Cigarette smoke elicits leukocyte adhesion to endothelium in hamsters: Inhibition by CuZn-SOD

Although cigarette smoking has been identified as a major risk factor for cardiovascular diseases, the underlying pathomechanism is largely unknown. Using a dorsal skinfold chamber model in Syrian golden hamsters for intravital microscopy on striated muscle microcirculation, we investigated whether cigarette smoke (CS) affects the adhesion of circulating leukocytes to the endothelium, a constant feature of early atherogenesis and a hallmark of ischemia-reperfusion injury. Awake hamsters were exposed for 5 min to the mainstream smoke of one cigarette (2R 1 research cigarette), inducing nicotine, cotinine, and carboxyhemoglobin plasma levels comparable to levels found in human smokers. In control animals (n = 7), CS exposure elicited the rolling and subsequent adhesion of fluorescently stained leukocytes to the endothelium of arterioles and postcapillary venules. Leukocyte/endothelium interaction was preceded by an early rise in xanthine oxidase activity and intravascular hemolysis. Leukocyte adhesion and xanthine oxidase (Xo) activation were significantly attenuated in hamsters pretreated with superoxide dismutase (5 mg/kg, 10 min prior to CS, n= 7), suggesting a key role of superoxide in this event. These in vitro results suggest a novel pathomechanism of CS-induced cardiovascular pathology.     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Cigarette smoke extract increases albumin flux across pulmonary endothelium in vitro

Cigarette smoking causes lung inflammation, and a characteristic of inflammation is an increase in vascular permeability. To determine if cigarette smoke could alter endothelial permeability, we studied flux of radiolabeled albumin across monolayers of porcine pulmonary artery endothelium grown in culture on microporous membranes. Extracts (in either dimethylsulfoxide or phosphate-buffered saline) of cigarette smoke in a range estimate of concentrations simulating cigarette smoke exposure to the lungs in vivo caused a dose-dependent increase in albumin flux that was dependent on extracellular divalent cations and associated with polymerization of cellular actin. The effect was reversible, independent of the surface of endothelial cells exposed (either luminal or abluminal), and due primarily to components of the vapor phase of smoke. The effects occurred without evidence of cell damage, but subtle morphological changes were produced by exposure to the smoke extracts. These findings suggest that cigarette smoke can alter permeability of the lung endothelium through effects on cytoskeletal elements.     

Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4

It is commonly accepted that thrombin exerts its proinflammatory properties through the activation of proteinase-activated receptor (PAR)-1, although two other thrombin receptors have been discovered: PAR-3 and PAR-4. In this study, we have investigated the mechanisms and the receptors involved in thrombin-induced leukocyte/endothelial cell interactions by using selective agonists and antagonists of thrombin receptors in an in vivo intravital microscopy system. Topical addition of selective PAR-1 agonists to rat mesenteric venules failed to reproduce the increased leukocyte rolling and adhesion observed after thrombin topical addition. When added together with the selective PAR-1 antagonist RWJ-56110, thrombin was still able to provoke increased leukocyte rolling and adherence. The thrombin-induced leukocyte rolling and adherence was not affected by pretreatment of rats with an anti-platelet serum. Selective PAR-4-activating peptide was able to reproduce the effects of thrombin on leukocyte rolling and adhesion. Intraperitoneal injection of PAR-4-activating peptide also caused a significant increase in leukocyte migration into the peritoneal cavity. In rat tissues, PAR-4 expression was detected both on endothelium and isolated leukocytes. Taken together, these results showed that in rat mesenteric venules, thrombin exerts proinflammatory properties inducing leukocyte rolling and adherence, by a mechanism independent of PAR-1 activation or platelet activation. However, PAR-4 activation either on endothelial cells or on leukocytes might be responsible for the thrombin-induced effects. These findings suggest that PAR-4 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence and recruitment, and that in addition to PAR-1, PAR-4 could be involved in proinflammatory properties of thrombin.     

Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels

Leukocyte transendothelial migration and sequestration are two distinct outcomes following leukocyte adhesion to endothelium during ischemia-reperfusion injury, in which platelets may play a pivotal role. In the present study, we established an in vitro hypoxia-reoxygenation model to mimic ischemia-reperfusion injury and found platelet pre-incubation significantly increased leukocyte adhesion to endothelial cells after hyoxia-reoxygenation (over 67%). Blockade of endothelial-cell-expressed adhesion molecules inhibited leukocyte direct adhesion to endothelial cells, while platelet-mediated leukocyte adhesion was suppressed by blockade of platelet-expressed adhesion molecules. Further experiments revealed platelets acted as a bridge to mediate leukocyte adhesion, and platelet-mediated adhesion was the predominant pattern in the presence of platelets. However, platelet pre-incubation significantly suppressed leukocyte transendothelial migration after hypoxia-reoxygenation (over 31%), which could be aggravated by blockade of endothelial-cell-expressed adhesion molecules, but alleviated by blockade of platelet- expressed adhesion molecules. This would indicate that platelet-mediated adhesion disrupted leukocyte transendothelial migration. An in vivo mesenteric ischemia-reperfusion model demonstrated leukocyte transfusion alone caused mild leukocyte adhesion to reperfused vessels and subsequent leukocyte infiltration, while simultaneous leukocyte and platelet transfusion led to massive leukocyte adhesion and sequestration within reperfused microvessels. Our studies revealed platelets enhanced leukocyte adhesion to endothelial cells, but suppressed leukocyte transendothelial migration. Overall, this leads to leukocyte sequestration in hypoxia-reoxygenated microvessels.     

Irradiation increases the interactions of platelets with the endothelium in vivo: analysis by intravital microscopy

Adhesion of platelets to the endothelium is believed to be a major factor contributing to thrombosis and vascular occlusion after radiotherapy or endovascular irradiation. In the present study, platelet-endothelium interactions were analyzed in vivo by intravital microscopy in mesenteric venules of mice according to three parameters: (1) platelet rolling, (2) platelet adhesion, and (3) the presence of platelet clusters. A 10-Gy total-body irradiation of mice resulted in an increase in the frequency of appearance of these three types of platelet-endothelium interactions in postcapillary venules 6 and 24 h after exposure, whereas only minor alterations were seen in large venules. In addition, the duration of platelet adhesion was increased 24 h after irradiation in both postcapillary and large venules. However, P-selectin was not up-regulated on the platelet membrane and platelet-leukocytes were not seen rolling together, suggesting that changes in platelet-endothelial cell interaction result from endothelial cell activation rather than platelet activation. Our data suggest that irradiation transforms resting endothelial cells to a pro-adhesive surface for platelets, which could ultimately lead to thrombosis.     

A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

Influence of cell deformation on leukocyte rolling adhesion in shear flow

Blood cell interaction with vascular endothelium is important in microcirculation, where rolling adhesion of circulating leukocytes along the surface of endothelial cells is a prerequisite for leukocyte emigration under flow conditions. HL-60 cell rolling adhesion to surface-immobilized P-selectin in shear flow was investigated using a side-view flow chamber, which permitted measurements of cell deformation and cell-substrate contact length as well as cell rolling velocity. A two-dimensional model was developed based on the assumption that fluid energy input to a rolling cell was essentially distributed into two parts: cytoplasmic viscous dissipation, and energy needed to break adhesion bonds between the rolling cell and its substrate. The flow fields of extracellular fluid and intracellular cytoplasm were solved using finite element methods with a deformable cell membrane represented by an elastic ring. The adhesion energy loss was calculated based on receptor-ligand kinetics equations. It was found that, as a result of shear-flow-induced cell deformation, cell-substrate contact area under high wall shear stresses (20 dyn/cm2) could be as much as twice of that under low stresses (0.5 dyn/cm2). An increase in contact area may cause more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy input may decrease due to the flattened cell shape. Our model predicts that leukocyte rolling velocity will reach a plateau as shear stress increases, which agrees with both in vivo and in vitro experimental observations.     

Mechanisms of platelet and leukocyte recruitment in experimental colitis

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 co-dependent processes.     

Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (betaS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, betaS, and chimeric mice produced by transplanting bone marrow from WT or betaS mice into WT or P-selectin-deficient (P-sel(-/-)) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic betaS mice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from betaS mice to P-sel(-/-) mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with betaS bone marrow transfer to WT mice. These findings indicate that betaS mice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.     

The LFA-1 integrin supports rolling adhesions on ICAM-1 under physiological shear flow in a permissive cellular environment

The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A beta2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.     

Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E

We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.     

Time-dependent platelet-vessel wall interactions induced by intestinal ischemia-reperfusion

Platelets roll and adhere in venules exposed to ischemia-reperfusion (I/R). This platelet-endothelial adhesion may influence leukocyte trafficking because platelet depletion decreases I/R-induced leukocyte emigration. The objectives of this study were 1) to assess the time course of platelet adhesion in the small bowel after I/R and 2) to determine the roles of endothelial and/or platelet P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) in this adhesion. The adhesion of fluorescently labeled platelets was monitored by intravital microscopy in postcapillary venules exposed to 45 min of ischemia and up to 8 h of reperfusion. Peak platelet adhesion was observed at 4 h of reperfusion. To assess the contributions of platelet and endothelial cell P-selectin, platelets from P-selectin-deficient and wild-type mice were infused into wild-type and P-selectin-deficient mice, respectively. Platelets deficient in P-selectin exhibited low levels of adhesion comparable to that in sham-treated animals. In the absence of endothelial P-selectin, platelet adhesion was reduced by 65%. Treatment with a blocking antibody against PSGL-1 reduced adhesion by 57%. These results indicate that I/R induces a time-dependent platelet-endothelial adhesion response in postcapillary venules via a mechanism that involves PSGL-1 and both platelet and endothelial P-selectin, with platelet P-selectin playing a greater role.     

Avidity modulation activates adhesion under flow and requires cooperativity among adhesion receptors

An early step in activation of leukocyte adhesion is a release of integrins from cytoskeletal constraints on their diffusion, leading to rearrangement and, consequently, increased avidity. Static adhesion assays using purified ligand as a substrate have demonstrated that very low doses of cytochalasin D disconnect beta2-integrins from their cytoskeletal links, allowing rearrangement and activating adhesion. The adhesion process in blood vessels is poorly simulated by these assays, however, for two reasons: leukocyte adhesion to endothelium 1), occurs in the presence of blood flow and 2), involves the simultaneous interactions of multiple sets of adhesion molecules. We investigated the effect of cytochalasin D, at concentrations that increase integrin diffusion but do not alter leukocyte shape and surface features, on adhesion of leukocytes to endothelial cells under flow. Cytochalasin D increased the number of rolling cells, the number of firmly adherent cells, and the duration of both rolling and firm adhesion. These effects required endothelial cell expression of ICAM-1, the ligand for leukocyte beta2-integrins. The beta2-integrin-ICAM-1 interaction alone was not sufficient, however. Experiments using purified substrates demonstrated that avidity effects on activation of adhesion under flow require functional cooperativity between integrins and other adhesion receptors.     

Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion.

The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.     

Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.     

Intracellular heterogeneity in adhesiveness of endothelium affects early steps in leukocyte adhesion

Endothelial cell junctions are thought to be preferential sites for transmigration. However, the factors that determine the site of transmigration are not well defined. Our data show that the preferential role of endothelial cell junctions is not limited to transmigration but extends to earlier steps of leukocyte recruitment, such as rolling and arrest. We used primary mouse neutrophils and mouse aortic endothelium in a flow chamber system to compare adhesive interactions near endothelial cell junctions to interactions over endothelial cell centers. We found differences in both rolling velocity and arrest frequency for neutrophils at endothelial cell junctions vs. more central areas of endothelial cells. Differences were governed by adhesion molecule interactions, not local topography. Interestingly, the role of particular adhesion molecules depended on their location on the endothelial cell surface. Although ICAM-1 stabilized and slowed rolling over central areas of the cell, it did not influence rolling velocity over endothelial cell junctions. P-selectin and VCAM-1 were more important for rolling near endothelial cell junctions than E-selectin. This demonstrates that adhesive properties of endothelial cell junctions influence early events in the adhesion cascade, which may help explain how leukocytes are localized to sites of eventual transmigration. endothelial cells; rolling; selectins; integrins     

High-temporal-resolution analysis demonstrates that ICAM-1 stabilizes WEHI 274.1 monocytic cell rolling on endothelium

Leukocyte rolling, adhesion, and migration on vascular endothelium involve several sets of adhesion molecules that interact simultaneously. Each of these receptor-ligand pairs may play multiple roles. We examined the role of ICAM-1 in adhesive interactions with mouse aortic endothelial cells (MAECs) in an in vitro flow system. Average rolling velocity of the monocytic cell line WEHI 274.1 was increased on ICAM-1-deficient MAECs compared with wild-type MAECs, both with and without TNF- stimulation. High-temporal-resolution analysis provided insights into the underlying basis for these differences. Without TNF- stimulation, average rolling velocity was slower on wild-type than on ICAM-1-deficient endothelium because of brief (<1 s) pauses. On TNF--stimulated ICAM-1-deficient endothelium, cells rolled faster because of transient accelerations, producing "jerky" rolling. Firm adhesion to ICAM-1-deficient MAECs was significantly reduced compared with wild-type MAECs, although the number of rolling cells was similar. These results demonstrate directly that ICAM-1 affects rolling velocity by stabilizing leukocyte rolling. intercellular adhesion molecule-1; cell adhesion; leukocytes; vascular endothelium; videomicroscopy     

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease², stroke³ and heart failure⁴. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species⁵. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers⁶ however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration^7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry¹⁰. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model⁹ which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter¹¹ as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.     

Neutrophil capture by selectins on endothelial cells exposed to cigarette smoke

We used a novel perfusion system to expose cultured human umbilical vein endothelial cells (HUVEC) to water-soluble components of cigarette smoke and study subsequent adhesion of flowing neutrophils. Neutrophils did not bind to HUVEC immediately after it had been exposed to cigarette smoke, but many adhered 90-150 min after exposure. The effect was reduced if the exposed medium was made serum-free, but this reduction was partially reversed if low density lipoprotein was added. Treatment of smoke-exposed HUVEC with antibodies against E-selectin or P-selectin reduced adhesion by approximately 50% or 75%, respectively; a combination of both antibodies essentially abolished adhesion. Enzyme-linked immunosorbent assay confirmed that exposure to smoke caused HUVEC to upregulate surface expression of E- and P-selectin. Thus, water-soluble constituent(s) of cigarette smoke cause efficient selectin-mediated capture of flowing neutrophils. This pro-inflammatory response may contribute to pathology associated with smoking, especially in tissues remote from the lung.