scholarly journals Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

2018 ◽  
Author(s):  
Yu Hisano ◽  
Mari Kono ◽  
Eric Engelbrecht ◽  
Koki Kawakami ◽  
Keisuke Yanagida ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lymph Nodes ◽  
Cross Talk ◽  
Transcriptional Activation ◽  
Endothelial Permeability ◽  
Lipid Mediator ◽  
Protein Signaling ◽  
A Genome ◽  
Sphingosine 1 Phosphate ◽  
A Site

AbstractLysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) activate G protein-coupled receptors (GPCRs) to regulate key pathobiological processes. Here we report a novel lipid mediator GPCR cross-talk mechanism that modulates lymphatic endothelial junctional architecture in lymph nodes. LPAR1 was identified as an inducer of S1PR1/ ß-arrestin coupling from a genome-wide CRISPR/ Cas9 transcriptional activation screen. LPAR1 activation induced S1PR1 ß-arrestin recruitment while suppressing Gαi protein signaling. Lymphatic endothelial cells from cortical and medullary sinuses of lymph nodes which express LPAR1 and S1PR1, exhibit porous junctional architecture and constitutive S1PR1 coupling to ß-arrestin which was suppressed by the LPAR1 antagonist AM095. In endothelial cells, LPAR1-activation increased trans-endothelial permeability and junctional remodeling from zipper-like structures to puncta of adhesion plaques that terminate at actin-rich stress fibers with abundant intercellular gaps. Cross-talk between LPA and S1P receptors regulates complex junctional architecture of lymphatic sinus endothelial cells, a site of high lymphocyte traffic and lymph flow.

scholarly journals Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes

2019 ◽  
Vol 216 (7) ◽  
pp. 1582-1598 ◽  
Author(s):  
Yu Hisano ◽  
Mari Kono ◽  
Andreane Cartier ◽  
Eric Engelbrecht ◽  
Kuniyuki Kano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lymph Nodes ◽  
Cross Talk ◽  
Loss Of Function ◽  
Genome Wide ◽  
A Genome ◽  
Sphingosine 1 Phosphate ◽  
Transendothelial Permeability ◽  
Cellular Phenotypes ◽  
Receptor Cross Talk

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein–coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9–based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/β-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/β-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.

Lysophosphatidylcholine increases endothelial permeability: role of PKCα and RhoA cross talk

2005 ◽  
Vol 289 (2) ◽  
pp. L176-L185 ◽  
Author(s):  
Fei Huang ◽  
Papasani V. Subbaiah ◽  
Oksana Holian ◽  
Jihang Zhang ◽  
Arnold Johnson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Induced Resistance ◽  
Cross Talk ◽  
Endothelial Permeability ◽  
Microvascular Endothelial Cells ◽  
Transendothelial Electrical Resistance ◽  
Microvascular Endothelial ◽  
Resistance Decrease ◽  
Stimulation Of

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid that can be generated by pathological activities. We investigated the hypothesis that LPC signals increase in endothelial permeability. Stimulation of human dermal microvascular endothelial cells and bovine pulmonary microvascular endothelial cells with LPC (10–50 μM) induced decreases (within minutes) in transendothelial electrical resistance and increase of endothelial permeability. LPC activated (within 5 min) membrane-associated PKC phosphotransferase activity in the absence of translocation. Affinity-binding analysis indicated that LPC induced increases (also by 5 min) of GTP-bound RhoA, but not Rac1 or Cdc42. By 60 min, both signaling pathways decreased toward baseline. Inhibition of RhoA with C3 transferase inhibited ∼50% of LPC-induced resistance decrease. Pretreatment with PKC inhibitor Gö-6983 (concentrations selective for classic PKC), PMA-induced depletion of PKCα, and transfection of antisense PKCα oligonucleotide each prevented 40–50% of the LPC-induced resistance decrease. Furthermore, these three PKC inhibition strategies inhibited 60–80% of the LPC-induced GTP-bound RhoA. These results show that LPC directly impairs the endothelial barrier function that was dependent, at least in part, on cross talk of PKCα and RhoA signals. The evidence indicates that elevated LPC levels can contribute to the activation of a proinflammatory endothelial phenotype.

scholarly journals Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and protection of anaphylaxis

2021 ◽  
Author(s):  
Thanh Nha Uyen Le ◽  
Toan Q Nguyen ◽  
Yen Thi Kim Nguyen ◽  
Clarissa Kai Hui Tan ◽  
Farhana Tukijan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessel ◽  
Cell Types ◽  
Lipid Mediator ◽  
Tight Junction Proteins ◽  
Severe Hemorrhage ◽  
Sphingosine 1 Phosphate ◽  
Vessel Integrity ◽  
Maximal Reduction ◽  
Junction Proteins

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types to induce signaling. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells, which contributes approximately 50% plasma S1P. To further determine the sources of plasma S1P, here, we report the characterizations of compound deletions of Mfsd2b and Spns2, another S1P transporter from endothelial cells. Global deletion of Mfsd2b and Spns2 (gDKO) resulted in embryonic lethality between E13.5 and E14.5 with severe hemorrhage that largely recapitulated the phenotypes from global S1P1 knockout mice, indicating that together with Mfsd2b, Spns2 also provides embryonic source of S1P for S1P1 stimulation. The hemorrhagic phenotypes in gDKO embryos were accompanied by increased angiogenesis and defects of tight junction proteins, indicating that S1P from Mfsd2b and Spns2 is essential for blood vessel integrity and maturation. The various sources of S1P in postnatal stages are yet to be fully understood. Postnatal ablation of S1P synthesis enzymes using Mx1Cre shows that Mx1Cre-sensitive cells provide most of plasma S1P. Interestingly, we showed that compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) resulted in maximal reduction of 80% plasma S1P. Thus, a small amount of plasma S1P is supplied from other sources independent of Mfsd2b and Spns2. Nevertheless, the vasculature in the lung of ctDKO-Mx1Cre mice was compromised. Furthermore, ctDKO-Mx1Cre mice also exhibited severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable during vascular stress. Together, our results show that Mfsd2b and Spns2 provide a critical source of S1P for embryonic development and they also provide a majority of plasma S1P for vascular homeostasis.

Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo

2012 ◽  
Vol 302 (10) ◽  
pp. C1460-C1468 ◽  
Author(s):  
Gor Sarkisyan ◽  
Stuart M. Cahalan ◽  
Pedro J. Gonzalez-Cabrera ◽  
Nora B. Leaf ◽  
Hugh Rosen
Keyword(s):  
Endothelial Cells ◽  
Real Time ◽  
Multiphoton Microscopy ◽  
Endothelial Permeability ◽  
Field Analysis ◽  
Lymphocyte Trafficking ◽  
Vascular Integrity ◽  
Sphingosine 1 Phosphate ◽  
Single Field

Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.

Hydrogen peroxide induces S1P1 receptors and sensitizes vascular endothelial cells to sphingosine 1-phosphate, a platelet-derived lipid mediator

2007 ◽  
Vol 292 (2) ◽  
pp. C740-C748 ◽  
Author(s):  
Junsuke Igarashi ◽  
Megumi Miyoshi ◽  
Takeshi Hashimoto ◽  
Yasuo Kubota ◽  
Hiroaki Kosaka
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Reactive Oxygen ◽  
Enzyme Activation ◽  
Lipid Mediator ◽  
Oxygen Species ◽  
Growth Responses ◽  
Sphingosine 1 Phosphate

Sphingosine 1-phosphate (S1P) is a platelet-derived angiogenic lipid growth factor, modulating G-protein-coupled S1P1 receptors (S1P1-R) to activate endothelial nitric oxide synthase (eNOS), as well as MAPK pathways in endothelial cells. We explored whether and how hydrogen peroxide (H2O2), a representative reactive oxygen species, alters S1P1-R expression and influences S1P signaling in cultured bovine aortic endothelial cells (BAECs). When BAECs are treated with pathophysiologically relevant concentrations of H2O2 (150 μM for 30 min), S1P1-R protein expression levels are acutely augmented by ∼30-fold in a dose-dependent fashion. When BAECs have been pretreated with H2O2, subsequent S1P stimulation (100 nM) leads to a higher degree of eNOS enzyme activation (assessed as intracellular cGMP content, 1.7 ± 0.2-fold vs. no H2O2 pretreatment groups, P < 0.05), associated with a higher magnitude of phosphorylation responses of eNOS and MAPK ERK1/2. PP2, an inhibitor of Src-family tyrosine kinase, abolished the effects of H2O2 on both S1P1-R protein upregulation and enhanced BAEC responses to S1P. H2O2 does not augment S1P1 mRNA expression, whereas VEGF under identical cultures leads to increases in S1P1 mRNA signals. Whereas H2O2 attenuates proliferation of BAECs, addition of S1P restores growth responses of these cells. These results demonstrate that extracellularly administered H2O2 increases S1P1-R expression and promotes endothelial responses for subsequent S1P treatment. These results may identify potentially important points of cross-talk between reactive oxygen species and sphingolipid pathways in vascular responses.

scholarly journals Chemerin, A Novel Adipokine in the Regulation of Angiogenesis

2010 ◽  
Vol 24 (4) ◽  
pp. 874-874
Author(s):  
Kiymet Bozaoglu ◽  
Joanne E. Curran ◽  
Claire J. Stocker ◽  
Mohamed S. Zaibi ◽  
David Segal ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mexican American ◽  
Large Family ◽  
Disease Status ◽  
Human Endothelial Cells ◽  
Gene Encoding ◽  
The Metabolic Syndrome ◽  
Heart Study ◽  
A Genome ◽  
Family Based

Abstract Context: Chemerin is a new adipokine associated with obesity and the metabolic syndrome. Gene expression levels of chemerin were elevated in the adipose depots of obese compared with lean animals and was markedly elevated during differentiation of fibroblasts into mature adipocytes. Objective: To identify factors that affect the regulation and potential function of chemerin using a genetics approach. Design, setting, patients and intervention: Plasma chemerin levels were measured in subjects from the San Antonio Family Heart Study (SAFHS), a large family-based genetic epidemiological study including 1354 Mexican American individuals. Individuals were randomly sampled without regard to phenotype or disease status. Main Outcome Measures: A genome wide association analysis using 542,944 SNPs in a subset of 523 of the same subjects was undertaken. The effect of chemerin on angiogenesis was measured using human endothelial cells and interstitial cells in co-culture in a specially formulated medium. Results: Serum chemerin levels were found to be highly heritable (h2 = 0.25; P = 1.4 x 10−9). The SNP showing strongest evidence of association (rs347344; P = 1.4 x 10−6) was located within the gene encoding EDIL3, which has a known role in angiogenesis. Functional angiogenesis assays in human endothelial cells confirmed that chemerin significantly mediated the formation of blood vessels to a similar extent as VEGF. Conclusion: Here we demonstrate for the first time that plasma chemerin levels are significantly heritable and identified a novel role for chemerin as a stimulator of angiogenesis.

scholarly journals Single-Cell Transcriptional Heterogeneity of Lymphatic Endothelial Cells in Normal and Inflamed Murine Lymph Nodes

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1371
Author(s):  
Eliane Sibler ◽  
Yuliang He ◽  
Luca Ducoli ◽  
Nadja Keller ◽  
Noriki Fujimoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lymph Nodes ◽  
Single Cell ◽  
In Silico Analysis ◽  
Skin Inflammation ◽  
Lymphatic Endothelial Cells ◽  
Depth Analysis ◽  
Subcapsular Sinus ◽  
Transcriptional Changes ◽  
Sinus Floor

The lymphatic system plays a crucial role in immunity and lymph nodes (LNs) undergo drastic remodeling during inflammation. Here, we used single-cell RNA sequencing to investigate transcriptional changes in lymphatic endothelial cells (LECs) in LNs draining naïve and inflamed skin. We found that subsets of LECs lining the different LN sinuses responded individually to skin inflammation, suggesting that they exert distinct functions under pathological conditions. Among the genes dysregulated during inflammation, we confirmed an up-regulation of CD200 in the LECs lining the subcapsular sinus floor with a possible function in immune regulation. Furthermore, by in silico analysis, we predicted numerous possible interactions of LECs with diverse immune cells in the LNs and found similarities in the transcriptional changes of LN LECs in different skin inflammation settings. In summary, we provide an in-depth analysis of the transcriptional landscape of LN LECs in the naïve state and in skin inflammation.

scholarly journals CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A. W. de Kok ◽  
Roel A. L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

scholarly journals The Endothelial Glycocalyx as a Target of Ischemia and Reperfusion Injury in Kidney Transplantation—Where Have We Gone So Far?

2021 ◽  
Vol 22 (4) ◽  
pp. 2157
Author(s):  
Anila Duni ◽  
Vassilios Liakopoulos ◽  
Vasileios Koutlas ◽  
Charalampos Pappas ◽  
Michalis Mitsis ◽  
...  
Keyword(s):  
Kidney Transplantation ◽  
Reperfusion Injury ◽  
Heparan Sulphate ◽  
Graft Function ◽  
Endothelial Permeability ◽  
Endothelial Glycocalyx ◽  
Ischemia And Reperfusion Injury ◽  
New Era ◽  
Sphingosine 1 Phosphate

The damage of the endothelial glycocalyx as a consequence of ischemia and/or reperfusion injury (IRI) following kidney transplantation has come at the spotlight of research due to potential associations with delayed graft function, acute rejection as well as long-term allograft dysfunction. The disintegration of the endothelial glycocalyx induced by IRI is the crucial event which exposes the denuded endothelial cells to further inflammatory and oxidative damage. The aim of our review is to present the currently available data regarding complex links between shedding of the glycocalyx components, like syndecan-1, hyaluronan, heparan sulphate, and CD44 with the activation of intricate immune system responses, including toll-like receptors, cytokines and pro-inflammatory transcription factors. Evidence on modes of protection of the endothelial glycocalyx and subsequently maintenance of endothelial permeability as well as novel nephroprotective molecules such as sphingosine-1 phosphate (S1P), are also depicted. Although advances in technology are making the visualization and the analysis of the endothelial glycocalyx possible, currently available evidence is mostly experimental. Ongoing progress in understanding the complex impact of IRI on the endothelial glycocalyx, opens up a new era of research in the field of organ transplantation and clinical studies are of utmost importance for the future.

scholarly journals Role of protein tyrosine phosphatase 1B (PTP1B) in the increased sensitivity of endothelial cells to a promigratory effect of erythropoietin in an inflammatory environment

2020 ◽  
Vol 401 (10) ◽  
pp. 1167-1180
Author(s):  
María Eugenia Chamorro ◽  
Romina Maltaneri ◽  
Agustina Schiappacasse ◽  
Alcira Nesse ◽  
Daniela Vittori
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Cross Talk ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Endothelial Cell Migration ◽  
Protein Tyrosine Phosphatase 1B ◽  
Vascular Events ◽  
Protein Tyrosine ◽  
Sensitizing Effect

AbstractThe proliferation and migration of endothelial cells are vascular events of inflammation, a process which can also potentiate the effects of promigratory factors. With the aim of investigating possible modifications in the activity of erythropoietin (Epo) in an inflammatory environment, we found that Epo at a non-promigratory concentration was capable of stimulating EA.hy926 endothelial cell migration when TNF-α was present. VCAM-1 and ICAM-1 expression, as well as adhesion of monocytic THP-1 cells to endothelial layers were also increased. Structurally modified Epo (carbamylation or N-homocysteinylation) did not exhibit these effects. The sensitizing effect of TNF-α on Epo activity was mediated by the Epo receptor. Inhibition assays targeting the PI3K/mTOR/NF-κB pathway, shared by Epo and TNF-α, show a cross-talk between both cytokines. As observed in assays using antioxidants, cell migration elicited by TNF-α + Epo depended on TNF-α-generated reactive oxygen species (ROS). ROS-mediated inactivation of protein tyrosine phosphatase 1B (PTP1B), involved in Epo signaling termination, could explain the synergistic effect of these cytokines. Our results suggest that ROS generated by inflammation inactivate PTP1B, causing the Epo signal to last longer. This mechanism, along with the cross-talk between both cytokines, could explain the sensitizing action of TNF-α on the migratory effect of Epo.

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