Peroxynitrite (ONOO Ϫ ) anion, formed by the interaction of superoxide with nitric oxide (NO), has previously been implicated as a cytotoxic agent. However, the effects of this free radical species on neutrophil (PMN)-endothelial cell interactions is largely unknown. We investigated the direct actions of ONOO Ϫ on PMN adhesion to endothelial cells in vitro and in vivo, as well as the effects of ONOO Ϫ on PMNmediated myocardial ischemia-reperfusion injury. In vitro, peroxynitrite (100-1,000 nM) inhibited the adhesion of rat PMNs to the endothelium of isolated thrombin-or H 2 O 2 -stimulated rat mesenteric artery ( P Ͻ 0.01 vs. thrombin or H 2 O 2 alone). In vivo, in the rat mesentery, thrombin (0.5 U/ ml) or N G -nitro-L -arginine-methyl ester (50 M) significantly increased venular leukocyte rolling and adherence, which were also significantly ( P Ͻ 0.01) attenuated by ONOO Ϫ (800 nM) accompanied by reduced P-selectin expression on the endothelial cell surface. Isolated perfused rat hearts were subjected to global ischemia and reperfusion with rat PMNs (10 8 cells), which resulted in profound cardiac depression (i.e., a marked reduction in left ventricular developed pressure and maximal rate of development of left ventricular pressure). Infusion of ONOO Ϫ reversed the myocardial contractile dysfunction of ischemic-reperfused rat hearts to near baseline levels, and markedly attenuated the accumulation of PMNs in the postischemic heart. The present study provides strong evidence that nanomolar concentrations of ONOO Ϫ both inhibit leukocyte-endothelial cell interactions and exert cytoprotective effects in myocardial ischemia-reperfusion injury. Furthermore, our results suggest that the inhibition of P-selectin expression by peroxynitrite is a key mechanism of the modulatory actions of ONOO Ϫ on leukocyte-endothelial cell interactions.

Reperfusion of the ischemic myocardium is associated with a cytokine cascade that reflects a cellular response to injury. We studied this cascade in the mouse and found that acute surgical trauma in sham-operated animals obscured early changes in cytokine induction that occur during myocardial ischemia-reperfusion (MI/R). Therefore, we utilized a new implantable device that allows occlusion and reperfusion of the left anterior descending coronary artery in a closed-chest mouse at any time after instrumentation. Induction of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha mRNA in the whole heart was examined by RNase protection assay and quantitated by Phosphor- Imager. At 3 h after instrumentation, levels of IL-6 mRNA in sham-operated animals increased above those of control naive hearts, whereas this increase did not occur until after 1 day for TNF-alpha mRNA. The surgical trauma led to exaggeration of I/R cytokine induction with greater variance in response. At 3 days and 1 wk after instrumentation, levels of both IL-6 and TNF-alpha mRNA in sham-operated animals were comparable to those of naive hearts and induction responses in I/R were much less variant. We also found that 1 h of ischemia and 2 h of reperfusion at all time points of recovery (i.e., 3 h and 1, 3, and 7 days after instrumentation) led to a significant increase in IL-6 and TNF-alpha mRNA levels. In addition, 3 h of permanent occlusion, which did not induce any mRNA increase after 1 wk postinstrumentation, caused marked upregulation of IL-6 mRNA in an acutely prepared animal. This study of early cytokine responses evoked by MI/R highlights the need for dissipation of acute surgical trauma by using a chronic, closed-chest mouse preparation.

These data suggest that physiologically relevant concentrations of ONOO- exert significant cardioprotective and vasculoprotective effects in MI/R in cats, at least partially by attenuating PMN-endothelium interactions.

Peroxynitrite (ONOO−), an intermediate formed from the equimolar interaction of nitric oxide (NO) and superoxide, is thought to be an important mediator of tissue injury in myocardial ischemia-reperfusion. However, physiologically relevant (i.e., maximally achievable) concentrations of ONOO− significantly decreased neutrophil-endothelium interactions in the rat mesentery. We therefore examined the dose-response relationship of infusion of different concentrations of ONOO− in a feline model of myocardial ischemia-reperfusion and provide data on the cellular mechanisms responsible for these observed effects. Cats subjected to 90 min of ischemia followed by 270 min of reperfusion were infused with different concentrations of ONOO− 10 min before reperfusion and continuing throughout reperfusion. We observed that infusion of 2 μM ONOO−provided significant cardioprotection, whereas either 0.2 or 20 μM ONOO− did not protect. ONOO− at 2 μM also preserved coronary endothelial function, decreased P-selectin expression, and attenuated polymorphonuclear leukocyte (PMN) adherence to the vascular endothelium. ONOO− did not exert its cardioprotective effects by acting as a direct NO donor in solution. However, in vitro, ONOO− can react with glutathione to form S-nitrosoglutathione, which can act as an NO carrier and exert beneficial effects. Thus only maximally achievable concentrations of ONOO− exert significant cardioprotective effects, in part by decreasing surface expression of P-selectin and decreasing PMN-endothelium interactions.

Early chemokine induction in the area at risk of an ischemic-reperfused (I/R) myocardium is first seen in the venular endothelium. Reperfusion is associated with several induction mechanisms including increased extracellular tumor necrosis factor (TNF)-alpha, reactive oxygen intermediate (ROI) species formation, and adhesion of leukocytes to the venular endothelium. To test the hypothesis that chemokine induction in cardiac venules can occur by ROIs in a TNF-alpha-independent manner, and in the absence of leukocyte accumulation, we utilized wild-type (WT) and TNF-alpha double-receptor knockout mice (DKO) in a closed-chest mouse model of myocardial ischemia (15 min) and reperfusion (3 h), in which there is no infarction. We demonstrate that a single brief period of I/R induces significant upregulation of the chemokines macrophage inflammatory protein (MIP) -1 alpha, -1 beta, and -2 at both the mRNA and protein levels. This induction was independent of TNF-alpha, whereas levels of these chemokines were increased in both WT and DKO mice. Chemokine induction was seen predominantly in the endothelium of small veins and was accompanied by nuclear translocation of nuclear factor-kappa B and c-Jun (AP-1) in venular endothelium. Intravenous infusion of the oxygen radical scavenger N-2-mercaptopropionyl glycine (MPG) initiated 15 min before ischemia and maintained throughout reperfusion obviated chemokine induction, but MPG administration after reperfusion had begun had no effect. The results suggest that ROI generation in the reperfused myocardium rapidly induces C-C and C-X-C chemokines in the venular endothelium in the absence of infarction or irreversible cellular injury.

P-selectin translocation to the surface of endothelial cells is increased after exposure to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), resulting in increased endothelial adhesiveness. L-NAME (3 mM) was added to human cultured iliac vein endothelial cells for 1, 2, 4, and 6 h, and P-selectin mRNA expression was quantified by a ribonuclease protection assay. In parallel experiments, the NO donor, SPM-5185 (10 microM), was added to human iliac venous endothelial cells, and P-selectin mRNA expression quantified. P-selectin protein synthesis was quantified by Western blot analysis. L-NAME caused increased expression of P-selection RNA at 2-4 h, whereas D-NAME, the stereoisomer lacking NO synthase-inhibitory activity, had no effect. The stimulatory effect of L-NAME was reversed by addition of 3 mM L-arginine. SPM-5185 decreased P-selectin mRNA over the same time period (P < 0.02). The increased P-selectin mRNA expression induced by L-NAME was paralleled by an increase in P-selectin protein synthesis. The effects of SPM-5185 and L-arginine were also paralleled by decreases in P-selectin protein synthesis and in decreased adherence of human neutrophils to human iliac venous endothelial cells. The peak effect of inhibition of NO synthesis or addition of exogenous NO occurred at 2-4 h. These results suggest a regulatory effect of NO on endothelial P-selectin expression that modulates early leukocyte-endothelial cell interactions to preserve vascular homeostasis.

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