Browsing by Author "Epshtein, Yulia"

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    Degradation of Group V Secretory Phospholipase A2 in Lung Endothelium is Mediated by Autophagy
    (Elsevier, 2020-05) Meliton, Lucille N.; Zhu, Xiangdong; Brown, Mary; Epshtein, Yulia; Kawasaki, Takeshi; Letsiou, Eleftheria; Dudek, Steven M.; Medicine, School of Medicine
    Group V secretory phospholipase A2 (gVPLA2) is a potent inflammatory mediator in mammalian tissues that hydrolyzes phospholipids and initiates eicosanoid biosynthesis. Previous work has demonstrated that multiple inflammatory stimuli induce its expression and secretion in several cell types, including the lung endothelium. However, little is known about the mechanism(s) by which gVPLA2 inflammatory signaling is subsequently downregulated. Therefore, in this study we characterized potential clearance mechanisms for gVPLA2 in lung endothelial cells (EC). We observed that exogenous gVPLA2 is taken up rapidly by nutrient-starved human pulmonary artery EC (HPAEC) in vitro, and its cellular expression subsequently is reduced over several hours. In parallel experiments performed in pulmonary vascular EC isolated from mice genetically deficient in gVPLA2, the degradation of exogenously applied gVPLA2 occurs in a qualitatively similar fashion. This degradation is significantly attenuated in EC treated with ammonium chloride or chloroquine, which are lysosomal inhibitors that block autophagic flux. In contrast, the proteasomal inhibitor MG132 fails to prevent the clearance of gVPLA2. Both immunofluorescence microscopy and proximity ligation assay demonstrate the co-localization of LC3 and gVPLA2 during this process, indicating the association of gVPLA2 with autophagosomes. Nutrient starvation, a known inducer of autophagy, is sufficient to stimulate gVPLA2 degradation. These results suggest that a lysosome-mediated autophagy pathway contributes to gVPLA2 clearance from lung EC. These novel observations advance our understanding of the mechanism by which this key inflammatory enzyme is downregulated in the lung vasculature.
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    UCHL1, a deubiquitinating enzyme, regulates lung endothelial cell permeability in vitro and in vivo
    (American Physiological Society, 2021) Mitra, Sumegha; Epshtein, Yulia; Sammani, Saad; Quijada, Hector; Chen, Weiguo; Bandela, Mounica; Desai, Ankit A.; Garcia, Joe G.N.; Jacobson, Jeffrey R.; Biochemistry and Molecular Biology, School of Medicine
    Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.