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It is known that hypertension is associated with endothelial dysfunction and that Angiotensin II (Ang II) is a key player in the pathogenesis of hypertension. We aimed to elucidate whether endothelial dysfunction is a specific feature of Ang II-mediated hypertension or a common finding of hypertension, independently of underlying etiology. We studied endothelial-dependent vasorelaxation in precapillary resistance arterioles and in various large-caliber conductance arteries in wild-type mice with Ang II-dependent hypertension (2-kidney 1-clip (2K1C) model) or Ang II-independent (volume overload) hypertension (1-kidney 1-clip model (1K1C)). Normotensive sham mice were used as controls. Aortic mechanical properties were also evaluated. Intravital microscopy of precapillary arterioles revealed a significantly impaired endothelium-dependent vasorelaxation in 2K1C mice compared with sham mice, as quantified by the ratio of acetylcholine (ACh)-induced over S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-induced vasorelaxation (2K1C: 0.49±0.12 vs. sham: 0.87±0.11, P=0.018). In contrast, the ACh/SNAP ratio in volume-overload hypertension 1K1C mice was not significantly different from sham mice, indicating no specific endothelial dysfunction (1K1C: 0.77±0.27 vs. sham: 0.87±0.11, P=0.138). Mechanical aortic wall properties and endothelium-dependent vasorelaxation, assessed ex vivo in rings of large-caliber conductance (abdominal and thoracic aorta, carotid and femoral arteries), were not different between 2K1C, 1K1C and sham mice. Endothelial dysfunction is an early feature of Ang II- but not volume-overload-mediated hypertension. This occurs exclusively at the level of precapillary arterioles and not in conduit arteries. Our findings, if confirmed in clinical studies, will provide a better understanding of the pathophysiological mechanisms of hypertension.

The 22 supersetnd Hohenheim Consensus Workshop took place in at the University of Stuttgart-Hohenheim. The subject of this conference was vitamin C and its role in the treatment of endothelial dysfunction. Scientists, who had published and reviewed scientific and regulatory papers on that topic were invited, among them basic researchers, toxicologists, clinicians and nutritionists. The participants were presented with eleven questions, which were discussed and answered at the workshop, with the aim of summarising the current state of knowledge. The explicatory text accompanying the short answers was produced and agreed on after the conference and was backed up by corresponding references. The therapeutic relevance of administration of the physiological antioxidant vitamin C in high parenteral doses in Endothelial Dependent Pathophysiological Conditions (EDPC) was discussed. Endothelial dysfunction is defined as including disturbed endothelial dependant relaxation of resistance vessels, breakdown of the microvascular endothelial barrier and/or loss of anti-adhesive function. It occurs in severe burn injury, intoxications, acute hyperglycemia, sepsis, trauma, and ischemic-reperfusion tissue injury and is induced by oxidative stress. Reduced plasma ascorbate levels are a hallmark of oxidative stress and occur in severe burns, sepsis, severe trauma, intoxication, chemotherapy/radiotherapy and organ transplantation. Vitamin C directly enhances the activity of nitric oxide synthase, the acyl CoA oxidase system and inhibits the actions of proinflammatory lipids. There is experimental evidence that parenteral high-dose vitamin C restores endothelial function in sepsis. In vitro, supraphysiological concentrations (> 1mM) of ascorbate restore nitric oxide bioavailability and endothelial function. Only parenterally, can enough vitamin C be administered to combat oxidative stress. There is no evidence that parenteral vitamin C exerts prooxidant effects in humans. Theoretical concerns in relation to competitive interactions between vitamin C and glucose cellular uptake are probably only relevant for oxidised vitamin C (dehydroascorbate).

The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected with a plasmid encoding the catalytic component of telomerase (hTERT) and a plasmid encoding the simian virus 40 (SV40) large T antigen. Cells transfected with either plasmid alone had an extended lifespan, but the cultures eventually entered crisis after several months of proliferation. Only those cells that were transfected with both plasmids acquired the capacity to grow in vitro without demonstrating major crisis, and these cells have been in culture for 24 months. HPMEC isolated from two different donors were used, generating two populations of immortalized cells, HPMEC-ST1 and HPMEC-ST2. Single cell-derived clones of the immortalized cells HPMEC-ST1 exhibited growth characteristics that were similar to those of the parental HPMEC. One selected clone, HPMEC-ST1.6R, displayed all major constitutively expressed and inducible endothelial phenotypic markers, including platelet endothelial cell adhesion molecule (PECAM-1, CD31), von Willebrand factor (vWF), and the adhesion molecules, intercellular adhesion molecule (ICAM-1), vascular adhesion molecule (VCAM-1), and E-selectin. In addition, an angiogenic response was demonstrated by sprout formation on a biological extracellular matrix (Matrigel). The HPMEC-ST1.6R cells did not form tumors in nude mice. The microvascular endothelial cell line, HPMEC-ST1.6R, will be a valuable tool for the study of microvascular endothelial physiology and pathology including gene expression, angiogenesis, and tumorigenesis.

Objective: The clinical complications of extracorporeal circulation (ECC) have been linked to a systemic activation of cellular and humoral components and to a dysregulation of the microcirculatory compartment. Since to date only in vitro methods exist for evaluation, we developed an animal model to study the effects of ECC on the microcirculation. To establish the model, we assessed whether these effects are dependent on the duration of ECC., Methods: Intravital fluorescence microscopy was used on the dorsal skinfold chamber preparation in chronically instrumented, awake Syrian golden hamsters. ECC was realized using a micro-rollerpump and a silicon tube shunting blood between the carotid artery and the jugular vein. ECC was performed in three groups for various times (2, 10 and 20 min) after application of heparin at 300 IU/kg body wt. In hamsters, the application of high-dose heparin releases endothelial bound superoxide dismutase (SOD), a natural scavenger of oxygen-derived free radicals. Protocol II assigned two groups receiving heparin at different doses of 50 and 2000 IU/kg body wt., Results: ECC for 2 min served as control to exclude effects from hemodilution and resulted in a minimal induction of leukocyte/endothelial cell interaction. Isovolemic ECC for 20 min resulted in an increase in rolling (from 11 +/- 3 to 38 +/- 20%, mean +/- S.D., P < 0.05) and adherent leukocytes (from 19 +/- 16 to 215 +/- 145 cells/mm2, mean +/- S.D., P < 0.05) in postcapillary venules. Microhemodynamic parameters and functional capillary density were not significantly affected. Arterial blood pressure and heart rate were stable. Heparin at 2000 IU/kg inhibited post-ECC leukocyte adhesion following ECC, whereas 50 IU/kg showed no protective effects., Conclusions: Leukocyte/endothelial cell interaction, induced by blood contact with synthetic surfaces, was directly visualized in vivo. The number of adherent leukocytes was dependent on the duration of ECC. The application of high-dose heparin followed by release of SOD almost prevented leukocyte activation, suggesting a formation of oxygen free radicals during ECC. The new application of the hamster model may allow to study the underlying pathomechanisms and to develop therapeutic/prophylactic strategies to avert problems associated with ECC.

Background: Human malignant tumors grown as xenografts in immunocompromised animals have been used extensively to study tumor growth and tumor response to therapy. The endothelium functions as an effective barrier between the intravascular space and the tumor cells. In a previous study we used species-specific monoclonal antibodies against endothelial cell adhesion molecules to demonstrate the host origin of the endothelium in xenotransplanted pancreatic islet grafts [Am J Pathol 1995;146:1397-1405]. We now investigated in this study whether the vascular endothelium of different xenografted human malignant tumors expresses mouse (= host)- or human (= graft)-specific CD31 (platelet endothelial cell adhesion molecule, PECAM-1) adhesion molecules., Methods and Results: Cultured human prostate, kidney, and colon cancer cells (passages 15-17) were transplanted subcutaneously into 8-week-old athymic nude mice and removed after another 8 weeks. The avidin biotin peroxidase method was utilized on frozen sections to demonstrate that the endothelium of the vasculature of all three human xenografts expressed mouse (= host)-specific CD31, but not human (= graft)-specific CD31., Conclusion: The presence between the intravascular space and the human tumor cells of a mouse-derived endothelium, expressing mouse-specific antigens, needs to be taken into careful consideration when evaluating results of antitumor therapies in these animal models. This caveat pertains particularly to the study of novel cell- or tissue-specific treatment modalities, such as antibody-targeted drugs, toxins or radionuclides, 'immuno'-liposomes, or tumor vaccines.

In previous studies we have demonstrated that syngeneic and xenogeneic pancreatic islet grafts are revascularized within a 10 to 14-day period after transplantation. With the combined use of intravital and electron microscopy, as well as immunohistochemistry using a set of species-specific or -crossreacting antibodies to endothelial cell antigens, we investigated 1) the origin of the endothelium of the newly formed capillaries in free pancreatic islet isografts (hamster-->hamster) and xenografts (rat-->hamster), and 2) the ultrastructural characteristics of these microvessels. Intravital microscopy demonstrated that newly formed microvessels grow from the vascular bed of the host muscle tissue into the islet grafts. Immunohistochemical analysis of host tissue and transplanted islets with antibodies against factor VIII (recognizing both hamster and rat factor VIII), bovine PECAM-1 (CD31; endoCAM, crossreacting with hamster but not rat PECAM-1), and rat ICAM-1 (CD54, non-crossreacting with hamster ICAM-1) showed that the transplanted rat islets were revascularized by endothelium of hamster (host) origin. At an ultrastructural level, the endothelial lining of the newly formed microvessels showed diaphragmatic fenestration, a characteristic feature of endothelial cells of pancreatic islets in situ. On the basis of these findings we suggest that pancreatic islet transplantation may take a unique position in the field of organ transplantation, since the generally proposed mechanisms of endothelial cell-dependent antigen recognition as a trigger of graft rejection may not be transferred to islet grafts, containing microvessels lined by endothelial cells of host origin.

Background: The ability of oxidized LDL (oxLDL) to stimulate leukocyte-endothelium interaction is considered to be an important aspect of its proatherogenic action. Using intravital fluorescence microscopy in the dorsal skinfold chamber model in hamsters, we have previously shown that systemic administration of oxLDL stimulates leukocyte adhesion to microvascular endothelium through a mechanism that involves the generation and action of reactive oxygen species (ROS)., Methods and Results: Through the combined use of scanning electron microscopy and intravital microscopy in the same animal model, we demonstrate that oxLDL-induced leukocyte adhesion is not confined to the microcirculation but can also be observed on aortic endothelium. OxLDL-induced leukocyte adhesion to both microvascular and macrovascular endothelium was almost entirely prevented by pretreatment of the hamsters with dietary or intravenous vitamin C, which has the capacity to scavenge and neutralize ROS (arterioles: 20.5 +/- 16.4 cells/mm2 [diet] and 16.3 +/- 23.8 cells/mm2 [IV] versus 74.2 +/- 47.5 cells/mm2 [control, P < .01]; aorta: 1.0 +/- 0.4 cells/mm2 [diet] and 1.1 +/- 0.5 cells/mm2 [IV] versus 14.7 +/- 6.0 cells/mm2 [control, P < .01], 15 minutes after oxLDL, n = 7 animals per group). Vitamin C pretreatment also completely prevented oxLDL-induced leukocyte-platelet aggregate formation in the blood-stream but did not affect leukocyte rolling along the microvascular endothelium. No inhibitory effect on any of the studied parameters was observed as a result of pretreatment of the animals with the lipid-soluble antioxidants vitamin E and probucol., Conclusions: The protective effects of vitamin C on oxLDL-induced leukocyte adhesion and aggregate formation were seen at vitamin C plasma levels that can easily be reached in humans by diet or supplementation, suggesting that this could be one of the mechanisms by which vitamin C contributes to the well-documented protraction of atherogenesis as observed in large epidemiological surveys.

Background: Oxidized low density lipoprotein (oxLDL) has been demonstrated to stimulate leukocyte/endothelium interaction, an early feature of atherogenesis. Using the skinfold chamber model for intravital microscopy in hamsters and mice, we have shown that oxLDL-induced leukocyte adhesion to microvascular endothelium shares many characteristics with leukocyte adhesion during inflammation and ischemia/reperfusion, including the involvement of beta 2 integrin adhesion molecules. In light of the two-step model of leukocyte adhesion, we have examined the contribution of P-selectin to oxLDL-induced leukocyte/endothelium interaction. P-selectin is an inducible adhesion molecule on platelets and endothelium, mediating the initial steps of leukocyte margination and rolling along the endothelial lining, as well as of aggregate formation between platelets and leukocytes., Experimental Design: For our studies, we used the dorsal skinfold chamber model for intravital fluorescence microscopy on awake Syrian golden hamsters. Hamsters were treated 10 minutes before oxLDL-injection (oxidized by Cu2+, 4 mg/kg body weight, intravenously) with blocking antibodies to P-selectin (2 mg/kg body weight intravenously, N = 7)., Results: In seven control animals (pretreated with an irrelevant IgG antibody), oxLDL injection elicited leukocyte rolling and adhesion on both venular and arteriolar endothelium, and also the formation of aggregates tumbling down the microvessels and firmly adhering to the microvascular endothelium. The aggregates consisted of leukocytes and activated, dendritic platelets, as assessed by scanning electron microscopy of the buffy coat isolated by density gradient centrifugation of whole blood taken from hamsters 15 minutes after injection of oxLDL. Leukocyte adhesion to venular and arteriolar endothelium, as well as the formation of leukocyte/platelet aggregates were significantly reduced by pretreatment of the animals with anti-P-selectin antibodies., Conclusions: These data emphasize the similarities between leukocyte adhesion in response to oxLDL and in other pathophysiologic conditions, identifying P-selectin as a crucial player in the interaction between leukocytes and microvascular endothelium as well as in the formation of circulating leukocyte/platelet aggregates.

Superoxide radicals formed during reperfusion of ischemic tissues have been identified as a key mediator in the microvascular manifestations of postischemic tissue damage. This understanding is based on studies in laboratory animals in which high doses of superoxide dismutase (SOD; 2.0-25.0 mg/kg body wt iv) were found to inhibit postischemic leukocyte adhesion and the leakage of fluid and macromolecules. Using a dorsal skinfold chamber model in hamsters, we demonstrate now that protection from reperfusion-induced leukocyte adhesion to venular endothelium after 4 h of ischemia to striated muscle can be attained by pretreatment of the animals with a significantly lower dose of exogenous CuZn-SOD (0.25 mg/kg body wt) or with heparin (2,000 IU/kg body wt), which induces a comparable increase in SOD plasma activity through the release of endogenous extracellular SOD from endothelial cell binding sites. This protective effect was maintained until 24 h after reperfusion. In contrast, CuZn-SOD or heparin failed to attenuate the postischemic shutdown of nutritional capillary perfusion, a phenomenon that is due to ischemia-induced endothelial cell swelling, rather than due to reperfusion-associated events, and hence is not susceptible to strategies directed against oxygen radicals generated during the reperfusion phase. The results of this study 1) imply that postischemic leukocyte/endothelium interaction can be attenuated by a low and clinically more relevant dose of SOD, and 2) caveat the administration of heparin in laboratory animals (i.e., to keep catheters patent) in studies of experimental ischemia/reperfusion injury or other oxygen radical-dependent pathomechanisms.

A common feature of cigarette-smoke (CS)-associated diseases such as atherosclerosis and pulmonary emphysema is the activation, aggregation, and adhesion of leukocytes to micro- and macrovascular endothelium. A previous study, using a skinfold chamber model for intravital fluorescence microscopy in awake hamsters, has shown that exposure of hamsters to the smoke generated by one research cigarette elicits the adhesion of fluorescently labeled leukocytes to the endothelium of arterioles and small venules. By the combined use of intravital microscopy and scanning electron microscopy, we now demonstrate in the same animal model that (i) CS-induced leukocyte adhesion is not confined to the microcirculation, but that leukocytes also adhere singly and in clusters to the aortic endothelium; (ii) CS induces the formation in the bloodstream of aggregates between leukocytes and platelets; and (iii) CS-induced leukocyte adhesion to micro- and macrovascular endothelium and leukocyte-platelet aggregate formation are almost entirely prevented by dietary or intravenous pretreatment with the water-soluble antioxidant vitamin C (venules, 21.4 +/- 11.0 vs. 149.6 +/- 38.7 leukocytes per mm2, P < 0.01; arterioles, 8.5 +/- 4.2 vs. 54.3 +/- 21.6 leukocytes per mm2, P < 0.01; aortas, 0.8 +/- 0.4 vs. 12.4 +/- 5.6 leukocytes per mm2, P < 0.01; means +/- SD of n = 7 animals, 15 min after CS exposure). No inhibitory effect was observed by pretreatment of the animals with the lipid-soluble antioxidants vitamin E or probucol. The protective effects of vitamin C on CS-induced leukocyte adhesion and aggregation were seen at vitamin C plasma levels (55.6 +/- 22.2 microM, n = 7) that can easily be reached in humans by dietary means or supplementation, suggesting that vitamin C effectively contributes to protection from CS-associated cardiovascular and pulmonary diseases in humans.

Leukocyte adhesion and subendothelial emigration, constant hallmarks of early atherogenesis, have been ascribed to the action of oxidized low-density lipoprotein (oxLDL). Using intravital fluorescence microscopy in the skinfold-chamber model in hamsters, we have previously shown that systemic administration of oxLDL stimulates leukocyte adhesion in vivo through a mechanism that depends on the generation and/or action of both leukotrienes and superoxide radicals. On the basis of the fact that oxygen radical-catalyzed peroxidation of phospholipids results in the generation of fragments with short sn2 residues, which besides authentic platelet-activating factor (PAF), activate the receptor for PAF on leukocytes and thereby induce leukocyte adhesion, we asked whether pretreatment of hamsters with a specific PAF receptor antagonist (WEB2170; 1 mg/kg of body weight IV, 10 minutes before oxLDL) attenuates leukocyte adhesion after injection of oxLDL (4 mg/kg of body weight IV, oxidized by 7.5 mumol/L Cu2+ for 18 hours at 37 degrees C). We demonstrate herein that in contrast to untreated control animals in which oxLDL elicited rolling and adhesion of circulating leukocytes to the endothelium of venules and arterioles, oxLDL-induced leukocyte adhesion was significantly attenuated in WEB2170-pretreated animals. These changes cannot be ascribed to alterations of microhemodynamic parameters and, hence, wall shear conditions. This finding indicates that oxLDL-induced leukocyte/endothelium interaction involves the PAF receptor, which may function both as a receptor for authentic PAF or for PAF-like lipids that are generated in a free radical-catalyzed peroxidation of phospholipids.

Although cigarette smoking has been identified as a major risk factor for cardiovascular diseases, the underlying pathomechanism is largely unknown. Using a dorsal skinfold chamber model in Syrian golden hamsters for intravital microscopy on striated muscle microcirculation, we investigated whether cigarette smoke (CS) affects the adhesion of circulating leukocytes to the endothelium, a constant feature of early atherogenesis and a hallmark of ischemia-reperfusion injury. Awake hamsters were exposed for 5 min to the mainstream smoke of one cigarette (2R1 research cigarette), inducing nicotine, cotinine, and carboxyhemoglobin plasma levels comparable to levels found in human smokers. In control animals (n = 7), CS exposure elicited the rolling and subsequent adhesion of fluorescently stained leukocytes to the endothelium of arterioles and postcapillary venules. Leukocyte/endothelium interaction was preceded by an early rise in xanthine oxidase activity and intravascular hemolysis. Leukocyte adhesion and xanthine oxidase (XO) activation were significantly attenuated in hamsters pretreated with superoxide dismutase (5 mg/kg, 10 min prior to CS, n = 7), suggesting a key role of superoxide in this event. These in vivo results suggest a novel pathomechanism of CS-induced cardiovascular pathology.

Background: The adhesion of leukocytes to the vascular endothelium has been identified as a constant, early feature of atherogenesis. Using a skinfold chamber model in hamsters for intravital microscopy, we have recently demonstrated the chemotactic and adhesion-promoting action of oxidatively modified low density lipoprotein (oxLDL) in vivo., Experimental Design: In order to rule out a species specificity of the experimental approach, and to investigate the involvement of leukocyte adhesion receptors in oxLDL-induced leukocyte/endothelium interaction, we have adapted the skinfold chamber model to hairless mice. Using intravital fluorescence microscopy, leukocyte/endothelium interaction in a fine striated skin muscle was assessed during the time course after intravenous injection of native human LDL (4 mg/kg body weight) and oxLDL (oxidized in vitro by 7.5 microM Cu2+, 6 hours, 37 degrees C) into control mice and into mice pretreated with either the selective inhibitor of leukotriene biosynthesis MK-886 (20 mumol/kg body weight, intravenously, 10 minutes before oxLDL) or with a monoclonal antibody directed towards the CD11b subunit of the CD11b/CD18 adhesion receptor complex on leukocytes (monoclonal antibody anti-Mac1, 0.5 mg/kg, iv, 10 minutes before oxLDL)., Results: We demonstrate in this study that injection of oxLDL (4 mg LDL-cholesterol/kg, intravenously, oxidized by 7.5 microM Cu2+, 6 hours, 37 degrees C), but not of native LDL or LDL-free Cu(2+)-solution, elicits the adhesion of fluorescently stained leukocytes to the endothelium of postcapillary venules (173 +/- 75 cells/mm2 at 20 minutes after oxLDL, mean +/- SD) and arterioles (119 +/- 74 cells/mm2 at 20 minutes after oxLDL, mean +/- SD) in the mice. OxLDL-induced leukocyte adhesion was entirely prevented by pretreatment of the animals with the inhibitor of leukotriene biosynthesis or with monoclonal antibody anti-Mac-1., Conclusions: These results demonstrate the involvement of leukotrienes and of the CD11b/CD18 adhesion receptor complex in oxLDL-induced leukocyte adhesion in vivo and rule out a species specificity of this pathophysiologic event.

Although cigarette smoke (CS) has been identified as an independent risk factor for atherogenesis and sudden cardiac death, the underlying pathomechanism has not been clarified. A common factor of both atherogenesis and ischemia/reperfusion damage associated with myocardial infarction is the adhesion of circulating leukocytes to the vascular endothelium. Searching for the mechanism by which CS exerts its deleterious effects on the cardiovascular system, we used a dorsal skinfold chamber model in hamsters for intravital microscopy to examine the effect of CS on the interaction of fluorescently stained leukocytes with the microvascular endothelium in striated muscle. Exposure of awake animals (n = 7) for 5 minutes to the mainstream smoke of one cigarette (2R1 research cigarette) elicited rolling and subsequent adhesion of circulating leukocytes to the endothelium of both arterioles and postcapillary venules with a maximum 30 minutes after CS-exposure. In order to test a putative mediator role of the chemotactic and adhesion-promoting leukotrienes in this event, we pretreated another group of 7 animals with MK-886, a potent and specific inhibitor of leukotriene biosynthesis (20 mumol/kg body weight, iv, 30 minutes prior to CS exposure). While no inhibitory effect was seen on CS-induced leukocyte rolling along the microvascular endothelium. MK-886 pretreatment significantly attenuated leukocyte adhesion to arterioles (5.2 +/- 13.7 cells/mm2 vs. 54.1 +/- 54.8 in control animals. P < 0.01) and venules (37.0 +/- 33.6 cells/mm2 vs. 161.6 +/- 91.1 in control animals, P < 0.01), 30 minutes after CS exposure, suggesting a key mediator role of leukotrienes in this event. We propose that the stimulation of leukocyte/endothelium interaction by CS may provide the pathophysiologic basis for--or at least contribute to--its deleterious effects on cardiovascular mortality and morbidity. The identification of the mediator role of leukotrienes in this event may open the way to novel pharmacologic and dietary approaches for the prophylaxis of CS-induced cardiovascular pathology.

The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg-1 min-1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p less than 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces post-ischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

Background: In vitro and in vivo experiments have demonstrated the role of oxidatively modified low density lipoprotein (oxLDL) in eliciting leukocyte/endothelium interaction during early atherogenesis., Methods and Results: In the present study we investigated the effect of dietary fish oil on oxLDL-induced leukocyte/endothelium interaction using intravital fluorescence microscopy in the dorsal skinfold chamber model in awake Syrian golden hamsters. Hamsters were fed for 4 weeks prior to the experiments with either standard laboratory chow or a diet supplemented with 5% of a fish oil concentrate (18% eicosapentaenoate, 12% docosahexaenoate). The efficacy of the fish oil diet was demonstrated by the incorporation of fish oil-derived omega-3 fatty acids into plasma, leukocyte, and erythrocyte lipids. In control hamsters (n = 7) and fish oil-fed hamsters (n = 7), leukocyte/endothelium interaction was assessed in the time course after intravenous injection of human LDL (4 mg/kg), oxidized by 7.5 microM Cu2+ (6 hours, 37 degrees C). In control hamsters, injection of oxLDL elicited the rolling and sticking of leukocytes to the endothelium of arterioles and postcapillary venules with a maximum 15 minutes after injection (arterioles: from 3 +/- 1 to 91 +/- 25 cells/mm2 at 15 minutes; venules: from 13 +/- 6 to 150 +/- 46 cells/mm2 at 15 minutes; mean +/- SD). This phenomenon was significantly reduced in fish oil-fed hamsters, where 15 minutes after injection of oxLDL leukocyte sticking reached a maximum of only 15 +/- 7 and 20 +/- 5 cells/mm2 in arterioles and postcapillary venules, respectively (p less than 0.01 versus control animals)., Conclusions: The results of the present study suggest that inhibition of leukocyte/endothelium interaction may be one of the mechanisms by which dietary fish oil exerts its protective effects on experimental and clinical atherogenesis.

The reduction of postischemic reperfusion injury by exogenous adenosine has been ascribed to reduced oxygen radical generation and adhesion of leukocytes to the vascular endothelium. To provide in vivo evidence for this concept we investigated the effects of adenosine (110 micrograms.kg-1.min-1 iv) on postischemic leukocyte-endothelium interaction in the dorsal skinfold chamber model in awake hamsters by intravital fluorescence microscopy. Leukocytes were stained in vivo with acridine orange and classified according to their interaction with the endothelium as nonadherent, rolling, or sticking leukocytes. In control animals, reperfusion after a 4-h pressure-induced ischemia to the striated muscle in the dorsal skinfold chamber elicited a marked increase in leukocyte rolling and sticking. This phenomenon was significantly attenuated in adenosine-treated animals 30 min after reperfusion. Postischemic changes in vessel diameters and red cell velocities were not affected by adenosine. The data suggest that systemic adenosine administration reduces reperfusion injury by the inhibition of postischemic leukocyte adherence to the microvascular endothelium.

In vitro studies indicate that oxidatively modified low-density lipoprotein (oxLDL) promotes leukocyte sticking to the vascular endothelium, a constant feature of early atherogenesis. Using intravital fluorescence microscopy in the dorsal skinfold chamber model in hamsters, we investigated whether systemic administration of human LDL, oxidized by Cu2+, elicited leukocyte/endothelium interaction in vivo. While no effect was seen after injection of native LDL, oxLDL administration resulted in an immediate induction of leukocyte rolling along the microvascular endothelium and subsequent firm sticking to the wall of postcapillary venules as well as arterioles. The presented model may provide an alternative experimental approach to long-term feeding studies with atherogenic diets for the in vivo investigation of leukocyte/endothelium interaction in early atherogenesis.