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    <title language="eng">An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept</title>
    <abstract language="eng">Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.</abstract>
    <parentTitle language="eng">International Journal of Molecular Sciences</parentTitle>
    <identifier type="issn">1422-0067</identifier>
    <identifier type="doi">https://doi.org/10.3390/ijms22158178</identifier>
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The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. 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    <author>Maria Bartosova</author>
    <author>David Ridinger</author>
    <author>Iva Marinovic</author>
    <author>Jana Heigwer</author>
    <author>Conghui Zhang</author>
    <author>Eszter Levai</author>
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    <author>Damir Krunic</author>
    <author>Felix Bestvater</author>
    <author>Michael Hausmann</author>
    <author>Claus Peter Schmitt</author>
    <author>Sotirios G. Zarogiannis</author>
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      <value>Endothelzelle</value>
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    <subject>
      <language>deu</language>
      <type>swd</type>
      <value>Nabelvene</value>
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    <collection role="institutes" number="">Medical Engineering and Data Science (Bachelor)</collection>
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    <title language="eng">Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis</title>
    <abstract language="eng">Rationale:&#13;
 Patients with chronic kidney disease (CKD) have an exceedingly high cardiovascular risk; which further increases in patients on peritoneal dialysis (PD). The pathophysiological role of reactive metabolites accumulating in CKD such as glucose degradation products (GDP) is uncertain.&#13;
 &#13;
 &#13;
 Objective:&#13;
 Delineating the impact of GDP present in PD fluids in accelerated vasculopathy development in patients with CKD.&#13;
 &#13;
 &#13;
 Methods and Results:&#13;
 Omental and parietal peritoneal tissues were obtained from 107 children with CKD before dialysis and 90 children on chronic PD with PD fluids containing very low or high concentrations of GDP. Omental arterioles, protected from local PD fluid exposure by surrounding fat, were microdissected for multiomics analyses. High-GDP exposed omental arterioles exhibited 3-fold higher advanced glycation endproduct concentrations and upregulated genes involved in cell death/apoptosis and suppressed genes related to cell viability/survival, cytoskeleton organization, and immune response biofunctions. Vasculopathy-associated canonical pathways concordantly regulated on gene and protein level with high-GDP exposure included cell death/proliferation, apoptosis, cytoskeleton organization, metabolism and detoxification, cell junction signaling, and immune response. Parietal peritoneal arterioles of patients exposed to high-GDP fluids exhibited lumen narrowing compared to patients with CKD stage 5 (end-stage kidney disease) and patients on low-GDP PD, intima thickness was increased. Protein quantification verified increased proapoptotic activity and cytoskeleton disintegration, single-molecule-localization microscopy demonstrated arteriolar endothelial ZO-1 (zonula occludens-1) disruption. Absolute and per endoluminal surface length, arteriolar endothelial cell counts inversely correlated with GDP exposure, caspase-3, TGF (transforming growth factor)-β–induced pSMAD2/3 (phosphorylated SMAD2/3), interleukin-6, ZO-1 abundance, and lumen narrowing. In vitro, 3,4-dideoxyglucosone-3-ene reduced lamin-A/C and membrane ZO-1 assembly, increased pSMAD2/3, and ionic and 4 and 10 kDa permeability of arterial endothelial cells.&#13;
 &#13;
 &#13;
 Conclusions:&#13;
 Our findings indicate a fundamental role of GDP in PD-associated vasculopathy, exerted by endothelial cell junction and cytoskeleton disruption, and induction of apoptosis. They should redirect the focus of research and intervention on targeting reactive metabolite overload in CKD and PD.&#13;
 &#13;
 &#13;
 Registration:&#13;
  URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01893710.</abstract>
    <parentTitle language="eng">Circulation Research</parentTitle>
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The pathophysiological role of reactive metabolites accumulating in CKD such as glucose degradation products (GDP) is uncertain.&lt;\/jats:p&gt;\n          &lt;\/jats:sec&gt;\n          &lt;jats:sec&gt;\n            &lt;jats:title&gt;Objective:&lt;\/jats:title&gt;\n            &lt;jats:p&gt;Delineating the impact of GDP present in PD fluids in accelerated vasculopathy development in patients with CKD.&lt;\/jats:p&gt;\n          &lt;\/jats:sec&gt;\n          &lt;jats:sec&gt;\n            &lt;jats:title&gt;Methods and Results:&lt;\/jats:title&gt;\n            &lt;jats:p&gt;Omental and parietal peritoneal tissues were obtained from 107 children with CKD before dialysis and 90 children on chronic PD with PD fluids containing very low or high concentrations of GDP. Omental arterioles, protected from local PD fluid exposure by surrounding fat, were microdissected for multiomics analyses. 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    <title language="eng">Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis</title>
    <abstract language="eng">Abstract&#13;
 &#13;
 Background and Aims&#13;
 Patients with chronic kidney disease patients (CKD) have an exceedingly high cardiovascular risk. While vasculopathy is further accelerated during peritoneal dialysis (PD), the pathophysiological role of reactive metabolites such as glucose degradation products (GDP) is uncertain.&#13;
 &#13;
 &#13;
 Method&#13;
 Omental and parietal peritoneal tissues from 100 non-CKD individuals, 107 children with CKD5, 60 children treated with neutral pH, low GDP, and 30 children treated with acidic pH, high GDP PD fluids underwent standardized digital histomorphometry. Omental arterioles localized within the fat tissue, protected from direct PD fluid exposure were microdissected for multi-omics analysis. Key regulated pathways were validated by quantitative immunostaining, with localization microscopy in peritoneal tissues of matched cohorts and in vitro in human umbilical vein endothelial cells.&#13;
 &#13;
 &#13;
 Results&#13;
 Arterioles from children with CKD5 exhibited reduced lumen to vessel ratio (L/V) and reduced endothelial telomere length compared to non-CKD individuals; gene ontology analysis identified enrichment of arteriolar genes associated with nuclear telomere cap complex and focal adhesion. Pathway analysis of arteriolar cross-omics identified top canonical pathways including telomere extension by telomerase, actin cytoskeleton, integrin and tight junction signalling.&#13;
 Peritoneal vasculopathy progressed with PD vintage and was more pronounced with high versus low GDP exposure (p&amp;amp;lt;0.001). Compared to CKD5, low GDP-PD upregulated 145/110 and downregulated 38/34 arteriolar genes/proteins, high GDP-PD upregulated 684/137 and supressed 1560/55 genes/proteins (p&amp;amp;lt;0.01). High GDP milieu induced upregulation of arteriolar genes involved in cell death/apoptosis and suppressed genes related to cell viability/survival, cytoskeleton organization and immune response biofunctions. Vasculopathy associated canonical pathways concordantly regulated on arteriolar gene and protein level with high GDP exposure included cell death/proliferation, apoptosis, cytoskeleton organization, metabolism and detoxification, cell junction signalling, and immune response.&#13;
 Quantitative validation in PD cohorts with similar PD vintage, dialytic glucose exposure and age (n=15 / group) verified increased proapoptotic activity and cytoskeleton disintegration with high-GDP exposure; single-molecule-localization microscopy demonstrated arteriolar endothelial zonula occludens-1 (ZO-1) disruption. Absolute and relative to endoluminal surface length, arteriolar endothelial cell counts were inversely correlated with GDP exposure, with apoptosis marker caspase-3, TGF-ß induced pSMAD2/3, interleukin-6, ZO-1 protein abundance and the degree of vasculopathy. In vitro, exposure to GDP 3,4-dideoxyglucosone-3-ene dose-dependently reduced nuclear endothelial lamin-A/C and membrane ZO-1 assembly. Transendothelial electrical resistance was decreased. ZO-1 and sealing tight junction claudin-5 protein abundance were decreased in cells after incubation with high GDP compared to low GDP PD fluid and culture media. On nanoscale level GDP reduced junction cluster formation in the membrane area.&#13;
 &#13;
 &#13;
 Conclusion&#13;
 Multi-omics analysis of omental arterioles from children without pre-existing vasculopathy and life-style related confounders identified key mechanisms of vascular aging in CKD5 and the major contribution of GDP to accelerated vasculopathy during PD, i.e. disruption of endothelial cell junctions and cytoskeleton and induction of apoptosis.</abstract>
    <parentTitle language="eng">Nephrology Dialysis Transplantation</parentTitle>
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While vasculopathy is further accelerated during peritoneal dialysis (PD), the pathophysiological role of reactive metabolites such as glucose degradation products (GDP) is uncertain.&lt;\/jats:p&gt;\n               &lt;\/jats:sec&gt;\n               &lt;jats:sec&gt;\n                  &lt;jats:title&gt;Method&lt;\/jats:title&gt;\n                  &lt;jats:p&gt;Omental and parietal peritoneal tissues from 100 non-CKD individuals, 107 children with CKD5, 60 children treated with neutral pH, low GDP, and 30 children treated with acidic pH, high GDP PD fluids underwent standardized digital histomorphometry. Omental arterioles localized within the fat tissue, protected from direct PD fluid exposure were microdissected for multi-omics analysis. 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