Stefan Bohr

Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure

Drug-induced liver injury (DILI) limits the development and application of many therapeutic compounds and presents major challenges to the pharmaceutical industry and clinical medicine. Acetaminophen-containing compounds are among the most frequently prescribed drugs and are also the most common cause of DILI. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and acetaminophen-induced hepatotoxicity. We demonstrate that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation and death caused by liver-toxic drugs. We identify a small-molecule inhibitor of Cx32 that protects against liver failure and death in wild-type mice when co-administered with known hepatotoxic drugs. These findings indicate that gap junction inhibition could provide a pharmaceutical strategy to limit DILI and improve drug safety.

Resolvin D2 prevents secondary thrombosis and necrosis in a mouse burn wound model.

Deep partial thickness burns are subject to delayed necrosis of initially viable tissues surrounding the primary zone of thermally induced coagulation, which results in an expansion of the burn wound, both in area and depth, within 48 hours postburn. Neutrophil sequestration and activation leading to microvascular damage is thought to mediate this secondary tissue damage. Resolvins, a class of endogenous mediators derived from omega‐3 polyunsaturated fatty acids, have been shown to regulate the resolution of inflammation. We hypothesized that exogenous resolvins could mitigate the deleterious impact of the inflammatory response in burn wounds. Using two different mouse burn injury models involving significant partial thickness injuries, we found that a systemically administered single dose of resolvin D2 (RvD2) as low as 25 pg/g bw given within an interval of up to 4 hours postburn effectively prevented thrombosis of the deep dermal vascular network and subsequent dermal necrosis. By preserving the microvascular network, RvD2 enhanced neutrophil access to the dermis, but prevented neutrophil‐mediated damage through other anti‐inflammatory actions, including inhibition of tumor necrosis factor‐α, interleukin‐1β, and neutrophil platelet–endothelial cell adhesion molecule‐1. In a clinical context, RvD2 may be therapeutically useful by reducing the need for surgical debridement and the area requiring skin grafting.

Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability.

Background & Aims Emerging data suggest that changes in intestinal permeability and increased gut microbial translocation contribute to the inflammatory pathway involved in nonalcoholic steatohepatitis (NASH) development. Numerous studies have investigated the association between increased intestinal permeability and NASH. Our meta-analysis of this association investigates the underlying mechanism. Methods A meta-analysis was performed to compare the rates of increased intestinal permeability in patients with NASH and healthy controls. To further address the underlying mechanism of action, we studied changes in intestinal permeability in a diet-induced (methionine-and-choline-deficient; MCD) murine model of NASH. In vitro studies were also performed to investigate the effect of MCD culture medium at the cellular level on hepatocytes, Kupffer cells, and intestinal epithelial cells. Results Nonalcoholic fatty liver disease (NAFLD) patients, and in particular those with NASH, are more likely to have increased intestinal permeability compared with healthy controls. We correlate this clinical observation with in vivo data showing mice fed an MCD diet develop intestinal permeability changes after an initial phase of liver injury and tumor necrosis factor-α (TNFα) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNFα production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNFα in altering intestinal permeability, we found that mice genetically resistant to TNFα-myosin light chain kinase (MLCK)–induced intestinal permeability changes fed an MCD diet still develop increased permeability and liver injury. Conclusions Our clinical and experimental results strengthen the association between intestinal permeability increases and NASH and also suggest that an early phase of hepatic injury and inflammation contributes to altered intestinal permeability in a fashion independent of TNFα and MLCK.

Non-thermal, pulsed electric field cell ablation: A novel tool for regenerative medicine and scarless skin regeneration

High voltage, short pulsed electric fields (PEF) is a non-thermal ablation method, in which defined PEF irreversibly destabilize cell membranes, while preserving other tissue components such as the extracellular matrix (ECM). In the present report, we show that PEF ablated rat skin retains its microvascular blood supply and ECM structure. Complete regeneration of epidermis, hair follicles, sebaceous glands, and the panniculus carnosusis observed two months after the ablation. Our results clearly indicate that non-thermal PEF has the potential to be a powerful and novel tool for scarless tissue regeneration.

Alternative erythropoietin-mediated signaling prevents secondary microvascular thrombosis and inflammation within cutaneous burns

Alternate erythropoietin (EPO)–mediated signaling via the heteromeric receptor composed of the EPO receptor and the β-common receptor (CD131) exerts the tissue-protective actions of EPO in various types of injuries. Herein we investigated the effects of the EPO derivative helix beta surface peptide (synonym: ARA290), which specifically triggers alternate EPO-mediated signaling, but does not bind the erythropoietic EPO receptor homodimer, on the progression of secondary tissue damage following cutaneous burns. For this purpose, a deep partial thickness cutaneous burn injury was applied on the back of mice, followed by systemic administration of vehicle or ARA290 at 1, 12, and 24 h postburn. With vehicle-only treatment, wounds exhibited secondary microvascular thrombosis within 24 h postburn, and subsequent necrosis of the surrounding tissue, thus converting to a full-thickness injury within 48 h. On the other hand, when ARA290 was systemically administered, patency of the microvasculature was maintained. Furthermore, ARA290 mitigated the innate inflammatory response, most notably tumor necrosis factor-alpha–mediated signaling. These findings correlated with long-term recovery of initially injured yet viable tissue components. In conclusion, ARA290 may be a promising therapeutic approach to prevent the conversion of partial- to full-thickness burn injuries. In a clinical setting, the decrease in burn depth and area would likely reduce the necessity for extensive surgical debridement as well as secondary wound closure by means of skin grafting. This use of ARA290 is consistent with its tissue-protective properties previously reported in other models of injury, such as myocardial infarction and hemorrhagic shock.

Endothelial Peroxisomal Dysfunction and Impaired Pexophagy Promotes Oxidative Damage in Lipopolysaccharide-Induced Acute Kidney Injury

AIMS We examined that (a) how the endotoxic stress affects peroxisomal function and autophagic degradation of peroxisomes-pexophagy, (b) how a superimposed dysfunction of lysosomes and pexophagy modifies responses to lipopolysaccharide (LPS), and (c) the mechanisms of peroxisomal contribution to renal injury. To accomplish this, we used lysosome-defective Lyst-mice in vivo and primary endothelial cells in vitro, and compared the responses with wild-type (WT) littermates. RESULTS LPS induced pexophagic degradation, followed by proliferation of peroxisomes in WT mice, which was abolished in Lyst-mice. Lyst-mice exhibited impaired activation of catalase, which together with preserved hydrogen peroxide-generating β-oxidation resulted in redox disequilibrium. LPS treatment induced a heightened inflammatory response, increased oxidative damage, and aggravated renal injury in Lyst-mice. Similarly, as in vivo, LPS-activated lysosomal (LYS) pexophagy and transiently repressed peroxisomes in vitro, supported by reduced peroxisomal density in the vicinity of lysosomes. Peroxisomal dynamics was also abolished in lysosome-defective cells, which accumulated peroxisomes with compromised functions and intraorganellar redox imbalance. INNOVATION We demonstrated that pexophagy is a default response to endotoxic injury. However, when LYS dysfunction (a frequent companion of chronic diseases) is superimposed, recycling and functioning of peroxisomes are impaired, and an imbalance between hydrogen peroxide-generating β-oxidation and hydrogen peroxide-detoxifying catalase ensues, which ultimately results in peroxisomal burnout. CONCLUSION Our data strongly suggest that pexophagy, a cellular mechanism per se, is essential in functional maintenance of peroxisomes during LPS exposure. Inhibition of pexophagy results in accumulation of impaired peroxisomes, redox disequilibrium, and aggravated renal damage.

Resolving cancer-stroma interfacial signalling and interventions with micropatterned tumour-stromal assays.

Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumor-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyze bulk and interfacial tumor-stromal signaling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human estrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumor-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumor-stromal interfacial interactions using ER+ MCF-7 breast cancer and bone marrow-derived stromal cells. Reversine suppresses MCF-7 tumor growth and bone metastasis in vivo by reducing tumor stromalization including collagen deposition and recruitment of activated stromal cells. This study advocates μTSA as a platform for studying tumor microenvironmental interactions and cancer field effects with applications in drug discovery and development.

Modulation of cellular stress response via the erythropoietin/CD131 heteroreceptor complex in mouse mesenchymal-derived cells

Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis.Key message• Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131.• ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor.• Oxidative stress and inflammation promote cell surface expression of CD131.• ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes.• Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.

A Novel Resolvin-Based Strategy for Limiting Acetaminophen Hepatotoxicity

Objectives:Acetaminophen (APAP)-induced hepatotoxicity is a major cause of morbidity and mortality. The current pharmacologic treatment for APAP hepatotoxicity, N-acetyl cysteine (NAC), targets the initial metabolite-driven injury but does not directly affect the host inflammatory response. Because of this, NAC is less effective if given at later stages in the disease course. Resolvins, a novel group of lipid mediators shown to attenuate host inflammation, may be a therapeutic intervention for APAP hepatotoxicity.Methods:The temporal patterns of liver injury and neutrophil activation were investigated in a murine model of APAP hepatotoxicity. In addition, the effect of neutrophil depletion and resolvin administration on the severity of liver injury induced by APAP was studied. In vitro studies to investigate the mechanism of resolvin effect on hepatocyte injury and neutrophil adhesion were performed.Results:We demonstrate that hepatic neutrophil activation occurs secondary to the initial liver injury induced directly by APAP. We also show that neutrophil depletion attenuates APAP-induced liver injury, and administration of resolvins hours after APAP challenge not only attenuates liver injury, but also extends the therapeutic window eightfold compared to NAC. Mechanistic in vitro analysis highlights resolvins’ ability to inhibit neutrophil attachment to endothelial cells in the presence of the reactive metabolite of APAP.Conclusions:This study highlights the ability of resolvins to protect against APAP-induced liver injury and extend the therapeutic window compared to NAC. Although the mechanism for resolvin-mediated hepatoprotection is likely multifactorial, inhibition of neutrophil infiltration and activation appears to play an important role.

The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model

In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.