Leukocyte-platelet aggregate adhesion and vascular permeability in intact microvessels: role of activated endothelial cells

2006 ◽  
Vol 291 (2) ◽  
pp. H591-H599 ◽  
Author(s):  
Pingnian He ◽  
Hong Zhang ◽  
Longkun Zhu ◽  
Yanyan Jiang ◽  
Xueping Zhou
Keyword(s):  
Endothelial Cells ◽  
Tissue Injury ◽  
Leukocyte Adhesion ◽  
Single Cells ◽  
Autologous Blood ◽  
Blood Perfusion ◽  
Endothelial Activation ◽  
Confocal Imaging ◽  
Platelet Aggregate ◽  
Microvessel Permeability

Leukocyte-platelet aggregation and aggregate adhesion have been indicated as biomarkers of the severity of tissue injury during inflammation or ischemic reperfusion. The objective of this study is to investigate the mechanisms of the aggregate adhesion and quantitatively evaluate its relationship with microvessel permeability. A combined autologous blood perfusion with single microvessel perfusion technique was employed in rat mesenteric venular microvessels. The aggregate adhesion was induced by systemic application of TNF-α plus local application of platelet-activating factor (PAF). Changes in permeability were determined by measurements of hydraulic conductivity ( Lp) before and after aggregate adhesion in the same individually perfused microvessels. The compositions of the adherent aggregates were identified with fluorescent labeling and confocal imaging. In contrast to leukocyte adhesion as single cells resulting in no increase in microvessel permeability, aggregate adhesion induced prolonged increases in microvessel Lp(6.1 ± 0.9 times the control, n = 9) indicated by the initial Lpmeasurements after 3 h of blood perfusion, which is distinct from the transient Lpincrease caused by PAF-induced endothelial activation in the absence of blood. Isoproteronol (Iso) attenuated aggregate adhesion-mediated Lpincreases if applied after autologous blood perfusion and prevented the aggregate adhesion if the initial endothelial activation is inhibited by applying Iso before PAF administration but showed less effect on single leukocyte adhesion. This study demonstrated that leukocyte-platelet aggregate adhesion via a mechanism different from that of single leukocyte adhesion caused a prolonged increase in microvessel permeability. Our results also indicate that the initial activation of endothelial cells by PAF plays a crucial role in the initiation of leukocyte-platelet aggregate adhesion.

Tumor necrosis factor-α-induced leukocyte adhesion and microvessel permeability

2002 ◽  
Vol 283 (6) ◽  
pp. H2420-H2430 ◽  
Author(s):  
Min Zeng ◽  
Hong Zhang ◽  
Clifford Lowell ◽  
Pingnian He
Keyword(s):  
Endothelial Cells ◽  
Intravenous Injection ◽  
Leukocyte Adhesion ◽  
Autologous Blood ◽  
Blood Perfusion ◽  
Solute Permeability ◽  
Tnf Α ◽  
Before And After ◽  
Microvessel Permeability ◽  
Tetramethylrhodamine Isothiocyanate

The objective of this study was to investigate whether leukocyte adhesion and/or emigration are critical steps in increased microvessel permeability during acute inflammation. To conduct this study, we combined autologous blood perfusion with a single microvessel perfusion technique, which allows microvessel permeability to be measured precisely after the endothelium has interacted with blood-borne stimuli. Experiments were carried out in intact venular microvessels in rat mesenteries. Firm attachment of leukocytes to endothelial cells was induced by intravenous injection of TNF-α (3.5 μg/kg) and resuming autoperfusion in a precannulated microvessel. Leukocyte emigration was facilitated by superfusion of formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as hydraulic conductivity ( Lp) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate-labeled α-lactalbumin before and after leukocyte adhesion and emigration in individually perfused microvessels. We found that perfusion of a microvessel with TNF-α did not affect basal microvessel permeability, but intravenous injection of TNF-α caused significant leukocyte adhesion. However, the significant leukocyte adhesion and emigration did not cause corresponding increases in either Lpor solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of endothelial cells. The appearance of the silver lines indicates that the TNF-α-induced firm adhesion of leukocyte to microvessel walls did not involve apparent changes in the junctional structure of endothelial cells, which is consistent with the results of permeability measurements.

fMLP-stimulated release of reactive oxygen species from adherent leukocytes increases microvessel permeability

2006 ◽  
Vol 290 (1) ◽  
pp. H365-H372 ◽  
Author(s):  
Longkun Zhu ◽  
Pingnian He
Keyword(s):  
Reactive Oxygen Species ◽  
Respiratory Burst ◽  
Leukocyte Adhesion ◽  
Autologous Blood ◽  
Blood Perfusion ◽  
Oxygen Species ◽  
Tnf Α ◽  
Microvessel Permeability ◽  
Adherent Leukocytes ◽  
Neutrophil Respiratory Burst

Our previous study ( Am J Physiol Heart Circ Physiol 288: H1331–H1338, 2005) demonstrated that TNF-α induced significant leukocyte adhesion without causing increases in microvessel permeability, and that formyl-Met-Leu-Phe-OH (fMLP)-stimulated neutrophils in the absence of adhesion increased microvessel permeability via released reactive oxygen species (ROS). The objective of our present study is to investigate the mechanisms that regulate neutrophil respiratory burst and the roles of fMLP-stimulated ROS release from adherent leukocytes in microvessel permeability. A technique that combines single-microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-α. Microvessel permeability was assessed by measuring hydraulic conductivity ( Lp). The 2-h autologous blood perfusion after TNF-α application increased leukocyte adhesion from 1.2 ± 0.2 to 13.3 ± 1.6 per 100 μm of vessel length without causing increases in Lp. When fMLP (10 μM) was applied to either perfusate ( n = 5) or superfusate ( n = 8) in the presence of adherent leukocytes, Lptransiently increased to 4.9 ± 0.9 and 4.4 ± 0.3 times the control value, respectively. Application of superoxide dismutase or an iron chelator, deferoxamine mesylate, after fMLP application prevented or attenuated the Lpincrease. Chemiluminescence measurements in isolated neutrophils demonstrated that TNF-α alone did not induce ROS release but that preexposure of neutrophils to TNF-α in vivo or in vitro potentiated fMLP-stimulated ROS release. These results suggest a priming role of TNF-α in fMLP-stimulated neutrophil respiratory burst and indicate that the released ROS play a key role in leukocyte-mediated permeability increases during acute inflammation.

scholarly journals Hepatic sinusoidal endothelium avidly binds platelets in an integrin-dependent manner, leading to platelet and endothelial activation and leukocyte recruitment

2013 ◽  
Vol 304 (5) ◽  
pp. G469-G478 ◽  
Author(s):  
Patricia F. Lalor ◽  
John Herbert ◽  
Roy Bicknell ◽  
David H. Adams
Keyword(s):  
Endothelial Cells ◽  
Liver Injury ◽  
Leukocyte Adhesion ◽  
Endothelial Activation ◽  
Dependent Manner ◽  
Hepatic Sinusoid ◽  
Public Expression ◽  
Platelet Binding ◽  
Sinusoidal Endothelium ◽  
Functional Relevance

Platelets have recently been shown to drive liver injury in murine models of viral hepatitis and promote liver regeneration through the release of serotonin. Despite their emerging role in inflammatory liver disease, little is known about the mechanisms by which platelets bind to the hepatic vasculature. Therefore, we referenced public expression data to determine the profile of potential adhesive receptors expressed by hepatic endothelium. We then used a combination of tissue-binding and flow-based endothelial-binding adhesion assays to show that resting platelets bind to human hepatic sinusoidal endothelial cells and that the magnitude of adhesion is greatly enhanced by thrombin-induced platelet activation. Adhesion was mediated by the integrins Gp1b, αIIbβIII, and αvβ3, as well as immobilized fibrinogen. Platelet binding to hepatic endothelial cells resulted in NF-κB activation and increased chemokine secretion. The functional relevance of platelet binding was confirmed by experiments that showed markedly increased binding of neutrophils and lymphocytes to hepatic endothelial cells under shear conditions replicating those found in the hepatic sinusoid, which was in part dependent on P-selectin expression. Thus the ability of platelets to activate endothelium and promote leukocyte adhesion may reflect an additional mechanism through which they promote liver injury.

Abstract 378: Contribution of Unfolded Protein Response in Arsenic-Induced Endothelial Activation and Atherosclerois

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Srinivas D Sithu ◽  
Nalinie S Wickramasinghe ◽  
Elena Vladykovskaya ◽  
Petra Haberzettl ◽  
Abhinav Agarwal ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Folding ◽  
Leukocyte Adhesion ◽  
Endothelial Activation ◽  
Aortic Endothelial Cells ◽  
Unfolded Protein ◽  
Atherosclerotic Lesions ◽  
Endothelial Migration ◽  
Nf Kappab ◽  
Protein Response

Epidemiological and animal studies suggest that exposure to arsenic contaminated water exacerbates atherosclerosis. However, the mechanisms by which arsenic exerts its atherogenic effects are not known. We observed that chronic (1 μM; 4 days) or acute (20 μM; 2-6 hours) exposure of sodium arsenite to human umbilical vein endothelial cells (HUVEC) increased the surface expression of adhesion molecules ICAM-1, VCAM-1 and E-Selectin by 1.2-1.5-fold; leukocyte adhesion by 1.5-3.5 fold; leukocyte trans-endothelial migration by 1.7-2.5-fold; and mRNA expression of IL-8 by 5-25-fold. Similarly, exposure of human aortic endothelial cells and mouse aortic endothelial cells to arsenic also caused endothelial activation. Arsenic also enhanced the unfolded protein response (UPR) in endothelial cells as evident by the phosphorylation of IRE-1 and activation of its downstream proteins JNK and NF-kappaB; nuclear translocation of ATF6, and increased expression of molecular chaperones GRP78 and HERP. SiRNA-mediated knockdown of IRE-1 attenuated arsenic-induced NF-kappaB activation and IL-8 expression, but did not affect JNK phosphorylation. Adenoviral transfection with ATF-6 upregulated GRP78 and decreased the expression of IL-8. Similarly, pre-incubation of endothelial cells with phenyl butyric acid (PBA; 5 mM, 16h), a chemical chaperone of protein folding, significantly (P<0.05) prevented arsenic-induced leukocyte adhesion, trans-endothelial migration and IL-8 expression. Feeding of apoE-null mice with PBA for 16 weeks inhibited the arsenic-induced UPR in atherosclerotic lesions; expression of adhesion molecules on endothelial cells lining the atherosclerotic lesions; lesional and systemic inflammation; and prevented the arsenic-induced exacerbation of lesion formation by 90% (P<0.05). Together, these data suggest that arsenic causes endothelial activation and exacerbates atherosclerosis by triggering UPR and chemical chaperones of protein folding prevent arsenic-induced exacerbation of atherogenesis.

Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between FcγRIIa and CXCR1/2

Blood ◽  
2007 ◽  
Vol 109 (9) ◽  
pp. 3881-3889 ◽  
Author(s):  
Oliver J. Florey ◽  
Michael Johns ◽  
Olubukola O. Esho ◽  
Justin C. Mason ◽  
Dorian O. Haskard
Keyword(s):  
Endothelial Cells ◽  
Lupus Erythematosus ◽  
Immune Complexes ◽  
Neutralizing Antibodies ◽  
Inflammatory Responses ◽  
Tissue Injury ◽  
Leukocyte Adhesion ◽  
Dependent Manner ◽  
Wegener Granulomatosis ◽  
Necessary And Sufficient

Abstract Antiendothelial cell antibodies (AECAs) are commonly detectable in diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Behçet syndrome, and transplant arteriosclerosis. Here, we explore the hypothesis that these antibodies might augment polymorphonuclear leukocyte (PMN) adhesion to endothelium in inflammation. Initially, we established that a mouse IgG mAb bound to endothelial cells (ECs) significantly increased PMN adhesion to cytokine-stimulated endothelium in an FcγRIIa-dependent manner. Neutralizing antibodies, and adenoviral transduction of resting ECs, demonstrated that the combination of E-selectin, CXCR1/2, and β2 integrins is both necessary and sufficient for this process. We observed an identical mechanism using AECA IgG isolated directly from patients with SLE. Assembled immune complexes also enhanced PMN adhesion to endothelium, but, in contrast to adhesion because of AECAs, this process did not require CXCR1/2, was not inhibited by pertussis toxin, and was FcγRIIIb rather than FcγRIIa dependent. These data are the first to demonstrate separate nonredundant FcγRIIa and FcγRIIIb-mediated mechanisms by which EC-bound monomeric IgG and assembled immune complexes amplify leukocyte adhesion under dynamic conditions. Furthermore, the observation that FcγRIIa and CXCR1/2 cooperate to enhance PMN recruitment in the presence of AECAs suggests a mechanism whereby AECAs may augment tissue injury during inflammatory responses.

scholarly journals Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca2+]i, nitric oxide, and gap formation in intact venules

2011 ◽  
Vol 301 (5) ◽  
pp. H1788-H1797 ◽  
Author(s):  
Xueping Zhou ◽  
Pingnian He
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Platelet Activating Factor ◽  
Cellular Level ◽  
Confocal Imaging ◽  
No Production ◽  
Molecular Signaling ◽  
Gap Formation ◽  
Microvessel Permeability ◽  
Endothelial Gaps

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca2+]i, NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca2+]i was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca2+]i, NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca2+]i preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca2+]i in each cell, suggesting that the initial levels of endothelial [Ca2+]i determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca2+ response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.

Antiphospholipid Antibodies Activate Vascular Endothelial Cells

Lupus ◽  
1996 ◽  
Vol 5 (5) ◽  
pp. 440-441 ◽  
Author(s):  
R Simantov ◽  
SK Lo ◽  
A Gharavi ◽  
LR Sammaritano ◽  
JE Salmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antiphospholipid Antibodies ◽  
Vascular Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Fetal Loss ◽  
Endothelial Activation ◽  
Vascular Endothelial ◽  
Endothelial Surface ◽  
Endotoxin Contamination ◽  
Leukocyte Adhesion Molecules

Antiphospholipid antibodies (aPL) are associated with a syndrome of arterial and venous thrombosis and recurrent fetal loss. We have shown that IgG purified from patients with aPL activate vascular endothelial cells (EC), converting the steady-state, non-thrombotic endothelial surface to a pro-thrombotic state. The aPL-activated EC are characterized by the expression of leukocyte adhesion molecules, including ICAM-1, VCAM, and E-selectin. EC activation is dependent upon the presence of β2-GP-I, a cofactor necessary for anticardiolipin reactivity. In addition, EC activation is not attributable to endotoxin contamination, Fc receptor interactions, or immune complexes, but rather is the result of the specific anticardiolipin reactivity of the IgG. Endothelial activation by aPL may be an important mechanism by which these antibodies cause a hypercoagulable state.

scholarly journals Haemodialysis through a cellulose membrane induces dephosphorylation of CD11b and promotes leukocyte adhesion to endothelial cells

2009 ◽  
Vol 32 (1) ◽  
pp. 48 ◽  
Author(s):  
E Rosa Hernandez ◽  
Berta Fuste ◽  
Aleix Cases ◽  
Raul Tonda ◽  
Gines Escolar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Blood Perfusion ◽  
Cellulose Membrane ◽  
Arterial Line ◽  
Leukocyte Activation ◽  
Cd11b Expression ◽  
Healthy Donors ◽  
Sds Page

Purpose: To explore modifications in signal mechanisms involving CD11b and leukocyte adhesion in patients under haemodialysis (HD). Methods: Samples were obtained from uremic patients at baseline, 15 and 120 min of HD from both arterial and venous lines. CD11b expression was studied by flow cytometry. To study signalling mechanisms, CD11b was immunoprecipitated using a specific antibody. Immunoprecipitates were resolved by 8% SDS-PAGE to measure phosphorylation in immunoblots. Leukocyte adhesion was measured after blood perfusion using endothelial cells (EC) as adhesive substrate. Parallel studies were performed with blood from healthy donors. Results: The percentage of CD11b+ cells increased during HD with a cellulose membrane in the venous line at 15 and 120 min (6.2±2.9% and 11.0±7.1%) and in the arterial line at 120 min (11.5±8.5 vs. 3.1±1.0% in control P < 0.05). After 120 min HD, CD11b phosphorylation decreased in leukocytes from both arterial (72.6±2.9) and venous lines (51.8±6.5) vs. basal samples (119.5±15.5 P < 0.005). Control leukocytes showed enhanced adhesion to uremic EC compared with control EC (3.0±0.3 vs. 2.3±1.0 leukocytes x100 EC-1 P < 0.05). Uremic leukocyte adhesion was enhanced after HD compared with basal samples 4.2±0.2 leukocytes/100 EC in the arterial and 4.4±0.3 in the venous line; after 120 min vs 2.3±1.0 (P < 0.005). Conclusion: Leukocyte activation during HD through a cellulose membrane occurs with decreases in CD11b phosphorylation. Activation also induces increases in CD11b expression associated with enhanced leukocyte adhesion to uremic endothelial cells.

scholarly journals Reduced endothelial activation upon human blood perfusion of pig kidney xenografts lacking MHC class I and three xenoantigens

2022 ◽  
Author(s):  
Lin Lin ◽  
Franca Witjas ◽  
Konrad Fischer ◽  
Marten Engelse ◽  
Annemarie de Graaf ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mhc Class I ◽  
Human Blood ◽  
Immune Activation ◽  
Whole Blood ◽  
Blood Perfusion ◽  
Endothelial Activation ◽  
Immune Rejection ◽  
Human Whole Blood ◽  
Activation Response

Abstract Genetically tailored pigs to eliminate human immune rejection of xenografts is one promising solution to the global donor organ shortage. The development of xenograft transplantation has however been hampered by incomplete understanding of its immune rejection and the inability to test this in a human transplantation setting. Here we use an ex vivo organ perfusion system with human whole blood to assess the initial immune activation within the xenograft endothelium at single cell transcriptome level. Renal injury, complement deposition, coagulation and lymphocyte influx are all strongly reduced in genetically modified pig kidneys with porcine MHC class I and three xenoantigens (GGTA1, CMAH, B4GALNT2) eliminated (4KO) compared to wildtype (WT) pig kidneys after 6-hours human blood perfusion. Single cell RNA sequencing of endothelial cells (EC) from 4KO and WT pig kidneys respectively reveal that there is a compartment (cortex, glomeruli and medulla) specific endothelial activation, with cortical and glomeruli endothelial cells being more affected. Differential gene expression analysis shows a downregulation of endothelial transcriptome activation response to human blood perfusion in the 4KO ECs. Pathway enrichment analysis further identify the NF-kB pathway as strongly activated in human blood perfused WT ECs but diminished in the 4KO. In conclusion, the 4KO pig model has strongly reduced endothelial immune activation response when perfused with human whole blood, that goes beyond prevention of humoral rejection. Our data support further development of the 4KO for use in clinical transplantation.

scholarly journals Mechanisms of platelet and leukocyte recruitment in experimental colitis

2007 ◽  
Vol 293 (5) ◽  
pp. G1054-G1060 ◽  
Author(s):  
Thorsten Vowinkel ◽  
Katherine C. Wood ◽  
Karen Y. Stokes ◽  
Janice Russell ◽  
Anitaben Tailor ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Sodium Sulfate ◽  
Platelet Adhesion ◽  
Tissue Injury ◽  
Leukocyte Adhesion ◽  
Microvascular Dysfunction ◽  
Experimental Colitis ◽  
Dss Colitis ◽  
Adherent Leukocytes

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes.

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