Donna Beer-Stolz

Surface Characterization of Extracellular Matrix Scaffolds

Extracellular matrix (ECM) scaffolds prepared from different tissue sources or using different methods have been demonstrated to have distinctive effects upon cell adhesion patterns and the ability to support and maintain differentiated phenotypes. It is unknown whether the molecular composition or the ultrastructure of the ECM plays a greater role in determining the phenotype of the cells with which it comes into contact. However, when implanted, the topology and ligand landscape of the material will determine the host molecules that bind and the type and behavior of cells that mediate the host response. Therefore, a comprehensive understanding of surface characteristics is essential in the design of scaffolds for specific clinical applications. The surface characteristics of ECM scaffolds derived from porcine urinary bladder, small intestine, and liver as well as the effects of two commonly used methods of chemical cross-linking upon UBM were investigated. Electron microscopy and time of flight secondary ion mass spectroscopy were used to examine the surface characteristics of the scaffolds. The results show that ECM scaffolds have unique morphologic and structural properties which are dependant on the organ or tissue from which the scaffold is harvested. Furthermore, the results show that the surface characteristics of an ECM scaffold are changed through chemical cross-linking.

Ethyl pyruvate decreases HMGB1 release and ameliorates murine colitis

Signals from stressed cells and the enteric microbiota activate macrophages and dendritic cells and mediate intestinal inflammation. HMGB1 serves as an immunogenic stimuli causing release of inflammatory cytokines by myeloid cells. Ethyl pyruvate inhibits secretion of HMGB1 and improves survival in models of endotoxemia and hemorrhagic shock. We reasoned that ethyl pyruvate may be protective in colitis, which involves similar inflammatory pathways. In IL‐10−/− mice with established chronic colitis, ethyl pyruvate administration ameliorated colitis and reduced intestinal cytokine production. IL‐10−/− mice demonstrated increased intestinal HMGB1 expression and decreased expression of RAGE compared with wild‐type mice. Fecal HMGB1 levels were decreased in ethyl pyruvate‐treated mice. Furthermore, ethyl pyruvate induced HO‐1 expression in intestinal tissue. In TNBS‐induced colitis, intrarectal administration of ethyl pyruvate resulted in amelioration of colitis and reduced intestinal cytokine production. In LPS‐activated murine macrophages, ethyl pyruvate decreased expression of IL‐12 p40 and NO production but did not affect IL‐10 levels. Ethyl pyruvate did not inhibit nuclear translocation of NF‐κB family members but attenuated NF‐κB DNA binding. Additionally, ethyl pyruvate induced HO‐1 mRNA and protein expression and HO‐1 promoter activation. Moreover, ethyl pyruvate prevented nuclear‐to‐cytoplasmic translocation of HMGB1. In conclusion, the HMGB1/RAGE pathway has pathophysiologic and diagnostic significance in experimental colitis. Ethyl pyruvate and other strategies to inhibit HMGB1 release and function represent promising interventions in chronic inflammatory diseases.

Histones Activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests that it also plays a role in inflammation driven by endogenous danger-associate molecular pattern molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. In this article, we report that both NLRP3 and its downstream target caspase-1 are activated during I/R and are essential for hepatic I/R injury, because both NLRP3 and caspase-1 knockout mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on caspase-1 expression in liver nonparenchymal cells. Although upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through TLR9. This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and the activation of innate immunity during sterile inflammation.

Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands

Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30–100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferonγ (IFNγ) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs.

Interleukin‐6 inhibits oxidative injury and necrosis after extreme liver resection

Extreme hepatectomy or resection of more than 80% of liver mass often leads to liver failure and death and is a major limitation to therapeutic liver resection for patients with liver tumors. We sought to define the mechanisms leading to liver failure and to determine the utility of interleukin‐6 (IL‐6) administration to improve outcomes. Mice were injected with Chinese hamster ovary cells expressing human IL‐6 or no recombinant protein, or were administered recombinant IL‐6 or carrier by osmotic mini‐pump. Mice were then subjected to 70% or 87% hepatectomy. Light and electron microscopy of liver sections after 87% hepatectomy showed ballooning hepatocytes, vacuolar changes, and mitochondrial abruption, with absence of anoikic nuclei. No significant activation of executor caspases or DNA laddering was observed, although a dramatic decrease in cellular adenosine triphosphate (ATP) stores was measured, suggesting cell death was by a necrotic pathway involving mitochondrial dysfunction. A large increase in protein oxidation was observed, indicative of significant oxidative stress. IL‐6 treatment before 87% hepatectomy resulted in less biochemical and histological evidence of liver injury as well as earlier proliferating chain nuclear antigen (PCNA) expression and accelerated recovery of liver mass. IL‐6 pretreatment induced the antioxidative injury proteins, ref‐1 and GPX1, decreased protein oxidation, vacuolar changes and leakage of mitochondrial products, improved ATP stores, and maintained cellular ultrastructure after 87% hepatectomy. Conclusion: Massive oxidative injury and mitochondrial dysfunction occurs in the liver after extreme hepatectomy. IL‐6 improves recovery and survival from extreme liver resection by enhancing pro‐growth pathways, reducing oxidative stress, and maintaining mitochondrial function. (HEPATOLOGY 2007.)

Hepatocyte-specific high-mobility group box 1 deletion worsens the injury in liver ischemia/reperfusion: a role for intracellular high-mobility group box 1 in cellular protection.

High‐mobility group box 1 (HMGB1) is an abundant chromatin‐associated nuclear protein and released into the extracellular milieu during liver ischemia‐reperfusion (I/R), signaling activation of proinflammatory cascades. Because the intracellular function of HMGB1 during sterile inflammation of I/R is currently unknown, we sought to determine the role of intracellular HMGB1 in hepatocytes after liver I/R. When hepatocyte‐specific HMGB1 knockout (HMGB1‐HC‐KO) and control mice were subjected to a nonlethal warm liver I/R, it was found that HMGB1‐HC‐KO mice had significantly greater hepatocellular injury after I/R, compared to control mice. Additionally, there was significantly greater DNA damage and decreased chromatin accessibility to repair with lack of HMGB1. Furthermore, lack of hepatocyte HMGB1 led to excessive poly(ADP‐ribose)polymerase 1 activation, exhausting nicotinamide adenine dinucleotide and adenosine triphosphate stores, exacerbating mitochondrial instability and damage, and, consequently, leading to increased cell death. We found that this was also associated with significantly more oxidative stress (OS) in HMGB1‐HC‐KO mice, compared to control. Increased nuclear instability led to a resultant increase in the release of histones with subsequently more inflammatory cytokine production and organ damage through activation of Toll‐like receptor 9. Conclusion: The lack of HMGB1 within hepatocytes leads to increased susceptibility to cellular death after OS conditions. (Hepatology 2014;59:1984–1997)

A Critical Role for TLR4 Induction of Autophagy in the Regulation of Enterocyte Migration and the Pathogenesis of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4ΔIEC) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7ΔIEC), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7ΔIEC mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

Eosinophils Oxidize Damage-Associated Molecular Pattern Molecules Derived from Stressed Cells

Eosinophils (Eos) are found at increased numbers within necrotic areas of tumors. We show that necrotic material from cell lysates containing damage-associated molecular pattern molecules induce eosinophil degranulation (release of major basic protein and eosinophil peroxidase) and enhance their oxidative burst while the stimulatory capacity of cell lysates is significantly diminished following oxidation. High mobility group box 1 (HMGB1), a prototypic damage-associated molecular pattern molecule, released following necrosis but not apoptosis, induced a similar effect on Eos. Additionally, we demonstrate that HMGB1 enhances eosinophil survival and acts as a chemoattractant. Consistently, we show that Eos express an HMGB1 receptor, the receptor for advanced glycation end product, and that anti-receptor for advanced glycation end product could diminish the HMGB1-mediated effects. Of all tested biologic activities, Eos respond most sensitively to the presence of necrotic material including HMGB1 with generation of peroxide. We postulate that Eos “sense” necrotic cell death, migrating to and responding to areas of tissue injury/necrosis. Oxidation of cell lysates reduces their biologic activity when compared with native lysates. We postulate that eosinophil-associated modulation of immunity within tumor and other damaged tissues may be primarily by promoting oxidative degradation of necrotic material. Novel therapeutic strategies may be considered by advancing oxidative denaturation of released necrotic material using Eos or other aerobic strategies.

Ethyl pyruvate administration inhibits hepatic tumor growth

EP is a potent inhibitor of HMGB1 release that has significant anti–inflammatory activities and exerts a protective effect in animal models of inflammation. As inflammation is linked to cancer growth, we hypothesized that EP would have anti–tumor activity and explored its effects in a liver tumor model. Mice injected intraportally with MC38 colorectal cancer cells led to the growth of visible hepatic tumors within 2 weeks. Pretreatment with EP 30 min prior to infusion of tumor cells and continuing daily for 9 days inhibited tumor growth significantly in a dose–dependent manner, with 80 mg/kg EP achieving >70% reduction in the number of tumor nodules when compared with untreated animals. Delayed treatment with EP also suppressed tumor growth significantly, although to a lesser extent. Tumors had early, marked leukocytic infiltrates, and EP administration decreased innate (NK cells, monocytes) and adaptive (T and B cell lymphocytic) immune cell infiltrates acutely and significantly in the liver. Serum IL–6 and HMGB1 levels, which were elevated following tumor injection, were decreased significantly in EP–treated animals. Tumors showed an increase in apoptosis in EP–treated mice, and tumor cells treated in vitro with EP had marked increases in LC3–II and cleaved PARP, consistent with enhanced autophagic flux and apoptosis. Thus, EP inhibition of tumor growth in the liver was mediated by tumor (induction of apoptosis) and host (decreased inflammation) effects. EP administration may have a therapeutic role in the treatment of cancer in conjunction with other therapeutic agents.

Mitochondrial Structure, Function and Dynamics Are Temporally Controlled by c-Myc

Although the c-Myc (Myc) oncoprotein controls mitochondrial biogenesis and multiple enzymes involved in oxidative phosphorylation (OXPHOS), the coordination of these events and the mechanistic underpinnings of their regulation remain largely unexplored. We show here that re-expression of Myc in myc−/− fibroblasts is accompanied by a gradual accumulation of mitochondrial biomass and by increases in membrane polarization and mitochondrial fusion. A correction of OXPHOS deficiency is also seen, although structural abnormalities in electron transport chain complexes (ETC) are not entirely normalized. Conversely, the down-regulation of Myc leads to a gradual decrease in mitochondrial mass and a more rapid loss of fusion and membrane potential. Increases in the levels of proteins specifically involved in mitochondrial fission and fusion support the idea that Myc affects mitochondrial mass by influencing both of these processes, albeit favoring the latter. The ETC defects that persist following Myc restoration may represent metabolic adaptations, as mitochondrial function is re-directed away from producing ATP to providing a source of metabolic precursors demanded by the transformed cell.