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ABSTRACT: OBJECTIVES: Patients with bicuspid aortic valves (BAV) are predisposed to developing ascending thoracic aortic aneurysms (TAA) at an earlier age than patients who develop degenerative TAAs and have a tricuspid aortic valve (TAV). The hypothesis tested is that BAV associated aortopathy is mediated by a mechanism of matrix remodeling that is distinct from that seen in TAAs of patients with tricuspid aortic valves. METHODS: Aortic specimens were collected during ascending aortic replacement, aortic valve replacement, and heart transplants from nonaneurysmal (NA) donors and recipients. Matrix architecture of the aortic media was assessed qualitatively using multiphoton microscopy followed by quantification of collagen and elastin fiber orientation. α-Elastin was determined and matrix maturity was assessed by quantifying immature and mature collagen and lysyl oxidase (Lox) expression and activity in aortic specimens. Matrix metalloproteinase-2/9 activity was quantified in aortic smooth muscle cells. RESULTS: Elastin and collagen fibers were more highly aligned in BAV-NA and BAV-TAA cases than in TAV-TAA cases, whereas TAV-TAA cases were more disorganized than TAV-NA cases. α-Elastin content was unchanged. Immature collagen was reduced in BAV-NA and BAV-TAA cases when compared with TAV-NA and TAV-TAA cases. Mature collagen was elevated in TAV-TAA cases compared with TAV-NA and BAV-TAA cases. There was a trend toward elevated Lox gene expression and activity and matrix metalloproteinase-2/9 activity for TAV-TAA, BAV-NA, and BAV-TAA specimens. CONCLUSIONS: The highly aligned matrix architecture in patients with BAVs indicates that wall remodeling is distinct from TAV-TAA. Altered matrix architecture and reduced collagen maturity suggest that the effector molecules mediating the remodeling of TAAs are different in BAV and TAV cases.
The Journal of thoracic and cardiovascular surgery 06/2013; · 3.41 Impact Factor
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ABSTRACT: Beclin-1 is a key regulator of autophagy that functions in the context of two phase-specific complexes in the initiation and maturation of autophagosomes. Its known interacting proteins include autophagy effectors, Bcl-2 family members, and organelle membrane anchor proteins. Here we report a newly identified interaction between Beclin-1 and the protein tyrosine kinase receptor Her2. We demonstrate that in Her2-expressing breast carcinoma cells that do not succumb to lapatinib, this Her1/2 inhibitor disrupts the cell surface interaction between Her2 and Beclin-1. The data suggest that the ensuing autophagic response is correlatively associated with the release of Beclin-1 from its complex with Her2 and with the subsequent increase in cytosolic Beclin-1. Upon its interaction with Her2, Beclin-1 upregulates the phosphorylation levels of Her2 and Akt. The Beclin-1 evolutionarily conserved domain, is required for both Beclin-1 interaction with Her2 and for the increased Her2 and Akt phosphorylation. These findings shed new light on mechanisms involved in lapatinib-mediated autophagy in Her2-expressing breast carcinoma cell lines and in Beclin-1 signaling in these cells.
Journal of Biological Chemistry 05/2013; · 4.77 Impact Factor
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ABSTRACT: iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro and in vivo. The structural determinants for iNOS localization are not known. One plausible mechanism for iNOS localization to the peroxisome is through the interaction with peroxisomal import proteins PEX5 or PEX7. siRNA knockdown of PEX7 reduced iNOS colocalization with the peroxisomal protein PMP70. Proteomic studies using MALDI-MS identified iNOS association with the 50-kD ezrin binding PDZ protein (EBP50). Confocal microscopy studies and immunoelectron microscopy confirmed iNOS association with EBP50, with greatest colocalization occurring at 8 hours of cytokine exposure. EBP50 associated with peroxisomes in a PEX5 and PEX7-dependent manner. iNOS localization to peroxisomes was contingent on EBP50 expression in LPS-treated mice. Thus, iNOS targeting to peroxisomes in hepatocytes involves interaction with PEX7 and EBP50. The targeting of iNOS protein to the peroxisome may shift the balance of metabolic processes that rely on heme proteins susceptible to modification by radical oxygen and nitrogen radicals.
Nitric Oxide 03/2013; · 3.55 Impact Factor
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ABSTRACT: The engineering foundation for novel approaches for the repair of congenital defects that involve the main pulmonary artery (PA) must rest on an understanding of changes in the structure-function relationship that occur during postnatal maturation. In the present study, we quantified the postnatal growth patterns in structural and biomechanical behavior in the ovine PA in the juvenile and adult stages. The biaxial mechanical properties and collagen and elastin fiber architecture were studied in four regions of the PA wall, with the collagen recruitment of the medial region analyzed using a custom biaxial mechanical-multiphoton microscopy system. Circumferential residual strain was also quantified at the sinotubular junction and bifurcation locations, which delimit the PA. The PA wall demonstrated significant mechanical anisotropy, except in the posterior region where it was nearly isotropic. Overall, we observed only moderate changes in regional mechanical properties with growth. We did observe that the medial and lateral locations experience a moderate increase in anisotropy. There was an average of about 24% circumferential residual stain present at the luminal surface in the juvenile stage that decreased to 16% in the adult stage with a significant decrease at the bifurcation, implying that the PA wall remodels toward the bifurcation with growth. There were no measurable changes in collagen and elastin content of the tunica media with growth. On average, the collagen fiber recruited more rapidly with strain in the adult compared to the juvenile. Interestingly, the PA thickness remained constant with growth. When this fact is combined with the observed stable overall mechanical behavior and increase in vessel diameter with growth, a simple Laplace Law wall stress estimate suggests an increase in effective PA wall stress with postnatal maturation. This observation is contrary to the accepted theory of maintenance of homeostatic stress levels in the regulation of vascular function and suggests alternative mechanisms regulate postnatal somatic growth. Understanding the underlying mechanisms, incorporating important structural features during growth, will help to improve our understanding of congenital defects of the PA and lay the basis for functional duplication in their repair and replacement.
Journal of Biomechanical Engineering 02/2013; 135(2):021022. · 1.90 Impact Factor
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ABSTRACT: Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca2+-activated K+ channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.
PLoS ONE 08/2012; 7(8). · 4.09 Impact Factor
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Jeremy S Tilstra,
Andria R Robinson,
Jin Wang,
Siobhán Q Gregg,
Cheryl L Clauson,
Daniel P Reay,
Luigi A Nasto,
Claudette M St Croix,
Arvydas Usas,
Nam Vo,
Johnny Huard,
Paula R Clemens,
Donna B Stolz,
Denis C Guttridge, Simon C Watkins,
George A Garinis,
Yinsheng Wang,
Laura J Niedernhofer,
Paul D Robbins
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ABSTRACT: The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.
The Journal of clinical investigation 06/2012; 122(7):2601-12. · 15.39 Impact Factor
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ABSTRACT: Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown).
Angewandte Chemie International Edition 03/2012; 51(20):4838-42. · 13.45 Impact Factor
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ABSTRACT: Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called "eat-me" signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6-null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered.
Molecular biology of the cell 03/2012; 23(9):1742-64. · 5.98 Impact Factor
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Thomas J Richards,
Chunghyun Park,
Yiliang Chen,
Kevin F Gibson,
Y Peter Di,
Annie Pardo, Simon C Watkins,
Augustine M K Choi,
Moises Selman,
Joseph Pilewski,
Naftali Kaminski,
Yingze Zhang
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ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a complex disease with poorly understood etiology. Previously, we reported upregulation of matrix metalloproteinase 7 (MMP7) in both lung and peripheral blood of IPF patients. Here we report evidence for genetic correlation of plasma levels and promoter polymorphisms (rs11568818 and rs11568819) of MMP7 in a well-characterized IPF cohort. Both the AA genotype of rs11568818 and the CT genotype of rs11568819 were found to be significantly associated with higher MMP7 plasma levels. These associations were observed only in IPF patients and not in healthy controls. The G-to-A transition of rs11568818 resulted in a novel binding site for the forkhead box A2 (FOXA2) transcription factor, a key regulator of embryonic lung development and proper function of the mature lung. In vitro, this transition led to increased sensitivity of the MMP7 promoter to FOXA2. In IPF lungs, FOXA2 was localized in the nucleus of epithelial cells that expressed MMP7 in the cytoplasm. These results suggest that increased sensitivity of the polymorphic MMP7 promoter to FOXA2 provides one of the genetic bases for the upregulation of MMP7 in IPF.
AJP Lung Cellular and Molecular Physiology 01/2012; 302(8):L746-54. · 3.66 Impact Factor
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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,
Adrian E Morelli
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ABSTRACT: Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate 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 01/2012; 119(3):756-66. · 9.90 Impact Factor
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ABSTRACT: Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type(1). In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines(2). Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms. Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism(3,4). This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation(4). In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines(5,6,7). A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes(8). To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.
Journal of Visualized Experiments 01/2012;
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ABSTRACT: The role of the endothelium in the pathogenesis of cardiovascular disease is an emerging field of study, necessitating the development of appropriate model systems and methodologies to investigate the multifaceted nature of endothelial dysfunction including disturbed barrier function and impaired vascular reactivity.
We aimed to develop and test an optimized high-speed imaging platform to obtain quantitative real-time measures of blood flow, vessel diameter and endothelial barrier function in order to assess vascular function in live vertebrate models.
We used a combination of cutting-edge optical imaging techniques, including high-speed, camera-based imaging (up to 1000 frames/second), and 3D confocal methods to collect real time metrics of vascular performance and assess the dynamic response to the thromboxane A(2) (TXA(2)) analogue, U-46619 (1 µM), in transgenic zebrafish larvae. Data obtained in 3 and 5 day post-fertilization larvae show that these methods are capable of imaging blood flow in a large (1 mm) segment of the vessel of interest over many cardiac cycles, with sufficient speed and sensitivity such that the trajectories of individual erythrocytes can be resolved in real time. Further, we are able to map changes in the three dimensional sizes of vessels and assess barrier function by visualizing the continuity of the endothelial layer combined with measurements of extravasation of fluorescent microspheres.
We propose that this system-based microscopic approach can be used to combine measures of physiologic function with molecular behavior in zebrafish models of human vascular disease.
PLoS ONE 01/2012; 7(8):e44018. · 4.09 Impact Factor
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ABSTRACT: Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca(2+)-activated K(+) channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.
PLoS ONE 01/2012; 7(8):e44150. · 4.09 Impact Factor
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ABSTRACT: Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.
Structure 11/2011; 19(11):1573-81. · 6.35 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H(2)O(2) was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.
Biochemical and Biophysical Research Communications 08/2011; 412(1):55-60. · 2.48 Impact Factor
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ABSTRACT: Cells survive exposure to bacterial pore-forming toxins, such as streptolysin O (SLO), through mechanisms that remain unclear. Previous studies have suggested that these toxins are cleared by endocytosis. However, the experiments reported here failed to reveal any evidence for endocytosis of SLO, nor did they reveal any signs of damage to endosomal membranes predicted from such endocytosis. Instead, we illustrate that SLO induces a characteristic form of plasma membrane blebbing that allows cells to shed SLO by the process known as ectocytosis. Specifically, 'deep-etch' electron microscopy of cells exposed to SLO illustrates that the toxin is rapidly sequestered into domains in the plasmalemma greatly enriched in SLO pores, and these domains bleb outwards and bud from the cell surface into the medium. Such ectocytosis is even observed in cells that have been chemically fixed before exposure to SLO, suggesting that it is caused by a direct physical action of the toxin on the cell membrane, rather than by an active cellular reaction. We conclude, therefore, that ectocytosis is an important means for SLO clearance and hypothesize that this is a primary method by which cells defend themselves generally against pore-forming toxins.
Journal of Cell Science 07/2011; 124(Pt 14):2414-23. · 6.11 Impact Factor
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Michelle L Manni,
Lauren P Tomai,
Callie A Norris,
L Michael Thomas,
Eric E Kelley,
Russell D Salter,
James D Crapo,
Ling-Yi L Chang, Simon C Watkins,
Jon D Piganelli,
Tim D Oury
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ABSTRACT: Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis.
American Journal Of Pathology 06/2011; 178(6):2752-9. · 4.89 Impact Factor
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ABSTRACT: Circulating inflammatory 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 identification 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 fibers 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 fibers within the adventitia through covalent thiolester bonds, leading to increased vascular stiffness.
Clinical and Translational Science 06/2011; 4(3):146-52. · 1.13 Impact Factor
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ABSTRACT: Circulating inflammatory 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 identification 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 fibers 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 fibers within the adventitia through covalent thiolester bonds, leading to increased vascular stiffness. Clin Trans Sci 2011; Volume 4: 146–152
Clinical and Translational Science 05/2011; 4(3):146 - 152. · 1.13 Impact Factor
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ABSTRACT: The multikinase inhibitor sunitinib malate (SUT) has been reported to reduce levels of myeloid suppressor cells and Treg cells in cancer patients, hypothetically diminishing intrinsic impediments for active immunization against tumor-associated antigens in such individuals. The goal of this study was to identify longitudinal immune molecular and cellular changes associated with tumor regression and disease-free status after the treatment of established day 7 s.c. MO5 (B16.OVA) melanomas with SUT alone (1 mg/day via oral gavage for 7 days), vaccination using ovalbumin (OVA) peptide-pulsed dendritic cell [vaccine (VAC)] alone, or the combination of SUT and VAC (SUT/VAC). We observed superior anti-tumor efficacy for SUT/VAC combination approaches, particularly when SUT was applied at the time of the initial vaccination or the VAC boost. Treatment effectiveness was associated with the acute loss of (and/or failure to recruit) cells bearing myeloid-derived suppressor cells or Treg phenotypes within the tumor microenvironment (TME) and the corollary, prolonged enhancement of Type-1 anti-OVA CD8+ T cell responses in the tumor-draining lymph node and the TME. Enhanced Type-1 T cell infiltration of tumors was associated with treatment-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and CXCR3 ligand chemokines in vascular/peri-vascular cells within the TME, with SUT/VAC therapy benefits conditionally negated upon adminsitration of CXCR3 or VCAM-1 blocking antibodies. These data support the ability of a short 7 day course of SUT to (re)condition the TME to become more receptive to the recruitment and prolonged therapeutic action of (VAC-induced) anti-tumor Tc1 cells.
International Journal of Cancer 05/2011; 129(9):2158 - 2170. · 5.44 Impact Factor
