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Superoxide has been implicated in the regulation of endothelial cell adhesion molecule expression and the subsequent initiation of leukocyte-endothelial cell adhesion in different experimental models of inflammation. The objective of this study was to assess the contribution of oxygen radicals to P-selectin expression in a murine model of whole body ischemia-reperfusion, i.e., hemorrhage-resuscitation (H/R), with the use of different strategies that interfere with either the production (allopurinol, CD11/CD18-deficient or p47phox−/− mice) or accumulation [intravenous superoxide dismutase (SOD), mutant mice that overexpress SOD] of oxygen radicals. P-selectin expression was quantified in different regional vascular beds by use of the dual-radiolabeled monoclonal antibody technique. H/R elicited a significant increase in P-selectin expression in all vascular beds. This response was blunted in SOD transgenic mice and in wild-type mice receiving either intravenous SOD or the xanthine oxidase inhibitor allopurinol. Mice genetically deficient in either a subunit of NADPH oxidase or the leukocyte adhesion molecule CD11/CD18 also exhibited a reduced P-selectin expression. These results implicate superoxide, derived from both xanthine oxidase and NADPH oxidase, as mediators of the increased P-selectin expression observed in different regional vascular beds exposed to hemorrhage and retransfusion.

Leukocyte-endothelial cell interactions play an important role in mediating organ dysfunctions observed after hemorrhagic shock. P-selectin is the first endothelial cell adhesion molecule to be upregulated after an ischemic insult. The objective of this study was to define kinetics of P-selectin expression in different regional vascular beds of mice exposed to hemorrhagic shock. In-vivo P-selectin expressions were determined using dual radiolabeled monoclonal antibody technique in lungs, heart, liver, kidneys, intestinal mesentery, stomach, small bowel, and colon 0.5, 1, 2, 5, 10, and 24 h after resuscitation of 40 mmHg hemorrhagic shock. In another group, P-selectin expression was determined in same organs 5 h after resuscitation of 30 mmHg hemorrhagic shock. Hemorrhagic shock of 40 mmHg caused significant upregulation of P-selectin in lungs and liver at 30 min after resuscitation (P < 0.001). There was a second and more pronounced upregulation of P-selectin in lungs and liver at 5 h after resuscitation (P < 0.001). In heart, intestinal mesentery, stomach, small bowel, and colon, P-selectin was not upregulated until 5 h after resuscitation from 40 mmHg hemorrhagic shock (P < 0.001). While hemorrhagic shock of 40 mmHg did not cause P-selectin upregulation in kidneys, hemorrhage to 30 mmHg did elicit a significant increase at 5 h after resuscitation (P < 0.001). We conclude that P-selectin is upregulated after resuscitation of hemorrhagic shock in lungs, liver, heart, stomach, and intestines. P-selectin upregulation in kidneys only takes place after more severe hemorrhagic shock.

The trafficking of leukocytes within the microcirculation is critical for normal immune surveillance of tissues. The process of leukocyte recruitment is tightly regulated by the sequential expression and activation of specific adhesion molecules on the surface of leukocytes and endothelial cells. These adhesion molecules mediate distinct steps in the recruitment of leukocytes in the microcirculation. The selectins mediate leukocyte rolling, whereas glycoproteins belonging to the integrin and immunoglobulin supergene families enable leukocytes to firmly adhere and emigrate in venules. The leukocyte-endothelial cell adhesion that is mediated by these adhesion molecules has been shown to alter the function of endothelial cells in all segments of the vasculature (ie, in arterioles, capillaries, and venules). Diseases such as ischemia-reperfusion, hypertension, and atherosclerosis exhibit vascular changes that are characteristic of acute or chronic inflammatory responses. These vascular alterations are associated with, and influenced by, changes in the avidity and density of adhesion molecules on the surface of either endothelial cells, leukocytes, or both. The activation and increased expression of these adhesion glycoproteins have been attributed to excessive production of cytokines and oxidants. The risk factors for cardiovascular disease, particularly diabetes mellitus and hypercholesterolemia, appear to sensitize the microvasculature to these inflammatory stimuli, thereby rendering tissues more vulnerable to the deleterious effects of ischemia and reperfusion. These findings raise the possibility of applying therapeutic strategies that are directed against adhesion molecules for the management of some cardiovascular diseases.

We have recently reported that hepatic ischemia/reperfusion (I/R) is associated with a biphasic increase in the expression of P-selectin in the liver microvasculature, with peak expression levels observed at 20 min and 5 h after reperfusion. This I/R-induced upregulation of P-selectin expression is accompanied by leukocyte-endothelial cell adhesion in terminal hepatic venules (THV). The objective of this study was to determine whether the early expression of P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in THV after liver I/R. Left hepatic lobe ischemia was induced for 30 min in anesthetized C57B1/6 and P-selectin knockout (KO) mice. The number of rolling, saltating, and adherent leukocytes in THV was measured at 0, 15, 30, 60, and 120 min after reperfusion using intravital video microscopy. Hepatic I/R elicited significant increases in the number of rolling, saltating, and adherent leukocytes, with peak values observed at 30 min after reperfusion. All of these responses were absent in P-selectin KO mice and in C57B1/6 mice treated with a blocking antibody to P-selectin. Our findings suggest that P-selectin is the primary determinant of leukocyte-endothelial cell adhesion observed in hepatic venules in the initial period after I/R. Hence, this adhesion molecule may represent a target for therapeutic intervention in liver transplantation and other conditions associated with hepatic I/R.

The essential role of endothelial CAMs in the recruitment of leukocytes to sites of inflammation has been extensively studied in recent years. The development of techniques such as the radiolabeled MAb method that is described in this chapter has enabled investigators to quantitatively assess the expression of endothelial CAMs in different regional vascular beds. Although the study of adhesion molecules in vitro has extended our knowledge of the mechanisms and mediators of endothelial CAM function, the newly acquired in vivo data would indicate that findings resulting from experiments on monolayers of cultured endothelial cells may not accurately depict inflammatory events that occur in vivo. Thus, additional work is needed to more fully define the conditions that account for the discrepencies noted between the in vivo and in vitro models of endothelial CAM expression.

The recruitment of leukocytes into postcapillary venules following reperfusion of ischemic tissues is a classical inflammatory response that is influenced by a balance of adhesive and hemodynamic forces. Initial periods of reperfusion are characterized by an elevation in low affinity adhesive interactions between leukocytes and endothelial cells, which permit the leukocytes to establish intimate contact with the vascular endothelial lining as they roll across it. This rolling behavior of leukocytes increases the probability that stronger adhesive interactions between leukocyte and endothelial cell will occur, allowing the leukocyte to firmly adhere to the endothelium and subsequently to emigrate across the venular wall into the interstitial compartment. Several factors appear to contribute to the leukocyte-endothelial cell adhesion that is elicited by ischemia/reperfusion, including: 1) the magnitude of adhesion molecule expression on leukocyte and/or endothelial cell surfaces, 2) reactive oxygen metabolites and lipid mediators released from stimulated leukocytes and endothelial cells and 3) hemodynamic dispersal forces that act to sweep leukocytes from the microvessel wall. Intravital microscopic techniques have allowed investigators to focus on inflammatory responses in postcapillary venules, which is the primary site of leukocyte adhesion and vascular protein leakage. This technology has also been employed to delineate the role of adhesive and hemodynamic factors in promoting leukocyte rolling, adhesion, and emigration in postcapillary venules exposed to ischemia/reperfusion.

SummaryThe reactive hyperemic response of the intestinal vasculature to graded arterial occlusion is often used to delineate mechanisms involved in the intrinsic regulation of intestinal blood flow. This study assesses if the reactive hyperemic response to 60- and 120-sec arterial occlusions occur uniformly throughout the wall of the small intestine. Radiolabeled microsphere injections were used to determine blood flow distribution in the mucosa-submucosa and muscu-laris layers of the small intestine under control conditions and during 60- and 120-sec arterial occlusion. The results of this study suggest that relatively uniform increases in intestinal wall blood flow occurs only for an occlusion period of 60 sec. Increasing the duration of the arterial occlusion to 120 sec results in a predominant hyperemia in the muscularis layer.

The hemodynamic effects of Ro 22-1327 [nat-(15R,16R)-16-fluoro-15-hydroxy-9-oxoprosta-5(Z),1 3(E)-dienoic acid], a prostaglandin E2 analog, were evaluated in anesthetized spontaneously hypertensive rats (SHR). Within 3-4 h of topical application (1 and 3 mg/kg), a dose-dependent decrease in mean arterial blood pressure was observed. At 6 h following drug application, steady-state mean arterial pressure was 79-87% of control (pretreatment) values. Increments in blood flow, as well as reductions in vascular resistance, were apparent in the kidneys, skin, and the majority of the splanchnic organs. The magnitude of the drug-induced hyperemia decreased from the duodenum to the ileum. Significant elevations in colonic blood flow were not apparent with either dose, suggesting a proximal-to-distal gradient of response of the intestinal vasculature to the drug. The results of this study indicate that Ro 22-1327 is an effective antihypertensive agent when applied topically in SHR. Our results also imply that the splanchnic vascular bed contributes heavily to the antihypertensive activity of the compound.

Reactive oxygen metabolites (ROMs) are partially reduced oxygen species that include Superoxide, hydrogen peroxide, hydroxyl radical, and hypohalous acids. Formation of Superoxide or hydrogen peroxide may be injurious to tissue directly; however, it is thought that the primary mediators of tissue damage are the secondarily derived oxidants such as hydroxyl radical and hypohalous acid. The gastrointestinal tract is particularly well endowed with the enzymatic machinery necessary to form large amounts of ROMs. Sources of ROMs in the gastrointestinal tract include mucosal oxidases such as xanthine oxidase, amine oxidase, and aldehyde oxidase as well as the NADPH oxidase found in the resident phagocytic leukocytes (macrophages, neutrophils, eosinophils) of the lamina propria. We have demonstrated that reperfusion of ischemic small intestine results in substantial mucosal injury that is mediated by oxy radicals generated from xanthine oxidase and inflammatory leukocytes. The final mediator of toxicity appears to be the hydroxyl radical derived from the iron-catalyzed interaction between Superoxide and hydrogen peroxide. Data from our laboratories as well as other laboratories suggest that reactive oxygen metabolites may play an important role in mediating mucosal injury during active episodes of ulcerative colitis. We present a working hypothesis which states that transient ischemic episodes in the bowel initiate a cascade of self-perpetuating cycles of ROM formation, inflammation and, ultimately, mucosal injury.