Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L-selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (-50%) attenuation in leukocyte adhesion in postischemic venules. (J. Clin. Invest. 1995Invest. . 95:2510Invest. -2519

This study demonstrates for the first time that thrombin plays an important role in ischemia-induced leukocyte rolling and adhesion and that ATIII can be used therapeutically postreperfusion to attenuate the leukocyte recruitment response in inflammation without the nonspecific effects associated with anti-adhesion molecule therapy.

Inhaled nitric oxide (NO) is being used more and more in intensive care units as a modality to improve the outcome of patients with pulmonary complications. Our objective was to demonstrate that inhaled NO could impact upon a distally inflamed microvasculature-improving perfusion, leukocyte adhesive interactions, and endothelial dysfunction. Using intravital microscopy to visualize ischemia/reperfusion of postcapillary venules, we were able to demonstrate that the reduction in perfusion, the dramatic increase in leukocyte rolling, adhesion, and emigration, and the endothelial dysfunction could all be significantly abrogated with 80 ppm, but not 20 ppm inhaled NO. Perfusing whole blood directly over an inert P-selectin and CD18 ligand substratum incorporated in a flow chamber recruited the same number of rolling and adhering leukocytes from NO-ventilated and non-NO-ventilated animals, suggesting that inhaled NO was not directly affecting leukocytes. To demonstrate that inhaled NO was actually reaching the peripheral microvasculature in vivo, we applied a NO synthase inhibitor locally to the feline mesentery and demonstrated that the vasoconstriction, as well as leukocyte recruitment, were essentially abolished by inhaled NO, suggesting that a NOdepleted peripheral microvasculature could be replenished with inhaled NO in vivo. Finally, inhaled NO at the same concentration that was effective in ischemia/reperfusion did not affect vascular alterations, leukocyte recruitment, and endothelial dysfunction associated with endotoxemia in the feline mesentery. In conclusion, our data for the first time demonstrate a role for inhaled NO as a therapeutic delivery system to the peripheral microvasculature, showing tremendous efficacy as an antiadhesive, antivasoconstrictive, and antipermeabilizing molecule in NO-depleted tissues, but not normal microvessels or vessels that have an abundance of NO (LPS-treated). The notion that blood borne molecules have NO carrying capacity is conceptually consistent with our observations. ( J. Clin. Invest. 1998. 101:2497-2505.)

Recent data have demonstrated that inhibition of nitric oxide synthesis exacerbated the mucosal injury associated with reperfusion of the postischemic intestine. In this study, using a feline 1-h intestinal ischemia followed by reperfusion model, we tested the possibility that exogenous sources of nitric oxide may prevent the reperfusion-induced mucosal barrier disruption and examined the mechanisms involved. Mucosal barrier integrity was assessed by determining 51Cr-EDTA clearance from blood to lumen. Intestinal blood flow and resistance were also determined. Reperfusion after 1 h of ischemia significantly increased 51Cr-EDTA clearance (0.05 +/- 0.01 to 0.35 +/- 0.07 ml.min-1.100 g-1) and decreased intestinal blood flow by 50%. Exogenous sources of nitric oxide including SIN-1, CAS-754, and nitroprusside as well as exogenous L-arginine all reduced reperfusion-induced mucosal barrier dysfunction without improving intestinal blood flow. Inhibition of endogenous nitric oxide with NG-nitro-L-arginine methyl ester between 1 and 2 h of reperfusion further augmented the rise in mucosal permeability associated with ischemia-reperfusion. Addition of the permeable analogue of guanosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, improved reperfusion-induced intestinal blood flow significantly but did not provide protection against mucosal barrier disruption associated with the first hour of ischemia-reperfusion. Exogenous sources of nitric oxide can reduce reperfusion-induced mucosal barrier dysfunction independent of alterations in intestinal blood flow.

Evidence shows that leukocyte recruitment into inflamed liver sinusoids does not require selectins, with one notable exception: ischemia-reperfusion (I/R). We used intravital microscopy to directly visualize the liver microcirculation during I/R and localized endotoxemia (liver superfused with lipopolysaccharide). General anti-selectin therapy (fucoidan) or anti-adhesion therapy with an antithrombin inhibitor (hirudin) was also used. Many neutrophils rolled and adhered in postsinusoidal vessels and sequestered in the sinusoids during I/R and local endotoxin superfusion. Although fucoidan blocked rolling in both forms of inflammation, leukocyte recruitment into sinusoids was only blocked in I/R. Adhesion was also inhibited in postischemic sinusoids with a second anti-adhesive agent (hirudin). Because liver I/R inevitably induces ischemia upstream in the intestine, anti-selectin therapy may prevent intestinal injury, which could prevent downstream liver inflammation. To test this hypothesis, we completely removed the intestine and rerouted blood flow from the superior mesenteric artery to the superior mesenteric vein. I/R was induced in the liver microcirculation, and many leukocytes rolled and adhered in postsinusoidal venules and adhered in sinusoids. Although fucoidan significantly reduced the rolling in postsinusoidal vessels, adhesion persisted in the sinusoids. Our data suggest that anti-adhesion therapy is effective in liver I/R in the sinusoids and postsinusoidal venules, perhaps in part due to its beneficial effect on the intestine.

The overproduction of nitric oxide in the small bowel has been invoked as a cytotoxic event in the vascular, mucosal, and whole organ dysfunction associated with inflammation. We assessed whether exogenous administration of nitric oxide in the form of nitric oxide donors (CAS 754, SIN-1) could cause microvascular and mucosal barrier dysfunction in vivo or epithelial and endothelial cell permeability alterations and cell injury in vitro. Increasing concentrations of CAS 754 or SIN-1 were infused locally into autoperfused segments of cat ileum at 30-min intervals. Baseline epithelial permeability (blood-to-lumen clearance of 51Cr-EDTA) was not affected by CAS 754, whereas vascular protein clearance was reduced. The latter effect could almost entirely be explained by a decrease in intestinal capillary hydrostatic pressure. Therefore, in some experiments venous pressure was elevated and the microvascular reflection coefficient for total proteins was estimated at filtration-independent rates. This direct measurement of microvascular permeability was unaffected by exogenous nitric oxide. CAS 754 did not increase permeability across monolayers of endothelial or epithelial cells and did not cause cell injury. Next, we assessed the possibility that excess nitric oxide may be detrimental, but only in inflamed intestine, by infusing CAS 754 with platelet-activating factor; the latter directly increases microvascular and mucosal permeability. CAS 754 did not exacerbate but rather reduced platelet-activating factor-induced rise in microvascular and mucosal permeability. These results suggest that high concentrations of nitric oxide do not cause breakdown of mucosal or microvascular barrier integrity under normal or inflammatory conditions.

Inhaled nitric oxide (NO) reduces pulmonary hypertension and dampens various aspects of lung inflammation; however, its effects are thought to be restricted to the lung because of its short half-life in biological systems. More recently, however, NO was shown to nitrosylate hemoglobin, albumin, and other plasma molecules to form stable nitrosothiol derivatives and could have an impact on the periphery. We examined whether inhaled NO could have an impact on the two compartments of distal organs, namely, the intravascular and extravascular spaces. The feline intestine was exposed to 1 h of ischemia and 1 h of reperfusion, and intestinal blood flow and mucosal dysfunction were measured in animals ventilated with room air and inhaling 0 or 80 ppm NO. A decrease in intestinal blood flow and an increase in mucosal barrier leakiness were noted in animals not exposed to inhaled NO. The intestinal blood flow impairment was entirely reversed in animals breathing 80 ppm NO, but the mucosal dysfunction was not affected. We further examined whether inhaled NO could reach the extravascular space by simply inhibiting NO in the intestine with the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME) that causes an increase in mucosal permeability that is rapidly reversed with NO donors. However, inhaled NO had no effect on the rise in mucosal permeability. l-NAME reduced lymph nitrosothiol concentrations, but inhaled NO could not replenish these levels. To further explore the intravascular impact of inhaled NO, we used intravital microscopy to visualize the microvasculature and demonstrated that inhaled NO could be initiated after reperfusion and still reduced microvascular disturbances, including reversing the impairment in blood flow and increasing leukocyte adhesion. The effects of inhaled NO persisted for an additional hour after termination of NO inhalation, consistent with a dramatic increase in nitrate within 1 h of NO inhalation, which persisted for 1 h after the termination of NO inhalation. These data suggest that inhaled NO can reach distal organs to dramatically improve reperfusion-induced microvascular but not extravascular dysfunction.

Because thrombin has been implicated in sepsis, it has been proposed that antithrombin III (AT III) is beneficial due to its anticoagulatory and antiadhesive effects. Using intravital microscopy, we visualized leukocyte-endothelium interactions in postcapillary venules of the feline mesentery exposed to lipopolysaccharide (LPS). At a concentration of AT III that blocks leukocyte adhesion in postischemic mesentery, we found no role for thrombin in LPS-induced rolling, adhesion and emigration, or microvascular dysfunction. Furthermore, AT III did not attenuate leukocyte-endothelial interactions after tumor necrosis factor-alpha superfusion of the mesentery. In contrast, fucoidan, a selectin inhibitor, prevented almost all LPS-induced rolling and reduced adhesion, emigration, and microvascular dysfunction. In a model of endotoxemia, leukocyte recruitment into mesentery or lungs was unaffected by AT III. Finally, in a human cell system that mimics the flow conditions in vivo, human neutrophils rolled, adhered, and emigrated similar to the feline postcapillary microvessels, and AT III had no effect on leukocyte recruitment induced by LPS. If AT III has beneficial effects in endotoxemia, it is not due to a direct effect upon leukocyte rolling, adhesion, or emigration in postcapillary venules in vivo.