Microvascular in vivo assessment of reperfusion injury: significance of prostaglandin E(1) and I(2) in postischemic "no-reflow" and "reflow-paradox"

BACKGROUND: Microvascular ischemia-reperfusion (I/R) injury is characterized by failure of capillary perfusion ("no-reflow") and reoxygenation-associated phenomena ("reflow-paradox"), including activation of leukocyte-endothelium interaction with cytotoxic mediator-induced loss of endothelial integrity. The objectives of this study were to elucidate the impact of both prostaglandins E(1) (PGE(1)) and I(2) (PGI(2)) in microvascular reperfusion injury, with special focus on the distinct pathophysiology of no-reflow- and reflow-paradox phenomena. MATERIALS AND METHODS: By use of the hamster dorsal skinfold preparation and in vivo fluorescence microscopy, the microcirculation of a striated skin muscle was assessed before 4 h of pressure-induced ischemia and 0.5, 2, and 24 h after onset of reperfusion. RESULTS: I/R was characterized by enhanced leukocyte-endothelium interaction in postcapillary venules, increase of macromolecular leakage, and reduction of functional capillary perfusion (P < 0.05). Intravenous 2-h infusion of PGE(1), starting with onset of reperfusion, reduced leukocyte adhesion and macromolecular leakage in postcapillary venules during early reperfusion (P < 0.05), while 6-h infusion, given during ischemia and early reperfusion, showed no significant effects. PGI(2) infusion also attenuated postischemic leukocyte adhesion, which was significant by a 6-h prolonged administration (P < 0.05), but did not influence the increase of microvascular permeability. Both prostaglandins were unable to prevent the postischemic failure of capillary perfusion (no-reflow). CONCLUSIONS: Both prostaglandins did not significantly influence postischemic no-reflow phenomena, but appeared as potent inhibitors of reflow-paradox under the experimental circumstances of this study.     

In vivo observation of leukocyte-endothelium interaction in ischemia reperfusion injury with the dorsal window chamber and the effects of pentoxifylline on reperfusion injury

OBJECTIVE: Ischemia reperfusion injury can cause failure in microsurgical operations. Interaction between leukocytes and endothelium is recognized as an integral step in ischemia reperfusion injury. Pentoxifylline is a methylxanthine derivative that has pharmacological properties that can be beneficial in ischemia reperfusion injury. The aim of this study was to investigate the in vivo effect of pentoxifylline on leukocyte-endothelium interaction in ischemia reperfusion injury. METHODS: Intravital fluorescent microscopy was used to observe leukocyte-endothelium interaction in a "dorsal window chamber" model. Twenty-eight postcapillary veins were analyzed in group 1, and twenty-two in group 2. Group 1 received 25 mg/kg pentoxifylline 20 min before reperfusion. Group 2 received an equivalent volume of 0.9% saline at the same time. The period of ischemia was 4 h. RESULTS: Quantification of leukocyte 'sticking' and 'rolling' was done before ischemia and at 30, 60, and 120 min after reperfusion. Offline video analysis was used for evaluating the results. Statistical evaluation showed that pentoxifylline significantly attenuated leukocyte 'sticking' and 'rolling' in postcapillary venules. It was also effective in preventing 'no-reflow' when compared with the control group. CONCLUSION: These results indicate pentoxifylline diminishes leukocyte-endothelium interaction, and may have a therapeutic role in preventing ischemia reperfusion injury in microsurgical operations.     

Leukocyte-endothelium interaction in arterioles after ischemia and reperfusion.

Leukocyte-endothelium interaction in postcapillary venules plays an important role in reperfusion injury, inflammation, shock, and sepsis. This phenomenon is poorly described in precapillary arterioles. In fact, many researchers have reported no evidence of leukocyte adherence in arterioles whatsoever. Most research has focused on venules of larger rodents, in which observation of the microcirculation, especially arterioles, is limited. We have developed a model which provides a clearer view of these microvessels using the mouse cremaster muscle. This muscle has an approximate thickness of 100 microm allowing images produced by transillumination to be very clear. After vascular isolation, the right cremaster muscle was subjected to 4 h of ischemia, followed by 2 h of reperfusion. The left muscle was not rendered ischemic, thereby allowing it to serve as the animal's own internal control. We observed leukocyte rolling in arterioles in both the ischemic and the nonischemic muscles. Leukocyte sticking was seen in arterioles and venules on both sides, except in control arterioles. The number of rolling and sticking leukocytes on the ischemic side was significantly higher than in controls (P < 0.05) for both arterioles and venules. During the reperfusion period, this number did not change significantly. Transmigration of leukocytes was observed only in venules, but not in arterioles. The number of perfused capillaries was reduced on the ischemic side compared to controls and did not change significantly during the 2 h of reperfusion. Our results demonstrate that leukocyte-endothelium interaction occurs in muscle arterioles of mice. This phenomenon is more pronounced after ischemia and reperfusion, i.e., depends on the extent of tissue insult.     

Hypoxia increases reepithelialization via an αvβ6-dependent pathway

Reepithelialization is an essential step in successful cutaneous wound healing. Human keratinocytes, integral in this process, have been shown to have increased motility in the hypoxic healing edge of wounds correlating with the clinical success of semiocclusive hypoxic dressings, although the underlying mechanisms remain poorly understood. Subconfluent human keratinocyte cell monolayers were exposed to 1% hypoxia for up to 24 hours or control conditions. Re-oxygenation studies were performed up to 72 hours. Cellular alphav subunit and alphavbeta6 integrin expression was measured by flow cytometry. Migration scratch assays on fibronectin following hypoxic exposure were performed over 24 hours. Relative matrix metallo-proteinase (MMP)-2, 9 activity was determined by gelatin zymography with TIMP-1 levels assayed by enzyme-linked immunoassay. Sustained increases in alphav and alphavbeta6 expression were shown up to 48 hours in re-oxygenation studies (P < 0.001). Standardized scratch assays confirmed increased migration in the hypoxic group (P < 0.05). This effect was attenuated by the addition of a specific inhibitor of the alphavbeta6 integrin. MMP-2 and -9 activity was up-regulated following hypoxic exposure (P < 0.001; P < 0.05, respectively), whereas increased MMP expression was significantly retarded by addition of an alphavbeta6 inhibitor (P < 0.05). Migration on fibronectin was attenuated by a specific gelatinase inhibitor. We conclude that integrin alphavbeta6-dependent MMP-2 and -9 up-regulation is an important feature of increased migration in hypoxic human keratinocytes.     

Compatibility of Different Colloid Plasma Expanders with Perflubron Emulsion: An Intravital Microscopic Study in the Hamster

Background: Perfluorocarbon-based oxygen carriers have been proposed as an adjunct to autologous blood conservation techniques during elective surgery. To date, the effects of perfluorocarbon emulsions at the microcirculatory level have not been studied extensively. In this study the effects of perflubron emulsion on the microcirculation after acute normovolemic hemodilution (ANH) were investigated using different colloid plasma expanders.

Methods: The dorsal skin fold chamber model and intravital fluorescence microscopy were used for analysis of the microcirculation in the thin striated skin muscle of conscious hamsters (body weight, 40-60 g). Measurements of microvascular perfusion and leukocyte adhesion (n = 6 animals per experimental group) were made before and at 10, 30, and 60 min after ANH (to hematocrit 0.3) with either 6% hydroxyethyl starch 200/0.6 (HES), 3.5% gelatin, 5% human serum albumin (HSA), or 6% dextran 60 (DX-60) followed by intravenous injection of 3 ml/kg body weight of a 60% weight/volume perfluorocarbon emulsion based on perflubron (perfluorooctyl bromide) emulsified with egg yolk lecithin.

Results: Acute normovolemic hemodilution with HES, gelatin, or HSA followed by injection of perflubron emulsion elicited no alterations of local microvascular perfusion or leukocyte-endothelium interaction as assessed in arterioles and postcapillary venules. However, ANH with DX-60 followed by injection of perflubron emulsion led to a significant reduction of erythrocyte velocity in postcapillary venules and an increase in venular leukocyte sticking that was never observed with DX-60 alone.     

Antithrombin III attenuates pulmonary tissue injury caused by mesenteric ischemia-reperfusion

BACKGROUND: Mesenteric ischemia-reperfusion (I/R) is a well-known event causing both local and remote organ injuries, including the lungs. Recently, several studies indicated that activated leukocyte-endothelial cell interactions play an important role in the mechanisms of these injuries. As a natural inhibitor of serine proteases, antithrombin was shown previously to attenuate the tissue damage after local I/R in several organ systems. Here, we examined the effects of antithrombin on pulmonary injury after mesenteric I/R. METHODS: Wistar albino rats underwent median laparotomy and were randomized into 3 groups: (1) sham-operated control (n = 12), (2) 60 minutes of mesenteric ischemia and 3 hours of reperfusion (n = 12), and (3) antithrombin-pretreated (250 U/kg) group before the I/R (n = 12). At the end of reperfusion, animals were killed and neutrophil sequestration, myeloperoxidase (MPO) activity, and Evans blue dye extravasation in the lung parenchyma were assessed and compared. RESULTS: There was a statistically significant increase in the quantity of Evans blue dye concentration, leukocyte sequestration, and MPO activity in the I/R group when compared with the control group. The pretreatment of animals with antithrombin significantly decreased the pulmonary injury characterized by increased Evans blue dye extravasation, leukocyte sequestration, and MPO activity. CONCLUSION: The data of the present study suggest that mesenteric ischemia and reperfusion induces pulmonary injury characterized by activated neutrophil sequestration and increased microvascular leakage in the lungs. A significant attenuation of intestinal I/R-related lung injury with the use of antithrombin concentrate warrants further studies to elucidate the potential role of this natural serine protease inhibitor in clinical settings.     

Beta3 integrin activation improves alphavbeta3-mediated retraction of fibrin matrices

BACKGROUND: Integrins are heterodimeric transmembrane glycoproteins that mediate cell interactions with the extracellular matrix. In vivo, integrin affinity can be modulated by intracellular signaling events. This can be simulated by a point mutation (D723R) in the cytoplasmic tail of the beta3 integrin subunit which results in constitutive activation. The effects of beta3 integrin activation on the function of alphavbeta3, an integrin which is important to the adhesive events of multiple cell types, were addressed using Chinese hamster ovary cells expressing either the wild-type alphavbeta3 integrin or the mutant alphavbeta3(D723R). The interactions of these cell lines with fibrin matrices were compared. METHODS: Receptor expression levels were confirmed by FACS analyses using a monoclonal anti-alphavbeta3 antibody. Cell attachment to fibrin-coated dishes was determined after 1 h by fixation and crystal violet staining followed by elution of the dye and OD measurement. Fibrin clot retraction was measured by culturing cells in fibrin clots for 24 h. The clots were detached from the dish and the surface area was calculated at individual time points. RESULTS: CHO alphavbeta3(D723R) cells displayed a greater than twofold increase in attachment to fibrinogen or to fibrin matrices when compared to wild-type transfectants. Further, CHO alphavbeta3(D723R) cell retraction of fibrin matrices was significantly greater at nearly all time points. CONCLUSION: Activation of the beta3 integrin subunit significantly improves the interaction of alphavbeta3 with fibrin and may play a role in the integrin-mediated signaling events which occur following vascular injury.     

Acute Cellular Rejection Following Human Heart Transplantation is Associated with Increased Expression of Vitronectin Receptor (Integrin αvβ3)

The vitronectin receptor (integrin alphavbeta3), a cell-surface adhesion receptor, has been shown to play a significant role in endothelial cell migration, apoptosis, atherosclerosis, and T-lymphocyte activation. This study was undertaken to test the hypothesis that cardiac allograft rejection is associated with increased expression of alphavbeta3. We also determined whether fibronectin receptor (alpha5beta1) and tissue factor are up-regulated in the presence of acute cellular rejection. We evaluated endomyocardial biopsy specimens with histologic evidence of different degrees of acute cellular rejection (grade 0, n = 10; grade 1A, n = 10; grade 2, n = 10; grade 3A, n = 10). Biopsies were obtained 2-4weeks after cardiac transplantation. Immunoperoxidase staining was performed for alphavbeta3, tissue factor, and alpha5beta1, and protein levels were further determined by Western blot analysis. Specimens with grade 2 and grade 3A rejection showed positive staining of alphavbeta3 in lymphocytic aggregates and vascular endothelial cells. By immunoblotting, we identified significantly increased expression of alphavbeta3 in the presence of acute rejection, grade 2 (3-fold, p = 0.01) and grade 3A (3.6-fold, p = 0.005) compared to grade 0 and 1 A specimens. There was no evidence of increased expression of alpha5beta1 or tissue factor. Acute cellular rejection, a process characterized by T-lymphocyte activation and release of inflammatory cytokines, is associated with increased expression of alphavbeta3.     

The integrin alpha5beta1 regulates alphavbeta3-mediated extracellular signal-regulated kinase activation

BACKGROUND: Integrin-mediated cell migration is essential for wound repair. Previous studies have shown that the interaction between integrins and the extracellular matrix (ECM) can initiate intracellular signaling pathways to regulate cell movement. Both the focal adhesion kinase (FAK) and the extracellular signal-regulated kinase/activated mitogen-activated protein kinase (ERK/MAPK) signaling pathways are required for efficient cell migration. Our previous work has shown that co-expression of the integrin alpha5beta1 inhibits alphavbeta3-mediated cell migration. We hypothesized that alpha5beta1 may regulate cell migration by modulating these alphavbeta3-mediated intracellular signaling events. METHODS: CHO B3 (alphavbeta3+) and B3C5 (alphavbeta3+/alpha5beta1+) cells were monitored by flow cytometry to determine integrin expression. Cells were allowed to migrate on fibrinogen (FBG)-coated transwells, with or without PD98059, an inhibitor of the ERK activator, mitogen-activated protein kinase kinase (MEK). Fixation, staining, and cell counting were used to quantify cell migration. Cells adherent to FBG were lysed and analyzed for FAK and ERK/MAPK activation by immunoblotting followed by image analysis densitometry. All experiments were repeated in triplicate. RESULTS: Treatment with PD98059 significantly decreased alphavbeta3-mediated cell migration on FBG (P = 0.0001) to a level comparable to untreated B3C5 cells. Following adhesion to FBG, B3 cells demonstrated a marked increase in ERK/MAPK activation compared to B3C5 cells. However, no significant difference was detected in FAK activation. CONCLUSION: Signaling through the ERK/MAPK pathway is required for efficient alphavbeta3-mediated migration on FBG. Inhibition of alphavbeta3-mediated migration by the integrin alpha5beta1 correlates with altered intensity and duration of ERK/MAPK activation, but not FAK activation, in response to adhesion. This suggests a mechanism for the regulatory effect of alpha5beta1 on alphavbeta3-mediated cell migration.     

Reduction of ischemia/reperfusion injury by antithrombin III after experimental pancreas transplantation

BACKGROUND: Graft pancreatitis is a major complication after pancreas transplantation. Antithrombin III (AT III) is an anticoagulatory and anti-inflammatory substance. The aim of our study was to evaluate a prophylactic application of AT III in experimental pancreas transplantation. METHODS: Pancreas transplantation was performed in rats. Cold ischemia time (University of Wisconsin solution at 4 degrees C) was 12 hours. After 4 hours of reperfusion, pancreatic enzymes were assessed and the pancreas was evaluated by intravital microscopy and histologic and immunohistochemical examination. Recipients were allocated randomly to 2 groups: 1 control group (n = 6) and 1 group in which recipients received 125 IU AT III/kg 30 minutes before reperfusion (n = 6). Six animals that did not undergo transplantation served as healthy controls. RESULTS: Enzyme levels showed no differences between the 2 transplantation groups but were significantly (P <.05) higher than in the control group. Histologic damage was significantly less evident in animals that received AT III compared with transplantation animals that did not receive AT III. During intravital microscopy, animals receiving AT III showed significantly higher capillary and venular erythrocyte velocities compared with untreated transplantation animals. The leukocyte-endothelium interaction in postcapillary venules was decreased significantly in animals with AT III treatment. CONCLUSIONS: AT III pretreatment decreases tissue damage by attenuating microcirculatory disturbances and leukocyte adherence in experimental graft pancreatitis by its anti-inflammatory and anticoagulatory properties.     

Compatibility of different colloid plasma expanders with perflubron emulsion: an intravital microscopic study in the hamster

BACKGROUND: Perfluorocarbon-based oxygen carriers have been proposed as an adjunct to autologous blood conservation techniques during elective surgery. To date, the effects of perfluorocarbon emulsions at the microcirculatory level have not been studied extensively. In this study the effects of perflubron emulsion on the microcirculation after acute normovolemic hemodilution (ANH) were investigated using different colloid plasma expanders. METHODS: The dorsal skin fold chamber model and intravital fluorescence microscopy were used for analysis of the microcirculation in the thin striated skin muscle of conscious hamsters (body weight, 40-60 g). Measurements of microvascular perfusion and leukocyte adhesion (n = 6 animals per experimental group) were made before and at 10, 30, and 60 min after ANH (to hematocrit 0.3) with either 6% hydroxyethyl starch 200/0.6 (HES), 3.5% gelatin, 5% human serum albumin (HSA), or 6% dextran 60 (DX-60) followed by intravenous injection of 3 ml/kg body weight of a 60% weight/volume perfluorocarbon emulsion based on perflubron (perfluorooctyl bromide) emulsified with egg yolk lecithin. RESULTS: Acute normovolemic hemodilution with HES, gelatin, or HSA followed by injection of perflubron emulsion elicited no alterations of local microvascular perfusion or leukocyte-endothelium interaction as assessed in arterioles and postcapillary venules. However, ANH with DX-60 followed by injection of perflubron emulsion led to a significant reduction of erythrocyte velocity in postcapillary venules and an increase in venular leukocyte sticking that was never observed with DX-60 alone. CONCLUSIONS: Hydroxyethyl starch, gelatin, and HSA are compatible with perflubron emulsion in the setting of ANH. Only DX-60 appeared to be incompatible with perflubron emulsion, as evidenced by impairment of capillary perfusion.     

C1-esterase inhibitor reduces reperfusion injury after lung transplantation

BACKGROUND: Activation of the complement system and polymorphonuclear neutrophilic leukocytes plays a major role in mediating reperfusion injury after lung transplantation. We hypothesized that early interference with complement activation would reduce lung reperfusion injury after transplantation. METHODS: Unilateral left lung autotransplantation was performed in 6 sheep. After hilar stripping the left lung was flushed with Euro-Collins solution and preserved for 2 hours in situ at 15 degrees C. After reperfusion the right main bronchus and pulmonary artery were occluded, leaving the animal dependent on the reperfused lung (reperfused group). C1-esterase inhibitor group animals (n = 6) received 200 U/kg body weight of C1-esterase inhibitor as a short infusion, half 10 minutes before, the other half 10 minutes after reperfusion. Controls (n = 6) underwent hilar preparation only. Pulmonary function was assessed by alveolar-arterial oxygen difference and pulmonary vascular resistance. The release of beta-N-acetylglucosaminidase served as indicator of polymorphonuclear neutrophilic leukocyte activation. Extravascular lung water was an indicator for pulmonary edema formation. Biopsy specimens were taken from all groups 3 hours after reperfusion for light and electron microscopy. RESULTS: In the reperfused group, alveolar-arterial oxygen difference and pulmonary vascular resistance were significantly elevated after reperfusion. All animals developed frank alveolar edema. The biochemical marker beta-N-acetylglucosaminidase showed significant leukocyte activation. In the C1-esterase inhibitor group, alveolar-arterial oxygen difference, pulmonary vascular resistance, and the level of polymorphonuclear neutrophilic leukocyte activation were significantly lower. CONCLUSIONS: Treatment with C1-esterase inhibitor reduces reperfusion injury and improves pulmonary function in this experimental model.     

The αvβ6 integrin plays a role in compromised epidermal wound healing

The alphavbeta6 integrin is an exclusively epithelial integrin that is highly expressed during fetal development. In adult tissue, alphavbeta6 integrin is expressed during inflammation, carcinogenesis, and in wound healing. We previously reported that alphavbeta6 integrin is highly expressed in poorly healing human wounds and its over-expression is associated with chronic wounds in a mouse model. The objective of this study was to investigate the role of alphavbeta6 integrin in compromised wound healing induced by hydrocortisone treatment or aging by using young and old mice deficient in or overexpressing the beta6 integrin subunit in the epidermis. Untreated aged beta6 integrin-deficient (beta6-/-) animals showed a significant delay in wound healing when compared to their age-matched controls or younger beta6-/- mice. The most significant delay was observed at the stages where granulation tissue deposition was occurring. Hydrocortisone treatment significantly delayed wound healing in wild-type and beta6 integrin-deficient mice in comparison with the untreated controls. However, hydrocortisone treatment in beta6 integrin overexpressing animals did not cause a significant delay in wound healing. The results of this study suggest that alphavbeta6 integrin plays an important role in wound healing in animals compromised by either age or stress mimicked by hydrocortisone.     

Both ischemic and pharmacological preconditioning decrease hepatic leukocyte/endothelial cell interactions

BACKGROUND: Ischemic preconditioning has been shown to protect some tissues from ischemia/reperfusion (I/R) injury. Adenosine is believed to play an important role by attenuating leukocyte-endothelial cell adhesive interactions. Dipyridamole increases adenosine bioavailability. The purpose of this study was to evaluate the effects of mechanical (MPC) and pharmacological preconditioning (PPC) on leukocyte endothelial cell interaction in hepatic I/R injury. METHODS: C57BL6 mice were subjected to 30 min of ischemia to the left lobe of the liver. Groups tested at 30 min, 2, 5, 12, and 24 hr of reperfusion had 1) sham laparotomy (n = 10, 2) I/R (n = 25), 3) ischemic preconditioning with 5 min of ischemia and 10 min reperfusion before I/R (n = 25), and 4) (PPC) with dipyridamole (n = 25). Intravital microscopic examination was used to assess leukocyte/endothelial cell adhesion. Blood was drawn for leukocyte counts and liver function tests. RESULTS: A significant decrease in leukocyte rolling was observed at 30-min and 5-hr reperfusion intervals in the PPC and ischemic preconditioning groups compared with the I/R group. A significant decrease in leukocyte saltation was also observed in the PPC and MPC groups at 2, 5, and 12 hr of reperfusion when compared with the I/R group. aspartate aminotransferase was significantly decreased in the 5-hr preconditioning groups. There was not a significant decrease in the white blood cell count because of PPC or MPC vs. I/R CONCLUSIONS: Preconditioning decreases endothelial/ leukocyte interaction and reduces liver damage as measured by aspartate aminotransferase. These data prove that IPC and PPC provide some degree of hepatic protection in I/R injury.     

Impact of enalapril on microvascular perfusion and leukocyte adherence in a model of rat liver transplantation assessed by in vivo microscopy

Abstract  ACE inhibitors have been proven to be effective in the reduction of ischemia/reperfusion damage after myocardial ischemia. In an attempt to investigate this effect in a model of syngeneic liver transplantation in the rat, we compared a control group with an ACE inhibitor treatment group, in which enalapril was given i.v. before and during re-perfusion. By means of in vivo microscopy, sinusoidal perfusion rate, permanent leukocyte sticking in sinusoids and postsinusoidal venules, and leukocyte rolling in postsinusoidal venules were assessed. Liver function was evaluated by measuring bile output. The sinusoidal perfusion rate was significantly improved by enalapril treatment. Leukocyte sticking in both sinusoids and postsinusoidal venules was found to be remarkably reduced in enalapril-treated animals; the fraction of rolling leukocytes remained unchanged. Bile output was increased in enala-pril-treated animals. These results demonstrated, in a model of rat liver transplantation, that ACE inhibition by enalapril is effective in reducing hepatic ischemidreperfusion damage as assessed by the leukocyte-en-dothelium interaction using in vivo microscopy and postreperfusion bile production.     

Functional antibodies to leukocyte adhesion molecules in antithymocyte globulins

BACKGROUND: Polyclonal antithymocyte globulins (ATG) induce T-cell depletion and functional impairment of nondeleted lymphocytes. Interference of ATG with the main leukocyte surface molecules involved in cellular adhesion and leukocyte-endothelium interaction was investigated in the present study. METHODS: In three rabbit ATG, the authors measured antibodies to integrins, beta2-integrin ligands, and chemokine receptors by flow cytometry; chemotactic responses; and down-modulation of cell surface expression on lymphocytes, monocytes, and neutrophils. RESULTS: Antibodies to CD11a/CD18 (leukocyte function-associated antigen-1 [LFA-1]) present in ATG induced a dose-dependent down-modulation of cell surface expression of this beta2 integrin on lymphocytes, monocytes, and neutrophils. In contrast, anti-LFA-1 monoclonal antibodies did not induce LFA-1 modulation unless cross-linked by a second antibody. ATG also contained functional antibodies to the beta1 integrin CD49d/CD29 (VLA-4), the alpha4beta7 integrin, CD50, CD54, and CD102 but not to CD62L. ATG were shown to bind to CXCR4 and CCR7 on lymphocytes, CXCR4, and CCR5 on monocytes; to down-modulate cell surface expression of CCR7; and to decrease monocyte chemotactic response to CCL5 (RANTES) and lymphocyte chemotactic response to CCL19 (MIP-3beta). CONCLUSION: These results show that ATG may interfere with leukocyte responses to chemotactic signals but mostly inhibit the expression of integrins required for firm cellular adhesion. The latter property of inhibition is not shared by monoclonal antibodies, and it may contribute to decreasing graft cellular infiltration during acute rejection and possibly after postischemic reperfusion.     

Effect of a cyclic adenosine monophosphate phosphodiesterase inhibitor, DN-9693, on myocardial reperfusion injury.

A new cyclic adenosine monophosphate phosphodiesterase inhibitor, DN-9693, was examined to see whether myocardial reperfusion injury could be reduced in a setting of cardioplegic arrest through its antiaggregation effect on leukocytes. Isolated rabbit heart models with whole blood perfusion were used, and 18 hearts were divided into three groups according to the reperfusion method: control (G-1, n = 5), DN-9693 (G-2, n = 7), and leukocyte depletion (G-3, n = 6). The hearts were subjected to 120 minutes of cold global ischemia under crystalloid cardioplegia followed by 30 minutes of reperfusion. A dose of 20 micrograms.kg-1.min-1 of DN-9693 was administered in G-2, and a leukocyte removal filter was used in G-3 during reperfusion. Ultrastructural changes in mitochondrial injuries, intracellular edema, and capillary injuries of the myocardium showed worse changes in G-1 than in G-2 and G-3. Under microscopic study, the intracapillary leukocyte count was significantly higher in G-1 than in G-2 and G-3. Recovery of rate-pressure product, left ventricular developed pressure, and coronary flow were significantly better in G-2 and G-3 than in G-1. There were no significant differences between G-2 and G-3 for all these indices. These results indicate that reperfusion with leukocyte-depleted blood attenuates reperfusion myocardial injury and DN-9693 has a comparable myocardial protective effect with possible inhibition of leukocyte aggregation.     

Integrin alphavbeta3-RGDS interaction mediates fibrin-induced morphological changes of glomerular endothelial cells.

BACKGROUND: In our previous studies, we found that intraglomerular deposition of fibrin and its metabolites was related to glomerular sclerosis and reduced renal function. It has been reported that both overlying and underlying fibrin may induce specific morphological changes of cultured endothelial cells from large blood vessels. The dependency of these morphological changes on the integrin alphavbeta3-arginyl-glycyl-aspartyl-serine (RGDS) interaction is still controversial. We hypothesized that glomerular endothelial cells (GECs) stimulated by fibrin might undergo morphological changes through an integrin alphavbeta3-RGDS interaction. Methods. In vitro studies were performed to examine the growing status of GECs stimulated by overlying and underlying fibrin gels in the presence or absence of the following: 50 microg/ml anti-alphavbeta3 integrin monoclonal antibody 23C6 or nonimmune mouse IgG, 1 mg/ml synthetic RGDS or arginyl-glycyl-glycyl-serine (RGGS) peptide, 10 mg/ml sodium heparin, 100 microg/ml cycloheximide, and 10 microM actinomycin D. Fast protein liquid chromatography (FPLC)-purified fibrinogen and the third to fifth passages of human GECs were also used in this study. RESULTS: GECs developed capillary tube structure after 60 hours of culturing on fibrin gels, and GECs cultured on gelatin-coated plates displayed a monolayer of cobblestone-like cells in the presence or absence of 23C6 and synthetic RGDS peptide. Fibrin-induced capillary tube formation was promoted by 23C6 and inhibited by RGDS peptide, cycloheximide, and actinomycin D. Disorganization of the GEC monolayer was induced by overlying fibrin, but was not induced by overlying agarose gels and glass cover slips or culturing in fibrinogen, 0.05 NIH U/ml thrombin, fibrin supernatants, as well as in fibrin degradation products. Disorganization of GEC monolayer can be induced by both des-AA-fibrin and des-AABB-fibrin and was unaffected by heparin. Furthermore, both 23C6 and synthetic RGDS peptide prevented disorganization of GECs induced by overlying fibrin, whereas nonimmune mouse IgG, synthetic RGGS peptide, cycloheximide, and actinomycin D had no similar effect. CONCLUSIONS: GECs cultured on fibrin gels may develop capillary structure spontaneously, and GECs covered by fibrin gels may undergo disorganization. Our data suggest that these GEC morphological changes are mediated by an integrin alphavbeta3-RGDS interaction.     

A leukocyte depleting filter reduces endothelial cell dysfunction and improves transplanted canine heart function.

BACKGROUND: To date leukocytes have been known to play a major role in reperfusion injury and have directed attention to leukocyte-endothelium interaction. This study was designed to investigate how much graft viability and the coronary microcirculatory function could be preserved by leukocyte depletion (LD) in a model of orthotopic cardiac transplantation. METHODS: The heart in 10 beagle dogs was arrested by introducing a 4 degrees C St. Thomas' cardioplegic solution. They were harvested, immersed in the cold saline for 3 hours, and then orthotopically transplanted. Five recipients underwent LD (LD group) at reperfusion with the use of a Pall BC1B leukocyte depleting filter inserted into the cardiopulmonary bypass (CPB) circuit. The other 5 dogs without filtration served as a control group. RESULTS: Leukocytes were about 80% filtrated and neutrophils were also 85% filtrated during the first 30 minutes of reperfusion in the LD group. A high level of adenosine triphosphate was maintained after transplantation in the LD group. The polymorphonuclear elastase level was significantly lower in the LD group. The cardiac function assessed by the slopes of the end-systolic pressure volume relation after transplantation was significantly higher in the LD group than in the control group (p < 0.05). The coronary vascular resistance responses to acetylcholine and nitroglycerin after transplantation were preserved significantly better in the LD group than in the control group (p < 0.05). CONCLUSIONS: These results suggest that a leukocyte depleting filter placed in the CPB circuit would prevent leukocyte-mediated endothelial cell injury, improve microcirculation of the myocardium, and lead to excellent graft function.     

Lung transplant reperfusion injury involves pulmonary macrophages and circulating leukocytes in a biphasic response

OBJECTIVE: Both donor pulmonary macrophages and recipient circulating leukocytes may be involved in reperfusion injury after lung transplantation. By using the macrophage inhibitor gadolinium chloride and leukocyte filters, we attempted to identify the roles of these two populations of cells in lung transplant reperfusion injury. METHODS: With our isolated, ventilated, blood-perfused rabbit lung model, all groups underwent lung harvest followed by 18-hour cold storage and 2-hour blood reperfusion. Measurements of pulmonary artery pressure, lung compliance, and arterial oxygenation were obtained. Group I (n = 8) served as a control. Group II (n = 8) received gadolinium chloride at 14 mg/kg 24 hours before lung harvest. Group III (n = 8) received leukocyte-depleted blood reperfusion by means of a leukocyte filter. RESULTS: The gadolinium chloride group had significantly improved arterial oxygenation and pulmonary artery pressure measurements compared with control subjects and an improved arterial oxygenation compared with the filter group after 30 minutes of reperfusion. After 120 minutes of reperfusion, however, the filter group had significantly improved arterial oxygenation and pulmonary artery pressure measurements compared with the control group and an improved arterial oxygenation compared with the gadolinium chloride group. CONCLUSIONS: Lung transplant reperfusion injury occurs in two phases. The early phase is mediated by donor pulmonary macrophages and is followed by a late injury induced by recipient circulating leukocytes.