Leslie A Mitchell

Emory University, Atlanta, GA, United States

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Publications (7)30.09 Total impact

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    ABSTRACT: Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A(-/-) mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A(-/-) mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
    The American journal of pathology. 11/2014;
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    Christian E Overgaard, Leslie A Mitchell, Michael Koval
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    ABSTRACT: Terminal airspaces of the lung, alveoli, are sites of gas exchange that are sensitive to disrupted fluid balance. The alveolar epithelium is a heterogeneous monolayer of cells interconnected by tight junctions at sites of cell-cell contact. Paracellular permeability depends on claudin (cldn)-family tight junction proteins. Of over a dozen alveolar cldns, cldn-3, cldn-4, and cldn-18 are the most highly expressed; other prominent alveolar claudins include cldn-5 and cldn-7. Cldn-3 is primarily expressed by type II alveolar epithelial cells, whereas cldn-4 and cldn-18 are expressed throughout the alveolar epithelium. Lung diseases associated with pulmonary edema, such as alcoholic lung syndrome and acute lung injury, affect alveolar claudin expression, which is frequently associated with impaired fluid clearance due to increased alveolar leak. However, recent studies have identified a role for increased cldn-4 in protecting alveolar barrier function following injury. Thus, alveolar claudins are dynamically regulated, tailoring lung barrier function to control the air-liquid interface.
    Annals of the New York Academy of Sciences 06/2012; 1257:167-74. · 4.38 Impact Factor
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    ABSTRACT: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury. We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.
    Circulation Research 05/2012; 111(2):201-11. · 11.86 Impact Factor
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    ABSTRACT: Claudins are a family of nearly two dozen transmembrane proteins that are a key part of the tight junction barrier that regulates solute movement across polarized epithelia. Claudin family members interact with each other, as well as with other transmembrane tight junction proteins (such as occludin) and cytosolic scaffolding proteins (such as zonula occludens-1 (ZO-1)). Although the interplay between all of these different classes of proteins is critical for tight junction formation and function, claudin family proteins are directly responsible for forming the equivalent of paracellular ion selective channels (or pores) with specific permeability and thus are essential for barrier function. In this review, we summarize current progress in identifying structural elements of claudins that regulate their transport, assembly, and function. The effects of oxidant stress on claudins are also examined, with particular emphasis on lung epithelial barrier function and oxidant stress induced by chronic alcohol abuse.
    Antioxidants & Redox Signaling 09/2011; 15(5):1179-93. · 8.20 Impact Factor
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    Am J Physiol Lung Cell Mol Physiol. 05/2011;
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    ABSTRACT: Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.
    AJP Lung Cellular and Molecular Physiology 04/2011; 301(1):L40-9. · 3.52 Impact Factor
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    Leslie A. Mitchell, Koval Michael
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    ABSTRACT: Clostridium perfringens enterotoxin (CPE), a major cause of food poisoning, forms physical pores in the plasma membrane of intestinal epithelial cells. The ability of CPE to recognize the epithelium is due to the C-terminal binding domain, which binds to a specific motif on the second extracellular loop of tight junction proteins known as claudins. The interaction between claudins and CPE plays a key role in mediating CPE toxicity by facilitating pore formation and by promoting tight junction disassembly. Recently, the ability of CPE to distinguish between specific claudins has been used to develop tools for studying roles for claudins in epithelial barrier function. Moreover, the high affinity of CPE to selected claudins makes CPE a useful platform for targeted drug delivery to tumors expressing these claudins.
    Toxins 01/2010; · 2.13 Impact Factor