Postischemic anti-inflammatory effects of bradykinin preconditioning.

We sought to determine the mechanisms whereby brief administration of bradykinin (bradykinin preconditioning, BK-PC) before prolonged ischemia followed by reperfusion (I/R) prevents postischemic microvascular dysfunction. Intravital videomicroscopic approaches were used to quantify I/R-induced leukocyte/endothelial cell adhesive interactions and microvascular barrier disruption in single postcapillary venules of the rat mesentery. I/R increased the number of rolling, adherent, and emigrated leukocytes and enhanced venular albumin leakage, effects that were prevented by BK-PC. The anti-inflammatory effects of BK-PC were largely prevented by concomitant administration of a B(2)-receptor antagonist but not by coincident B(1) receptor blockade, nitric oxide (NO) synthase inhibition, or cyclooxygenase blockade. However, NO synthase blockade during reperfusion after prolonged ischemia was effective in attenuating the anti-inflammatory effects of BK-PC. Pan protein kinase C (PKC) inhibition antagonized the beneficial effects of BK-PC but only when administered during prolonged ischemia. In contrast, specific inhibition of the conventional PKC isotypes failed to alter the effectiveness of BK-PC. These results indicate that bradykinin can be used to pharmacologically precondition single mesenteric postcapillary venules to resist I/R-induced leukocyte recruitment and microvascular barrier dysfunction by a mechanism that involves B(2) receptor-dependent activation of nonconventional PKC isotypes and subsequent formation of NO.

Experimental models to investigate inflammatory processes in chronic venous insufficiency.

Chronic venous insufficiency (CVI) is characterized by leukocyte adhesion and infiltration, venous hypertension and dilatation, and valvular dysfunction. The fact that activated white cells can direct a powerful cytotoxic arsenal at parenchymal cells following their extravasation into the tissues led to the original proposal that leukocytes may play a causative role in the pathogenesis of venous disease. A large body of subsequent work indicates that white blood cells are indeed activated in CVI. However, identification of the factors responsible for initiating leukosequestration and activation in such disorders and determination of whether these activated cells then contribute to the progression of venous disease have been hampered by the lack of appropriate animal models that accurately mimic the human condition. Tantalizing evidence suggesting that cyclical periods of ischemia and reperfusion (I/R) may occur in diseased regions of the skin is beginning to accumulate. As is the case with CVI, leukocyte infiltration is a prominent feature in I/R and activated neutrophils play a causative role in the reperfusion component of tissue injury via the targeted release of reactivate oxygen metabolites and hydrolytic enzymes. In light of these considerations, many investigators have suggested that examining the mechanisms of I/R injury in skin and skeletal muscle, where ischemia is produced by arterial occlusion, may provide a relevant model for studying the pathogenesis of CVI. Others have suggested that venous occlusion may represent a more appropriate model, as this approach also produces the venous hypertension that is characteristic of the disease. The purpose of this review is to summarize the evidence pointing to the involvement of I/R and venous hypertension as causative factors in CVI-induced leukocyte recruitment. In addition, we will describe the evidence in favor of the view that white blood cells contribute to the pathogenesis of CVI. Finally we will describe several different experimental models that have been used to examine the role of I/R-induced microvascular dysfunction as it may pertain to the development of CVI, together with a discussion of the relative advantages and limitations of the various models.

Ischemic preconditioning prevents postischemic P-selectin expression in the rat small intestine.

Ischemic preconditioning (IPC) prevents the deleterious effects of prolonged ischemia and reperfusion (I/R). Because leukocyte infiltration is required to produce the microvascular dysfunction induced by I/R in the small intestine, and P-selectin-dependent leukocyte rolling is a requisite step in this process, we hypothesized that IPC would attenuate postischemic P-selectin expression. To address this postulate, P-selectin expression was evaluated in nonischemic (control) rat jejunum and in rat jejunum subjected to I/R alone (20 min ischemia/60 min reperfusion), or IPC (5 min ischemia/10 min reperfusion) + I/R using a dual radiolabeled monoclonal antibody approach. I/R was associated with a sevenfold increase in jejunal P-selectin expression, an effect that was completely abolished by IPC. Exposing the bowel to adenosine deaminase or an adenosine A1, but not an A2, receptor antagonist during the period of preconditioning ischemia or to selective PKC antagonists during prolonged ischemia prevented the beneficial effect of IPC to limit I/R-induced P-selectin expression. Our data indicate that P-selectin expression is a novel downstream effector target of the adenosine-initiated, PKC-dependent, anti-inflammatory signaling pathway in IPC.

PR-39, a proline/arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction.

We and others have previously demonstrated that intestinal ischemia-reperfusion (I/R) is associated with a large increase in oxidant production that contributes to microvascular barrier disruption in the small bowel. It has been suggested that the bulk of tissue damage during reperfusion can be attributed to adherent, activated neutrophils. From these observations, we hypothesized that pretreatment with PR-39, an endogenous neutrophil antibacterial peptide that is also a potent inhibitor of the neutrophil NADPH oxidase, would prevent postischemic oxidant production and the development of oxidant-dependent sequelae to I/R such as increased venular protein leakage. To test this postulate, oxidant production, venular protein leakage, leukocyte adhesion, and leukocyte emigration were monitored during reperfusion in control (no ischemia) rat mesenteric venules and in mesenteric venules subjected to I/R alone or PR-39 + I/R. Treatment with a single intravenous bolus injection of PR-39 (administered at a dose to achieve an initial blood concentration of 5 microM) abolished I/R-induced leukocyte adhesion and emigration in vivo. In vitro studies indicated that PR-39 prevents platelet-activating factor-induced neutrophil chemotaxis as well as phorbol myristate acetate (PMA)-stimulated intercellular adhesion molecule-1 expression by cultured endothelial cells. PR-39 pretreatment of rat neutrophils also blocked PMA-stimulated neutrophil adhesion to activated endothelial monolayers. In vivo, I/R was associated with a marked and progressive increase in oxidant production and venular protein leakage during reperfusion, effects that were abolished by PR-39 treatment. The results of this study indicate that PR-39 completely abolishes postischemic leukocyte adhesion and emigration. The time course for inhibition of oxidant production by PR-39 suggests that its antiadhesive properties account for this effect of the peptide. PR-39 may thus be therapeutically useful for prevention of neutrophil adhesion and activation during the postischemic inflammatory response.

Adhesion molecule expression in postischemic microvascular dysfunction: activity of a micronized purified flavonoid fraction.

Ischemia and reperfusion (I/R) induces neutrophil infiltration in skeletal muscle that is localized to the ischemic region. To transmigrate at ischemic regions, granulocytes must first arrest in the postcapillary venular segment of the microcirculation. Initially, leukocytes roll along the endothelium of these venules, a weak adhesive interaction that is mediated by the selectins (L-, E-, and P-selectin). Leukocyte rolling functions to slow the neutrophil during its transit through the microcirculation, thereby allowing it to monitor its local environment for the presence of activating factors arising from the ischemic tissues. When activated, the rolling granulocyte is rendered capable of forming the stronger adhesive interactions that allow the cell to become arrested in postcapillary venules in the ischemic region. These adhesive interactions are mediated by a leukocyte glycoprotein complex designated CD11/CD18 and intercellular adhesion molecule-1 (ICAM-1) expressed on endothelial cells. The stationary neutrophil uses the gradient in concentration of soluble chemoattractants liberated from ischemic tissues as a directional cue to move from the vascular to extravascular compartment, being guided in its transit across the endothelium by interactions with platelet endothelial cell adhesion molecule-1 (PECAM-1), an adhesive molecule localized to the interendothelial cleft. This paper reviews current understanding of the mechanisms underlying the establishment of leukocyte/endothelial cell interactions in postischemic skeletal muscle in terms of specific adhesion molecules that participate in neutrophil sequestration after I/R. Discovery of the molecular determinants of neutrophil/endothelial cell adhesion has uncovered potential mechanisms whereby agents exhibiting anti-adhesive properties may act. The micronized purified flavonoid fraction (450 mg diosmin, 50 mg hesperidin) prevents I/R-induced leukocyte adhesion in skeletal muscle. This anti-adhesive effect appears to be mediated at least in part by inhibition of induced expression of ICAM-1.

Concentration-dependent effects of bradykinin on leukocyte recruitment and venular hemodynamics in rat mesentery.

The results of several recent studies indicate that bradykinin protects tissues against the deleterious effects of ischemia-reperfusion (I/R). However, other studies indicate that bradykinin can act as a proinflammatory agent, inducing P-selectin expression, the formation of chemotactic stimuli, and endothelial barrier disruption. In the present study, we used intravital microscopic techniques to examine the dose-dependent effects of bradykinin on leukocyte-endothelial cell interactions, the formation of platelet-leukocyte aggregates, and venular hemodynamics in rat mesentery in an attempt to explain these divergent findings. Superfusion of the mesentery with low concentrations of bradykinin (/=10(-6) M) decreased V(RBC), increased the number of platelet-leukocyte aggregates, and induced leukocyte adhesion in single postcapillary venules. The formation of platelet-leukocyte aggregates and increased leukocyte adhesion induced by high-dose bradykinin were attenuated by administration of a B(2)-receptor (HOE-140) or a platelet-activating factor (PAF, WEB-2086) antagonist. Thus these adhesive interactions induced by high-dose bradykinin appear to be mediated by a mechanism that is dependent on B(2)-receptor activation and the formation of PAF or PAF-like lipids. The effects of bradykinin on venular V(RBC) and blood flow were also concentration dependent, with low doses producing nitric oxide-mediated vasodilation, whereas high doses decreased V(RBC) by a mechanism that is PAF independent.

Bradykinin prevents postischemic leukocyte adhesion and emigration and attenuates microvascular barrier disruption.

Although a number of recent reports indicate that bradykinin attenuates ischemia- reperfusion (I/R)-induced tissue injury, the mechanisms underlying its protective actions are not fully understood. However, because bradykinin induces endothelial nitric oxide (NO) production and NO donors have been shown to attenuate postischemic leukocyte adhesion, endothelial barrier disruption, and tissue injury, we hypothesized that bradykinin may act to reduce I/R-induced tissue injury by preventing leukocyte recruitment and preserving microvascular barrier function. To address this postulate, we used intravital videomicroscopic approaches to quantify leukocyte-endothelial cell interactions and microvascular barrier function in single postcapillary venules in the rat mesentery. Reperfusion after 20 min of ischemia significantly decreased wall shear rate and leukocyte rolling velocity, increased the number of rolling, adherent, and emigrated leukocytes, and disrupted the microvascular barrier as evidenced by enhanced venular albumin leakage. Superfusion of the mesentery with bradykinin (10 nM) during I/R significantly reduced these deleterious effects of I/R. Although these inhibitory effects of bradykinin were not affected by cyclooxygenase blockade with indomethacin (10 microM), coadministration with NO synthase (N(omega)-nitro-L-arginine methyl ester, 10 microM) or bradykinin B(2)-receptor (HOE-140, 1 microM) antagonists abolished the protective actions of bradykinin. Plasma NO concentration was measured in the mesenteric vein and was significantly decreased after I/R, an effect that was prevented by bradykinin treatment. These results indicate that bradykinin attenuates I/R-induced leukocyte recruitment and microvascular dysfunction by a mechanism that involves bradykinin B(2)-receptor-dependent NO production.

Inflammatory responses to ischemia and reperfusion in skeletal muscle.

Skeletal muscle ischemia and reperfusion is now recognized as one form of acute inflammation in which activated leukocytes play a key role. Although restoration of flow is essential in alleviating ischemic injury, reperfusion initiates a complex series of reactions which lead to neutrophil accumulation, microvascular barrier disruption, and edema formation. A large body of evidence exists which suggests that leukocyte adhesion to and emigration across postcapillary venules plays a crucial role in the genesis of reperfusion injury in skeletal muscle. Reactive oxygen species generated by xanthine oxidase and other enzymes promote the formation of proinflammatory stimuli, modify the expression of adhesion molecules on the surface of leukocytes and endothelial cells, and reduce the bioavailability of the potent antiadhesive agent nitric oxide. As a consequence of these events, leukocytes begin to form loose adhesive interactions with postcapillary venular endothelium (leukocyte rolling). If the proinflammatory stimulus is sufficient, leukocytes may become firmly adherent (stationary adhesion) to the venular endothelium. Those leukocytes which become firmly adherent may then diapedese into the perivascular space. The emigrated leukocytes induce parenchymal cell injury via a directed release of oxidants and hydrolytic enzymes. In addition, the emigrating leukocytes also exacerbate ischemic injury by disrupting the microvascular barrier during their egress across the vasculature. As a consequence of this increase in microvascular permeability, transcapillary fluid filtration is enhanced and edema results. The resultant increase in interstitial tissue pressure physically compresses the capillaries, thereby preventing microvascular perfusion and thus promoting the development of the no-reflow phenomenon. The purpose of this review is to summarize the available information regarding these mechanisms of skeletal muscle ischemia/reperfusion injury.

Mechanisms of ischemic preconditioning.

Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia by previous exposure to brief periods of vascular occlusion. While the beneficial effects of IPC were first demonstrated in the myocardium, it is now clear that preconditioning protects postischemic skeletal muscle, brain, and small intestine and may also occur in humans. Although first described over a decade ago, the mechanisms underlying the powerful protective effects of IPC remain uncertain. However, a growing body of evidence indicates that the beneficial actions of IPC involve the activation of adenosine A1 receptors during the period of preconditioning ischemia in most organs and species. Adenosine A1 receptor stimulation is thought to promote the translocation and activation of specific isoforms of protein kinase C1 which in turn phosphorylate as yet unidentified cellular effector molecules. In the heart, it has been suggested that ATP-sensitive potassium channels may represent important effectors of the preconditioning phenomenon. In contrast, ATP-sensitive potassium channel activation does not seem to contribute to the beneficial effects of IPC in the small bowel and seems to play only a limited role in skeletal muscle. In these peripheral tissues, the beneficial effects of IPC are related to inhibition of leukocyte adhesion and emigration. In the small intestine, IPC seems to prevent postischemic leukocyte adhesion by maintaining the bioavailability of nitric oxide (a potent endogenous anti-adhesive agent) and preventing, the expression of P-selectin (an adhesive molecule expressed by endothelial cells that is thought to modulate leukocyte rolling). In skeletal muscle, these actions are mediated by an effect of IPC to augment the production of adenosine (another potent endogenous anti-adhesive agent) during reperfusion. Thus, although adenosine-induced protein kinase C activation seems to play an important role in initiating the beneficial actions of IPC in most tissues, the effector of the preconditioning phenomenon seems to differ among tissues. Understanding the mechanisms of IPC has led to the recognition that tissues may also be preconditioned by administration of agents that act via the same signaling cascade (e.g., adenosine, bradykinin, alpha 1-adrenergic agonists). The purpose of this review is to summarize the evidence regarding the mechanisms of IPC in different organs.

Melanoma cell adhesion to injured arterioles: mechanisms of stabilized tethering.

An isolated perfused vessel model was used to examine the mechanisms underlying the adhesive interactions between circulating tumor cells and subendothelial matrix in denuded arterioles. Arterioles ranging from 70 to 100 microm in diameter were isolated from rat mesentery, transferred to an isolated vessel chamber, cannulated on both ends with glass micropipettes, and perfused with media containing 10(6) hamster melanoma (RPMI 1856) cells/ml. In a second group of arterioles, the endothelium was denuded by running 2 ml of air through the vessel lumen. Since the tumor cells did not adhere to the vessel wall when perfused at physiologically relevant shear rates, perfusate flow was stopped and the tumor cells were allowed to settle onto the vessel wall for 20 min. After counting the number of tumor cells that settled onto the arteriolar wall, perfusate flow was re-initiated and unattached cells were washed away. The number of cells remaining adherent were counted and the percentage of adherent cells (relative to the total number of cells that settled on to the vessel wall during the period of no-flow) were calculated and compared among different groups. We observed that tumor cells are much more adhesive to denuded arterioles than to intact arterioles. To determine the mechanisms responsible for the adhesive interactions that become established and stabilized during the period of flow reduction, denuded arterioles were treated with fibronectin antiserum or Arg-Gly-Asp (RGD) peptides. Both treatments significantly reduced tumor cell adhesion to denuded arterioles. In subsequent studies, melanoma cells were treated with a transglutaminase inhibitor, monodansylcadaverine (MDC), which reduced the ability of adherent tumor cells to withstand the anti-adhesive effects of a subsequent increase in perfusate flow rate after the period of no-flow. Our data suggest that tumor cells adhere to fibronectin in the subendothelial matrix in denuded arterioles by an RGD-dependent mechanism. Moreover, our observations are consistent with the concept that a transglutaminase-catalysed reaction acts to stabilize the adhesive interactions between subendothelial matrix components and melanoma cells during the period of flow stasis such that the cells are able to withstand subsequent substantial increases in wall shear rate and remain adherent.

Neutrophil mediated microvascular injury in acute, experimental compartment syndrome.

The purpose of this study was to determine the contribution of neutrophils and tissue xanthine oxidase to the skeletal muscle microvascular dysfunction in an ex vivo model of acute compartment syndrome. Adult dogs were rendered neutropenic or depleted of tissue xanthine oxidase before gracilis muscle isolation. Compared with continuously perfused, nonischemic muscles, acute, experimental compartment syndrome resulted in a dramatic increase in microvascular permeability, muscle neutrophil content, and muscle vascular resistance. Neutropenia prevented, whereas xanthine oxidase depletion had no effect on, the microvascular dysfunction and muscle neutrophil infiltration elicited by experimental compartment syndrome. These results suggest that neutrophils contribute to the microvascular dysfunction and blood flow distribution abnormalities elicited by acute, experimental compartment syndrome.

Postischemic leukocyte/endothelial cell interactions and microvascular barrier dysfunction in skeletal muscle: cellular mechanisms and effect of Daflon 500 mg.

A growing body of evidence indicates that neutrophils play a critical role in disrupting the microvascular barrier in skeletal muscle. Recent studies from our laboratory and by others indicate that administration of antibodies directed against P-selectin, ICAM-1, or the common subunit (CD18) of CD11/CD18 was as effective as neutrophil depletion in attenuating ischemia/reperfusion (I/R)-induced microvascular barrier disruption and edema formation in skeletal muscle. These studies have important implications with regard to the pathogenesis of leg ulceration in view of our more recent work indicating that the increase in tissue pressure induced by edema formation secondary to microvascular barrier disruption may lead to the development of capillary no-reflow. The resulting maldistribution of blood flow during reperfusion exacerbates muscle injury induced by ischemia. Daflon 500 mg is a purified, micronized flavonoid fraction that exhibits a number of anti-inflammatory properties and is used clinically to treat venous insufficiency. In view of these actions and the demonstrated role of neutrophil adhesion in the pathogenesis of I/R, we sought to determine whether this agent would prevent leukocyte adhesion and microvascular barrier disruption in postischemic rat cremaster muscles and small bowel. Rats were treated with Daflon 500 mg (80 mg/kg/day by gavage) or its vehicle for 2 (cremaster studies) or 10 (mesenteric studies) days prior to the experiments. Leukocyte/endothelial cell interactions and venular protein leakage were quantitated using intravital microscopic techniques in rat cremaster muscles and mesenteries subjected to ischemia (60 min for cremaster, 20 min for mesentery) and reperfusion (60 min). The results indicated that Daflon 500 mg was as effective as the anti-adhesive monoclonal antibodies in reducing postischemic leukocyte adhesion and emigration and venular protein leakage in these models.

Hypoxia/reoxygenation selectively impairs alpha 1b-adrenoceptor function in small mesenteric arteries.

The present study examined whether hypoxia/reoxygenation (H/R) attenuates norepinephrine (NE) effectiveness in small arteries by interfering with function of alpha 1a- and/or alpha 1b- adrenoceptor subtypes. Small mesenteric arteries (approximately 150 microns) were obtained from rats mounted on a small vessel myograph in oxygenated physiological salt solution (PSS), and the relationship between NE concentrations and contractile tension was assessed. Hypoxia was induced by bubbling the vessels with 95% N2-5% CO2 for 15 min. Vessels were then reoxygenated for 30 min, and NE responses were reevaluated. Superoxide dismutase (SOD) and catalase (CAT) were added to the PSS in one group of vessels to investigate the role of reactive oxygen metabolites. In other groups, alpha 1b-receptors were blocked with chloroethylclonidine and alpha 1a-receptors were blocked with 5-methylurapidil or WB-4101 to produce exclusive alpha 1a- or alpha 1b-responses to NE. H/R decreased the NE negative logarithm of the mean effective concentration (pD2: i.e., -log[EC50], where EC50 is mean effective concentration) from 6.26 +/- 0.24 to 5.84 +/- 0.12 (P < 0.05). SOD and CAT prevented the H/R-induced contractile dysfunction. alpha 1a-Receptor responses to NE were not altered by H/R. In contrast, alpha 1b-receptor responses were significantly attenuated after H/R. The results indicate that alterations in NE responsiveness after H/R are due to dysfunction of the alpha 1b signal transduction pathway.

Protease inhibition attenuates microvascular dysfunction in postischemic skeletal muscle.

Neutrophils accumulate in skeletal muscle subjected to ischemia-reperfusion and appear to contribute to reperfusion-induced microvascular dysfunction. The overall objective of this study was to assess the role of the neutrophilic hydrolytic enzyme elastase in ischemia-reperfusion-induced granulocyte accumulation and microvascular dysfunction in skeletal muscle. We examined the effect of three structurally unrelated elastase inhibitors [eglin C, MeOsuc-Ala-Ala-Val-CH2Cl (MAAPV), or L-658758], administered at the onset of reperfusion, on neutrophil content and the increase in microvascular permeability induced by 4 h of ischemia and 0.5 h of reperfusion in the isolated canine gracilis muscle. Changes in vascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in the following groups: 1) 4.5 h of continuous perfusion (nonischemic), 2) ischemia-reperfusion alone, 3) ischemia-reperfusion + eglin C, 4) ischemia-reperfusion + MAAPV, and 5) ischemia-reperfusion + L-658758. Muscle neutrophil content was monitored by assessing tissue myeloperoxidase (MPO) activity in biopsies obtained at the end of the experiments. In nonischemic muscles, 1 - sigma and MPO activity averaged 0.13 +/- 0.03 and 0.7 +/- 0.2 units/g wet wt, respectively. Ischemia-reperfusion was associated with marked increases in microvascular permeability (1 - sigma = 0.39 +/- 0.02) and muscle MPO activity (8.9 +/- 1.2 units/g wet wt) that were attenuated by eglin C, MAAPV, and L-658758 (1 - sigma = 0.21 +/- 0.01, 0.22 +/- 0.02, and 0.21 +/- 0.03, respectively; MPO activity = 2.7 +/- 0.4, 2.1 +/- 0.8, and 2.8 +/- 1.8 units/g wet wt, respectively). These results suggest that granulocyte accumulation in postischemic skeletal muscle is dependent on the release of elastase from activated phagocytic cells. Moreover, neutrophilic elastase appears to play a major role in reperfusion-induced increases in microvascular permeability in skeletal muscle.

Ischemic preconditioning attenuates postischemic leukocyte adhesion and emigration.

Intravital microscopy was used to determine whether ischemic preconditioning (IPC; 5 min ischemia and 10 min reperfusion) would attenuate leukocyte adhesion and emigration induced by subsequent prolonged ischemia (60 min) and reperfusion (60 min) (I/R) in murine cremaster muscle and whether adenosine produced during IPC and/or reperfusion contributed to these beneficial effects. I/R elicited a marked increase in the number of adherent and emigrated leukocytes compared with the nonischemic control muscles, an effect that was largely prevented by IPC. Superfusion of the cremaster with adenosine deaminase only during IPC or only during 60-min reperfusion attenuated the inhibitory effect of IPC on postischemic leukocyte adhesion and emigration. However, the beneficial effects of IPC were mimicked in cremaster muscles preconditioned with adenosine (topical application for 10 min beginning 20 min before the onset of prolonged ischemia). Similar results were obtained in experiments in which adenosine was topically applied to the cremaster only during the 60-min reperfusion period. Our findings suggest that the ability of IPC to attenuate postischemic leukocyte adhesion and emigration may be mediated by adenosine released during IPC and during reperfusion after prolonged ischemia.

Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules.

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mM), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 microM), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of L-arginine was reversed by administration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 microM), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3',5'-cyclic monophosphate (8-B-cGMP) (1 mM), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETA/NO) and endogenously generated (L-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.

Hypoxia-reoxygenation impairs endothelium-dependent relaxation in isolated rat aorta.

The effects of hypoxia followed by reoxygenation on endothelium-dependent relaxation in isolated rat aorta were investigated. Acetylcholine (ACh, 3 nM-10 microM) and calcium ionophore A-23187 (3 nM-300 nM)-induced endothelium-dependent vasorelaxation of isolated rate aortic vessel rings was impaired after 15 min of hypoxia followed by 30 min of reoxygenation. Impairment of ACh-induced relaxation was prevented by pretreatment with the combination of superoxide dismutase (200 U/ml) and catalase (1,000 U/ml). Hypoxia-reoxygenation did not affect sodium nitroprusside (0.1 nM-1 microM)-induced endothelium-independent relaxation nor the dissociation constant of ACh to endothelial M3 muscarinic receptors. Propidium iodide staining of the vascular endothelium revealed a significant increase in the number of dead endothelial cells on the aortic vessel rings following hypoxia-reoxygenation, but not on those pretreated with superoxide dismutase and catalase. These results suggest that hypoxia-reoxygenation impairs endothelium-dependent relaxation of rat aorta by a mechanism that involves oxidant-mediated endothelial cell death.

Mechanisms of postischemic injury in skeletal muscle: intervention strategies.

Reperfusion of ischemic skeletal muscle leads to adverse local and systemic effects. These detrimental effects may be attenuated by interfering with or modulating the pathophysiological processes that are set in motion during ischemia and/or reperfusion. The purpose of this paper is to review the different intervention strategies that have been employed in an attempt to elucidate the mechanisms involved in the pathogenesis of skeletal muscle ischemia-reperfusion injury. The results of these studies indicate that the postischemic injury processes that lead to cell dysfunction and death are multifactorial in nature and include oxidant generation, elaboration of proinflammatory mediators, infiltration of leukocytes, Ca2+ overload, phospholipid peroxidation and depletion, impaired nitric oxide metabolism, and reduced ATP production. Although the etiopathogenesis of skeletal muscle ischemia-reperfusion is complex, careful delineation of the mechanisms that contribute to postischemic microvascular dysfunction and muscle necrosis has progressed to the point where rational intervention strategies may be proposed and implemented as potential treatments for skeletal muscle dysfunction associated with ischemia-reperfusion.

Effects of hypoxia/reoxygenation on aortic vasoconstrictor responsiveness.

The purpose of the present study was to assess the effects of hypoxia/reoxygenation (H/R) on vasoconstrictor effectiveness, in vitro. Aortic rings were obtained from rats and placed on isometric force transducers in oxygenated Krebs buffer (95% O2/5% CO2, PO2 > 500 torr). Cumulative concentration/effect relationships to norepinephrine, G-protein activation by AlICl3/NaF, depolarization by KCl or BayK-8644, mobilization of intracellular calcium by caffeine, and protein kinase C activation by l-indolactam were evaluated. Hypoxia (PO2 < 5 torr) was induced by rapidly bubbling the Krebs buffer with 95% N2/5% CO2 for 15 min. Vessel rings were reoxygenated for 30 min and concentration/effect relationships reevaluated. The dissociation constant (KA) for norepinephrine was also determined. The pD2 for maximal norepinephrine responsiveness decreased from 7.7 to 7.3 following H/R. Maximal tension generation was significantly decreased following H/R. Endothelium denudation or nitric oxide synthesis inhibition did not prevent the right shift in norepinephrine concentration/effect relationship caused by H/R. The combination of superoxide dismutase and catalase prevented the dextral shift in the concentration/effect curve. The dissociation constant for norepinephrine increased from 0.16 to 0.32 microM following H/R, suggesting decreased affinity of adrenergic receptor. H/R did not alter AlCl3/NaF, KCl, BayK-8644 or l-indolactam-induced vasoconstriction. Caffeine-induced vasoconstriction was significantly impaired following H/R, suggesting that release of calcium from the sarcoplasmic reticulum is compromised. These results suggest that H/R leads to an endothelium independent, oxidant-mediated decrease in vascular norepinephrine responsiveness that may be related to defects in the mobilization of intracellular calcium from the sarcoplasmic reticulum pool.

Fructose-1,6-diphosphate or adenosine attenuate leukocyte adherence in postischemic skeletal muscle.

The purpose of this study was to determine whether fructose-1,6-diphosphate (FDP) or adenosine (Ado), administered at the onset of reperfusion, would prevent ischemia/reperfusion (I-R)-induced leukocyte adherence and microvascular dysfunction in skeletal muscle. Changes in vascular permeability and tissue neutrophil content were assessed by measurement of the solvent drag reflection coefficient (delta) for total plasma proteins and muscle myeloperoxidase (MPO) activity, respectively, in continuously perfused, isolated canine gracilis muscles and in muscles subjected to I-R alone, I-R + FDP, and I-R + Ado. To determine whether FDP or Ado would attenuate leukocyte-endothelial cell adhesive interactions induced by I-R, leukocyte adherence and emigration were assessed in postischemic mouse cremaster muscles, using intravital microscopy in the presence and absence of FDP or Ado during reperfusion. I-R was associated with a marked increase in microvascular permeability and muscle MPO activity relative to nonischemic controls. These increases were attenuated by FDP and Ado. I-R also increased the number of adherent and emigrated leukocytes relative to control. I-R-induced leukocyte adherence and emigration were significantly attenuated by either FDP or Ado. These results indicate that FDP and Ado attenuate postischemic microvascular barrier dysfunction in skeletal muscle by a mechanism that may be related to their ability to inhibit leukocyte adhesion and emigration.