Hemodynamic Environments from Opposing Sides of Human Aortic Valve Leaflets Evoke Distinct Endothelial Phenotypes In Vitro

The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the “atheroprotective” transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.National Institutes of Health (U.S.) (Molecular, Cellular, and Tissue Biomechanics training grant (T32 EB006348))National Institutes of Health (U.S.) (NHLBI RO1-HL7066686)Charles Stark Draper Laboratory (Fellowship

The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression

Effect of rejection on electrophysiologic function of canine intestinal grafts: Correlation with histopathology and na-k-ATPase activity

To investigate whether electrophysiologic changes can detect the early onset and progress of intestinal rejection, changes in in vitro electrophysiologic function, intestinal histopathology, and Na-K-ATPase activity were studied in dogs. Adult mongrel dogs of both sexes, weighing 18-24 kg, were used for auto and allo small bowel transplantation. The entire small bowels, except for short segments at the proximal and distal ends, were snitched between a pair of dogs (allograft). Animals receiving intestinal autotransplantation were used as controls. AIIograji recipients were sacrificed 3, 4, 5, 7, or 9 days after transplantation, and autograft recipients were sacrificed 3, 7, or 14 days afier transplantation. Immunosuppression was not used. Electrophysiologic measurements were done with an Ussing chamber. Histological analysis was performed blindly using whole thickness sections. Na-K-ATPase activity in the mucosal tissue, which is said to regulate the potential difference, was also measured. Potential difference, resistance, and Na-K-ATPase activity of the allografi intestine decreased with time and were significantly lower 7 and 9 days after transplantation compared to host intestine, normul intestine, and graft intestine of controls (autograft). Potential difference, resistance, and Na-K-ATPase activity of the native intestinal tissue and the autografts did not decrease with time. Detection of histologically mild rejection of the intestine, which is important for appropriate immunosup-pressive treatment in clinical cases, could not be achieved based on electrophysiology or Na-K-ATPase activity. Deterioration of electrophysiologic function during rejection correlated with the histological rejection process and Na-K-ATPase activity; however, electrophysiology my not be a reliable tool for monitoring grafrs, since it cannot detect early intestinal rejection. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

The HIV protease inhibitor saquinavir inhibits HMGB1 driven inflammation by targeting the interaction of cathepsin V with TLR4/MyD88

Extracellular HMGB1 (disulfide form), via activation of Toll-Like-Receptor (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report, that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1 induced TLR4 activation, as assayed by the production of TNF-alpha by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334, or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver I/R, models both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4 driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside

Amine functionalization of cholecyst-derived extracellular matrix with generation 1 PAMAM dendrimer

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Biomacromolecules, copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/pdf/10.1021/bm701055k.A method to functionalize cholecyst-derived extracellular matrix (CEM) with free amine groups was established in an attempt to improve its potential for tethering of bioactive molecules. CEM was incorporated with Generation-1 polyamidoamine (G1 PAMAM) dendrimer by using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide cross-linking system. The nature of incorporation of PAMAM dendrimer was evaluated using shrink temperature measurements, Fourier transform infrared (FTIR) assessment, ninhydrin assay, and swellability. The effects of PAMAM incorporation on mechanical and degradation properties of CEM were evaluated using a uniaxial mechanical test and collagenase degradation assay, respectively. Ninhydrin assay and FTIR assessment confirmed the presence of increasing free amine groups with increasing quantity of PAMAM in dendrimer-incorporated CEM (DENCEM) scaffolds. The amount of dendrimer used was found to be critical in controlling scaffold degradation, shrink temperature, and free amine content. Cell culture studies showed that fibroblasts seeded on DENCEM maintained their metabolic activity and ability to proliferate in vitro. In addition, fluorescence cell staining and scanning electron microscopy analysis of cell-seeded DENCEM showed preservation of normal fibroblast morphology and phenotype

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. © 2013 Namas et al

Development and stability of Th17 cells in ovarian cancer requires nitric oxide and endogenous NOS2 activity in cancer-associated CD4+ T cells

Th17 cells play reciprocal roles in different forms and at different stages of cancer. We report that the presence of Th17 cells in ovarian cancer ascites correlates with local expression of nitric oxide synthase-2 (NOS2). Furthermore, the development of RORγt+IL-23R+IL-17+ Th17 cells from human naive-, memory- or tumor-infiltrating CD4+ T cells critically depends on NO and endogenous NOS2 induced in CD4+ T cells by Th17-inducing cytokines (IL-1β/IL-6/IL-23) or by cancer-associated IL-1β/IL-6/IL-23/NO-producing MDSCs. Inhibition of NOS2 or its downstream cGMP/cGK signaling pathway abolishes de novo induction of Th17 cells. Moreover, even short-term blockade of NOS/cGMP suppresses the IL-17 production by established Th17 cells isolated from ovarian cancer patients, demonstrating the novel key role of NOS/cGMP in Th17 cell physiology and providing for new therapeutic targets to manipulate Th17- and NOS/cGMP-associated immunity in precancerous lesions and advanced cancer

An improved parameter estimation and comparison for soft tissue constitutive models containing an exponential function

Motivated by the well-known result that stiffness of soft tissue is proportional to the stress, many of the constitutive laws for soft tissues contain an exponential function. In this work, we analyze properties of the exponential function and how it affects the estimation and comparison of elastic parameters for soft tissues. In particular, we find that as a consequence of the exponential function there are lines of high covariance in the elastic parameter space. As a result, one can have widely varying mechanical parameters defining the tissue stiffness but similar effective stress–strain responses. Drawing from elementary algebra, we propose simple changes in the norm and the parameter space, which significantly improve the convergence of parameter estimation and robustness in the presence of noise. More importantly, we demonstrate that these changes improve the conditioning of the problem and provide a more robust solution in the case of heterogeneous material by reducing the chances of getting trapped in a local minima. Based upon the new insight, we also propose a transformed parameter space which will allow for rational parameter comparison and avoid misleading conclusions regarding soft tissue mechanics

Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor kappaB, degradation of inhibitor of kappaB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor kappaB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage

Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch