Evaluation of novel epoxy-tigliane compounds as modulators of dermal fibroblast-myofibroblast differentiation, scar tissue resolution and fibrosis; and elucidation of their underlying mechanisms of action

EBC-46 and EBC-211 are novel epoxy-tiglianes found to occur naturally within seeds of the Fontain’s Blushwood Tree, indigenous to the Queensland tropical rainforest in Australia. The Australian biotechnology company, QBiotics Group, has demonstrated that EBC-46 stimulates enhanced dermal wound healing responses in vivo, following cancer treatment and tumour destruction in domesticated animals. Consequently, QBiotics is developing EBC-46 as both a human and veterinary anti-cancer therapy. However, little is known on how the epoxy-tiglianes induce their exceptional healing effects; manifested as rapid wound re-epithelialisation, wound contraction/closure and minimal scarring. This study aimed to determine how EBC-46 and EBC-211 mediate these exceptional wound healing effects in vitro, via analysis of dermal fibroblast (DF)/myofibroblast genotypic and phenotypic responses; following epoxy-tigliane treatment (0.001 - 10 μg/mL). A number of wound healing responses were assessed, such as transforming growth factor-β1 (TGF-β1)-driven DF-myofibroblast differentiation, extracellular matrix (ECM) synthesis/turnover, global gene expression and underlying signalling pathways. Studies exhibited that EBC-46 and EBC-211 significantly inhibited α-smooth muscle actin (αSMA) expression, stress fibre formation and myofibroblast formation at 0.1 μg/mL and 10 μg/mL, respectively. Such concentrations were also shown to reduce type I/III collagen; and up-regulate matrix metalloproteinase-1 (MMP-1) gene and protein levels. The epoxy-tiglianes also increased elastin, hyaluronan and pericellular coat synthesis, via up-regulated hyaluronan synthase (HAS) expression. Microarray analysis and protein level validation identified numerous differentially expressed genes in epoxy-tigliane-treated DFs. Up-regulated genes included proteinases and other ‘anti-fibrotic’ genes; while down-regulated genes included protease inhibitors, myofibroblast- and cytoskeletal-related genes; as well as other ‘pro-fibrotic’ genes. Inhibitory effects upon TGF-β1-driven, DF-myofibroblast differentiation were shown to be protein kinase C (PKC)-dependent. This study has provided evidence to explain the reduced scarring responses observed in epoxy-tigliane-treated skin; highlighting the potential of epoxy-tiglianes as novel therapeutics for excessive scarring situations

Hepatocyte growth factor mediates enhanced wound healing responses and resistance to transforming growth factor-β1-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-β1 (TGF-β1) 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-β1 also significantly inhibited proliferation and migration, concomitant with significantly down-regulated HGF expression. In addition, knockdown abrogated OMF resistance to TGF-β1-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-β1 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-β1-driven differentiation into myofibroblasts

Role of oxidative stress in impaired type II diabetic bone repair: scope for antioxidant therapy intervention?

Impaired bone healing is a significant complication observed in individuals with type 2 diabetes mellitus (T2DM), leading to prolonged recovery, increased risk of complications, impaired quality of life, and increased risk of patient morbidity. Oxidative stress, resulting from an imbalance between the generation of reactive oxygen species (ROS) and cellular/tissue antioxidant defence mechanisms, has been identified as a critical contributor to the pathogenesis of impaired bone healing in T2DM. Antioxidants have shown promise in mitigating oxidative stress and promoting bone repair, particularly non-enzymic antioxidant entities. This comprehensive narrative review aims to explore the underlying mechanisms and intricate relationship between oxidative stress, impaired bone healing and T2DM, with a specific focus on the current preclinical and clinical evidence advocating the potential of antioxidant therapeutic interventions in improving bone healing outcomes in individuals with T2DM. From the ever-emerging evidence available, it is apparent that exogenously supplemented antioxidants, especially non-enzymic antioxidants, can ameliorate the detrimental effects of oxidative stress, inflammation, and impaired cellular function on bone healing processes during uncontrolled hyperglycaemia; and therefore, hold considerable promise as novel efficacious therapeutic entities. However, despite such conclusions, several important gaps in our knowledge remain to be addressed, including studies involving more sophisticated enzymic antioxidant-based delivery systems, further mechanistic studies into how these antioxidants exert their desirable reparative effects; and more extensive clinical trial studies into the optimisation of antioxidant therapy dosing, frequency, duration and their subsequent biodistribution and bioavailability. By enhancing our understanding of such crucial issues, we can fully exploit the oxidative stress-neutralising properties of these antioxidants to develop effective antioxidant interventions to mitigate impaired bone healing and reduce the associated complications in such T2DM patient populations

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

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

Evaluation of Cypholophus macrocephalus sap as a treatment for infected cutaneous ulcers in Papua New Guinea

Cypholophus macrocephalus sap is used to treat bacterially infected cutaneous leg ulcers in Papua New Guinea. High resolution LC-MS analysis of the sap revealed it to be rich in sulphated flavonoids. We assessed the effects of the sap on the differentiation and pro-inflammatory anti-microbial responses of M1 macrophages using IL-6 and TNF-α ELISAs and found significant increases in M1 macrophage IL-6 expression with concentrations as low as 243 ng/ml sap. Neutrophil IL-6 and TNF-α expression was also significantly increased but to a lesser degree. Matrix metalloproteinases (MMPs) 1, 2, 8 and 9 which are known to contribute to the toxic nature of wound exudates were inhibited by the sap at 24 μg/ml. The sap was tested with several bacterial species known to colonize cutaneous ulcers in Papua New Guinea but proved not to be active. Cypholophus sap stimulates pro-inflammatory, anti-microbial M1 macrophage and neutrophil responses at very low concentrations, whilst also inhibiting MMPs. The combination of an enhanced innate immune response and inhibition of MMPs in ulcer exudate, may contribute to the eradication of bacteria and healing of these infected ulcers. The sap concentrations used in these assays are readily achievable in an in vivo context

Assessment of Lab4P Probiotic Effects on Cognition in 3xTg-AD Alzheimer’s Disease Model Mice and the SH-SY5Y Neuronal Cell Line

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer’s disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with β-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies

Ficus septica exudate, a traditional medicine used in Papua New Guinea for treating infected cutaneous ulcers: in vitro evaluation and clinical efficacy assessment by cluster randomised trial

Background and objectives: Infected cutaneous ulcers are major health problems for children living in rural areas of Papua New Guinea. The inaccessibility of affected populations and lack of access to basic healthcare, make a local plant-based therapy an attractive treatment option. We assessed Ficus septica exudate in biological assays relevant to wound healing. We then carried out a clinical trial to determine the exudate's efficacy in healing small cutaneous ulcers compared with Savlon antiseptic cream, and soap and water washing. Methods: Pre-clinical in vitro assessment of the exudate was carried out using assays to monitor the pro-inflammatory responses of M1 macrophages and neutrophils, antibacterial assays using known ulcer pathogens, an Ames test for mutagenicity and LC-MS chemical analysis of the exudate. An open label cluster-randomised clinical trial was performed, enrolling participants from three different clusters with skin lesions less than 1 cm in diameter. Each cluster comprising 50 participants was randomly assigned to one of three treatment arms namely topical exudate, topical Savlon antiseptic cream, and standard care (soap and water treatment), all administered daily for 2 days. The primary outcome was clinical healing/improvement measured at days 7 and 14, assessed by three dermatologists using blinded photographs. The primary analysis was assessed as non-inferiority of F. septica treatment based on the risk difference for healing/improvement. Results: In vitro, the exudate which is rich in the alkaloid ficuseptine, was found to be non-mutagenic whilst also inhibiting pro-inflammatory responses and exhibiting antibacterial activity. When administered to participants enrolled in the clinical trial, no significant differences were observed between the healing efficacy of F. septica exudate and the two comparator treatments (Savlon antiseptic cream and soap/water treatment). At day 14, but not at day 7, the efficacy of F. septica exudate for healing/improving the ulcers was non-inferior to Savlon antiseptic cream or water/soap treatment. Conclusions: F. septica exudate is non-mutagenic and has both bactericidal and anti-inflammatory properties. When applied topically to small cutaneous ulcers, the exudate has a healing effect that is non-inferior to Savlon antiseptic cream and standard treatment with soap and water at day 14. Our findings, which should be confirmed in larger clinical trials, have important public health implications