CD99 and CD99L2 act at the same site as, but independently of, PECAM-1 during leukocyte diapedesis

Abstract Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1β-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-α, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.

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L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System

Blood ◽

10.1182/blood.v89.9.3228 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3228-3235 ◽

Cited By ~ 45

Author(s):

A. Zakrzewicz ◽

M. Gräfe ◽

D. Terbeek ◽

M. Bongrazio ◽

W. Auch-Schwelk ◽

...

Keyword(s):

Endothelial Cells ◽

Time Course ◽

Leukocyte Adhesion ◽

Flow Chamber ◽

Shear Stresses ◽

Selectin Ligands ◽

Tnf Α ◽

Factor Α

Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

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Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage

Molecules ◽

10.3390/molecules25133010 ◽

2020 ◽

Vol 25 (13) ◽

pp. 3010 ◽

Cited By ~ 3

Author(s):

Anna Kicinska ◽

Rafał P. Kampa ◽

Jan Daniluk ◽

Aleksandra Sek ◽

Wieslawa Jarmuszkiewicz ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Damage ◽

Herbal Medicines ◽

Pharmacological Action ◽

Bkca Channel ◽

Inner Mitochondrial Membrane ◽

Beneficial Effects ◽

Factor Α

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at −40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor α (TNF-α) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.

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Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽

10.1182/blood.v83.11.3206.3206 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3206-3217 ◽

Cited By ~ 8

Author(s):

N Dubois-Stringfellow ◽

A Jonczyk ◽

VL Bautch

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Basement Membrane ◽

Organizational Behavior ◽

Cell Lines ◽

Primary Cultures ◽

Cyst Formation ◽

Fibrin Gels

Abstract Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

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The endothelial basement membrane acts as a checkpoint for entry of pathogenic T cells into the brain

Journal of Experimental Medicine ◽

10.1084/jem.20191339 ◽

2020 ◽

Vol 217 (7) ◽

Cited By ~ 3

Author(s):

Xueli Zhang ◽

Ying Wang ◽

Jian Song ◽

Hanna Gerwien ◽

Omar Chuquisana ◽

...

Keyword(s):

T Cells ◽

Basement Membrane ◽

Immune Cell ◽

Cell Phenotype ◽

T Cell Infiltration ◽

And Migration ◽

Endothelial Basement Membrane ◽

Laminin 411

The endothelial cell basement membrane (BM) is a barrier to migrating leukocytes and a rich source of signaling molecules that can influence extravasating cells. Using mice lacking the major endothelial BM components, laminin 411 or 511, in murine experimental autoimmune encephalomyelitis (EAE), we show here that loss of endothelial laminin 511 results in enhanced disease severity due to increased T cell infiltration and altered polarization and pathogenicity of infiltrating T cells. In vitro adhesion and migration assays reveal higher binding to laminin 511 than laminin 411 but faster migration across laminin 411. In vivo and in vitro analyses suggest that integrin α6β1- and αvβ1-mediated binding to laminin 511–high sites not only holds T cells at such sites but also limits their differentiation to pathogenic Th17 cells. This highlights the importance of the interface between the endothelial monolayer and the underlying BM for modulation of immune cell phenotype.

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Capillary endothelial cell cultures: phenotypic modulation by matrix components.

The Journal of Cell Biology ◽

10.1083/jcb.97.1.153 ◽

1983 ◽

Vol 97 (1) ◽

pp. 153-165 ◽

Cited By ~ 417

Author(s):

J A Madri ◽

S K Williams

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

Membrane Surface ◽

Phenotypic Expression ◽

Aortic Endothelial Cells ◽

Capillary Endothelial Cells ◽

Endothelial Cell Cultures ◽

Cell Densities

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.

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Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane

International Journal of Molecular Sciences ◽

10.3390/ijms222313120 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13120

Author(s):

Skadi Lau ◽

Manfred Gossen ◽

Andreas Lendlein

Keyword(s):

Basement Membrane ◽

Cell Types ◽

Adjacent Cell ◽

Design Strategies ◽

Graft Thrombosis ◽

Future Design ◽

Early Graft ◽

Endothelial Basement Membrane

Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure–function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field.

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Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes

Blood ◽

10.1182/blood-2007-05-089565 ◽

2008 ◽

Vol 111 (8) ◽

pp. 4096-4105 ◽

Cited By ~ 102

Author(s):

Marc Tjwa ◽

Lola Bellido-Martin ◽

Yuan Lin ◽

Esther Lutgens ◽

Stéphane Plaisance ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Heart Transplantation ◽

Mouse Models ◽

Inflammatory Stimuli ◽

Leukocyte Extravasation

AbstractThe role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

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Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽

10.1182/blood.v83.11.3206.bloodjournal83113206 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3206-3217 ◽

Cited By ~ 1

Author(s):

N Dubois-Stringfellow ◽

A Jonczyk ◽

VL Bautch

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Basement Membrane ◽

Organizational Behavior ◽

Cell Lines ◽

Primary Cultures ◽

Cyst Formation ◽

Fibrin Gels

Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

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Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro

Blood ◽

10.1182/blood.v69.6.1563.bloodjournal6961563 ◽

1987 ◽

Vol 69 (6) ◽

pp. 1563-1569

Author(s):

J Doukas ◽

D Shepro ◽

HB Hechtman

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

Polymorphonuclear Leukocyte ◽

Cytochalasin B ◽

Membrane Surface ◽

Stress Fiber ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells

Bovine aortic endothelial cells were cultured on the basement membrane surface of amnionic membrane and used as a substrate for polymorphonuclear leukocyte (PMN) diapedesis in vitro. Norepinephrine (NE), serotonin (5HT), or phalloidin treatment of the endothelial cells (ECs) reduces, whereas histamine or cytochalasin B increases, the number of PMNs migrating across the ECs and amnionic membrane. In contrast, amine treatment of PMNs or acellular amnionic membrane does not alter PMN diapedesis or chemotaxis. The NE and histamine effects are blocked by appropriate receptor antagonists, but the 5HT effect is not. All the agents' effects are also reversible. Qualitatively similar effects on EC permeability to Evan's blue-labeled albumin occur with all agents; however, PMN adhesion to ECs is not affected. Previously, we reported that NE and 5HT increase stress fiber numbers and decrease EC permeability to macromolecules in vitro, whereas histamine has the opposite effects, and that NE and 5HT eliminate the erythrocyte extravasation associated with thrombocytopenia in vivo. In this study, we propose that these vasoactive amines also alter PMN diapedesis in vitro through a direct effect on the EC, in part due to alterations in the EC cytoskeleton.

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A 3D-Printed, Modular, and Parallelized Microfluidic System with Customizable ECM Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells

10.26434/chemrxiv.12995846.v1 ◽

2020 ◽

Author(s):

Curtis G. Jones ◽

Tianjiao Huang ◽

Jay H. Chung ◽

Chengpeng Chen

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

3D Structure ◽

Microfluidic System ◽

Fully Integrated ◽

Membrane Topography ◽

3D Printed ◽

Easy Integration

Because dysfunctions of endothelial cells are involved in many pathologies, in vitro endothelial cell models for pathophysiological and pharmaceutical studies have been a valuable research tool. Although numerous microfluidic-based endothelial models have been reported, they had the cells cultured on a flat surface without considering the possible 3D structure of the native ECM. Endothelial cells rest on the basement membrane in vivo, which contains an aligned microfibrous topography. To better understand and model the cells, it is necessary to know if and how the fibrous topography can affect endothelial functions. With conventional fully integrated microfluidic apparatus, it is difficult to include additional topographies in a microchannel. Therefore, we developed a modular microfluidic system by 3D-printing and electrospinning, which enabled easy integration and switching of desired ECM topographies. Also, with standardized designs, the system allowed for high flow rates up to 4000 µL/min, which covered the full shear stress range for endothelial studies. We found that the aligned fibrous topography on the ECM altered arginine metabolism in endothelial cells, and thus increased nitric oxide production. To the best of our knowledge, this is the most versatile endothelial model that has been reported, and the new knowledge generated thereby lays a groundwork for future endothelial research and modeling.

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