Ethanol-Induced Leukocyte Adherence and Albumin Leakage in Rat Mesenteric Venules: Role of CD18/Intercellular Adhesion Molecule-1

The adherence and emigration of leukocytes have been implicated as a rate-limiting step in the microvascular disturbance in a variety of pathogenic events. The objective of the present study was to investigate leukocyte-endothelial cell adhesion and endothelial barrier function in rat mesenteric microvessels exposed to ethanol, which is known to cause inflammation and injury in various organs. Mesentery of male Wistar rats was used for intravital microscopic observations. Leukocyte adherence and albumin leakage were monitored in single postcapillary venules using the intravital fluorescence microscope. Supervision of 50 mM ethanol elicited the leukocyte adherence and albumin leakage within 60 min. Pretreatment with a monoclonal antibody directed against either CD18 or intercellular adhesion molecule-1 (ICAM-1) significantly prevented the ethanol-induced increase in leukocyte adherence and decrease in barrier function of endothelium. These results suggest that ethanol-induced leukocyte adherence is mediated by CD18 on leukocytes and ICAM-1 on endothelial cells. The present study further supports that CD18/ ICAM-1-dependent leukocyte-endothelial adhesive interactions lead to macromolecular leakage in the postcapillary venules exposed to ethanol.     

The role of T-lymphocytes in hypercholesterolemia-induced leukocyte-endothelial interactions

OBJECTIVE: Hypercholesterolemia promotes the adhesion of leukocytes to vascular endothelium in large and microscopic blood vessels. Lymphocytes that can modulate endothelial cell adhesion molecule expression have been implicated in the altered structure and function of large arterial vessels associated with chronic hypercholesterolemia. This study assesses the contribution of CD4(+) and CD8(+) T-cells to acute inflammatory responses observed in the microcirculation of hypercholesterolemic mice. METHODS: Intravital microscopy was used to quantify baseline leukocyte-endothelial cell interactions in cremasteric postcapillary venules of wild-type (WT) and severe combined immunodeficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. A group of SCID-HC mice received splenocytes from WT-HC mice (WT-->SCID). Separate WT-HC groups were depleted of neutrophils or CD4(+) and/or CD8(+) T-cells. RESULTS: WT-HC mice, compared with WT-ND, exhibited exaggerated leukocyte adherence and emigration. These leukocytes were predominantly granulocytes. These responses were absent in neutropenic WT-HC mice. SCID-HC mice also showed significantly less leukocyte adherence and emigration than WT-HC mice. Elevated leukocyte adherence and emigration were restored in WT-->SCID mice, despite a continued absence of circulating blood lymphocytes. Selective depletion of either CD4(+) or CD8(+) cell populations attenuated HC-induced leukocyte adhesion but not emigration. However, simultaneous depletion of both CD4(+) and CD8(+) cells attenuated both leukocyte adhesion and emigration to ND levels. DISCUSSION: These findings indicate that both CD4(+) and CD8(+) T-cells contribute to granulocyte adhesion and emigration elicited in postcapillary venules by hypercholesterolemia.     

T-lymphocytes modulate the microvascular and inflammatory responses to intestinal ischemia-reperfusion

OBJECTIVE: The overall objective of this study was to define the contribution of T-lymphocytes to the microvascular and inflammatory responses of the intestine to ischemia/reperfusion (I/R). METHODS: The superior mesenteric artery of wild-type (WT) and SCID mice was occluded for 45 minutes, followed by 30 minutes or 6 hours of reperfusion. Intravital fluorescence microscopy was used to monitor the extravasation of FITC-labeled albumin or the adhesion of carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled T-lymphocytes in mucosal venules of the postischemic intestine. Tissue myeloperoxidase (MPO) was used to monitor neutrophil accumulation in the intestine of WT and SCID mice. RESULTS: Although the number of adherent T-cells was not increased above baseline at 1 hour after reperfusion, significant T-cell adhesion (both CD4(+) and CD8(+)) was noted at 6 hours of reperfusion. The latter response was prevented by pretreatment with a blocking antibody directed against MAdCAM-1, but not ICAM-1 or VCAM-1. A significant increase in MAdCAM-1 expression was noted in both lymphoid (Peyer's patch) and nonlymphoid regions of the postischemic small bowel. The early (30 minutes after reperfusion) albumin extravasation elicited by gut I/R in WT mice was reduced in SCID mice. Reconstitution of SCID mice with T-lymphocytes restored the albumin leakage response to WT levels. The increased intestinal MPO caused by I/R (6 hours of reperfusion) in WT mice was attenuated in SCID mice; with reconstitution of SCID mice with T-cells the MPO response was restored. CONCLUSIONS: These findings indicate that intestinal I/R is associated with the recruitment of CD4+ and CD8+ T-cells, which is mediated by endothelial MAdCAM-1. T-cells seem to modulate the recruitment of neutrophils that occurs hours after reperfusion as well as the increased albumin extravasation that occurs within minutes after reperfusion.     

Oxidative Stress During Platelet-Activating Factor-Induced Microvascular Dysfunction

Objective : To assess the potential contribution of hydrogen peroxide (H₂O₂) to the leukocyte-endothelial cell adhesion and increased microvascular permeability (to fluorescein isothiocyanate [FITC]-albumin) observed in rat mesenteric venules exposed to platelet-activating factor (PAF). Methods : The production of oxidants derived from H₂O₂ in mesenteric tissue was monitored using the H₂O₂-sensitive fluorochrome, dihydrorhodamine 123 (DHR). PAF elicited a rapid increase in both the rate of albumin extravasation and oxidation of DHR, which was followed by an increased adherence and emigration of leukocytes in postcapillary venules. Results : The PAF-induced oxidation of DHR, leukocyte-endothelial cell interactions, and albumin leakage were attenuated by treatment with either catalase or dimethylthiourea. Treatment with monoclonal antibody directed against either CD11b/CD18 on leukocytes or ICAM-1 on endothelial cells attenuated the PAF-induced oxidative stress, albumin leakage, and leukocyte-endothelial cell adhesion. Conclusions : These findings indicate that most of the oxidants generated in mesenteric tissue exposed to PAF results from the accumulation of activated leukocytes.     

Protective effects of N-acetyl-L-cysteine and platelet activating factor inhibition are not linked to intercellular adhesion molecule-1 expression after intestinal ischemia and reperfusion injury in rats

BACKGROUND: Intestinal ischemia and reperfusion (I/R) injury may result in development of the systemic inflammatory response syndrome (SIRS). The interactions between activated leukocytes and endothelial cells, mediated by adhesion molecules, seem to be pivotal in these conditions, leading as they do to extravasation of circulating leukocytes within the inflamed tissue. The intercellular adhesion molecule-1 (ICAM-1) mediating firm adhesion of activated leukocytes is upregulated in many organs after I/R injury, but the regulatory mechanisms are complex and have not been fully investigated. METHODS: We evaluated whether ICAM-1 expression was linked with a potential protective effect of N-acetyl-L-cysteine (NAC) and the platelet activating factor (PAF) inhibitor (Lexipafant), administered 15 min after the start of reperfusion, in a model of intestinal ischemia (40 min) and reperfusion (12 h) in the rat. RESULTS: ICAM-1 expression increased significantly in the ileum, colon, lungs and pancreas after intestinal I/R. Treatments with NAC and the PAF inhibitor did not affect this response. An increased endothelial albumin-leakage was observed in the same organs after I/R. Treatment with NAC reduced the endothelial leakage of albumin in the ileum, colon and lungs, whereas administration of the PAF inhibitor alone demonstrated a protective effect only in the ileum. Furthermore, neutrophil sequestration in the lungs and IL-1beta levels in plasma increased significantly after I/R, and these changes were markedly reduced by both treatment regimes. CONCLUSION: The protective effect of NAC and the PAF inhibitor Lexipafant in intestinal I/R injury is not due to a decreased expression of ICAM-1.     

AT1 receptor blockade prevents microvascular dysfunction induced by ischemia/reperfusion injury

Ischemia/reperfusion (I/R) in post-arterior post-capillary venules induces an acute inflammatory response, characterized by increased adherence and emigration of leukocytes and vascular permeability, all of which play important roles in cardiovascular disease. The aim of this study was to determine the roles of angiotensin II and AT1 receptor blockade in microvascular I/R injury in rats. Rats were anesthetized and intubated, then the peritoneum was opened and the mesentery was revealed. Small post-capillary venules were examined by in vivo fluorescence microscopy. The flow of erythrocytes and leukocytes was observed under the microscope and video recorded for later dynamic analyses. The superior mesenteric artery (SMA) was ligated with polyethylene tubing and released to induce I/R (20 min of ischemia/60 min of reperfusion). Subsequently, leukocyte adhesion, emigration and albumin leakage were compared with those of non-I/R controls. I/R injury was significantly suppressed by superfusing tissues with the AT1 receptor antagonist losartan (LO; 10 microM). The beneficial effects of LO were inhibited by topical application of either the bradykinin B2 receptor antagonist HOE140 (10 nM) or nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME 10 microM). The effects of LO were lost in the presence of AT2 receptor blocker PD 123319 (PD).In conclusion, LO suppressed and protected against I/R injuries. The possible interaction between AT1 and AT2 receptors was also suggested.     

Protective Effects of N-Acetyl- L -Cysteine and Platelet Activating Factor Inhibition Are Not Linked to Intercellular Adhesion Molecule-1 Expression after Intestinal Ischemia and Reperfusion Injury in Rats

Background: Intestinal ischemia and reperfusion (I/R) injury may result in development of the systemic inflammatory response syndrome (SIRS). The interactions between activated leukocytes and endothelial cells, mediated by adhesion molecules, seem to be pivotal in these conditions, leading as they do to extravasation of circulating leukocytes within the inflamed tissue. The intercellular adhesion molecule-1 (ICAM-1) mediating firm adhesion of activated leukocytes is upregulated in many organs after I/R injury, but the regulatory mechanisms are complex and have not been fully investigated. Methods: We evaluated whether ICAM-1 expression was linked with a potential protective effect of N-acetyl- l -cysteine (NAC) and the platelet activating factor (PAF) inhibitor (Lexipafant), administered 15 &#114 min after the start of reperfusion, in a model of intestinal ischemia (40 &#114 min) and reperfusion (12 &#114 h) in the rat. Results: ICAM-1 expression increased significantly in the ileum, colon, lungs and pancreas after intestinal I/R. Treatments with NAC and the PAF inhibitor did not affect this response. An increased endothelial albumin-leakage was observed in the same organs after I/R. Treatment with NAC reduced the endothelial leakage of albumin in the ileum, colon and lungs, whereas administration of the PAF inhibitor alone demonstrated a protective effect only in the ileum. Furthermore, neutrophil sequestration in the lungs and IL-1 &#35 levels in plasma increased significantly after I/R, and these changes were markedly reduced by both treatment regimes. Conclusion: The protective effect of NAC and the PAF inhibitor Lexipafant in intestinal I/R injury is not due to a decreased expression of ICAM-1.     

Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats

Recent evidence suggests an active role of endothelial cells and inflammatory cells in radiation-induced vascular dysfunction and organ damage. The aim of this study was to characterize the endothelial cell-leukocyte interactions, their molecular mechanisms, and the associated microvascular dysfunction in postcapillary venules exposed to ionizing radiation. Leukocyte rolling, adherence, and emigration and leakage of fluorescein isothiocyanate albumin in rat mesenteric venules were measured in control conditions and at 2, 4, and 6 hours after abdominal irradiation. Some animals were treated with monoclonal antibodies against leukocyte (CD18) or endothelial cell (intercellular adhesion molecule 1, P-selectin) adhesion molecules before radiation and 5 hours thereafter. In comparison with controls, irradiated animals had a marked increase in the number of rolling leukocytes at 2 hours after radiation. In animals studied 6 hours after radiation, a significant increment in the number of adherent and emigrated leukocytes was observed. This was associated with an increased permeability to fluorescein isothiocyanate albumin. Treatment with antibodies against either CD18 or intercellular adhesion molecule 1, but not P-selectin, significantly attenuated leukocyte adherence, emigration, and the increase in permeability induced by radiation. Radiation-induced leukocyte adherence and emigration involves an interaction between CD11/CD18 on leukocytes and intercellular adhesion molecule 1 on vascular endothelium. These interactions are implicated in the early increase in vascular permeability after irradiation.     

Compound Danshen injection improves endotoxin-induced microcirculatory disturbance in rat mesentery

AIM： To investigate the effect of compound Danshen injection on lipopolysaccharide （LPS）-induced rat mesenteric microcirculatory dysfunctions and the underlying possible mechanism by an inverted intravital microscope and high-speed video camera system. METHODS： LPS was continuously infused through the jugular artery of male Wistar rats at the dose of 2 mg/kg per hour. Changes in mesenteric microcirculation, such as diameters of arterioles and venules, velocity of RBCs in venules, leukocyte rolling, adhesion and emigration, free radicals released from post-capillary venules, FITC- albumin leakage and mast cell degranulation, were observed through an inverted intravital microscope assisted with CCD camera and SIT camera. Meanwhile, the expression of adhesion molecules CD11b/CD18 and the production of free radical in neutrophils, and the expression of intercellular adhesion molecule 1 （ICAM-1） in human umbilical vein endothelial cells （HUVECs） were quantified by flow cytometry （FACS） in vitro. RESULTS： The continuous infusion with LPS resulted in a number of responses in microcirculation, including a significant increase in the positive region of venulestained with Monastral blue B, rolling and adhesion of leukocytes, production of oxygen radical in venular wall, albumin efflux and enhanced mast cell degranulation in vivo, all of which, except for the leukocyte rolling, were attenuated by the treatment with compound Danshen injection. Experiments performed in vitro further revealed that the expression of CD11b/CD18 and the production of oxygen free radical in neutrophils, and the expression of ICAM-1 in HUVECs were increased by exposure to LPS, and they were attenuated by compound Danshen injection. CONCLUSION： These results suggest that compound Danshen injection is an efficient drug with multi-targeting potential for improving the microcirculatory disturbance.     

ICAM-1 expression and leukocyte behavior in the microcirculation of chronically ischemic rat skeletal muscles

In muscle microcirculation, short periods of ischemia followed by reperfusion are known to upregulate leukocyte and endothelial adhesion molecules, but little is known about leukocyte adherence and ICAM-1 expression during chronic ischemia or any likely effect of muscle activity which is recommended in chronic ischemia due to peripheral arterial disease. Leukocyte rolling and stationary adhesion were observed in post-capillary venules in ischemic and contralateral rat extensor digitorum longus (EDL) muscles 3 and 7 days after unilateral ligation of the common iliac artery and in 3-day ischemic EDLs that were electrically stimulated on days 1 and 2 post-ligation (7 x 15 min per day). ICAM-1 was localized immunohistochemically to venular vessels in all muscles. Following ligation, use of the ischemic leg was observed to be restricted for the first 3 days, returning to normal by 7 days. After 3 days, leukocyte rolling/adherence and ICAM-1 expression were no different in ischemic than control muscles, but all were increased in contralateral muscles. In ischemic muscles, electrical stimulation doubled the numbers of rolling leukocytes and upregulated ICAM-1 expression. After 7 days, increased muscle activity as a result of natural movement also resulted in greater ICAM-1 expression, a 4- to 5-fold increase in rolling leukocyte numbers and a 3-fold increase in stationary adherent leukocytes. Chronic ischemia thus increases ICAM-1 and leukocyte adherence in muscle microcirculation only when combined with contractile activity. Post-capillary venular endothelium may be modified by muscle acidosis when contractions are performed under low flow conditions or by changes in rheological (shear force) factors.     

Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium

Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte beta2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte beta1 integrin (alpha4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against alpha4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.     

In vivo evaluation of retinal injury after transient ischemia in hypertensive rats

A number of studies have suggested that hypertension affects the pathogenesis of inflammatory reactions in various organs. The objective of this study was to evaluate the effects of hypertension on leukocyte-endothelial interactions after transient retinal ischemia. Transient retinal ischemia was induced for 60 minutes in spontaneously hypertensive rats (SHR) and in age-matched normotensive Wistar-Kyoto rats (WKY). At 4, 12, 24, 48, and 72 hours after reperfusion, flat-mount retinas were prepared to evaluate the density of leukocytes that had been accumulated in the retina. Intercellular adhesion molecule-1 (ICAM-1) mRNA expression was studied by semiquantitative polymerase chain reaction and ICAM-1 protein levels were studied by enzyme-linked immunosorbent assay. At 14 days after reperfusion, the retinal damage and the effect of superoxide dismutase on the damage were evaluated histologically. In SHR, the number of accumulated leukocytes peaked at 48 hours after reperfusion, and it was upregulated to 5.2-fold, as compared with that of WKY (P<0.001). ICAM-1 mRNA expression and ICAM-1 protein levels were increased significantly in the ischemia-reperfused retina in SHR compared with WKY (P<0.05). Histological examination demonstrated marked increase in the retinal ischemia/reperfusion damage in SHR (P<0.01) and a significant amelioration of the damage by treatment with superoxide dismutase in SHR (P<0.05). Oxidative stress may thus be an important mechanism for the deterioration seen in ischemia/reperfusion injury in the SHR retina.     

Leucocyte-endothelial cell adhesion in a model of intestinal inflammation.

Leucocyte-endothelial cell adhesion is modulated by a variety of adhesion glycoprotein expressed on the surface of leucocytes and endothelial cells. Although in vitro studies show that these adhesion molecules mediate the decrease in leucocyte rolling velocity and the increase in leucocyte adherence and emigration associated with inflammation, there are few in vivo data to support this hypothesis. The aim of this study was to assess the role of leucocyte (CD11b/CD18) and endothelial cell (P- and E-selectin) adhesion molecules in mediating the leucocyte-endothelial cell adhesion elicited in rat mesenteric venules during a model of longlasting intestinal inflammation. Indomethacin was injected 48 and 24 hours before the experiment. The mesenteric microcirculation was observed by intravital microscopy in animals treated with monoclonal antibodies (MAb) directed against either P-selectin, E-selectin, or CD11b/CD18. Leucocyte rolling velocity, and the number of adherent and emigrated leucocytes as well as vessel diameter and erythrocyte velocity were monitored in roughly 30 micron diameter postcapillary venules. Indomethacin treatment resulted in mucosal ulceration and granulocyte infiltration, and a corresponding inflammatory response in the mesentery, which was characterised by an increase in the number of adherent (eightfold) and emigrated (sixfold) leucocytes and a reduction (80%) in leucocyte rolling velocity. The indomethacin induced leucocyte-endothelial cell adhesion in mesenteric venules was significantly reduced by treatment with MAbs against either CD11b/CD18 or E-selectin, but not by the P-selectin MAb. These results suggest that both leukocyte (CD11b/CD18) and endothelial cell (E-selectin) adhesion molecules contribute to the granulocyte accumulation in a chronic model of intestinal inflammation.     

Modulation of leukocyte adhesion in rat mesenteric venules by aspirin and salicylate.

Erythrocyte velocity, vessel diameter, leukocyte rolling velocity, and number of adherent and emigrated leukocytes were measured in postcapillary venules both before and during superfusion of rat mesentery with either aspirin or sodium salicylate. In some experiments, animals were treated with either a leukotriene (LT)-synthesis inhibitor (L-663,536), an LTD4 antagonist (MK-571), an LTB4 antagonist (SC-41930), misoprostol, or prostaglandin (PG) I2, then the aspirin protocol was repeated. Superfusion of aspirin but not sodium salicylate resulted in increased leukocyte adherence and a reduced leukocyte rolling velocity but did not affect leukocyte emigration. Aspirin-induced leukocyte adhesion was effectively prevented by the LT-synthesis inhibitor and LTB4 antagonist but not by the LTD4 antagonist. Misoprostol and PGI2 also prevented the aspirin-induced adhesion responses. Superfusion of the mesentery with either platelet-activating factor (PAF) or LTB4 enhanced leukocyte adherence and emigration while reducing leukocyte rolling velocity. Sodium salicylate prevented all of the adhesion responses elicited by LTB4. Although salicylate did not affect the PAF-induced leukocyte adherence and rolling responses, it completely prevented the increased leukocyte emigration. These results indicate that aspirin promotes, whereas sodium salicylate inhibits, leukocyte-endothelial cell adhesive interactions at therapeutically relevant concentrations.     

Neutrophil-derived oxidants promote leukocyte adherence in postcapillary venules.

The objective of this study was to determine whether hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and monochloramine (NH2Cl), at concentrations produced by activated neutrophils, promote leukocyte adherence to microvascular endothelium in post-capillary venules. Cat mesenteric venules (30-45 microns diameter) were examined using intravital video microscopy. Red blood cell velocity (VRBC), venular diameter (DV), and the number of adherent leukocytes (NWBC) were measured in postcapillary venules. Venular blood flow and wall shear rate (tau) were calculated from the measured values of VRBC and DV. Different concentrations (0.01-1.0 mM) of H2O2, HOCl, or NH2Cl were superfused on the mesentery. In some experiments, the contributions of the leukocyte adhesive glycoprotein CD11/CD18 and platelet-activating factor (PAF) in the oxidant-induced leukocyte adherence were determined using a CD18-specific antibody (IB4) and a PAF-receptor antagonist (WEB 2086), respectively. The results of our in vivo experiments indicate that H2O2 and NH2Cl, but not HOCl, promote leukocyte adhesion to venular endothelium. Incubation of isolated cat neutrophils with either NH2Cl or H2O2 resulted in activation of CD11/CD18, as assessed by flow cytometry. Although the leukocyte adhesion induced by both H2O2 and NH2Cl was associated with a reduction in venular wall shear rate, corresponding decrements in shear rate induced by partial occlusion of the mesenteric artery did not lead to similar levels of leukocyte adherence. The leukocyte adherence induced by H2O2 and NH2Cl was largely prevented by monoclonal antibody IB4, indicating that both oxidants promote leukocyte adherence via activation of CD11/CD18. The H2O2-induced, CD18-mediated leukocyte adherence appears to be elicited by PAF and by a direct effect of the oxidant on CD11/CD18 expression. The mechanism underlying the NH2Cl-induced leukocyte adherence remain unclear.     

Mouse cremaster venules are predisposed to light/dye-induced thrombosis independent of wall shear rate, CD18, ICAM-1, or P-selectin

OBJECTIVE: Microvascular adhesion of platelets to endothelium occurs in response to various inflammatory stimuli, and in venules is often accompanied by adherent leukocytes. In a light/dye injury model, platelet adhesion and thrombi occur preferentially in venules, though the reasons for this predisposition are unknown. The authors sought to determine whether lower wall shear rates or leukocyte-endothelial interactions accounted for preferential platelet thrombi formation in venules relative to arterioles. METHODS: A light/dye injury model of microvascular thrombosis was used in the mouse cremaster microcirculation. RESULTS: In wild-type mice (n = 17), the time to form microvascular platelet aggregates was delayed in arterioles by 3.1-fold relative to venules (p <.0001). However, arterioles with spontaneously low wall shear rates, as well as arterioles manipulated to reduce wall shear rate to venous levels, still had delayed thrombosis as compared to venules. Similarly, in animals deficient in CD18, P-selectin, or ICAM-1, the time to form platelet thrombi in arterioles was >3.0-fold higher than in venules. CONCLUSIONS: Mouse cremaster venules are predisposed to light/dye-induced microvascular thrombosis. The data suggest that functional differences between arteriolar and venular endothelial cells (independent of wall shear rate and of CD18, P-selectin, and ICAM-1) account for the venular predisposition to thrombosis.     

Nitric oxide: an endogenous modulator of leukocyte adhesion.

The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.     

In Vivo Platelet Thrombus Formation in Microvessels of Spontaneously Hypertensive Rats

Sustained high blood pressure causes functional changes in both vascular endothelial cells and platelets. Therefore, we hypothesized that in vivo platelet thrombus formation would be increased in the cremaster muscle microvessels of rats during genetic hypertension. Experiments were carried out on spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) at 12 weeks of age. Fluorescein isothiocyanate tagged to bovine serum albumin (FITC-BSA) was injected intraarterially and 450 to 490 nm light was used to activate the FITC-BSA and induce a thrombus within the vasculature. In vivo television microscopy was used to quantitate thrombus formation and microvascular diameter changes. The time of platelet thrombus initiation and subsequent time of thrombus growth were studied at wall shear rates of approximately 2000 sec⁻¹ and 270 sec⁻¹ in third-order arterioles and venules, respectively. In SHR, times for platelet thrombus initiation and vessel occlusion were significantly less in both arterioles and venules, whereas time for platelet thrombus growth following initiation was significantly prolonged. Greater shear rates in arterioles compared to venules decreased platelet adhesion and subsequently decreased the rate of thrombus formation in both WKY and SHR groups. However, the ratio of WKY to SHR platelet thrombus growth (platelet aggregation) time remained similar (0.83 ± 0.06 in arterioles and 0.79 ± 0.06 in venules). These results indicate that there is increased thrombus formation during hypertension and that the platelet adhesion processes may be of greater importance than platelet aggregation in producing this increase.     

Early in Reperfusion Following Myocardial Ischemia,Leukocyte Activation Is Necessary for Venular Adhesion But Not Capillary Retention

Objective : The pathobiology of leukocyte sequestration in the coronary microcircu-lation following ischemia is unclear. We examined the location(s) and persistence of leukocyte sequestration of unactivated and preactivated blood in the coronary microcirculation early during reperfusion following ischemia. Methods : Isolated rat hearts were subjected to 30 min of 37°C, no-flow ischemia. Hearts were initially reperfused with diluted whole blood containing fluorescent leukocytes (DWB*). At 5, 20, and 35 min of reperfusion (R), the deposition of leukocytes in the coronary capillaries and venules was observed directly using intravital fluorescence microscopy. Four groups were studied: a nonischemic control group (Gr I), and postischemic groups reperfused with DWB* treated with vehicle (Gr II) or preactivated with 10^?8 M N-formylmethionyl-leucyl-phenylala-nine (fMLP) (Gr III) or 10^?6 M fMLP (Gr IV). Results : At R5, postischemic reperfusion with unactivated blood caused a significant trapping of leukocytes in coronary capillaries (Gr I = 2.2 ± 0.4 versus Gr II = 5.6 ± 0.6 leukocytes per capillary field, P < 0.05). Hearts in Gr IV exhibited significantly greater leukocyte retention in capillaries compared to all other groups at R5 (R5, Gr IV = 8.8 leukocytes per capillary field, P < 0.05) and at R35. At R5, although more leukocytes were observed adhered to the venules in Gr II compared to Gr I, the difference was not statistically significant (Gr I = 1.7 ± 0.7 versus Gr II = 3.4 ± 0.5 leukocytes per 100 |μm venule, P = 0.23). DWB* preactivated with the lower concentration of fMLP (10^?8 M) resulted in a significant increase in venular leukocyte adhesion at R5 compared to Gr I and Gr II (Gr III 6.1 ± 0.5, P < 0.05). After 35 min of reperfusion, a greater percentage of leukocytes remained in the capillaries than in the venules. Conclusions : These direct observations suggest that early in reperfusion after ischemia, both leukocyte and endothelial activation are necessary for venular adhesion, but that ischemia-induced coronary microvascular alterations are sufficient to promote leukocyte retention in coronary capillaries. These results also indicate that during 35 min of reperfusion, the degree of leukocyte washout is greater in the venules than in the capillaries. These results suggest that the mechanisms contributing to leukocyte retention early in reperfusion following myocardial ischemia are, indeed, different in the capillaries and venules and that the mechanisms affecting retention in capillaries are more persistent than those in the venules.