A recombinant signalling-selective activated protein C that lacks anticoagulant activity is efficacious and safe in cutaneous wound preclinical models.

Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent. 3K3A-APC, like wild-type APC, demonstrated positive effects on proliferation of human skin cells (keratinocytes, endothelial cells and fibroblasts). Similarly it also increased matrix metollaproteinase-2 activation in keratinocytes and fibroblasts. Topical 3K3A-APC treatment at 10 or 30 µg both accelerated mouse wound healing when culled on Day 11. And at 10 µg, it was superior to APC and had half the dermal wound gape compared to control. Further testing was conducted in excisional porcine wounds due to their congruence to human skin. Here, 3K3A-APC advanced macroscopic healing in a dose-dependent manner (100, 250 and 500 µg) when culled on Day 21. This was histologically corroborated by greater collagen maturity, suggesting more advanced remodelling. A non-interference arm of this study found no evidence that topical 3K3A-APC caused either any significant systemic side-effects or any significant leakage into the circulation. However the female pigs exhibited transient and mild local reactions after treatments in week three, which did not impact healing. Overall these preclinical studies support the hypothesis that 3K3A-APC merits future human wound studies.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1ß, IL-6 and TGF-ß1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Deficiency of protease-activated receptor (PAR) 1 and PAR2 exacerbates collagen-induced arthritis in mice via differing mechanisms.

OBJECTIVES: Protease-activated receptor (PAR) 1 and PAR2 have been implicated in RA, however their exact role is unclear. Here, we detailed the mechanistic impact of these receptors on the onset and development of inflammatory arthritis in murine CIA and antigen-induced arthritis (AIA) models. METHODS: CIA or AIA was induced in PAR1 or PAR2 gene knockout (KO) and matched wild type mice. The onset and development of arthritis was monitored clinically and histologically. Immune cells, cytokines and MMPs were detected by ELISA, zymography, flow cytometry, western blot or immunohistochemistry. RESULTS: In CIA, PAR1KO and PAR2KO exacerbated arthritis, in opposition to their effects in AIA. These deficient mice had high plasma levels of IL-17, IFN-γ, TGF-ß1 and MMP-13, and lower levels of TNF-α; T cells and B cells were higher in both KO spleen and thymus, and myeloid-derived suppressor cells were lower only in PAR1KO spleen, when compared with wild type cells. Th1, Th2 and Th17 cells were lower in PAR1KO spleens cells, whereas Th1 and Th2 cells were lower and Th17 cells higher in both KO thymus cells, when compared with wild type cells. PAR1KO synovial fibroblasts proliferated faster and produced the most abundant MMP-9 amongst three type cells in the control, lipopolysaccharides or TNF stimulated conditions. CONCLUSION: This is the first study demonstrated that deficiency of PAR1 or PAR2 aggravates inflammatory arthritis in CIA. Furthermore, the protective functions of PAR1 and PAR2 in CIA likely occur via differing mechanisms involving immune cell differentiation and cytokines/MMPs.

Activated Protein C in Cutaneous Wound Healing: From Bench to Bedside.

Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions, anti-apoptosis, and endothelial and epithelial barrier stabilisation. Such beneficial effects have made APC an attractive target of research in a plethora of physiological and pathophysiological processes. Of note, the past decade or so has seen the emergence of its roles in cutaneous wound healing-a complex process involving inflammation, proliferation and remodelling. This review will highlight APC's functions and mechanisms, and detail its pre-clinical and clinical studies on cutaneous wound healing.

Inflammation in Chronic Wounds.

Non-healing chronic wounds present a major biological, psychological, social, and financial burden on both individual patients and the broader health system. Pathologically extensive inflammation plays a major role in the disruption of the normal healing cascade. The causes of chronic wounds (venous, arterial, pressure, and diabetic ulcers) can be examined through a juxtaposition of normal healing and the rogue inflammatory response created by the common components within chronic wounds (ageing, hypoxia, ischaemia-reperfusion injury, and bacterial colonisation). Wound bed care through debridement, dressings, and antibiotics currently form the basic mode of treatment. Despite recent setbacks, pharmaceutical adjuncts form an interesting area of research.

Dermal Fibroblast Heterogeneity and Its Contribution to the Skin Repair and Regeneration.

Significance: Dermal fibroblasts are the major cell type in the skin's dermal layer. These cells originate from distinct locations of the embryo and reside in unique niches in the dermis. Different dermal fibroblasts exhibit distinct roles in skin development, homeostasis, and wound healing. Therefore, these cells are becoming attractive candidates for cell-based therapies in wound healing. Recent Advances: Human skin dermis comprises multiple fibroblast subtypes, including papillary, reticular, and hair follicle-associated fibroblasts, and myofibroblasts after wounding. Recent studies reveal that these cells play distinct roles in wound healing and contribute to diverse healing outcomes, including nonhealing chronic wound or excessive scar formation, such as hypertrophic scars (HTS) and keloids, with papillary fibroblasts having antiscarring and reticular fibroblast scar-forming properties. Critical Issues: The identities and functions of dermal fibroblast subpopulations in many respects remain unknown. In this review, we summarize the current understanding of dermal fibroblast heterogeneity, including their defined cell markers and dermal niches, dynamic changes, and contributions to skin wound healing, with the emphasis on scarless healing, healing with excessive scars (HTS and keloids), chronic wounds, and the potential application of this heterogeneity for developing cell-based therapies that allow wounds to heal faster with less scarring. Future Directions: Heterogeneous dermal fibroblast populations and their functions are poorly characterized. Refining and advancing our understanding of dermal fibroblast heterogeneity and their participation in skin homeostasis and wound healing may create potential therapeutic applications for nonhealing chronic wounds or wounds that heal with excessive scarring.

Early protein C activation is reflective of burn injury severity and plays a critical role in inflammatory burden and patient outcomes.

BACKGROUND: Navigating the complexities of a severe burn injury is a challenging endeavour where the natural course of some patients can be difficult to predict. Straddling both the coagulation and inflammatory cascades that feature strongly in the burns systemic pathophysiology, we propose the pleiotropic protein C (PC) system may produce a viable biomarker to assist traditional evaluation methods for diagnostic and prognostic purposes. METHODS: We enrolled 86 patients in a prospective observational cohort study. Over three weeks, serial blood samples were taken and measured for PC, activated (A)PC, their receptor endothelial protein C receptor (EPCR), and a panel of inflammatory cytokines including C-reactive protein (CRP), tumour necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-8, and IL-17. Their temporal trends were analysed alongside clinical factors including burn size, burn depth, presence of inhalational injury, and a composite outcome of requiring increased support. RESULTS: (i) APC increased from a nadir on Day 3 (2.3±2.1ng/mL vs 4.1±2.5ng/mL by Day 18, p<0.0005), only becoming appropriately correlated to PC from Day 6 onwards (r=0.412-0.721, p<0.05 for all Days 6-21). (ii) This early disturbance in the PC system was amplified in the more severe burns (≥30% total body surface area, predominantly full thickness, or with inhalational injury), which were characterised by a marked fall in PC activation (approximated by APC/PC ratio) and APC levels during Days 0-3 with low unchanged PC levels. Critically low levels of this cytoprotective agent was associated with greater inflammatory burden, as reflected by significantly elevated CRP, IL-6, and IL-8 levels in the more severe compared to less severe burns, and by negative correlations between both PC and APC with most inflammatory cytokines. (iii) Alongside clinical markers of severity at admission (burn size, burn depth, and presence of inhalational injury), only Day 0 APC/PC ratio (OR 1.048 (1.014-1.083), p=0.006), APC (OR 1.364 (1.032-1.803), p=0.029), PC (OR 0.899 (0.849-0.953), p<0.0005), and not any inflammatory cytokines were predictive markers of requiring increased support. Uniquely, decreased Day 0 PC was further individually associated with each increased total length of stay, ICU length of stay, intravenous fluid resuscitation, and total surgeries, as well as possibly mortality. CONCLUSION: An early functional depletion of the cytoprotective PC system provides a physiological link between severe burns and the cytokine storm, likely contributing to worse outcomes. Our findings on the changes in APC, PC and PC activation during this pathological state support APC and PC as early diagnostic and prognostic biomarkers, and provides a basis for their therapeutic potential in severe burn injuries.

A Critical Update of the Assessment and Acute Management of Patients with Severe Burns.

Significance: Burns are debilitating, life threatening, and difficult to assess and manage. Recent advances in assessment and management have occurred since a comprehensive review of the care of patients with severe burns was last published, which may influence research and clinical practice. Recent Advances: Recent advances have occurred in the understanding of burn pathophysiology, which has led to the identification of potential biomarkers of burn severity, such as protein C. There is new evidence about the potential superiority of natural colloids over crystalloids during fluid resuscitation, and new evidence about components of initial and perioperative management, including an improved understanding of pain following burns. Critical Issues: The limitations of the clinical examination highlight the need for imaging and biomarkers to assist in estimations of burn severity. Fluid resuscitation reduces mortality, although there is conjecture over the ideal method. The subsequent perioperative period is associated with significant morbidity and the evidence for preventing and treating pain, infection, and fluid overload while maximizing wound healing potential is described. Future Directions: Promising developments are ongoing in imaging technology, histopathology, biomarkers, and wound healing adjuncts such as hyperbaric oxygen therapy, topical negative pressure therapy, stem cell treatments, and skin substitutes. The greatest benefit from further research on management of patients with burns would most likely be derived from the elucidation of optimal fluid resuscitation protocols, pain management protocols, and surgical techniques from randomized controlled trials.

Plasma protein C levels are directly associated with better outcomes in patients with severe burns.

Protein C circulates in human plasma to regulate inflammation and coagulation. It has shown a crucial role in wound healing in animals, and low plasma levels predict the presence of a wound in diabetic patients. However, no detailed study has measured protein C levels in patients with severe burns over the course of a hospital admission. A severe burn is associated with dysfunction of inflammation and coagulation as well as a significant risk of morbidity and mortality. The current methods of burn assessment have shortcomings in reliability and have limited prognostic value. The discovery of a biomarker that estimates burn severity and predicts clinical events with greater accuracy than current methods may improve management, resource allocation and patient counseling. This is the first study to assess the potential role of protein C as a biomarker of burn severity. We measured the plasma protein C levels of 86 patients immediately following a severe burn, then every three days over the first three weeks of a hospital admission. We also analysed the relationships between burn characteristics, blood test results including plasma protein C levels and clinical events. We used a primary composite outcome of increased support utilisation defined as: a mean intravenous fluid administration volume of five litres or more per day over the first 72 h of admission, a length of stay in the intensive care unit of more than four days, or greater than four surgical procedures during admission. The hypothesis was that low protein C levels would be negatively associated with increased support utilisation. At presentation to hospital after a severe burn, the mean plasma protein C level was 76 ± 20% with a range of 34-130% compared to the normal range of 70-180%. The initial low can be plausibly explained by impaired synthesis, increased degradation and excessive consumption of protein C following a burn. Levels increased gradually over six days then remained at a steady-state until the end of the inpatient study period, day 21. A multivariable regression model (Nagelkerke's R2 = 0.83) showed that the plasma protein C level on admission contributed the most to the ability of the model to predict increased support utilisation (OR = 0.825 (95% CI = 0.698-0.977), P = 0.025), followed by burn size (OR = 1.252 (95% CI = 1.025-1.530), P = 0.027), burn depth (partial thickness was used as the reference, full thickness OR = 80.499 (1.569-4129.248), P = 0.029), and neutrophil count on admission (OR = 1.532 (95% CI = 0.950-2.473), P = 0.08). Together, these four variables predicted increased support utilisation with 93.2% accuracy, 83.3% sensitivity and 97.6% specificity. However if protein C values were disregarded, only 49.5% of the variance was explained, with 82% accuracy, 63% sensitivity and 91.5% specificity. Thus, protein C may be a useful biomarker of burn severity and study replication will enable validation of these novel findings.

Delivery systems of current biologicals for the treatment of chronic cutaneous wounds and severe burns.

While wound therapy remains a clinical challenge in current medical practice, much effort has focused on developing biological therapeutic approaches. This paper presents a comprehensive review of delivery systems for current biologicals for the treatment of chronic wounds and severe burns. The biologicals discussed here include proteins such as growth factors and gene modifying molecules, which may be delivered to wounds free, encapsulated, or released from living systems (cells, skin grafts or skin equivalents) or biomaterials. Advances in biomaterial science and technologies have enabled the synthesis of delivery systems such as scaffolds, hydrogels and nanoparticles, designed to not only allow spatially and temporally controlled release of biologicals, but to also emulate the natural extracellular matrix microenvironment. These technologies represent an attractive field for regenerative wound therapy, by offering more personalised and effective treatments.

The differential expression of protease activated receptors contributes to functional differences between dark and fair keratinocytes.

BACKGROUND: Dark skin has different properties in comparison to fair skin. Melanocytes have been shown to partly contribute to these differences, however, the involvement of keratinocytes from dark or fair skin is not well demonstrated. OBJECTIVES: This study investigated the proliferation and barrier function of dark keratinocytes (DK) and fair keratinocytes (FK), and the role of protease activated receptor (PAR)1 and PAR2. METHODS: DK and FK were isolated from human neonatal foreskins. Cells were treated with PAR1/PAR2 agonists or antagonists, proliferation was measured by BrdU assay; permeability by the flux of FITC-dextran; protein expression by immunostaining or western blot. RESULTS: When compared to FK, DK proliferated significantly slower; had higher cell permeability; expressed less phosphorylated (P)-ERK/ERK, caspase-14, E-cadherin, tissue growth factor (TGF)-ß3 and PAR1; and expressed more PAR2, and matrix metalloproteinase (MMP)-9. Activation of PAR1 or inhibition of PAR2 stimulated cell proliferation and ERK activation, and in concordance inhibition of PAR1 or activation of PAR2 suppressed cell proliferation and ERK activation in both DK and FK. Inhibition of PAR2 decreased and inhibition of PAR1 increased cell permeability. In foreskin sections, the epidermis of dark foreskin expressed less caspase-14 and the same level but different distribution of E-cadherin, when compared to fair foreskin. CONCLUSIONS: These data highlight functional differences in proliferation and barrier integrity between HK and FK that are partly associated with their differential expression of PAR1 and PAR2.