Endothelial Protein C Receptor and Its Impact on Rheumatic Disease.

Endothelial Protein C Receptor (EPCR) is a key regulator of the activated protein C anti-coagulation pathway due to its role in the binding and activation of this protein. EPCR also binds to other ligands such as Factor VII and X, γδ T-cells, plasmodium falciparum erythrocyte membrane protein 1, and Secretory group V Phospholipases A2, facilitating ligand-specific functions. The functions of EPCR can also be regulated by soluble (s)EPCR that competes for the binding sites of membrane-bound (m)EPCR. sEPCR is created when mEPCR is shed from the cell surface. The propensity of shedding alters depending on the genetic haplotype of the EPCR gene that an individual may possess. EPCR plays an active role in normal homeostasis, anti-coagulation pathways, inflammation, and cell stemness. Due to these properties, EPCR is considered a potential effector/mediator of inflammatory diseases. Rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus are autoimmune/inflammatory conditions that are associated with elevated EPCR levels and disease activity, potentially driven by EPCR. This review highlights the functions of EPCR and its contribution to rheumatic diseases.

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.

EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

OBJECTIVES: Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. METHODS: CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. RESULTS: EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro, EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. CONCLUSION: Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

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.

Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

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.

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.

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.

Skin Barrier Dysregulation in Psoriasis.

The skin barrier is broadly composed of two elements-a physical barrier mostly localised in the epidermis, and an immune barrier localised in both the dermis and epidermis. These two systems interact cooperatively to maintain skin homeostasis and overall human health. However, if dysregulated, several skin diseases may arise. Psoriasis is one of the most prevalent skin diseases associated with disrupted barrier function. It is characterised by the formation of psoriatic lesions, the aberrant differentiation and proliferation of keratinocytes, and excessive inflammation. In this review, we summarize recent discoveries in disease pathogenesis, including the contribution of keratinocytes, immune cells, genetic and environmental factors, and how they advance current and future treatments.

Limited utility of novel serological biomarkers in patients newly suspected of having giant cell arteritis.

AIM: Diagnosing and monitoring vascular activity in giant cell arteritis (GCA) is difficult due to the paucity of specific serological biomarkers. We assessed the utility of 8 novel biomarkers in an inception cohort of newly suspected GCA patients. METHOD: Consecutive patients were enrolled between May 2016 and December 2017. Serum was collected within 72 hours of commencing corticosteroids and at 6 months. It was analyzed for levels of intra-cellular adhesion molecule 1, vascular endothelial growth factor (VEGF), pentraxin 3, von Willebrand factor and procalcitonin (5-plex R&D Systems multiplex assay) and interleukin (IL)6, IL12 and interferon-γ (high-sensitivity 3-plex ProcartaPlex multiplex assay). A GCA specific positron emission tomography / computed tomography (PET/CT) scan was performed at enrolment with uptake in each vascular territory graded and summed to derive a total vascular score (TVS). RESULTS: For the 63 patients enrolled, 12 (19%) had a final diagnosis of biopsy-positive GCA and a further 9 had a clinical diagnosis of biopsy-negative GCA. None of the 8 biomarkers was significantly higher in GCA patients compared with those with alternative diagnoses, or demonstrated a positive correlation with the PET/CT TVS. This was in contrast to the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) which were higher in the biopsy-positive GCA cohort (P < .04) and showed weak positive correlations with the TVS (correlation coefficient 0.34, P < .01). Procalcitonin did not distinguish between GCA and infection. Concentrations of CRP, ESR, VEGF and pentraxin 3 decreased between diagnosis and 6 months in GCA patients. CONCLUSION: This study did not identify new serological biomarkers to assist in diagnosing or assessing the vasculitis burden in GCA.

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.

Printability, Durability, Contractility and Vascular Network Formation in 3D Bioprinted Cardiac Endothelial Cells Using Alginate-Gelatin Hydrogels.

BACKGROUND: 3D bioprinting cardiac patches for epicardial transplantation are a promising approach for myocardial regeneration. Challenges remain such as quantifying printability, determining the ideal moment to transplant, and promoting vascularisation within bioprinted patches. We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural self-organisation into networks. METHODS: We evaluated 3D-bioprinted double-layer patches using alginate/gelatine (AlgGel) hydrogels and three extrusion bioprinters (REGEMAT3D, INVIVO, BIO X). Bioink contained either neonatal mouse cardiac cell spheroids or free (not-in-spheroid) human coronary artery endothelial cells with fibroblasts, mixed with AlgGel. To test the effects on durability, some patches were bioprinted as a single layer only, cultured under minimal movement conditions or had added fibroblast-derived extracellular matrix hydrogel (AlloECM). Controls included acellular AlgGel and gelatin methacryloyl (GELMA) patches. RESULTS: Printability was similar across bioprinters. For AlgGel compared to GELMA: resolutions were similar (200-700 µm line diameters), printing accuracy was 45 and 25%, respectively (AlgGel was 1.7x more accurate; p < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); p = 0.36. For durability, AlgGel patch median survival in culture was 14 days (IQR:10-27) overall which was not significantly affected by bioprinting system or cellular content in patches. We identified three factors which reduced durability in culture: (1) bioprinting one layer depth patches (instead of two layers); (2) movement disturbance to patches in media; and (3) the addition of AlloECM to AlgGel. Cells were viable after bioprinting followed by 28 days in culture, and all BIO X-bioprinted mouse cardiac cell spheroid patches presented contractile activity starting between day 7 and 13 after bioprinting. At day 28, endothelial cells in hydrogel displayed organisation into endothelial network-like structures. CONCLUSION: AlgGel-based 3D bioprinted heart patches permit cardiomyocyte contractility and endothelial cell structural self-organisation. After bioprinting, a period of 2 weeks maturation in culture prior to transplantation may be optimal, allowing for a degree of tissue maturation but before many patches start to lose integrity. We quantify AlgGel printability and present novel factors which reduce AlgGel patch durability (layer number, movement, and the addition of AlloECM) and factors which had minimal effect on durability (bioprinting system and cellular patch content).

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.

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.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

OBJECTIVES: To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. METHODS: RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. RESULTS: In vitro, APC inhibited IL-1ß, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1ß, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. CONCLUSION: APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Isolation of Human Skin Epidermal Stem Cells Based on the Expression of Endothelial Protein C Receptor.

Skin epidermis is a continuous self-renewal tissue maintained by interfollicular epidermal stem cells (IESCs) that reside in the basal layer of epidermis. IESCs also contribute to the repair and regeneration of the epidermis during wound healing. The great plasticity and easy accessibility afforded by IESCs make them a promising source of stem cells for scientific research and clinical applications. Thus, simple methods to isolate and define pure and viable IESCs are a valuable resource. Here, we provide a method for isolating IESCs from human skin epidermis. This method relies exclusively on selecting cells with a higher expression of the endothelial protein C receptor, using fluorescence-activated cell sorting.

Interleukin 29 inhibits RANKL-induced osteoclastogenesis via activation of JNK and STAT, and inhibition of NF-κB and NFATc1.

Interleukin (IL)-29 is known to modulate immune functions of monocytes or macrophages. In this study, we investigated the effect and its underlying mechanism of IL-29 on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis using murine macrophage cell line RAW264.7 cells and bone-marrow-derived monocyte/macrophage precursor cells (BMMs), and human peripheral blood mononuclear cells (PBMCs). In response to human recombinant IL-29, cell viability and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry; the osteoclast formation and activity by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay, respectively; the expression and activation of molecules that associated with osteoclastogenesis by real time-PCR, immunoblotting or immunofluorescent analysis. IL-28 receptor α (IL-28Rα), a specific receptor of IL-29 was expressed on RAW264.7 cells. Although IL-29 did not affect the viability and apoptosis of RAW264.7 cells, it inhibited multinucleated cells in the differentiation of osteoclastogenesis, the bone-resorbing activity of mature osteoclasts and osteoclastic specific genes expression including TRAP, cathepsin K (CTSK), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), C-Fos and matrix metallopeptidase 9 (MMP-9). This inhibitory effect of IL-29 was confirmed on BMMs and PBMCs and mediated via IL-28Rα through the activation of Stat1 and 3 and the suppression of nuclear factor kappa B (NF-κB) and NFATc1 nuclear translocation in RAW264.7 cells. In conclusion, IL-29 inhibited osteoclastogenesis via activation of STAT signaling pathway, prevention of NF-κB activation and NFATc1 translocation, and suppression of downstream osteoclastogenic genes expression.

Lupeol, a Pentacyclic Triterpene, Promotes Migration, Wound Closure, and Contractile Effect In Vitro: Possible Involvement of PI3K/Akt and p38/ERK/MAPK Pathways.

Skin wound healing is a dynamic and complex process involving several mediators at the cellular and molecular levels. Lupeol, a phytoconstituent belonging to the triterpenes class, is found in several fruit plants and medicinal plants that have been the object of study in the treatment of various diseases, including skin wounds. Various medicinal properties of lupeol have been reported in the literature, including anti-inflammatory, antioxidant, anti-diabetic, and anti-mutagenic effects. We investigated the effects of lupeol (0.1, 1, 10, and 20 µg/mL) on in vitro wound healing assays and signaling mechanisms in human neonatal foreskin keratinocytes and fibroblasts. Results showed that, at high concentrations, Lupeol reduced cell proliferation of both keratinocytes and fibroblasts, but increased in vitro wound healing in keratinocytes and promoted the contraction of dermal fibroblasts in the collagen gel matrix. This triterpene positively regulated matrix metalloproteinase (MMP)-2 and inhibited the NF-κB expression in keratinocytes, suggesting an anti-inflammatory effect. Lupeol also modulated the expression of keratin 16 according to the concentration tested. Additionally, in keratinocytes, lupeol treatment resulted in the activation of Akt, p38, and Tie-2, which are signaling proteins involved in cell proliferation and migration, angiogenesis, and tissue repair. These findings suggest that lupeol has therapeutic potential for accelerating wound healing.

Author Correction: Bisphosphonate enhances TRAIL sensitivity to human osteosarcoma cells via death receptor 5 upregulation.

After publication of this article, the authors noticed an error in the figure section.

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.