The role of RAGE in the pathogenesis of intestinal barrier dysfunction after hemorrhagic shock

The receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of numerous conditions associated with excessive inflammation. To determine whether RAGE-dependent signaling is important in the development of intestinal barrier dysfunction after hemorrhagic shock and resuscitation (HS/R), C57Bl/6, rage(-/-), or congenic rage(+/+) mice were subjected to HS/R (mean arterial pressure of 25 mmHg for 3 h) or a sham procedure. Twenty-four hours later, bacterial translocation to mesenteric lymph nodes and ileal mucosal permeability to FITC-labeled dextran were assessed. Additionally, samples of ileum were obtained for immunofluorescence microscopy, and plasma was collected for measuring IL-6 and IL-10 levels. HS/R in C57Bl/6 mice was associated with increased bacterial translocation, ileal mucosal hyperpermeability, and high circulating levels of IL-6. All of these effects were prevented when C57Bl/6 mice were treated with recombinant human soluble RAGE (sRAGE; the extracellular ligand-binding domain of RAGE). HS/R induced bacterial translocation, ileal mucosal hyperpermeability, and high plasma IL-6 levels in rage(+/+) but not rage(-/-) mice. Circulating IL-10 levels were higher in rage(-/-) compared with rage(+/+) mice. These results suggest that activation of RAGE-dependent signaling is a key factor leading to gut mucosal barrier dysfunction after HS/R.     

Anti-HMGB1 neutralizing antibody ameliorates gut barrier dysfunction and improves survival after hemorrhagic shock

Intestinal barrier dysfunction occurs following hemorrhagic shock and resuscitation (HS/R). High-mobility group B1 (HMGB1) has been shown to increase the permeability of Caco-2 human enterocyte-like epithelial monolayers in vitro. In this study, we found that serum concentrations of HMGB1 were higher in blood samples obtained from 25 trauma victims with hemorrhagic shock than in 9 normal volunteers. We also studied whether treatment with anti-HMGB1 antibody can ameliorate HS/R-induced gut barrier dysfunction in mice. Animals were shocked by withdrawal of blood to maintain mean arterial pressure at 25 to 30 mmHg for 2 h. After resuscitation with shed blood plus Ringer's lactate solution, the mice were treated with either anti-HMGB1 antibody or nonimmune rabbit IgG. Serum HMGB1 concentrations were significantly higher in trauma victims than control mice. Treatment with anti-HMGB1 antibody improved survival at 24 h and ameliorated the development of ileal mucosal hyperpermeability to FITC-labeled dextran. At 24 h after HS/R, treatment with anti-HMGB1 antibody decreased bacterial translocation to mesenteric lymph nodes and was associated with lower circulating concentrations of IL-6 and IL-10. These data support the notion that HMGB1 is a mediator of HS/R-induced gut barrier dysfunction and suggest that anti-HMGB1 antibodies warrant further evaluation as a therapeutic to ameliorate the morbidity of HS/R in trauma patients.     

Cyclic AMP and Cyclic GMP suppress TNFα-induced hepatocyte apoptosis by inhibiting FADD up-regulation via a protein kinase A-dependent pathway

Cyclic AMP (cAMP) and cyclic GMP (cGMP) suppress apoptosis in many cell types, including hepatocytes. We have previously shown that membrane-permeable cAMP and cGMP analogs attenuate tumor necrosis factor α plus actinomycin D (TNFα/ActD)-induced apoptosis in hepatocytes at a step upstream of caspase activation and cytochrome c release. Recently we have also shown that FADD levels increase 10 folds in response to TNFα/ActD. Therefore we hypothesized that cAMP and cGMP would inhibit FADD upregulation. We show here that cyclic nucleotide analogs dibutyryl cAMP (db-cAMP) and 8-bromo-cGMP (Br-cGMP) inhibit cell death and the cleavages of multiple caspases including caspase-10, -9, -8, -3, and -2, as well as suppress FADD protein up-regulation in TNFα/ActD-induced apoptosis. The inhibitory effects of cAMP were seen at lower concentrations than cGMP. Both cAMP and cGMP prevented FADD overexpression and cell death in hepatocytes transfected with the FADD gene. A protein kinase A (PKA) inhibitor, KT 5720, reversed the inhibition of FADD protein levels induced by cAMP or cGMP. In conclusion, our findings indicate that cAMP and cGMP prevent TNFα/ActD-induced apoptosis in hepatocytes and that this occurs in association with a near complete inhibition of the upregulation of FADD via a PKA-dependent mechanism. Supported by the National Institutes of Health Grant GM-44100 (to T.R.B).     

Applying controlled non-uniform deformation for in vitro studies of cell mechanobiology

Cells within connective tissues routinely experience a wide range of non-uniform mechanical loads that regulate many cell behaviors. In this study, we developed an experimental system to produce complex strain patterns for the study of strain magnitude, anisotropy, and gradient effects on cells in culture. A standard equibiaxial cell stretching system was modified by affixing glass coverslips (5, 10, or 15 mm diameter) to the center of 35 mm diameter flexible-bottomed culture wells. Ring inserts were utilized to limit applied strain to different levels in each individual well at a given vacuum pressure thus enabling parallel experiments at different strain levels. Deformation fields were measured using high-density mapping for up to 6% applied strain. The addition of the rigid inclusion creates strong circumferential and radial strain gradients, with a continuous range of stretch anisotropy ranging from strip biaxial to equibiaxial strain and radial strains up to 24% near the inclusion. Dermal fibroblasts seeded within our 2D system (5 mm inclusions; 2% applied strain for 2 days at 0.2 Hz) demonstrated the characteristic orientation perpendicular to the direction of principal strain. Dermal fibroblasts seeded within fibrin gels (5 mm inclusions; 6% applied strain for 8 days at 0.2 Hz) oriented themselves similarly and compacted their surrounding matrix to an increasing extent with local strain magnitude. This study verifies how inhomogeneous strain fields can be produced in a tunable and simply constructed system and demonstrates the potential utility for studying gradients with a continuous spectrum of strain magnitudes and anisotropies.     

High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.     

Combined In Silico,In Vivo,and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO₂ ^−/NO₃ ⁻ data from “middle-aged” (6–8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for “young” (2–3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging.     

Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.     

Directed cellular self-assembly to fabricate cell-derived tissue rings for biomechanical analysis and tissue engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.(1) Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.(2-5) Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues. To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.(6,7) These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength(8) which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (<30 kPa).(9,10) Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.     

Complement C3 deficiency attenuates chronic hypoxia-induced pulmonary hypertension in mice

Background

Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.

Methodology/Principal Findings

Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3−/− hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3−/− mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3−/− mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3−/− mice.

Conclusions

Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.