Protective effect of ischaemic preconditioning on acute and chronic renal damage following ischaemia reperfusion injury: characterisation of fibrosis development after inflammation resolution.

OBJECTIVES: Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD) are increasingly recognised as one disease continuum, rather than distinct entities, and are associated with a huge burden to healthcare services. The leading cause of AKI worldwide is Ischaemia Reperfusion Injury (IRI), most commonly seen in clinical settings of sepsis-driven hypotension. Ischaemic Preconditioning (IPC) is a strategy aimed at reducing the deleterious effects of IRI. The objectives of this study were to demonstrate an efficacious in vivo model of Kidney IRI, and the protective influence of IPC in attenuating AKI and development of renal fibrosis. METHODS: A rat model of bilateral kidney IRI was used: Male Lewis rats (n=84) were assigned to IRI, sham or IPC. In IRI, renal pedicles were clamped for 45 minutes. IPC groups underwent pulsatile IPC prior to IRI. Kidneys were retrieved at 24 hours, 48 hours, 7 days, 14 days and 28 days, and assessed histologically. RESULTS: IRI led to marked AKI (24-48 h) and renal fibrosis development by 28 days. IPC attenuated this damage, with 66% less fibrosis. Interestingly, at 14-days, the histological appearance of both IRI and IPC kidneys was rather similar, potentially representing an important transitional point at which kidneys commit to either fibrosis or recovery. This may provide a suitable inflexion point for introduction of novel anti-fibrotic therapies. CONCLUSIONS: In conclusion, we have characterised a model of kidney injury from acute to chronic phases, allowing detailed mechanistic understanding and which can be manipulated by effective treatment strategies such as IPC.

Role of Enzymic Antioxidants in Mediating Oxidative Stress and Contrasting Wound Healing Capabilities in Oral Mucosal/Skin Fibroblasts and Tissues.

Unlike skin, oral mucosal wounds are characterized by rapid healing and minimal scarring, attributable to the "enhanced" healing properties of oral mucosal fibroblasts (OMFs). As oxidative stress is increasingly implicated in regulating wound healing outcomes, this study compared oxidative stress biomarker and enzymic antioxidant profiles between patient-matched oral mucosal/skin tissues and OMFs/skin fibroblasts (SFs) to determine whether superior oral mucosal antioxidant capabilities and reduced oxidative stress contributed to these preferential healing properties. Oral mucosa and skin exhibited similar patterns of oxidative protein damage and lipid peroxidation, localized within the lamina propria/dermis and oral/skin epithelia, respectively. SOD1, SOD2, SOD3 and catalase were primarily localized within epithelial tissues overall. However, SOD3 was also widespread within the lamina propria localized to OMFs, vasculature and the extracellular matrix. OMFs were further identified as being more resistant to reactive oxygen species (ROS) generation and oxidative DNA/protein damage than SFs. Despite histological evaluation suggesting that oral mucosa possessed higher SOD3 expression, this was not fully substantiated for all OMFs examined due to inter-patient donor variability. Such findings suggest that enzymic antioxidants have limited roles in mediating privileged wound healing responses in OMFs, implying that other non-enzymic antioxidants could be involved in protecting OMFs from oxidative stress overall.

Strategies to maximise study retention and limit attrition bias in a prospective cohort study of men reporting a history of injecting drug use released from prison: the prison and transition health study.

BACKGROUND: There are significant challenges associated with studies of people released from custodial settings, including loss to follow-up in the community. Interpretation of findings with consideration of differences between those followed up and those not followed up is critical in the development of evidence-informed policies and practices. We describe attrition bias in the Prison and Transition Health (PATH) prospective cohort study, and strategies employed to minimise attrition. METHODS: PATH involves 400 men with a history of injecting drug use recruited from three prisons in Victoria, Australia. Four interviews were conducted: one pre-release ('baseline') and three interviews at approximately 3, 12, and 24 months post-release ('follow-up'). We assessed differences in baseline characteristics between those retained and not retained in the study, reporting mean differences and 95% confidence intervals (95% CIs).  RESULTS: Most participants (85%) completed at least one follow-up interview and 162 (42%) completed all three follow-up interviews. Retained participants were younger than those lost to follow-up (mean diff - 3.1 years, 95% CI -5.3, - 0.9). There were no other statistically significant differences observed in baseline characteristics. CONCLUSION: The high proportion of participants retained in the PATH cohort study via comprehensive follow-up procedures, coupled with extensive record linkage to a range of administrative datasets, is a considerable strength of the study. Our findings highlight how strategic and comprehensive follow-up procedures, frequent contact with participants and secondary contacts, and established working relationships with the relevant government departments can improve study retention and potentially minimise attrition bias.

Myofibroblasts: Function, Formation, and Scope of Molecular Therapies for Skin Fibrosis.

Myofibroblasts are contractile, α-smooth muscle actin-positive cells with multiple roles in pathophysiological processes. Myofibroblasts mediate wound contractions, but their persistent presence in tissues is central to driving fibrosis, making them attractive cell targets for the development of therapeutic treatments. However, due to shared cellular markers with several other phenotypes, the specific targeting of myofibroblasts has long presented a scientific and clinical challenge. In recent years, myofibroblasts have drawn much attention among scientific research communities from multiple disciplines and specialisations. As further research uncovers the characterisations of myofibroblast formation, function, and regulation, the realisation of novel interventional routes for myofibroblasts within pathologies has emerged. The research community is approaching the means to finally target these cells, to prevent fibrosis, accelerate scarless wound healing, and attenuate associated disease-processes in clinical settings. This comprehensive review article describes the myofibroblast cell phenotype, their origins, and their diverse physiological and pathological functionality. Special attention has been given to mechanisms and molecular pathways governing myofibroblast differentiation, and updates in molecular interventions.

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-ß1.

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-ß1 and proinflammatory interleukin (IL)-1ß are widely associated with fibrotic progression. TGF-ß1 induces myofibroblast differentiation, while IL-1ß induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-ß1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-ß1- and IL-1ß-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-ß1-induced fibroblast/myofibroblast differentiation and IL-1ß-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.

Hyaluronidase-2 Regulates RhoA Signaling, Myofibroblast Contractility, and Other Key Profibrotic Myofibroblast Functions.

Hyaluronidase (HYAL)-2 is a weak, acid-active, hyaluronan-degrading enzyme broadly expressed in somatic tissues. Aberrant HYAL2 expression is implicated in diverse pathology. However, a significant proportion of HYAL2 is enzymatically inactive; thus the mechanisms through which HYAL2 dysregulation influences pathobiology are unclear. Recently, nonenzymatic HYAL2 functions have been described, and nuclear HYAL2 has been shown to influence mRNA splicing to prevent myofibroblast differentiation. Myofibroblasts drive fibrosis, thereby promoting progressive tissue damage and leading to multimorbidity. This study identifies a novel HYAL2 cytoplasmic function in myofibroblasts that is unrelated to its enzymatic activity. In fibroblasts and myofibroblasts, HYAL2 interacts with the GTPase-signaling small molecule ras homolog family member A (RhoA). Transforming growth factor beta 1-driven fibroblast-to-myofibroblast differentiation promotes HYAL2 cytoplasmic relocalization to bind to the actin cytoskeleton. Cytoskeletal-bound HYAL2 functions as a key regulator of downstream RhoA signaling and influences profibrotic myofibroblast functions, including myosin light-chain kinase-mediated myofibroblast contractility, myofibroblast migration, myofibroblast collagen/fibronectin deposition, as well as connective tissue growth factor and matrix metalloproteinase-2 expression. These data demonstrate that, in certain biological contexts, the nonenzymatic effects of HYAL2 are crucial in orchestrating RhoA signaling and downstream pathways that are important for full profibrotic myofibroblast functionality. In conjunction with previous data demonstrating the influence of HYAL2 on RNA splicing, these findings begin to explain the broad biological effects of HYAL2.

The Prison and Transition Health (PATH) Cohort Study: Study Protocol and Baseline Characteristics of a Cohort of Men with a History of Injecting Drug Use Leaving Prison in Australia.

People who inject drugs (PWID) are disproportionately represented among individuals who experience imprisonment and often have more complex physical and mental health needs than people in prison without injecting histories. The trajectories of PWID after prison release are poorly understood, hampering the development of effective strategies to address their distinct health needs. The Prison and Transition Health (PATH) Cohort Study is characterising the post-release trajectories of incarcerated male PWID in Victoria, Australia. We outline study methodology and baseline characteristics of participants prior to their release. Four hundred participants were recruited from three prisons and completed researcher-administered baseline interviews covering socio-demographics, social supports, physical health, mental health, alcohol and other drug use, and pre-release and transitional service utilisation. The median age among participants was 36 years (IQR 30-42), and they reported a median of five (IQR 3-9) previous adult incarcerations. Almost half (49%) were reliant on government payments prior to incarceration. One quarter (25%) of participants reported removal from their parents' care as children and 64% reported being a parent or primary caregiver to children. Most participants (81%) reported a previous mental health diagnosis and 44% reported three or more diagnoses. The most common drugs injected prior to incarceration were crystal methamphetamine (80%) and heroin (62%), and most (85%) reported being under the influence of drugs at the time of committing offences for which they were currently incarcerated. Injecting drug use during their current sentence was reported by 40% of participants, and 48% reported engaging with some form of drug treatment during their current sentence. Study participants are characterised by significant mental health and substance use morbidities, social disadvantage and criminogenic histories that present challenges for the provision of post-release support services. Data from the PATH Cohort Study will help inform strategies to improve the health and social outcomes of this population.

Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype.

The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of CD44 splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine-rich (SR) protein-mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and CD44 pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating CD44 alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis.

Hepatocyte Growth Factor Mediates Enhanced Wound Healing Responses and Resistance to Transforming Growth Factor-ß1-Driven Myofibroblast Differentiation in Oral Mucosal Fibroblasts.

Oral mucosal wounds are characterized by rapid healing with minimal scarring, partly attributable to the "enhanced" wound healing properties of oral mucosal fibroblasts (OMFs). Hepatocyte growth factor (HGF) is a pleiotropic growth factor, with potential key roles in accelerating healing and preventing fibrosis. HGF can exist as full-length or truncated (HGF-NK), NK1 and NK2 isoforms. As OMFs display elevated HGF expression compared to dermal fibroblasts (DFs), this study investigated the extent to which HGF mediates the preferential cellular functions of OMFs, and the influence of pro-fibrotic, transforming growth factor-ß1 (TGF-ß1) on these responses. Knockdown of HGF expression in OMFs by short-interfering RNA (siHGF) significantly inhibited OMF proliferative and migratory responses. Supplementation with exogenous TGF-ß1 also significantly inhibited proliferation and migration, concomitant with significantly down-regulated HGF expression. In addition, knockdown abrogated OMF resistance to TGF-ß1-driven myofibroblast differentiation, as evidenced by increased α-smooth muscle actin (α-SMA) expression, F-actin reorganisation, and stress fibre formation. Responses were unaffected in siHGF-transfected DFs. OMFs expressed significantly higher full-length HGF and NK1 levels compared to patient-matched DFs, whilst NK2 expression was similar in both OMFs and DFs. Furthermore, NK2 was preferentially expressed over NK1 in DFs. TGF-ß1 supplementation significantly down-regulated full-length HGF and NK1 expression by OMFs, while NK2 was less affected. This study demonstrates the importance of HGF in mediating "enhanced" OMF cellular function. We also propose that full-length HGF and HGF-NK1 convey desirable wound healing properties, whilst fibroblasts preferentially expressing more HGF-NK2 readily undergo TGF-ß1-driven differentiation into myofibroblasts.

Tumor Necrosis Factor-stimulated Gene 6 (TSG-6)-mediated Interactions with the Inter-α-inhibitor Heavy Chain 5 Facilitate Tumor Growth Factor ß1 (TGFß1)-dependent Fibroblast to Myofibroblast Differentiation.

Fibroblasts are central to wound healing and fibrosis through TGFß1-triggered differentiation into contractile, α-smooth muscle actin (α-SMA)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent co-localization of CD44 with the epidermal growth factor receptor (EGFR). Interactions of HA with hyaladherins, such as inter-α-inhibitor (IαI) and tumor necrosis factor-stimulated gene-6 (TSG-6), are also essential for differentiation. This study investigated the mechanisms involved. TSG-6 and α-SMA had different kinetics of induction by TGFß1, with TSG-6 peaking before α-SMA Si CD44 or EGFR inhibition prevented differentiation but had no effect on TSG-6 expression. TSG-6 was essential for differentiation, and mAb A38 (preventing IαI heavy chain (HC) transfer), HA-oligosaccharides, cobalt, or Si bikunin prevented TSG-6 activity, preventing differentiation. A38 also prevented the EGFR/CD44 association. This suggested that TSG-6/IαI HC interaction was necessary for the effect of TSG-6 and that HC stabilization of HA initiated the CD44/EGFR association. The newly described HC5 was shown to be the principal HC expressed, and its cell surface expression was prevented by siRNA inhibition of TSG-6 or bikunin. HC5 was released by hyaluronidase treatment, confirming its association with cell surface HA. Finally, HC5 knockdown by siRNA confirmed its role in myofibroblast differentiation. The current study describes a novel mechanism linking the TSG-6 transfer of the newly described HC5 to the HA-dependent control of cell phenotype. The interaction of HC5 with cell surface HA was essential for TGFß1-dependent differentiation of fibroblasts to myofibroblasts, highlighting its importance as a novel potential therapeutic target.

The effects of pH on wound healing, biofilms, and antimicrobial efficacy.

It is known that pH has a role to play in wound healing. In particular, pH has been shown to affect matrix metalloproteinase activity, tissue inhibitors of matrix metalloproteinases activity, fibroblast activity, keratinocyte proliferation, microbial proliferation, and also immunological responses in a wound; the patient's defense mechanisms change the local pH of a wound to effect microorganism invasion and proliferation; this pH change has been found to affect the performance of antimicrobials, and therefore the efficacy in biological environments directly relevant to wound healing. Based on the available body of scientific evidence to date, it is clear that pH has a role to play in both the healing of and treatment of chronic and acute wounds. It is the purpose of this review to evaluate the published knowledge base that concerns the effect of pH changes, the role it plays in wound healing and biofilm formation, and how it can affect treatment efficacy and wound management strategies.

Biofilm evidence and the microbial diversity of horse wounds.

Evidence of biofilms in human chronic wounds are thought to be responsible for preventing healing in a timely manner. However, biofilm evidence in horse wounds has not yet been documented. Consequently, this study aimed to determine whether biofilms could be detected in wounds, and to investigate the microbiology of chronic wounds in horses. Prior to analysis, wound surfaces were irrigated with 5 mL of sterile saline to remove debris. All wounds were swabbed twice (1 cm2 area) using sterile cotton-tipped swabs. In addition to this, 2 tissue biopsies were taken to investigate evidence of biofilm and the microbiology richness of the wounds. All swabs and 1 biopsy sample were transported to the laboratory in Robertson's cooked meat broth. Traditional culturable techniques and denaturing gradient gel electrophoresis with PCR were utilized to identify common bacteria isolated in all wounds. Following analysis of a number of the biopsy samples, biofilms could be clearly seen. The most common bacteria isolated from each wound analysed included Pseudomonas aeruginosa, Staphylococcus epidermidis, Serratia marcescens, Enterococcus faecalis, and Providencia rettgeri. Sequencing of the 16S ribosmonal DNAs, selected on the basis of DGGE profiling, enabled identification of bacterial species not identified using culturable technology. This study is the first to identify biofilms in the chronic wounds of horses. In addition, this study also demonstrated the importance of combining DGGE-PCR with culture techniques to provide better microbiology analysis of chronic wounds.

Silver resistance in MRSA isolated from wound and nasal sources in humans and animals.

Methicillin-resistant Staphylococcus aureus (MRSA) colonises skin, nasal passages and dermal wounds. Methods used to manage wounds infected and colonised with MRSA often include the use of topical antiseptics such as ionic silver and iodine. The objectives of this study were to determine the prevalence of silver-resistance (sil) genes in MRSA and methicillin-resistant coagulase-negative staphylococci (MR-CNS) isolated from wounds and nasal cavities of humans and animals, and also to determine the susceptibility of sil-positive and sil-negative MRSA isolates to a silver-containing Hydrofiber (SCH) wound dressing, on planktonic silE-positive and silE-negative MRSA. Polymerase chain reaction was used to determine the presence of three silver-resistance (sil) genes, silE, silP and silS in 33 MRSA and 8 methicillin-resistant staphylococci (MR-CNS). SilP and silS genes were absent in all isolates tested; however, two MRSA strains were found to contain the silE gene, together with one isolate of MR-CNS. Phenotypic resistance of the silE-positive strains and their susceptibility to the SCH dressing was evaluated using the zone of inhibition test on Mueller Hinton agar, and confocal laser microscopy using a live/dead fluorescent stain. Results confirmed that the SCH dressing was effective in killing all MRSA strains with and without the silE gene. First, this study showed that the prevalence of sil genes was low in the isolates investigated; and secondly, that the presence of a silver-resistance gene (silE) in MRSA and MR-CNS did not afford protection to the organism in the presence of a SCH wound dressing. The use of topical antiseptics in chronic wound care should be considered before the use of antibiotics that can result in their overuse and the risk of further resistance.

Prevalence of silver resistance in bacteria isolated from diabetic foot ulcers and efficacy of silver-containing wound dressings.

Silver dressings are used to manage wounds at risk of infection or locally infected. This in vitro study was conducted to assess the prevalence of silver resistance genes in 112 bacterial isolates obtained from the diabetic foot ulcers of patients attending the Diabetic Foot Clinic at Tameside General Hospital, UK. Using polymerase chain reaction to screen for three silver-resistance transcriptional units--silE, silS and silP--two silver-resistant bacteria were identified; both are strains of Enterobacter cloacae, an organism rarely implicated as a primary pathogen in chronic wounds. No recognized wound pathogens (Staphylococcus aureus-24 isolates and Pseudomonas aeruginosa-nine isolates) were found to contain silver-resistant genes. Analysis of the efficacy of silver-containing dressings on the silver-resistant strains of Enterobacter cloacae using confocal laser microscopy showed that, despite evidence of genetic resistance to silver, all strains were killed following a maximum of 48 hours of exposure to the dressings. Results suggest that presence of silver resistance genes is rare and that genetic resistance does not necessarily translate to phenotypic resistance to silver. While silver resistance in wound care should be monitored, the threat of widespread resistance is low and silver-containing dressings remain an extremely important tool in managing wound infection.

Assessing the effect of an antimicrobial wound dressing on biofilms.

To date the effect of silver-containing wound dressings on biofilms, known to be present in chronic wounds, has not been determined or documented. In this current study, we aimed to determine the antimicrobial effect of a silver-containing dressing on biofilms grown in a chambered slide model. Before the addition of a wound dressing onto a 24-hour biofilm, composed of either Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus, or a mixed bacterial community, a fluorescent dye was applied. This enabled the viability of sessile bacteria to be monitored in real-time, using a rapid form of confocal laser scanning microscopy over a contact time period of 48 hours. By analyzing all the three-dimensional data generated from the confocal time-lapse sequences, 90% of all sessile bacteria within the biofilm were observed to progressively turn red (i.e., died) within 24 hours. Total bacterial kill in the biofilm was achieved after 48 hours. This research has shown that the dressing was effective in killing the tested bacteria evident in both the tested mono and polymicrobial biofilms, which provides valuable evidence that this dressing may have an effect on biofilms found in recalcitrant chronic wounds.