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Continuous Microfiber Wire Mandrel-Less Biofabrication for Soft Tissue Engineering Applications
Arianna Adamo, Joseph G. Bartolacci, Drake D. Pedersen, Marco G. Traina, Seungil Kim, Antonio Pantano, Giulio Ghersi, Simon C. Watkins, William R. Wagner, Stephen F. Badylak, and Antonio D’Amore
Suture materials are the most common bioimplants in surgical and clinical practice, playing a crucial role in wound healing and tendon and ligament repair. Despite the assortment available on the market, sutures are still affected by significant disadvantages, including failure in mimicking the mechanical properties of the tissue, excessive fibrosis, and inflammation. This study introduces a mandrel-less electrodeposition apparatus to fabricate continuous microfiber wires of indefinite length. The mandrel-less biofabrication produces wires, potentially used as medical fibers, with different microfiber bundles, that imitate the hierarchical organization of native tissues, and tailored mechanical properties. Microfiber wire morphology and mechanical properties are characterized by scanning electron microscopy, digital image processing, and uniaxial tensile test. Wires are tested in vitro on monocyte/macrophage stimulation and in vivo on a rat surgical wound model. The wires produced by mandrel-less deposition show an increased M2 macrophage phenotype in vitro. The in vivo assessment demonstrates that microfiber wires, compared to the medical fibers currently used, reduce pro-inflammatory macrophage response and preserve their mechanical properties after 30 days of use. These results make this microfiber wire an ideal candidate as a suture material for soft tissue surgery, suggesting a crucial role of microarchitecture in more favorable host response.
Hepatocytes Are Resistant to Cell Death From Canonical and Non-Canonical Inflammasome-Activated Pyroptosis
Ping Sun, Jie Zhong, Hong Liao, Patricia Loughran, Joud Mulla, Guang Fu, Da Tang, Jie Fan, Timothy R. Billiar, Wentao Gao, and Melanie J. Scott
Caspase-1/11 activation induces macrophage pyroptosis, but hepatocytes are resistant to pyroptosis, which we show is mainly due to low levels of caspase-1/11 even after stimulation. Overexpression of caspase-1/11 p10/p20 can induce hepatocyte pyroptosis and cell shrinkage, but cell structure remains relatively intact differently from macrophage pyroptosis.
P-selectin–deficient mice to study pathophysiology of sickle cell disease
Margaret F. Bennewitz, Egemen Tutuncuoglu, Shweta Gudapati, Tomasz Brzoska, Simon C. Watkins, Satdarshan P. Monga, Tirthadipa Pradhan-Sundd,and Prithu Sundd
Sickle cell disease (SCD) is an autosomal-recessive genetic disorder that affects millions of people worldwide.1,2 Vaso-occlusion and hemolysis are the 2 predominant vascular events that contribute to the pathogenesis of SCD.3 Vaso-occlusion is believed to trigger acute systemic painful vaso-occlusive episode, which is the primary reason for emergency medical treatment of SCD patients.1,4 A role for P-selectin in promoting vaso-occlusion in the cremaster venules of SCD mice was first demonstrated by Kaul and Hebbel.5 Later, sickle erythrocytes were shown to adhere to P-selectin in vitro and undergo P-selectin–mediated rolling adhesion in postcapillary venules of mice in vivo, and P-selectin deletion or inhibition was shown to prevent adhesion of adoptively transferred sickle erythrocytes and vasoocclusion in nonsickle mice in vivo.6-9 Also, platelet P-selectin–dependent neutrophil-plateleterythrocyte heterocellular aggregates were shown to be significantly elevated in SCD patient blood. Key Points: • P-selectin–deficient SCD mice are protected from lung vaso-occlusion. P-selectin–deficient SCD mice will be useful in assessing the benefits of anti–P-selectin therapy in diverse complications of SCD.
Modeling Therapeutic Antibody–Small Molecule Drug-Drug Interactions Using a Three-Dimensional Perfusable Human Liver Coculture Platform
Thomas J. Long, Patrick A. Cosgrove, Robert T. Dunn, II, Donna B. Stolz, Hisham Hamadeh, Cynthia Afshari, Helen McBride, and Linda G. Griffith
Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)–mediated inflammation over 2 weeks. Hepatocyte cultures remained stable over 2 weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble interleukin-6 receptor (IL-6R) levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL- 6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to desuppress CYP3A4 activity while reducing the CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and area under the curve up to the last measurable concentration (AUClast) of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well suited for these types of long-term treatment studies and show promise for improved drug safety assessment.
Skeletal Muscle Triacylglycerol Hydrolysis Does Not Influence Metabolic Complications of Obesity
Mitch T. Sitnick, Mahesh K. Basantani, Lingzhi Cai, Gabriele Schoiswohl, Cynthia F. Yazbeck, Giovanna Distefano, Vladimir Ritov, James P. DeLany, Renate Schreiber, Donna B. Stolz, Noah P. Gardner, Petra C. Kienesberger, Thomas Pulinilkunnil, Rudolf Zechner, Bret H. Goodpaster, Paul Coen, and Erin E. Kershaw
Intramyocellular triacylglycerol (IMTG) accumulation is highly associated with insulin resistance and metabolic complications of obesity (lipotoxicity), whereas comparable IMTG accumulation in endurance-trained athletes is associated with insulin sensitivity (the athlete’s paradox). Despite these findings, it remains unclear whether changes in IMTG accumulation and metabolism per se influence muscle-specific and systemic metabolic homeostasis and insulin responsiveness. By mediating the rate-limiting step in triacylglycerol hydrolysis, adipose triglyceride lipase (ATGL) has been proposed to influence the storage/production of deleterious as well as essential lipid metabolites. However, the physiological relevance of ATGL-mediated triacylglycerol hydrolysis in skeletal muscle remains unknown. To determine the contribution of IMTG hydrolysis to tissue-specific and systemic metabolic phenotypes in the context of obesity, we generated mice with targeted deletion or transgenic overexpression of ATGL exclusively in skeletal muscle. Despite dramatic changes in IMTG content on both chow and high-fat diets, modulation of ATGL-mediated IMTG hydrolysis did not significantly influence systemic energy, lipid, or glucose homeostasis, nor did it influence insulin responsiveness or mitochondrial function. These data argue against a role for altered IMTG accumulation and lipolysis in muscle insulin resistance and metabolic complications of obesity. Diabetes 62:3350–3361, 2013
CD14 contributes to warm hepatic ischemia-reperfusion injury in mice
Changchun Cai, Xiaolian Shi, Sebastian Korff, Jinxiang Zhang, Patricia A Loughran, Xiangcai Ruan, Yong Zhang, Li Liu, and Timothy R. Billiar
Introduction—Ischemia/reperfusion (I/R) of the liver contributes to the pathobiology of liver injury in transplantation, liver surgery, and hemorrhagic shock. I/R induces an inflammatory response that is driven, in part, by TLR4 signaling. CD14 is known to participate in the function of TLR4. We hypothesized that CD14 would be involved in the pathobiology of warm hepatic I/R. Methods—Using a 70% liver inflow inclusion model, CD14 knockout and wild type mice were subjected to 1h warm ischemia followed by reperfusion. CD14 mRNA levels, circulating transaminase, Interleukin-6, soluble CD14 and HMGB1 levels were measured. CD14 neutralizing antibody or isotype control antibody was given before ischemia or reperfusion for CD14 blockade in wild type mice. Recombinant HMGB1 was given before reperfusion in some experiments to test if liver injury worsens. Results—There was an upregulation of CD14 mRNA in reperfused livers together with increased sCD14 levels in the circulation. Compared with wild type control mice, CD14 knockout mice had much lower ALT and IL-6 levels at 6h and 24h following I/R, and much less liver necrosis by histology. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining displayed less apoptosis at 24h in the absence of CD14. CD14 blockage by neutralizing antibody also attenuated liver injury and the inflammatory response in C57BL/6 mice following I/ R, but did not provide additional protection to toll-like receptor 4 mutant C3H/Hej mice. CD14 deficiency did not change circulating HMGB1 levels following I/R (6h). A dose of recombinant HMGB1 which worsened hepatic injury when given before reperfusion in wild type mice did not increase liver damage in CD14 deficient mice. Conclusions—CD14 is actively involved in hepatic I/R injury. Its deficiency or blockade ischemia attenuates liver injury and inflammatory response. CD14 mediates liver damage and inflammatory responses in the setting of warm hepatic I/R in mice. Keywords CD14; sCD14; High mobility group box 1; hepatic ischemia reperfusion injury; toll-like receptor 4
The Tyrosine Phosphatase SHP2 Regulates Sertoli Cell Junction Complexes
Pawan Puri and William H. Walker
The blood-testis barrier (BTB) is a large junctional complex composed of tight junctions, adherens junctions, and gap junctions between adjacent Sertoli cells in the seminiferous tubules of the testis. Maintenance of the BTB as well as the controlled disruption and reformation of the barrier is essential for spermatogenesis and male fertility. Tyrosine phosphorylation of BTB proteins is known to regulate the integrity of adherens and tight junctions found at the BTB. SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) and a key regulator of growth factor-mediated tyrosine kinase signaling pathways. We found that SHP2 is localized to Sertoli-Sertoli cell junctions in rat testis. The overexpression of a constitutive active SHP2 mutant, SHP2 Q79R, up-regulated the BTB disruptor ERK1/2 via Src kinase in primary rat Sertoli cells in culture. Furthermore, focal adhesion kinase (FAK), which also supports BTB integrity, was found to interact with SHP2 and constitutive activation of SHP2 decreased FAK tyrosine phosphorylation. Expression of the SHP2 Q79R mutant in primary cultured Sertoli cells also resulted in the loss of tight junction and adherens junction integrity that corresponded with the disruption of the actin cytoskeleton and mislocalization of adherens junction and tight junction proteins N-cadherin, b-catenin, and ZO-1 away from the plasma membrane. These results suggest that SHP2 is a key regulator of BTB integrity and Sertoli cell support of spermatogenesis and fertility. blood-testis barrier, spermatogenesis, testis
Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro
Victor Tapias, J. Timothy Greenamyre, Simon C. Watkins
Quantitation of neurons using stereologic approaches reduces bias and systematic error, but is timeconsuming and labor-intensive. Accurate methods for quantifying neurons in vitro are lacking; conventional methodologies are limited in reliability and application. The morphological properties of the soma and neurites are a key aspect of neuronal phenotype and function, but the assays commonly used in such evaluations are beset with several methodological drawbacks. Herein we describe automated techniques to quantify the number and morphology of neurons (or any cell type, e.g., astrocytes) and their processes with high speed and accuracy. Neuronal quantification from brain tissue using a motorized stage system yielded results that were statistically comparable to those generated by stereology. The approach was then adapted for in vitro neuron and neurite outgrowth quantification. To determine the utility of our methods, rotenone was used as a neurotoxicant leading to morphological changes in neurons and cell death, astrocytic activation, and loss of neurites. Importantly, our technique counted about 8 times as many neurons in less than 5–10% of the time taken by manual stereological analysis.
Dendritic Cells Promote Macrophage Infiltration and Comprise a Substantial Proportion of Obesity-Associated Increases in CD11c+ Cells in Adipose Tissue and Liver
Maja Stefanovic-Racic, Xiao Yang, Michael S. Turner, Benjamin S. Mantell,1 Donna B. Stolz, Tina L. Sumpter, Ian J. Sipula, Nikolaos Dedousis, Donald K. Scott, Penelope A. Morel, Angus W. Thomson, and Robert M. O’Doherty
Obesity-associated increases in adipose tissue (AT) CD11c+ cells suggest that dendritic cells (DC), which are involved in the tissue recruitment and activation of macrophages, may play a role in determining AT and liver immunophenotype in obesity. This study addressed this hypothesis. With the use of flow cytometry, electron microscopy, and loss-and-gain of function approaches, the contribution of DC to the pattern of immune cell alterations and recruitment in obesity was assessed. In AT and liver there was a substantial, high-fat diet (HFD)–induced increase in DC. In AT, these increases were associated with crown-like structures, whereas in liver the increase in DC constituted an early and reversible response to diet. Notably, mice lacking DC had reduced AT and liver macrophages, whereas DC replacement in DC-null mice increased liver and AT macrophage populations. Furthermore, delivery of bone marrow–derived DC to lean wild-type mice increased AT and liver macrophage infiltration. Finally, mice lacking DC were resistant to the weight gain and metabolic abnormalities of an HFD. Together, these data demonstrate that DC are elevated in obesity, promote macrophage infiltration of AT and liver, contribute to the determination of tissue immunophenotype, and play a role in systemic metabolic responses to an HFD. Diabetes 61:2330–2339, 2012
Xiaoyan Liang , Michael E. De Vera , William J. Buchser , Antonio Romo de Vivar Chavez, Patricia Loughran, Donna Beer Stolz , Per Basse , Tao Wang, Bennett Van Houten, Herbert J. Zeh III , and Michael T. Lotze
Administration of high-dose interleukin-2 (HDIL-2) has durable antitumor effects in 5% to 10% of patients with melanoma and renal cell carcinoma. However, treatment is often limited by side effects, including reversible, multiorgan dysfunction characterized by a cytokine-induced systemic autophagic syndrome. Here, we hypothesized that the autophagy inhibitor chloroquine would enhance IL-2 immunotherapeutic efficacy and limit toxicity. In an advanced murine metastatic liver tumor model, IL-2 inhibited tumor growth in a dose-dependent fashion. These antitumor effects were significantly enhanced upon addition of chloroquine. The combination of IL-2 with chloroquine increased long-term survival, decreased toxicity associated with vascular leakage, and enhanced immune cell proliferation and infiltration in the liver and spleen. HDIL-2 alone increased serum levels of HMGB1, IFN-g, IL-6, and IL-18 and also induced autophagy within the liver and translocation of HMGB1 from the nucleus to the cytosol in hepatocytes, effects that were inhibited by combined administration with chloroquine. In tumor cells, chloroquine increased autophagic vacuoles and LC3-II levels inhibited oxidative phosphorylation and ATP production and promoted apoptosis, which was associated with increased Annexin-Vþ/propidium iodide (PI) cells, cleaved PARP, cleaved caspase-3, and cytochrome c release from mitochondria. Taken together, our findings provide a novel clinical strategy to enhance the efficacy of HDIL-2 immunotherapy for patients with cancer. Cancer Res; 72(11); 2791–801. 2012 AACR.
Mitochondrial hyperfusion induced by loss of the fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress
Wei Qian, Serah Choi, Gregory A. Gibson, Simon C. Watkins, Christopher J. Bakkenist and Bennett Van Houten
Mitochondrial fission and fusion cycles are integrated with cell cycle progression. In this paper, we demonstrate that the inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by a hyperfused mitochondrial structure and unscheduled expression of cyclin E in the G2 phase. This persistent replication stress then induces an ATM-dependent activation of the G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and reactive oxygen species (ROS) generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates the DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics. Key words: Drp1, Cell cycle defects, Genome instability, Mitochondrial fission, Replication stress
Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes
Angela Montecalvo, Adriana T. Larregina, William J. Shufesky, Donna Beer Stolz, Mara L. G. Sullivan, Jenny M. Karlsson, Catherine J. Baty, Gregory A. Gibson, Geza Erdos, Zhiliang Wang, Jadranka Milosevic, Olga A. Tkacheva, Sherrie J. Divito, Rick Jordan, James Lyons-Weiler, Simon C. Watkins, and Adrian E. Morelli
Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs downregulate T-cell responses to induce/ maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs. (Blood. 2012;119(3):756-766)
Kelly J Shields PhD, Donna Stolz PhD, Jonathan Franks, MS, Simon C Watkins PhD, Joseph M Ahearn MD
Vasa vasorum within the perivascular adipose tissue containing crenated erythrocytes. Sectioned adipocytes to the right of the vasa vasorum include the lipid droplet and the surrounding cytoplasm.
Complement Proteins C3 and C4 Bind to Collagen and Elastin in the Vascular Wall: A Potential Role in Vascular Stiffness and Atherosclerosis
Kelly J. Shields , Ph.D., Donna Stolz , Ph.D. , Simon C. Watkins , Ph.D. , and Joseph M. Ahearn , M.D.
Circulating infl ammatory mediators including complement activation products participate in the pathogenesis of cardiovascular diseases. As such, previous reports demonstrating the presence of complement proteins within atherosclerotic plaque and on the luminal surface would be anticipated. In contrast, we have recently made the unexpected observation that complement proteins also deposit along the external elastic lamina of mouse aortas in the absence of luminal deposition or plaque development. This suggests that complement activation may play a critical role in the pathogenesis of vascular stiffness and atherosclerosis through a mechanism initiated within the adventitia rather than on the endothelial surface. This hypothesis was tested in the current study by ultrastructural identifi cation of the C3- and C4-binding targets within the adventitia of the mouse aorta. The results demonstrate extensive binding of C3 and C4 to both collagen and elastin fi bers within the adventitia in both ApoE(−/−) and C57Bl/6J control mice, as well as the presence of C3 and C4 within perivascular adipose tissue. These observations suggest a potential “outside-in” mechanism of vascular stiffness during which perivascular adipose may produce C3 and C4 that bind to collagen and elastin fi bers within the adventitia through covalent thiolester bonds, leading to increased vascular stiffness. Clin Trans Sci 2011; Volume 4: 146–152 Keywords: atherosclerosis, aorta, immunology, immunohistochemistry, infl ammation, vasculature.
A ‘‘tool box’’ for rational design of degradable controlled release formulations
Sam N. Rothsteinab and Steven R. Little
Controlled release technology could provide a universal solution to the problems of patient compliance and sub-optimal dosing that often plague modern pharmaceuticals. Yet, harnessing this potential requires the ability to design drug delivery formulations which satisfy specific dosing schedules. This review intends to portray how material properties, processing methods and mathematical models can serve as effective tools for rationally tuning the duration and rate of drug release from biodegradable polymer matrices.
Serpins Flex Their Muscle I. PUTTING THE CLAMPS ON PROTEOLYSIS IN DIVERSE BIOLOGICAL SYSTEMS
Gary A. Silverman, James C. Whisstock, Stephen P. Bottomley, James A. Huntington, Dion Kaiserman, Cliff J. Luke, Stephen C. Pak, Jean-Marc Reichhart, and Phillip I. Bird
Serpins compose the largest superfamily of peptidase inhibitors and are well known as regulators of hemostasis and thrombolysis. Studies using model organisms, from plants to vertebrates, now show that serpins and their unique inhibitory mechanism and conformational flexibility are exploited to control proteolysis in molecular pathways associated with cell survival, development, and host defense. In addition, an increasing number of non-inhibitory serpins are emerging as important elements within a diversity of biological systems by serving as chaperones, hormone transporters, or anti-angiogenic factors.
A proteome investigation of roxarsone degradation by Alkaliphilus oremlandii strain OhILAs
Peter Chovanec, John F. Stolz and Partha Basu
Clostridial species predominate in both chicken gastrointestinal tract as well as litter where the organoarsenical roxarsone (3-nitro 4-hydroxybenzenearsonic acid) is anaerobically transformed releasing the more recognized toxic inorganic arsenic. 2D-gel electrophoresis and mass spectrometry were used to evaluate the changes in protein expression of Alkaliphilus oremlandii in response to different growth conditions (e.g., terminal electron acceptors) in order to explore the mechanism of microbial biotransformation of roxarsone. Aldehyde ferredoxin oxidoreductase, the enzyme that belongs to the xanthine oxidase family of molybdoenzymes was significantly overexpressed in the presence of roxarsone suggesting a role in the anaerobic metabolism of this substituted nitrophenol.
Ethyl pyruvate decreases HMGB1 release and ameliorates murine colitis
Shaival H. Dave, Jeremy S. Tilstra, Katsuyoshi Matsuoka, Fengling Li, Richard A. DeMarco, Donna Beer-Stolz, Antonia R. Sepulveda, Mitchell P. Fink, Michael T. Lotze, and Scott E. Plevy
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 - (_k^)B family members but attenuated NF - (_k^)B 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. J. Leukoc. Biol. 86: 633–643; 2009.
The Werner Syndrome Protein Functions in Repair of Cr(VI)-Induced Replication-Associated DNA Damage
Fu-Jun Liu, Aaron Barchowsky, and Patricia L. Opresko
Werner syndrome is a premature aging disorder characterized by cancer predisposition that is caused by loss of the Werner syndrome protein (WRN) helicase/exonuclease DNA repair protein. Hexavalent chromium is an environmental carcinogen and genotoxicant that is associated with respiratory cancers and induces several forms of DNA damage, including lesions that interfere with DNA replication. Based on the evidence that WRN protein facilitates repair of stalled and collapsed replication forks, we hypothesized that WRN functions in the cellular response to and recovery from Cr(VI)-induced genotoxicity and genomic instability. Here we report that human cells deficient in WRN protein are hypersensitive to Cr(VI) toxicity, and exhibit a delayed reduction in DNA breaks and stalled replication forks, indicated by gH2AX foci, during recovery from Cr(VI) exposure. Cr(VI)- induced WRN protein translocation from the nucleoli into nucleoplasmic foci in S-phase cells, and these foci colocalized with gH2AX foci indicating WRN responds to replicationassociated DNA damage. As further evidence that Cr(VI) triggers stalled DNA replication, we observed Cr(VI) treatment induced an accumulation of cells in S-phase that exhibited high levels of gH2AX foci. Therefore, these data demonstrate a novel role for WRN protein in cellular protection against the environmental genotoxicant Cr(VI) and further provide evidence that Cr(VI) induces DNA replicative stress which has implications for aging and cancer. Key Words: Werner syndrome protein; chromium; DNA damage; replication stress; DNA repair
Sinusoidal Endothelial Cell Repopulation Following Ischemia/Reperfusion Injury in Rat Liver Transplantation
Donna Beer Stolz, Mark A. Ross, Atsushi Ikeda, Koji Tomiyama, Takashi Kaizu, David A. Geller, and Noriko Murase
We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold-stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re-endothelialize within 48 hours of transplantation, and repopulation is coincident with up-regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor-2 expression on large vessel endothelial cells and repopulating LSECs. Although re-endothelialization occurs rapidly, we show here, using several high-resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow– derived cells into functioning LSECs is routinely between 1% and 5%. Conclusion: Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow– derived LSECs after cold ischemia and warm reperfusion. (HEPATOLOGY 2007;46:1464-1475.)
An Intracellular Serpin Regulates Necrosis by Inhibiting the Induction and Sequelae of Lysosomal Injury
Cliff J. Luke, Stephen C. Pak, Yuko S. Askew, Terra L. Naviglia, David J. Askew, Shila M. Nobar, Anne C. Vetica, Olivia S. Long, Simon C. Watkins, Donna B. Stolz, Robert J. Barstead, Gary L. Moulder, Dieter Bro¨ mme, and Gary A. Silverman,
Extracellular serpins such as antithrombin and a1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.
Dendritic cell image on cover taken by Stuart Shand
Stuart Shand
Dendritic cell image on cover taken by Stuart Shand at CBI. Membrane marker green, exosomes phagocytosed by the dendritic cell labeled red, nucleus labeled blue.
Proteolysis Consistent with Activation of Caspase-7 after Severe Traumatic Brain Injury in Humans
XIAOPENG ZHANG, SEAN ALBER, SIMON C. WATKINS, PATRICK M. KOCHANEK, DONALD W. MARION, STEVEN H. GRAHAM, and ROBERT S.B. CLARK
The expression and proteolysis of caspase family proteins are involved in the initiation and execution of apoptosis, which has been reported to occur in human and experimental traumatic brain injury (TBI). Caspase-3, -6, and -7 belong to the group of executioner caspases, which are cleaved and activated at the late, irreversible stage of apoptosis. Our previous studies demonstrated roles for caspase-1, -3, and -8 in humans after severe TBI. Here we report expression of caspase-7 mRNA and protein in humans after TBI (n = 16) and control brain-bank tissue (n = 6). Semiquantitative reverse transcription polymerase chain reaction showed no differences between caspase-7 mRNA in TBI patients versus controls (73 ± 24 vs. 85 ± 56 relative optical density [ROD], respectively). In contrast, Western blot analysis showed increased pro-caspase-7 in TBI patients versus controls (214 ± 30 vs. 1 ± 1 ROD, respectively), as well as an increase in the ~20 kD proteolytic fragment in TBI patients versus controls (86 ± 13 vs. 22 ± 12 ROD, respectively), consistent with activation of caspase-7 after TBI in humans. Immunohistochemical analysis showed that cells expressing caspase-7 included astrocytes and neurons and possibly other glial cell types and infiltrated inflammatory cells. These data show that caspase-7 and its cleavage product are increased in human brain after TBI in many central nervous systems, as well as noncentral nervous system, cell types. Thus, caspase-7 may play a role in the glial and inflammatory responses, and possibly neuronal death, after TBI in humans.
Essential Role of Integrin-Linked Kinase in Podocyte Biology: Bridging the Integrin and Slit Diaphragm Signaling
Chunsun Dai, Donna B. Stolz, Sheldon I. Bastacky, Rene St.-Arnaud, Chuanyue Wu, Shoukat Dedhar, and Youhua Liu
Integrin-linked kinase (ILK) has been implicated in the pathogenesis of proteinuria and congenital nephrotic syndrome. However, the function of ILK in glomerular podocyte in a physiologic setting remains unknown. In this study, a mouse model was generated in which ILK gene was selectively disrupted in podocytes by using the Cre-LoxP system. Podocyte-specific ablation of ILK resulted in heavy albuminuria, glomerulosclerosis, and kidney failure, which led to animal death beginning at 10 wk of age. Podocyte detachment and apoptosis were not observed at 4 wk of age, when albuminuria became prominent, indicating that they are not the initial cause of proteinuria. Electron microscopy revealed an early foot process effacement, as well as morphologic abnormality, in ILK-deficient podocytes. ILK deficiency caused an aberrant distribution of nephrin and -actinin-4 in podocytes, whereas the localization of podocin and synaptopodin remained relatively intact. Co-immunoprecipitation demonstrated that ILK physically interacted with nephrin to form a ternary complex, and -actinin-4 participated in ILK/nephrin complex formation. Therefore, ILK plays an essential role in specifying nephrin and -actinin-4 distribution and in maintaining the slit diaphragm integrity and podocyte architecture. These results also illustrate that the integrin and slit diaphragm signals in podocytes are intrinsically coupled through an ILK-dependent mechanism.
3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury
Hideyoshi Toyokawa, Atsunori Nakao, Donna B Stolz, Anna J Romanosky, Michael A Nalesnik, Joao Seda Neto, Takashi Kaizu, Anthony J Demetris and Noriko Murase
Bone marrow cells (BMC) have been shown to migrate into injured sites for parenchymal repair. However, the extent of BMC involvement is controversial. To determine whether and to what extent BMC contribute to renal parenchymal repair, we employed three-dimensional (3D) fluorescent confocal microscopy/video in renal warm and cold ischemia/reperfusion (I/R) injury using enhanced green fluorescent protein transgenic rats and their radiation chimeras. After induction of renal warm I/R injury in chimeras, BM-derived renal tubular cells were found in 2D microscopy as isolated single cells or clusters of 2–3 cells. Likewise, cold I/R injury resulted in hostderived tubular cells with frequencies B0.2%. However, stringent confocal microscopic analysis and 3D image construction revealed that BM-derived tubules identified in 2D images were frequently artifacts of overlapping cells separately stained with different markers. The actual frequency in 3D analysis was approximately onefourth of that seen in 2D analysis. 3D confocal imaging precisely detected BM-derived tubular epithelial cells and could be useful to study BMC contribution to tissue repair.
The fruit fly, Drosophila melanogaster, is a powerful model genetic organism that has been used since the turn of the previous century in the study of complex biological problems. In the last decade, numerous researchers have focused their attention on understanding neurodegenerative diseases by utilizing this model system. Numerous Drosophila mutants have been isolated that profoundly affect neural viability and integrity of the nervous system with age. Additionally, many transgenic strains have been developed as models of human disease conditions. We review the existing Drosophila neurodegenerative mutants and transgenic disease models, and discuss the role of the fruit fly in therapeutic development for neurodegenerative diseases.
Functional Connectivity between Immune Cells Mediated by Tunneling Nanotubules
Simon C. Watkins and Russell D. Salter2, Department of Cell Biology and Physiology, Department of Immunology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15213
Intercellular signals can be transmitted through neuronal synapses or through gap junctions, with the latter mediating transmission of calcium fluxes and small molecules between cells. We show here that a third form of communication between cells can be mediated by tunneling nanotubules (TNT). When myeloid-lineage dendritic cells and monocytes are triggered to flux calcium by chemical or mechanical stimulation, the signal can be propagated within seconds to other cells at distances hundreds of microns away via TNT. A complex and transient network of TNT is seen in live cells, with individual tubules exhibiting substantial variation in length and diameter. In addition to calcium fluxes, microinjected dye tracers can be transferred through these connections. Following TNT-mediated stimulation, spreading of lamellipodia occurs in dendritic cells characteristic of that seen during the phagocytic response to bacteria. These results demonstrate that nonneuronal cells can transmit signals to distant cells through a physically connected network.
The Role of Hepatic Type 1 Plasminogen Activator Inhibitor (PAI-1) During Murine Hemorrhagic Shock
Claudio E. Lagoa, Yoram Vodovotz, Donna B. Stolz, Franck Lhuillier, Carol McCloskey, David Gallo, Runkuan Yang, Elena Ustinova, Mitchell P. Fink, Timothy R. Billiar, and Wendy M. Mars
Hemorrhagic shock (HS) followed by resuscitation (HS-R) is characterized by profound physiological changes. Even if the patient survives the initial blood loss, these poorly understood changes can lead to morbidity. One of the tissues most often affected is liver. We sought to recognize specific hepatic changes induced by this stressor to identify targets for therapeutic intervention. Gene array analyses using mouse liver mRNAs were used to identify candidate genes that contribute to hepatic damage. To verify the role of one of the genes identified using the arrays, mice were subjected to HS-R, and multiple parameters were analyzed. A profound increase in plasminogen activator inhibitor type 1 (PAI-1) mRNA was observed using hepatic mRNAs from C57Bl/6 mice after HS, both with and without resuscitation. Constitutive loss of PAI-1 resulted in notable tissue preservation and lower (P < .05) alanine aminotransferase (ALT) levels. Fibrin degradation products (FDPs) and interleukins 6 and 10 (IL-6 and IL-10) were unaffected by loss of PAI-1; however, enhanced urokinase activity, an elevation of active hepatocyte growth factor (HGF), an increase in unprocessed transforming growth factor-1 (TGF-1), and retention of ERK phosphorylation after HS-R were associated with improved hepatic function. In conclusion, PAI-1 protein is a negative effector of hepatic damage after HS-R through its influence on classic regulators of hepatic growth, as opposed to its role in fibrinolysis. (HEPATOLOGY 2005;42: 390-399.)
Imaging of Biological Systems with Fluorescent Technologies
Claudette M. St. Croix, Bruce R. Pitt, and Simon C. Watkins
Fluorescence microscopy imaging has become one of the most useful techniques in biomedical research, used to assess the activity of individual cells as well as subcellular processes. The past 2 decades have seen tremendous advances in the rational design and development of probes to serve as reporters of the intracellular environment of cells. New fluorescent molecules have been engineered to follow signaling traffic, sense ionic and nonionic secondary messengers, and report various kinase activities. In particular, the advent of GFP cloning has provided a means of making specific proteins visible in living cells, allowing the study of molecular behavior and interactions with high spatial and temporal resolution. It is now possible to examine these events in intact tissue, enabling a more complete understanding of complex physiologic processes. SCI & MED 10(1):16-29, 2005.
Physical and functional association of migfilin with cell-cell adhesions
Vasiliki Gkretsi, Yongjun Zhang, Yizeng Tu, Ka Chen, Donna B. Stolz, Yanqiang Yang, Simon C. Watkins and Chuanyue Wu
Cell-cell junctions are essential for epithelial and endothelial tissue formation and communication between neighboring cells. We report here that migfilin, a recently identified component of cell-extracellular matrix adhesions, is recruited to cell-cell junctions in response to cadherin-mediated cell-cell adhesions. Migfilin is detected at cell-cell junctions in both epithelial and endothelial cells. It forms detergent-resistant, discrete clusters that associate with actin bundles bridging neighboring cells. Immunoelectron microscopic analyses reveal that migfilin is closely associated with β-catenin, but not desmosomes, at cell-cell junctions. Furthermore, we show that the Cterminal LIM domains, but not its N-terminal domain, mediates migfilin localization to cell-cell junctions. The site mediating the localization of migfilin to cell-cell junctions at least partially overlaps with that mediating the localization of migfilin to cell-ECM adhesions. Finally, siRNA-mediated depletion of migfilin compromised the organization of adherens junctions and weakened cell-cell association. These results identify migfilin as a component of adherens junctions and suggest an important role for migfilin in the organization of the cell-cell adhesion structure.
Gene Combination Transfer to Block Autoimmune Damage in Transplanted Islets of Langerhans
Suzanne Bertera, Angela M. Alexander, Megan L. Crawford, Glenn Papworth, Simon C. Watkins, Paul D. Robbins, and Massimo Trucco1
Islet transplantation therapy would be applicable to a wider range of diabetic patients if donor islet acceptance and protection were possible without systemic immunosuppression of the recipient. To this aim, gene transfer to isolated donor islets ex vivo is one method that has shown promise. This study examines the combined effect of selected immunomodulatory and anti-inflammatory genes known to extend the functional viability of pancreatic islet grafts in an autoimmune system. These genes, indoleamine 2,3-dioxygenase (IDO), manganese superoxide dismutase (MnSOD), and interleukin (IL)-1 receptor antagonist protein (IRAP), were transferred to isolated NOD donor islets ex vivo then transplanted to NODscid recipients and evaluated in vivo after diabetogenic T-cell challenge. The length of time the recipient remained euglycemic was used to measure the ability of the transgenes to protect the graft from autoimmune destruction. Although the results of these cotransfections gave little evidence of a synergistic relationship, they were useful to show that gene combinations can be used to more efficiently protect islet grafts from diabetogenic T cells.
Adenoviral Gene Transfer of the Human Inducible Nitric Oxide Synthase Gene Enhances the Radiation Response of Human Colorectal Cancer Associated with Alterations in Tumor Vascularity
Zifa Wang, Tracy Cook, Sean Alber, Kaihong Liu, Imre Kovesdi, Simon K. Watkins, Yoram Vodovotz, Timothy R. Billiar, and David Blumberg
Nitric oxide is a potent radiosensitizer of tumors, but its use clinically is limited by serious side effects when administered systemically. We have demonstrated previously that gene transfer of the inducible nitric oxide synthase gene (iNOS) into colorectal cancer cells enhances radiationinduced apoptosis in vitro. The objectives of this study were to further characterize the effects of iNOS gene transfer on the radiosensitivity of human colorectal cancer cells in vitro and tumors grown in athymic nude mice. Adenoviral gene transfer of iNOS (AdiNOS) into human colorectal cancer cell lines (HCT-116 and SNU-1040 cells) significantly enhanced the effects of radiation with sensitizing enhancement ratios (0.1) of 1.65 and 1.6, respectively. The radiation enhancement induced by iNOS was associated with increased iNOS expression and nitric oxide production and prevented by L-NIO, an enzymatic inhibitor of iNOS. AdiNOS treatment of HCT-116 tumors combined with radiation (2 Gy × three fractions) led to a 3.4-fold greater (P < 0.005) tumor growth delay compared with radiation (RT) alone. AdiNOS plus RT also caused significant (P < 0.01) tumor regression with 63% of tumors regressing compared with only 6% of tumors treated with RT. AdiNOS plus RT significantly (P < 0.001) increased the percentage of apoptotic cells (22 ± 4%) compared with either tumors treated with control vector plus RT (9 ± 1%), AdiNOS alone (9 ± 3%), or no treatment (2 ± 1%). These radiosensitizing effects of AdiNOS occurred at low infection efficiency (4% of tumor infected), indicating a significant bystander effect.
Rapid and extensive membrane reorganization by dendritic cells following exposure to bacteria revealed by highresolution imaging
Russell D. Salter, Renee J. Tuma-Warrino, Paul Q. Hu, and Simon C. Watkins
Using live cell imaging, we demonstrate that immature dendritic cells (DC) derived from human peripheral blood monocytes undergo pronounced morphologic changes in vitro within minutes of exposure to unopsonized Escherichia coli, developing extensive membrane veils that ef- ficiently capture additional bacteria. Internalization does not occur in the veils, but instead, bacteria are transported to the central region of the cell, where they sink directly into the plasma membrane. In contrast, exposure to polystyrene beads does not induce notable changes in cell morphology, and DC do not efficiently capture beads when introduced alone or mixed with bacteria. Long dendritic processes were also visualized in some cells that allowed capture of clumps of bacteria at a distance of more than 100 m. These results demonstrate that immature DC can distinguish between inert particles and bacteria and alter their shape and phagocytic capacity in response to the latter. J. Leukoc. Biol. 75: 240–243; 2004.
Intratumoral Delivery of Dendritic Cells Engineered to Secrete Both Interleukin (IL)-12 and IL-18 Effectively Treats Local and Distant Disease in Association with Broadly Reactive Tc1-type Immunity1
Tomohide Tatsumi, Jian Huang, William E. Gooding, Andrea Gambotto, Paul D. Robbins, Nikola L. Vujanovic, Sean M. Alber, Simon C. Watkins, Hideho Okada, and Walter J. Storkus2
Dendritic cells (DCs) were adenovirally engineered to constitutively and durably secrete the potent Th1-biasing cytokines interleukin (IL)-12 (AdIL12DC) and/or IL-18 (AdIL18DC) and evaluated for their ability to promote therapeutic antitumor immunity in murine sarcoma models. Injection of either AdIL12DC or AdIL18DC into day 7 CMS4 or MethA tumors resulted in tumor rejection or slowed tumor growth when compared with control cohorts. Importantly, intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 (AdIL12/IL18DC) resulted in complete and the most acute rejection of any treatment group analyzed. This strategy was also effective in promoting the regression of contralateral, untreated tumors. Both CD4+ and CD8+ T cells were required for tumor rejection. CD8+ splenic T cells from mice treated with AdIL12/ IL18DC produced the highest levels of IFN- γ in response to tumor rechallenge in vitro and displayed the broadest repertoire of Tc1-type reactivity to acid-eluted, tumor-derived peptides among all treatment cohorts. This apparent enhancement in cross-presentation of tumor-associated epitopes in vivo may result from the increased capacity of engineered DCs to kill tumor cells, survive tumor-induced apoptosis, and present immunogenic MHC/tumor peptide complexes to T cells after intratumoral injection. In support of this hypothesis, cytokine gene-engineered DCs expressed higher levels of MHC and costimulatory molecules, as well as Fas ligand and membrane-bound tumor necrosis factor α, with the latter markers associated with elevated tumoricidal activity in vitro. Cytokine gene-engineered DCs appeared to have a survival advantage in situ when injected into tumor lesions, to be found in approximation with regions of tumor apoptosis, and to have the capacity to ingest apoptotic tumor bodies. These results support the ability of combined cytokine gene transfer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accelerated immune-mediated rejection of tumors.
Carbon Monoxide Inhalation Protects Rat Intestinal Grafts from Ischemia/Reperfusion Injury
Atsunori Nakao, Kei Kimizuka, Donna B. Stolz, Joao Seda Neto,Takashi Kaizu, Augustine M. K. Choi, Takashi Uchiyama, Brian S. Zuckerbraun, Michael A. Nalesnik, Leo E. Otterbein, and Noriko Murase
Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to exert anti-inflammatory effects. This study examines the cytoprotective efficacy of inhaled CO during intestinal cold ischemia/reperfusion injury associated with small intestinal transplantation. Orthotopic syngenic intestinal transplantation was performed in Lewis rats after 6 hours of cold preservation in University of Wisconsin solution. Three groups were examined: normal untreated controls, control intestinal transplant recipients kept in room air, and recipients exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. In air grafts, mRNA levels for interleukin-6, cyclooxygenase-2, intracellular adhesion molecule (ICAM-1), and inducible nitric oxide synthase rapidly increased after intestinal transplant. Histopathological analysis revealed severe mucosal erosion, villous congestion, and inflammatory infiltrates. CO effectively blocked an early up-regulation of these mediators, showed less severe histopathological changes, and resulted in significantly improved animal survival of 92% from 58% in air-treated controls. CO also significantly reduced mRNA for proapoptotic Bax, while it up-regulated anti-apoptotic Bcl-2. These changes in CO-treated grafts correlated with well-preserved CD31^+ vascular endothelial cells, less frequent apoptosis/necrosis in intestinal epithelial and capillary endothelial cells, and improved graft tissue blood circulation. Protective effects of CO in this study were mediated via soluble guanylyl cyclase, because 1H-(1,2,4)oxadiazole (4,3 - α) quinoxaline-1-one (soluble guanylyl cyclase inhibitor) completely reversed the beneficial effect conferred by CO. Perioperative CO inhalation at a low concentration resulted in protection against ischemia/ reperfusion injury to intestinal grafts with prolonged cold preservation. (Am J Pathol 2003, 163:1587–1598)
Intra-mitochondrial Poly(ADP-ribosylation) Contributes to NAD Depletion and Cell Death Induced by Oxidative Stress
Lina Du, Xiaopeng Zhang, Yong Y. Han, Nancy A. Burke, Patrick M. Kochanek, Simon C. Watkins, Steven H. Graham, Joseph A. Carcillo, Csaba Szabo, and Robert S. B. Clark
Poly(ADP-ribosylation), primarily via poly(ADP-ribose) polymerase-1 (PARP-1), is a pluripotent cellular process important for maintenance of genomic integrity and RNA transcription in cells. However, during conditions of oxidative stress and energy depletion, poly- (ADP-ribosylation) paradoxically contributes to mitochondrial failure and cell death. Although it has been presumed that poly(ADP-ribosylation) within the nucleus mediates this pathologic process, PARP-1 and other poly(ADP-ribosyltransferases) are also localized within mitochondria. To this end, the presence of PARP-1 and poly(ADP-ribosylation) were verified within mitochondrial fractions from primary cortical neurons and fibroblasts. Inhibition of poly(ADP-ribosylation) within the mitochondrial compartment preserved transmembrane potential, NAD+ content, and cellular respiration, prevented release of apoptosis-inducing factor, and reduced neuronal cell death triggered by oxidative stress. Treatment with liposomal NAD+ also preserved m and cellular respiration during oxidative stress. Furthermore, inhibition of poly(ADP-ribosylation) prevented intranuclear localization of apoptosis-inducing factor and protected neurons from excitotoxic injury; and PARP-1 null fibroblasts were protected from oxidative stress-induced cell death. Collectively these data suggest that poly(ADP-ribosylation) compartmentalized to the mitochondria can be converted from a homeostatic process to a mechanism of cell death when oxidative stress is accompanied by energy depletion. These data implicate intra-mitochondrial poly(ADP-ribosylation) as an important therapeutic target for central nervous system and other diseases associated with oxidative stress and energy failure.
CHARACTERIZATION OF THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN RAT AND HUMAN LIVER DURING HEMORRHAGIC SHOCK
Joy L. Collins, Yoram Vodovotz, Christian Hierholzer, Raphael T. Villavicencio, Shubing Liu, Sean Alber, David Gallo, Donna B. Stolz, Simon C. Watkins, Anthony Godfrey, William Gooding, Edward Kelly, Andrew B. Peitzman, and Timothy R. Billiar
It has been previously shown that the inducible nitric oxide (NO) synthase (iNOS; NOS-2) is elevated after hemorrhage, and that iNOS-derived NO participates in the upregulation of inflammation as well as lung and liver injury postresuscitation from shock. The purpose of this study was to elucidate the time course of iNOS mRNA expression, as well as the cellular and subcellular localization of iNOS protein in the liver posthemorrhage in rats subjected to varying durations of hemorrhagic shock (HS; mean arterial blood pressure [MAP] = 40 mmHg) with or without resuscitation. Expression of iNOS mRNA in rat liver by real-time reverse transcriptase (RT)-PCR demonstrated iNOS upregulation in shocked animals as compared with their sham counterparts as early as 60 min after the initiation of hemorrhage. By 1 h of HS, iNOS protein was detectable in rat liver by immunofluorescence, and this expression increased with time. Immunofluorescence localized iNOS primarily to the hepatocytes, and in particular to hepatocytes in the centrilobular regions. This analysis, confirmed by immunoelectron microscopy, revealed that iNOS colocalizes with catalase, a peroxisomal marker. Furthermore, we determined that iNOS mRNA is detectable by RT-PCR in liver biopsies from human subjects with HS (MAP < 90 mmHg) associated with trauma (n = 18). In contrast, none of the seven nontrauma surgical patients studied had detectable iNOS mRNA in their livers. Collectively, these results suggest that hepatic iNOS expression, associated with peroxisomal localization, is an early molecular response to HS in experimental animals and possibly in human patients with trauma with HS.
Lipid-Mediated Delivery of Oligonucleotide to Pulmonary Endothelium
Zheng Ma, Junlan Zhang, Sean Alber, John Dileo, Yoichi Negishi, Donna Stolz, Simon Watkins, Leaf Huang, Bruce Pitt, and Song Li
Pulmonary endothelium plays an important role in the maintenance of normal pulmonary physiology and its dysfunction is involved in a number of pulmonary diseases. Correction of endothelial dysfunction via antisense oligodeoxyonucleotides (ODN) is dependent on the development of a delivery vehicle that can efficiently deliver the ODN to pulmonary endothelium with minimal toxicity. To this end, we have developed a lipidic vector (LPD) that is composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, protamine, and ODN. This formulation is highly efficient in delivering ODN to the lung via the vascular route. The efficiency of delivery is a function of lipid composition and of the charge ratio between lipid and ODN. Immunofluorescence staining of BrdU-labeled ODN suggested efficient accumulation of ODN in the alveolar capillary region. Transmission electron microscopy of immunogold localization of BrdU-labeled ODN confirmed that pulmonary endothelial cells were indeed targeted by the vector. Furthermore, this formulation is associated with minimal proinflammatory cytokine response and other hematologic toxicities when the ODN lack a potent unmethylated CpG motif. Pretreatment of mice with LPD containing an ODN against intercellular adhesion molecule-1 (ICAM-1) significantly decreased ICAM-1 expression in the lung following LPS challenge. These results provide a basis for lipid-mediated delivery of ODN for the treatment of pulmonary diseases.
Donna Stolz’s 1998 Christmas Card compiled with pseudo‐colored SEM images of pollen from her garden
Donna Stolz
Donna Stolz’s 1998 Christmas Card compiled with pseudo - colored SEM images of pollen from her garden. Trees are SEM images of pine needles, arborvitae and tansy leaves. The ground is paper.
Improved DNA/Emulsion Complex Stabilized by poly(ethylene glycol) Conjugated Phospholipid
Sophie Chesnoy, Dominique Durand, Jean Doucet, Donna Beer Stolz, and Leaf Huang
Recently, finding correlations between the structure of lipid-DNA complexes and their biological activity is gaining more interest (1–3). One major drawback associated with in vivo lipid mediated gene delivery is relatively low transfection efficiency due to poor stability of the complex upon contact with serum (4,5). Given that the transfection efficiency of lipid-DNA complexes highly depends on its structural and physico-chemical properties, a detailed description of these particles is necessary. Strong electrostatic interactions between positively charged lipid-DNA complexes and negatively charged proteins in the blood are responsible for the rapid aggregation of lipid-DNA complexes upon contact with serum. One way to overcome the problem of serum instability and to prolong their circulation time in the blood is to protect their surface by adding PE-PEG (6). Hong et al. (1) reported reduced aggregation of cationic liposome/DNA complex by incorporating a small amount of PE-PEG into the formulation, but the size of their particles is still very large. Recently, Blessing et al. (7) obtained small and negatively charged plasmid-detergent particles, which because of the absence of targeting ligand, failed to show any biological activity. More recently, Monck et al. (8) reported extended circulation time of small, stabilized plasmid-lipid particles following intravenous administration. In vitro transfection studies performed by Liu et al. (9) showed that the transfection activity of tween 80-containing emulsions was not affected by the presence of serum. More recently, Yi et al. (10) developed a physically stable and serum-resistant, cationic oil-in-water emulsion containing DNA for in vitro delivery. Previously, we reported that intraportal injection of cationic DNA/emulsion resulted in high level of gene expression in the liver but poor transfection efficiency following intravenous administration (11). Based on this study, we report the first physico-chemical study on the development of a new DNA/emulsion formulation stabilized by PE-PEG for in vivo administration.
Changes in Wnt/β-Catenin Pathway During Regulated Growth in Rat Liver Regeneration
SATDARSHAN P. S. MONGA, PETER PEDIADITAKIS, KAREN MULE, DONNA BEER STOLZ, AND GEORGE K. MICHALOPOULOS
The wnt/β-catenin pathway is important during embryogenesis and carcinogenesis. β-Catenin interaction with Ecadherin has been shown to be crucial in cell-cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt-1 and β-catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in β-catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of β-catenin (decrease in serine phosphorylated β-catenin) as seen by immunoprecipitation studies. We observed activation of β-catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine-phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E-cadherin protein during liver regeneration are, in general, reverse to those seen in β-catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine-phosphorylated β-catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic β-catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell-cell adhesion for structural reorganization during the process of liver regeneration. (HEPATOLOGY 2001;33:1098-1109.)
Sinusoidal Ultrastructure Evaluated During the Revascularization of Regenerating Rat Liver
KATHRYN E. WACK, MARK A. ROSS, VASTHY ZEGARRA, LAURA R. SYSKO, SIMON C. WATKINS, AND DONNA BEER STOLZ
Sinusoidal endothelial cell (SEC) porosities were compared between the periportal (zone 1) and pericentral (zone 3) regions of the rat liver during regeneration following partial hepatectomy (PHx). SEC porosities and fenestration diameters were measured in control livers, as well as at 5 minutes, 24, 48, 72, 96, 120 hours, and 14 days following PHx. Bimodal maximums in both porosity and fenestration diameters were observed in both zones at 5 minutes and 5 days following PHx. SEC porosities increased signifi- cantly in both zones 1 and 3 within 5 minutes following PHx, but the increase was maintained only in zone 1 at 24 hours after resection. Following the initial rise, both zones displayed a gradual decrease to less than half their porosity values at 72 hr post-PHx. After 72 hours, porosities increased to over control levels and remained elevated until 14 days after PHx. The decrease in porosity at 72 hr post-PHx is accompanied by ultrastructural changes within the sinusoid at this time. Vascular corrosion casting and transmission electron microscopy (TEM) show sinusoid compression resulting from increased hepatic plate widths due to hepatocyte proliferation in the absence of SEC proliferation. Also at this time, we observed many SEC completely enveloped by stellate cells. The zonal variations observed for porosities throughout regeneration did not correlate with changes in laminin, collagen I and IV, or fibronectin deposition within the space of Disse. Taken together, the data reveal that SEC are dynamic regulators of porosity that respond rapidly and locally to environmental zonal stimuli during liver regeneration. (HEPATOLOGY 2001;33:363-378.)
p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes
Beatrice B. Yaroslavskiy, Donna B. Stolz, Simon C. Watkins, Sean M. Alber, Neil A. Bradbury and Richard A. Steinman
Background: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These “rafts” provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. Methods: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. Results: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. Conclusions: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling.
Expression of Inducible Nitric Oxide Synthase and Interleukin-12 in Experimental Necrotizing Enterocolitis
Evan P. Nadler, M.D., Eva Dickinson, M.D., Alex Knisely, M.D., Xiao-Ru Zhang, Patricia Boyle, Donna Beer-Stolz, Ph.D., Simon C. Watkins, Ph.D., and Henri R. Ford, M.D
Background. Previous investigators have relied on administration of pro-inflammatory cytokines or invasive surgical procedures to reproduce the morphologic changes of necrotizing enterocolitis (NEC) in rats. However, these artificial insults do not mimic the human disease. We developed a reproducible model of NEC in rats that more closely resembles human NEC and determined the pattern of inflammatory cytokine expression in this model. Materials and methods. Newborn rats were randomized into four groups. Groups 1 and 2 were breast-fed, while Groups 3 and 4 were gavaged with formula thrice daily. In addition, Groups 2 and 4 were subjected to 3 min of hypoxia thrice daily, prior to each feeding. The rats were killed on day 4 and the distal 2 cm of terminal ileum was harvested for morphological studies and analysis of inflammatory cytokine mRNA expression. Results. Nearly 70% of formula-fed neonatal rats displayed moderate or severe morphological abnormalities resembling human NEC. Breast-fed pups had normal histology. The terminal ileum from rats with abnormal histology demonstrated increased inducible nitric oxide synthase (iNOS) expression, decreased interleukin-12 (IL-12) mRNA expression, and enterocyte apoptosis. There was a trend toward upregulation of IFN-g mRNA, but no difference in expression of TNF-a mRNA. Hypoxia did not significantly alter intestinal morphology or mRNA expression. Conclusions. Formula-fed neonatal rats, with or without hypoxia, exhibit morphological changes in the intestinal epithelium similar to those seen in patients with acute NEC. The mechanism likely involves upregulation of iNOS mRNA, enterocyte apoptosis, and decreased IL-12 production in the intestinal epithelium. This model may offer a simple reproducible method for inducing experimental NEC.
Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis.
Lee PC, Wang ZL, Qian S, Watkins SC, Lizonova A, Kovesdi I, Tzeng E, Simmons RL, Billiar TR, Shears LL 2nd.
Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS: We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS: Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS: We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.
Clemens Esche, Anna Lokshin, Galina V. Shurin, Brian R. Gastman, Hannah Rabinowich, Simon C. Watkins, Michael T. Lotze, and Michael R. Shurin
The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co-culture of murine or human DC with different tumor cell lines for 4–48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase-3-like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time- and dose-dependent induction of apoptosis in DC by tumor-derived factors. This finding is both effector and target specific. The mechanism of tumorinduced DC apoptosis involved regulation of Bcl-2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumorassociated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC-based immunotherapies for cancer and other immune diseases. J. Leukoc. Biol. 66: 336–344; 1999.
Morphogenetic Events in Mixed Cultures of Rat Hepatocytes and Nonparenchymal Cells Maintained in Biological Matrices in the Presence of Hepatocyte Growth Factor and Epidermal Growth Factor
GEORGE K. MICHALOPOULOS, WILLIAM C. BOWEN, VALERIE F. ZAJAC, DONNA BEER-STOLZ, SIMON WATKINS, VSEVOLOD KOSTRUBSKY, AND STEVEN C. STROM
Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagencoated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endotheliumforming vascular structures. Desmin-positive cells surrounded hepatocytes. Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane. When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts. Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters. HGF, EGF, and transforming growth factor-a (TGF-a) enhance these phenomena. HGF plus EGF elicited maximal response. TGF-b1 suppressed formation of the ducts and plates. Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well-delineated canalicular network. The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1. Following implantation of the cell clusters in Matrigel, there was decreased expression of c-met, urokinase, urokinase receptor, and TGF-b1. Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure. The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo-deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15%. These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate. (HEPATOLOGY 1999;29: 90-100.)
R Campbell, SE Wright, EA Repasky, SC Watkins and MT Lotze
Early polar spectrin aggregation following CD45RA+ T - cell activation by human dendritic cells. Human dendritic cells derived from macrophage precursors after culture in 1000 U/ml of rGM - CSF and rIL - 4 for 7 days were admixed with allogenic T - cells within the peripheral blood mononuclear cells with anti - CD3 for 24 hr and stained with FITC - phalloidin to stain f - actin within the T - cells and DCs. Cells were then satined with rabbit polyclonal anti - spectrin antibody and visualized with a donkey anti - rabbit TRITC.Shown are spectrin aggregates within the activated T - cells, some with uropods primarily distal to the site of DC/T - cell interaction. Other experiments demonstrate that this polarization of T - cell - DC interactions occurs predominantly in naïve CD45RA+ CD4+ cells.
Expression of Inducible Nitric Oxide Synthase and Interleukin - 12 in Experimental Necrotizing Enterocolitis
Evan P. Nadler, M.D., Eva Dickinson, M.D., Alex Knisely, M.D., Xiao-Ru Zhang, Patricia Boyle, Donna Beer-Stolz, Ph.D., Simon C. Watkins, Ph.D., and Henri R. Ford, M.D.
Background. Previous investigators have relied on administration of pro-inflammatory cytokines or invasive surgical procedures to reproduce the morphologic changes of necrotizing enterocolitis (NEC) in rats. However, these artificial insults do not mimic the human disease. We developed a reproducible model of NEC in rats that more closely resembles human NEC and determined the pattern of inflammatory cytokine expression in this model. Materials and methods. Newborn rats were randomized into four groups. Groups 1 and 2 were breast-fed, while Groups 3 and 4 were gavaged with formula thrice daily. In addition, Groups 2 and 4 were subjected to 3 min of hypoxia thrice daily, prior to each feeding. The rats were killed on day 4 and the distal 2 cm of terminal ileum was harvested for morphological studies and analysis of inflammatory cytokine mRNA expression. Results. Nearly 70% of formula-fed neonatal rats displayed moderate or severe morphological abnormalities resembling human NEC. Breast-fed pups had normal histology. The terminal ileum from rats with abnormal histology demonstrated increased inducible nitric oxide synthase (iNOS) expression, decreased interleukin-12 (IL-12) mRNA expression, and enterocyte apoptosis. There was a trend toward upregulation of IFN-g mRNA, but no difference in expression of TNF-a mRNA. Hypoxia did not significantly alter intestinal morphology or mRNA expression. Conclusions. Formula-fed neonatal rats, with or without hypoxia, exhibit morphological changes in the intestinal epithelium similar to those seen in patients with acute NEC. The mechanism likely involves upregulation of iNOS mRNA, enterocyte apoptosis, and decreased IL-12 production in the intestinal epithelium. This model may offer a simple reproducible method for inducing experimental NEC.
The Subcellular Distribution of Chromosome 6-encoded Dystrophin-related Protein in the Brain
Tejvir S. Khurana, Simon C. Watkins and Louis M. Kunkel
Chromosome 6-encoded dystrophin-relatedprotein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.
