Number Of Adherent Leukocytes Research Articles

Adhesion of human leukocytes on mixtures of hydroxyl‐ and methyl‐terminated self‐assembled monolayers: Effect of blood protein adsorption

The adhesion of human leukocytes to nanostructured surfaces with different chemical properties and the effect of protein adsorption were investigated. Self-assembled monolayers (SAMs) prepared with mixtures of methyl- and hydroxyl-terminated alkanethiols in different percentages on gold were used. The surfaces were pre-immersed in distinct protein solutions (human serum albumin, human fibrinogen, and autologous plasma). Adherent leukocytes were analyzed both by light and SEM. SAMs submitted to pre-immersion in plasma presented higher numbers of adherent leukocytes in the pure OH-terminated SAM, whereas methyl-terminated surfaces accounted for the lowest number of adherent cells. We observed a general increase in the number of adherent human leukocytes as the percentage of OH groups on the surface of the SAMs increased for all the pre-immersion conditions investigated. The number of adherent human leukocytes is highly influenced by the pre-immersion conditions used, and this observation is particularly relevant in the case of the methyl-terminated SAMs. The results obtained demonstrate that surface chemistry has a major influence in leukocyte adhesion to biomaterials, and that pre-immersion in protein solutions has a determinant effect in leukocyte adhesion.

Septic impairment of capillary blood flow requires activated coagulation pathway and is reversed by ascorbate through eNOS‐dependent dislodging of platelets in capillaries

Sepsis impairs capillary blood flow, leading to organ failure. We hypothesized that activation of the coagulation pathway in sepsis contributes to this impairment and that ascorbate reverses impairment by blocking the pathway. Sepsis in mice was induced by feces injection into peritoneum (FIP). Impaired flow in the hindlimb skeletal muscle was assessed with intravital microscopy. At time 0, mice were injected i.v. bolus of saline, platelet‐depleting Ab, P‐selectin blocking Ab or antithrombin, and then assessed at 6 h post‐FIP. Alternatively, eptifibatide (GPIIb/IIIa inhibitor) or ascorbate was injected at 6 h and impairment assessed at 7 h post‐FIP. In some mice, we injected rhodamine 6 G to view platelet and leukocyte adhesion in capillaries at 7 h. Sepsis impaired flow (complete stoppage) in 40 % of capillaries. In wild type mice, platelet depletion, P‐selectin blocking, antithrombin, eptifibatide, and ascorbate all reduced impairment to ~20 %. Sepsis increased the number of adhering platelets ~8 fold (the number of adhering leukocytes was negligible). Blocking of P‐selectin, eptifibatide, and ascorbate reduced this increase down to 3.2 ‐ 4.7 fold. Reduction in impairment and adhesion by ascorbate was not seen in eNOS‐/‐ mice. Thus, impairment of flow requires platelets, is inhibited by anticoagulants, and reversed by ascorbate through eNOS‐dependent dislodging of platelets in capillaries. (Support: HSFO)

DOCA‐salt hypertension promotes leukocyte (but not platelet) adhesion in cerebral venules

Although hypertension is a major risk factor for the development of ischemic stroke, the mechanisms underlying the influence of increased blood pressure on stroke risk remain poorly understood. Since both platelets and inflammatory cells have been implicated in the pathogenesis of ischemic stroke, we examined whether DOCA‐salt induced hypertension alters the behavior of platelets and leukocytes in cerebral venules in male mice. A 50 mg DOCA pellet was implanted via s.c. in uninephrectomized C57Bl/6 mice placed on 1 % saline for a period of 3 weeks, which produced a significant increase in blood pressure (from 103±2 to 127±2 mmHg) as well as cardiac and renal hypertrophy (relative to controls). Platelets isolated from matched donor mice were labeled ex vivo with CSFE while rhodamine was administered i.v. for in vivo labeling of leukocytes. Intravital microscopic observation of blood cell trafficking in pial venules revealed a large increase in the number of adherent leukocytes in DOCA‐salt mice (558±58 per mm2 vs 183±39 per mm2). However, platelet adhesion within venules was unaffected by DOCA‐salt (12±7 per mm2 vs 4±4 per mm2). These findings indicate that DOCA‐salt induced hypertension induced a pro‐inflammatory, but not a pro‐thrombogenic, phenotype in the cerebral microvasculature. The accumulation of inflammatory cells in the brain during hypertension may predispose this tissue to ischemic tissue injury. (Supported by HL26441).

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO 2), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO 2 was capable to deliver free radical nitric oxide ( ·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO 2 for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO 2 were observed in vivo by intravital microscopy assays. LNO 2 decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO 2 reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO 2 on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO 2 involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO 2 inhibited adhesion molecules expression and promoted ·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.

Phosphomannopentaose sulfate (PI-88) inhibits retinal leukostasis in diabetic rat

Retinal leukostasis, mediated by intracellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF), has been implicated in the pathogenesis of early diabetic retinopathy. Phosphomannopentaose sulfate (PI-88) is a highly sulfonated oligosaccharide which inhibits heparanase activity and competes with heparan sulfate binding to growth factors. In this study, we evaluated whether PI-88 could inhibit retinal leukostasis in strepotzotocin(STZ)-induced diabetic rat and elucidated the possible mechanisms. Diabetes was induced in Sprague–Dawley rats by intraperitoneal injection (i.p.) of STZ. Three months after induction, diabetic rats were administered PI-88 (25 mg/kg body weight) or vehicle solution daily via i.p. for 14 consecutive days. Leukostasis was analyzed on retinal flatmounts by concanavalin A and CD45 immunofluorescence staining. Retinal function was analyzed by electroretinography (ERG). ICAM-1 and VEGF levels in retinas were studied by Western blot and enzyme-linked immunosorbent assay (ELISA) respectively. The systemic administration of PI-88, but not vehicle, significantly decreased the number of adherent leukocytes in retinas by 52.24% ( P < 0.001) and led to significant preservation (about 50%, P < 0.001) of scotopic ERG a- and b-wave amplitudes in treated diabetic rats as compared to those of diabetic control rats. These changes were associated with downregulation of ICAM-1 (45%, P < 0.001) and VEGF (26.83 ± 2.01 versus 40.8 ± 3.24 pg/mg, P < 0.01) in retinas of PI-88 treated diabetic rats as compared to those of diabetic control rats. PI-88 significantly inhibited retinal leukostasis and reversed retinal dysfunction by a mechanism that may include decreased ICAM-1 and VEGF expression in diabetic rats. Our data suggests that PI-88 is a promising agent for the treatment of diabetic retinopathy.

Potential of 3,4-dihydroxy-phenyl lactic acid for ameliorating ischemia-reperfusion-induced microvascular disturbance in rat mesentery

This study intended to examine the effect of 3,4-dihydroxy-phenyl lactic acid (DLA), a major ingredient of Salvia miltiorrhiza, on ischemia-reperfusion (I/R)-induced rat mesenteric microcirculatory injury. DLA (5 mg.kg(-1).h(-1)), superoxide dismutase (SOD, 12,000 U.kg(-1).h(-1)), or catalase (CAT, 20 mg/kg) was continuously infused either starting from 10 min before the ischemia or 10 min after the initiation of reperfusion. The venule diameter, number of adherent leukocytes, FITC-albumin leakage, dihydrorhodamine 123 fluorescence, and mast cell degranulation were determined using an intravital microscope. The production of hydrogen peroxide (H(2)O(2)) and the expression of adhesion molecules CD11b/CD18 in neutrophils were evaluated by in vitro experiments. The results showed that pretreatment with DLA significantly reduced peroxide production in and leukocyte adhesion to venular wall, albumin leakage, and mast cell degranulation induced by I/R. The DLA posttreatment exerted an ameliorating effect on I/R-induced disorders as well, characterized by inhibiting further increase in peroxide production in venular wall and albumin leakage and diminishing the number of leukocytes that had adhered to the venular wall. In vitro experiments revealed that treatment with DLA significantly attenuated TNF-alpha plus fMLP-evoked production of H(2)O(2) and the H(2)O(2)-elicited expression of CD11b/CD18 on neutrophils. SOD and CAT manifested similarly but with the exception that either SOD or CAT were unable to retrieve the adherent leukocytes if administrated after initiation of reperfusion and to depress the H(2)O(2)-induced expression of CD11b/CD18 on neutrophils. It is concluded that DLA protects from and ameliorates the I/R-induced microcirculatory disturbance by interfering with both peroxide production and adhesion molecule expression.

Kinin B2receptor regulates chemokines CCL2 and CCL5 expression and modulates leukocyte recruitment and pathology in experimental autoimmune encephalomyelitis (EAE) in mice

BackgroundKinins are important mediators of inflammation and act through stimulation of two receptor subtypes, B1 and B2. Leukocyte infiltration contributes to the pathogenesis of autoimmune inflammation in the central nervous system (CNS), occurring not only in multiple sclerosis (MS) but also in experimental autoimmune encephalomyelitis (EAE). We have previously shown that the chemokines CCL2 and CCL5 play an important role in the adhesion of leukocytes to the brain microcirculation in EAE. The aim of the present study was to evaluate the relevance of B2 receptors to leukocyte-endothelium interactions in the cerebral microcirculation, and its participation in CNS inflammation in the experimental model of myelin-oligodendrocyte-glycoprotein (MOG)35–55-induced EAE in mice.MethodsIn order to evaluate the role of B2 receptor in the cerebral microvasculature we used wild-type (WT) and kinin B2 receptor knockout (B2-/-) mice subjected to MOG35–55-induced EAE. Intravital microscopy was used to investigate leukocyte recruitment on pial matter vessels in B2-/- and WT EAE mice. Histological documentation of inflammatory infiltrates in brain and spinal cords was correlated with intravital findings. The expression of CCL5 and CCL2 in cerebral tissue was assessed by ELISA.ResultsClinical parameters of disease were reduced in B2-/- mice in comparison to wild type EAE mice. At day 14 after EAE induction, there was a significant decrease in the number of adherent leukocytes, a reduction of cerebral CCL5 and CCL2 expressions, and smaller inflammatory and degenerative changes in B2-/- mice when compared to WT.ConclusionOur results suggest that B2 receptors have two major effects in the control of EAE severity: (i) B2 regulates the expression of chemokines, including CCL2 and CCL5, and (ii) B2 modulates leukocyte recruitment and inflammatory lesions in the CNS.

BURN PLASMA TRANSFER INDUCES BURN EDEMA IN HEALTHY RATS

Thermal injuries greater than 20% body surface area (BSA) result in systemic shock with generalized edema in addition to local tissue destruction. Burn shock is induced by a variety of mediators, mainly immunomodulative cytokines. This experimental study evaluates if burn shock can be induced in healthy rats by transfer of burn plasma (BP) with mediators. Thermal injury was induced by hot water (100 degrees C water, 12 s, 30% BSA) in male syngenic Wistar rats. Donor rats were killed 4 h posttrauma, and BP was harvested. Burn plasma was transferred to healthy animals by continuous intravenous infusion in three types of dilution (100%, 10%, and 1%). Positive controls were directly examined 4 h after thermal injury, and negative control rats had a continuous infusion done with sham burn (SB) plasma (37 degrees C water, 12 s, 30% BSA). Afterwards, intravital fluorescence microscopy was performed in postcapillary mesenteric venules at 0, 60, and 120 min. Edema formation was assessed by relative changes over time in fluorescence intensity of fluorescein isothiocyanate-albumin in the intravascular versus the extravascular space. The interactions of leucocytes and endothelium were evaluated by quantification of leukocyte sticking. Additionally, microhemodynamic (volumetric blood flow, erythrocyte velocity, venular wall shear rate, venular diameters) and macrohemodynamic parameters (blood pressure, heart frequency, temperature) were assessed online (arterial catheter). For statistics, an ANOVA was performed with Bonferroni adjustment procedure. Differences were considered significant when P < 0.05. There are no statistically significant differences in microhemodynamics or macrohemodynamics between study groups. Burn plasma infusion and thermal injury lead to significant increases in fluorescein isothiocyanate-albumin extravasation, whereas SB plasma shows no significant changes. Even BP diluted in 0.9% saline (10% and 1%) results in a similar transvascular flux of plasma proteins as direct thermal injury. Differences between positive controls and BP infusion are not significant, whereas all groups are statistically different from the SB group (P<0.05). Leukocyte sticking is significantly increased in all groups except the SB group, and the number of adherent leukocytes is dose dependent. The present study demonstrates that as early as 4 h after thermal injury, there are sufficient factors (e.g., cytokines) in BP to induce systemic burn shock in healthy rats even in diluted plasma (1%). However, the "key" cytokines are not identified at this point. The burned tissue is no longer required for burn shock induction, and the pathophysiologic process seems to be self-perpetuating as early as 4 h posttrauma. Leukocytes are activated by thermal injury and BP infusion. The role of leukocyte-endothelium interactions for edema formation remains uncertain and requires further investigation.

TNF-α INDUCES THROMBOXANE RECEPTOR SIGNALING-DEPENDENT MICROCIRCULATORY DYSFUNCTION IN MOUSE LIVER

TNF-alpha is a critical mediator of hepatic microcirculatory dysfunction during endotoxemia. The present study was to investigate the role of thromboxane A2 (TXA2) and the biological significance of thromboxane prostanoid (TP) receptor signaling in TNF-alpha-mediated hepatic microcirculatory dysfunction in male C57Bl/6 mice. The number of leukocytes adhering to the endothelial cells of the hepatic microvessels (the portal venules, sinusoids, and central venules) and the percentage of nonperfused sinusoids were determined using in vivo fluorescence microscopy. FR167653, an inhibitor of TNF-alpha, was administered 0 and 2 h after LPS injection. A TXA2 synthase inhibitor, OKY-046, was administered 30 min before TNF-alpha injection. Thromboxane prostanoid receptor knockout mice were used to investigate whether TNF-alpha-induced hepatic microcirculatory dysfunction is mediated by endogenously produced TXA2. FR167653 reduced LPS-induced leukocyte adhesion (50%-80%) and the percentage of nonperfused sinusoids (55%). The leukocyte adhesion was increased in the portal venules (8-fold), sinusoids (51-fold), and central venules (73-fold) in TNF-alpha-treated mice, accompanied with an increase in sinusoidal perfusion deficits (8-fold). Alanine aminotransferase levels rose as the adhesion of leukocytes increased. OKY-046 administration before TNF-alpha administration reduced leukocyte adhesion (41%-49% decrease) and sinusoid perfusion deficits (34% decrease). In TP receptor knockout mice, the number of adhering leukocytes, the percentage of nonperfused sinusoids, and alanine aminotransferase levels were lower (by 43%-56%, 41%, and 29%, respectively) than in wild-type counterparts. The results suggest that TP receptor signaling may promote hepatic microcirculatory dysfunction elicited by TNF-alpha. Blockade of TNF-alpha generation and TP receptor signaling may be a good strategy for managing endotoxin-induced hepatic injury.

Prevention of Leukocyte Activation by the Neutrophil Elastase Inhibitor, Sivelestat, in the Hepatic Microcirculation After Ischemia-Reperfusion

Liver ischemia-reperfusion (I/R) injury is one of the most serious complications of hepatic surgery. However, no effective treatment is yet clinically available. Although neutrophil elastase inhibitor (NEI) has been used clinically in acute lung injury, the effect of NEI on leukocyte dynamics in the liver microcirculation after hepatic I/R remained unclear. The purpose of this study was to use intravital microscopy (IVM) to evaluate the effect of NEI on leukocyte dynamics in the liver microcirculation after hepatic I/R. Hepatic ischemia was induced in male Sprague-Dawley (SD) rats. Sivelestat, a specific NEI, or normal saline (NS) was given as a continuous intravenous infusion before ischemia. The number of adherent leukocytes and the disturbances of sinusoidal perfusion in hepatic microcirculation were observed up to 120 min after reperfusion. Samples of liver tissue and blood were taken for histological examination and measurement of liver enzymes and tissue malondialdehyde (MDA). Compared with NS, sivelestat significantly decreased the number of adherent leukocytes and prevented perfusion disturbance. In addition, sivelestat obviously improved liver injury as assessed by histological findings and liver enzymes, and prevented the increase of MDA. Administration of sivelestat before ischemia effectively suppressed the activation of leukocytes and lipid peroxide, and it consequently prevented hepatic I/R injury.

Shiga-toxin-induced firm adhesion of human leukocytes to endothelium is in part mediated by heparan sulfate

Shiga toxin (Stx) is the main pathogenic factor in the haemolytic-uraemic syndrome (HUS). Stx damages the renal endothelium, which leads to inflammation and coagulation. Endothelial heparan sulfate proteoglycans (HSPG), and heparan sulfate in particular, play an important role in the inflammatory process by acting as a ligand for l-selectin. Furthermore, leukocytes are able to interact with chemokines bound to HSPG (examples are IL-8, RANTES and MCP-1). This leads to an activation of integrins on leukocytes and results in more stable leukocyte-endothelial wall adhesion. In this study, we have evaluated the effect of a subtoxic dose of Stx1 and Stx2 on the HSPG and its role in adhesion of leukocytes. Primary human umbilical venous endothelial cells (HUVEC) and primary human glomerular microvascular endothelial cells (GMVEC) were incubated for 24 h with a subtoxic dose of Stx1 or Stx2. Then, cells were treated with heparan sulfate-degrading enzyme heparitinase I or left untreated, followed by determination of binding leukocytes to endothelial cells in a parallel plate flow chamber. In both cell types, Stx increased the amount of firmly adherent leukocytes. After removal of endothelial heparan sulfate, the number of adhering leukocytes decreased. HSPG have a distinctive role in adhesion of leukocytes to endothelial cells stimulated by a subtoxic dose of Stx.

Prolene–Monocryl-composite meshes do not increase microvascular Staphylococcus aureus adherence and do not sensitize for leukocytic inflammation

Mesh implantation for hernia repair bears the risk of bacterial mesh infection. In this study, we analyzed whether this complication is supported by an increased interaction of bacteria and leukocytes with the microvascular endothelium at the implantation site. Ultrapro meshes were implanted into the dorsal skinfold chamber of Syrian golden hamsters. After 12 days, fluorescein isothiocyanate (FITC)-labeled staphylococci were injected in the animals. Subsequently, we analyzed bacterial adherence, leukocyte-endothelial cell interaction, and microhemodynamics in venules of the mesh border zone and of distant control tissue under baseline conditions and during TNF-alpha-induced inflammation using intravital fluorescence microscopy. The results were compared to animals which did not receive any bacteria. Under baseline conditions, leukocyte-endothelial cell interaction and bacterial adherence were not affected by the implanted biomaterial. TNF-alpha-induced inflammation significantly increased numbers of adherent leukocytes and bacteria in venules located in direct vicinity to the mesh however without any differences to control tissue. Comparable results were found for the leukocyte-endothelial cell interaction when animals were not exposed to bacteria. Implanted Ultrapro meshes do neither increase microvascular Staphylococcus aureus adherence nor sensitize for leukocytic inflammation. Thus, we suggest that a mesh-induced increase of bacterial adherence in vessels of the implantation site cannot be considered as a primary cause for the development of mesh infection.

Improving effect of post-treatment with Panax notoginseng saponins on lipopolysaccharide-induced microcirculatory disturbance in rat mesentery

Panax notoginseng saponin (PNS) is the collective of the major effective components of Panax notoginseng. The present study intended to explore the effect of post-treatment of PNS on rat mesentery microcirculatory disturbance induced by lipopolysaccharide (LPS) continuous challenge. By virtue of a microcirculation observation system, the vascular hemodynamics were determined continuously until 60 min of LPS (2 mg/kg/h) infusion through the left femoral vein. After observation, blood was taken for assessment of the expression of CD11b/CD18 in neutrophils and the concentration of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interferon-gamma (INF-gamma) in plasma with flow-cytometry. The number of leukocytes adherent to venular wall, the intensity of hydrogen peroxide dependent dihydrorhodamine 123 (DHR) fluorescence in the venular walls and albumin leakage from venules were increased impressively after 20 min of LPS infusion, the RBCs velocity diminished after 30 min, and degranulated mast cells increased remarkably after 60 min. Post-treatment with PNS (5 mg/kg/h) through the left jugular vein from 20 min of LPS exposure resulted in significant reduction in the number of adherent leukocytes, degranulation of mast cell, expression of CD11b and the concentration of IL-6, INF-gamma, while had no influence on the intensity of DHR fluorescence and albumin leakage. The results suggested that post-treatment with PNS significantly attenuated microcirculatory disturbance induced by LPS.

Time course of anti-inflammatory effect of low-dose radiotherapy: Correlation with TGF-β 1 expression

Background and Purpose Low-dose radiotherapy (LD-RT) has a potent anti-inflammatory effect, and transforming growth factor (TGF)-β 1 is a potential mediator of this effect. The objectives of this study were to characterize the in vivo effects of LD-RT on leukocyte recruitment over time, and its relationship with TGF-β 1 production. Materials and methods Mice were submitted to abdominal irradiation with a dose of 0.3 Gy, or to sham radiation and studied 5, 24, 48 or 72 h after irradiation. Four hours before the study a proinflammatory stimulus consisting of LPS or placebo was administered. Leukocyte–endothelial cell interactions in intestinal venules were assessed using intravital microscopy. Circulating levels and intestinal tissue production of TGF-β 1 were determined. Results Compared to non-irradiated LPS-challenged group, the number of adherent leukocytes was significantly reduced 5, 24 and 48 h, but not 72 h after irradiation in LPS-challenged mice. Rolling leukocytes were significantly decreased at all time points analyzed. Plasma TGF-β 1 levels were increased 5 and 24 h after irradiation. Increased intestinal production during this period was corroborated by in vitro culture experiments. Conclusions LD-RT has a sustained inhibitory effect on leukocyte recruitment for 48 h, which is initially associated with an increase in TGF-β 1 intestinal production.

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

Abstract 1193: Mast Cell Activation Promotes Leukocyte Recruitment And Adhesion To The Atherosclerotic Plaque

Activated mast cells have been identified in the perivascular tissue of human coronary artery plaques. As mast cells have been described to release a whole array of chemokines including interleukin 8 (IL-8) and MIP1 α, we propose that activated mast cells play a pivotal role in leukocyte recruitment at advanced stages of atherosclerotic plaque development. Peritoneal mast cells of either C57Bl/6 or mast cell deficient Kit(W −sh /W −sh ) mice were activated by injection of compound 48/80 (1.2 mg/kg). Interestingly, mast cell activation led to a massive neutrophil influx into the peritoneal cavity at 3 hours after activation (controls: 1 ± 0.7*10 4 Gr1 + -neutrophils/ml up to 8 ± 0.2*10 4 Gr1 + neutrophils/ml at 3 hours after activation, *P&lt;0.05), while neutrophil numbers in Kit(W −sh /W −sh ) mice were not affected by compound 48/80 administration. Moreover, increased levels of CXCR2 + Gr1 + neutrophils (t=0: 0.55 ± 0.07% versus t=3 hours: 1.00 ± 0.12%, *P&lt;0.05) were observed after mast cell activation. Next, we investigated whether mast cell activation also translated in induced leukocyte adhesion to advanced atherosclerotic plaques. Adventitial mast cells of advanced collar aided carotid artery plaques were activated by local application of a dinitrophenyl-BSA (DNP) challenge in ApoE −/− mice. Three days later, the carotid artery segments carrying the plaques were isolated and perfused ex vivo with rhodamine labeled leukocytes, showing a dramatically increased number of adherent leukocytes after mast cell activation (49 ± 6 versus 19 ± 4 leukocytes/microscopic field for DNP versus control plaques, respectively, **P&lt;0.001). Strikingly, antibody blockade of either the CXCR2 or VCAM-1 receptor VLA-4 on labeled leukocytes completely inhibited leukocyte adhesion to the atherosclerotic plaque (*P&lt;0.05), while blockade of CCR1, -3 and -5 with Met-RANTES had no effect. In conclusion, our data suggest that chemokines such as IL-8 released from activated perivascular mast cells induce leukocyte recruitment and adhesion to the atherosclerotic plaque, aggravating the ongoing inflammatory response and thus effecting plaque destabilization. We propose that mast cell stabilization could be a new therapeutic approach in the prevention of acute coronary syndromes.

Pial Microvascular Responses to Transient Bilateral Common Carotid Artery Occlusion: Effects of Hypertonic Glycerol

Objective: The aim of the study was to assess the rat pial microvessel alterations due to transient bilateral common carotid artery occlusion (BCCAO) and to investigate the mechanism of 10% hypertonic glycerol neuroprotection. Our suggestion was that 10% glycerol solution infusion could dilate pial arterioles through nitric oxide release and/or stimulation of ATP-sensitive potassium (K_ATP) channels. Therefore, we studied the effects of hypertonic glycerol after inhibition of nitric oxide synthase, with N^G-nitro-L-arginine-methyl ester or N^G-nitro-L-arginine, or K_ATP channels with glibenclamide. Methods: Pial microcirculation of male Wistar rats was visualized by a fluorescent microscopy technique through an open cranial window, using fluorescein isothiocyanate bound to dextran (molecular weight 70 kDa). BCCAO was induced for 30 min and reperfusion lasted 60 min. The arterioles were classified according to the Strahler ordering scheme. Permeability increase was quantified by normalized grey levels (NGL). Leucocytes were stained with rhodamine 6G. Perfused capillary length and capillary red blood cell (RBC) velocity were measured by computer-assisted methods. Results: The arterioles were assigned 5 orders of branchings, from order 1 (diameter 16.0 ± 2.5 µm) to order 5 (62.0 ± 5.0 µm). BCCAO caused inhomogenous changes in diameter of arterioles and leakage of fluorescent dextran, that was further enhanced by reperfusion (0.45 ± 0.05 NGL, p < 0.01). Adhesion of leukocytes to venules was marked and capillary perfusion was reduced by 39.2 ± 6.0% of baseline as well as capillary RBC velocity. 10% glycerol solution caused an increase in diameter of all arterioles within 25 ± 2 min of administration (by 20 ± 5% in order 4, 25 ± 4% in order 3 and 18 ± 3% in order 2; p < 0.01). Leakage (0.19 ± 0.03 NGL, p < 0.01), leukocyte adhesion (2.0 ± 1.0/100 µm of venular length, p < 0.01) and capillary occlusion (reduction by 13.0 ± 5.5% of baseline) were prevented compared with controls. Capillary RBC velocity increased compared with controls. N^G-nitro-L-arginine-methyl ester or N^G-nitro-L-arginine infused prior to glycerol caused vasoconstriction and reduced the protective effects of hypertonic glycerol on permeability increase. The number of adherent leukocytes and perfused capillary length decreased, while capillary RBC velocity was higher than baseline. Glibenclamide prior to 10% glycerol solution blunted glycerol-induced vasodilatation, but did not affect protection by hypertonic glycerol on blood-brain barrier disruption, leukocyte adhesion and capillary perfusion, preserving high capillary RBC velocity. Papaverine (20 mg/kg body weight) induced an increase in arteriolar diameter, enhancing interstitial edema; adhesion of leukocytes was marked as well as capillary occlusion, while capillary RBC velocity increased. Conclusions: 10% glycerol solution was able to prevent microvascular alterations due to BCCAO protecting cerebral tissue. The effects appear to be due to hyperosmolality causing stimulation of K_ATP channels, increase in vessel wall shear stress and release of nitric oxide.

Protective effects of ginsenoside Rb1, ginsenoside Rg1, and notoginsenoside R1 on lipopolysaccharide-induced microcirculatory disturbance in rat mesentery

Ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), and notoginsenoside R1 (R1) are major active components of Panax notoginseng, a Chinese herb that is widely used in traditional Chinese medicine to enhance blood circulation and dissipate blood stasis. To evaluate the effect of these saponins on microcirculatory disturbance induced by lipopolysaccharide (LPS), vascular hemodynamics in rat mesentery was observed continuously during their administration using an inverted microscope and a high speed video camera system. LPS administration decreased red blood cell velocity but Rb1, Rg1, and R1 attenuated this effect. LPS administration caused leukocyte adhesion to the venular wall, mast cell degranulation, and the release of cytokines. Rb1, Rg1, and R1 reduced the number of adherent leukocytes, and inhibited mast cell degranulation and cytokine elevation. In vitro experiments using flow cytometry further demonstrated that a) the LPS-enhanced expression of CD11b/CD18 by neutrophils was significantly depressed by Rb1 and R1, and b) hydrogen peroxide (H 2O 2) release from neutrophils in response to LPS stimulation was inhibited by treatment with Rg1 and R1. These results suggest that the protective effect of Rb1 and R1 against leukocyte adhesion elicited by LPS may be associated with their suppressive action on the expression of CD11b/CD18 by neutrophils. The protective effect against mast cell degranulation by Rb1 and R1, and the blunting of H 2O 2 release from neutrophils by Rg1 and R1 suggest mechanistic diversity in the effects of Panax notoginseng saponins in the attenuation of microcirculatory disturbance induced by LPS.

Real-time imaging of mechanically injured femoral artery in mice reveals a biphasic pattern of leukocyte accumulation

Wire injury of an artery has been recognized as a standard model of vascular inflammation and atherosclerosis; however, the mechanism of leukocyte recruitment has not been studied in this model. In this study, we documented the recruitment of leukocytes to the murine femoral artery after a wire injury. A transluminal mechanical injury was generated by insertion of a wire into the femoral artery of male C57BL/6J mice. The mice were anesthetized and ventilated after tracheotomy and protected from hypothermia by a warming lamp. Body temperature and blood pH did not significantly change during the experiment. The interaction between rhodamine 6G-labeled leukocytes and the injured femoral artery was monitored using an epifluorescent microscope, and the images were evaluated using a computer-assisted image analysis program. In the absence of injury, virtually no leukocyte adhesion was observed. In contrast, the number of adherent leukocytes increased 4 and 24 h after injury and declined 72 h after injury. The rolling flux of leukocytes increased 4 h after injury and remained high up to 7 days, but it was faster 72 h after injury. We identified another peak of leukocyte adhesion 7 days after injury. Injection of anti-P-selectin antibody significantly reduced leukocyte adhesion at the early and later phases. In conclusion, we have established a novel experimental system for direct observation of leukocyte recruitment to the injured femoral artery. Our system revealed a previously undetected, unique profile of leukocyte recruitment during vascular injury.

Long-term effects of oral vitamin C supplementation on the endothelial dysfunction in the iris microvessels of diabetic rats

The current study was aimed to investigate effects of long-term supplementation of vitamin C on the iris microcirculation in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar-Furth rats by intravenous injection of STZ (55 mg/kg b.w.). The rats were divided into three groups: control rats (CON), STZ-induced diabetic rats (STZ), and STZ rats supplemented with vitamin C (STZ-vitC). For supplementation of vitamin C, ascorbic acid (1 g/l) was added into the drinking water. The experiments were performed at different periods (8, 12, 24 and 36 weeks) after injection of STZ. Blood glucose, tissue lipid peroxidation and plasma vitamin C were measured. To examine the endothelial function, leukocyte adhesion to the venular endothelium was evaluated in the iris post-capillaries by means of counting the number of leukocytes labeled with rhodamine 6G. Blood flow perfusion in the iris was monitored using a laser Doppler flow meter. In the STZ rats, hyperglycemia was induced with an increase in HbA 1c and lipid peroxidation but with a decrease in the plasma vitamin C level which improved by vitamin C supplementation. The number of adherent leukocytes increased significantly, associated with reduction in the iris blood flow perfusion, at 8, 12, 24 and 36 weeks after injection of STZ. In the STZ-vitC rats, the iris blood flow perfusion was significantly increased in comparison with the STZ rats, while the leukocyte adhesion was decreased at 24 and 36 weeks. The statistical analysis shows that the leukocyte adhesion decreased with increase in the iris blood flow perfusion in STZ and STZ-vitC rats. In conclusion, vitamin supplementation suppressed leukocyte adhesion and thus endothelial dysfunction, associated with increase in iris blood flow perfusion in diabetes. The antioxidant vitamin C may be a therapeutic agent for preventing diabetic retinopathy.