of suture skills by novice medical students?

OBJETIVO: Embora vários modelos de bancada inanimados tenham sido descritos para o treinamento de habilidades de sutura, até o momento, não existe um método ideal para esse ensino e aprendizagem durante a formação médica. O objetivo foi avaliar se a fidelidade dos modelos de bancada interfere na aquisição de habilidades de sutura em estudantes de medicina iniciantes na prática cirúrgica. MÉTODOS: 36 estudantes de medicina sem exposição prévia a habilidades cirúrgicas foram randomizados em três grupos (n = 12): treinamento de suturas baseado em materiais didáticos (controle); treinamento de suturas em modelo de baixa-fidelidade (modelo de bancada de etileno vinil acetato); ou treinamento de suturas em modelo de alta-fidelidade (modelo de bancada de pele de pata de porco). Foram aplicados pré e pós-testes (realização de pontos simples e pontos subdérmicos invertidos em língua de boi). Três ferramentas (Global Rating Scale com avaliação cega, tamanho do efeito e autopercepção da confiança baseada em uma escala de Likert) foram utilizadas para mensurar todas as performances de sutura. RESULTADOS: A análise após o treinamento demonstrou que os estudantes que treinaram nos modelos tiveram um melhor (p < 0.0000) desempenho na avaliação pela Global Rating Scale, quando comparados com o controle, independente da fidelidade do modelo. A magnitude do efeito (treinamento) foi considerada grande (> 0.80) em todas as mensurações. Após o treinamento os alunos sentiram-se mais confiantes (p < 0.0000) para executarem os dois tipos de suturas. CONCLUSÃO: A aquisição de habilidades de suturas no modelo de baixa fidelidade foi semelhante à prática no modelo de alta fidelidade, sendo que a melhora no desempenho dos participantes que treinaram nesses dois modelos foi superior à aprendizagem baseada em materiais didáticos.

The essential role of annexin A1 mimetic peptide in the skin allograft survival

Immunosuppressive drugs have a critical role in inhibiting tissue damage and allograft rejection.Studies have demonstrated the anti-infl ammatory effects of the annexin A1 (AnxA1) in the regulationof transmigration and apoptosis of leucocytes. In the present study, an experimental skin allograftmodel was used to evaluate a potential protective effect of AnxA1 in transplantation survival. Micewere used for the skin allograft model and pharmacological treatments were carried out using eitherthe AnxA1 mimetic peptide Ac2-26, with or without cyclosporine A (CsA), starting 3 days beforesurgery until rejection. Graft survival, skin histopathology, leucocyte transmigration and expressionof AnxA1 and AnxA5 post-transplantation were analysed. Pharmacological treatment with Ac2-26increased skin allograft survival related with inhibition of neutrophil transmigration and inductionof apoptos is, thereby reducing the tissue damage compared with control animals. Moreover, AnxA1and AnxA5 expression increased after Ac2-26 treatment in neutrophils. Interestingly, thecombination of Ac2-26 and cyclosporine A showed similar survival of transplants when compared withthe cyclosporine A group, which could be attributed to a synergistic effect of both drugs. Investigationsin vitro revealed that cyclosporine A inhibited extracellular-signal-regulated kinase (ERK) phosphory-lation induced by Ac2-26 in neutrophils. Overall, the results suggest that AnxA1 has an essential role inaugmenting the survival of skin allograft, mainly owing to inhibition of neutrophil transmigration andenhancement of apoptosis. This effect may lead to the development of new therapeutic approachesrelevant to transplant rejection.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Molecular events associated with trisomy of chromosome 7 in mouse skin chemical carcinogenesis

The primary objective of this study has been to investigate the effects at the molecular level of trisomy of mouse chromosome 7 in chemically induced skin tumors. It was previously proposed that the initiation event in the mouse skin carcinogenesis model is a heterozygous mutation of the Ha-ras-1 gene, mapped to chromosome 7. Previous studies in this laboratory identified trisomy 7 as one of the primary nonrandom cytogenetic abnormalities found in the majority of severely dysplastic papillomas and squamous cell carcinomas induced in SENCAR mice by an initiation-promotion protocol. Therefore, the first hypothesis tested was that trisomy 7 occurs by specific duplication of the chromosome carrying a mutated Ha-ras-1 allele. Results of a quantitative analysis of normal/mutated allelic ratios of the Ha-ras-1 gene confirmed this hypothesis, showing that most of the tumors exhibited overrepresentation of the mutated allele in the form of 1/2, 0/3, and 0/2 (normal/mutated) ratios. In addition, histopathological analysis of the tumors showed an apparent association between the degree of malignancy and the dosage of the mutated Ha-ras-1 allele. To determine the mechanism for loss of the normal Ha-ras-1 allele, found in 30% of the tumors, a comparison of constitutional and tumor genotypes was performed at different informative loci of chromosome 7. By combining Southern blot and polymerase chain reaction fragment length polymorphism analyses of DNAs extracted from squamous cell carcinomas, complete loss of heterozygosity was detected in 15 of 20 tumors at the Hbb locus, and in 5 of 5 tumors at the int-2 locus, both distal to Ha-ras-1. In addition, polymerase chain reaction analysis of DNA extracted from papillomas indicated that loss of heterozygosity occurs in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion, suggesting that this event may be associated to the acquisition of the malignant phenotype. Allelic dosage analysis of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1, indicated that loss of heterozygosity on mouse chromosome 7 occurs by a mitotic recombination mechanism. Overall, these findings suggest the presence of a putative tumor suppressor locus on the 7F1-ter region of mouse chromosome 7. Thus, loss of function by homozygosis at this putative suppressor locus may complement activation of the Ha-ras-1 gene during tumor progression, and might be associated with the malignant conversion stage of mouse skin carcinogenesis. ^

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.