Brief murine myocardial I/R induces chemokines in a TNF-alpha-independent manner: role of oxygen radicals.

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.

A chronic mouse model of myocardial ischemia-reperfusion: essential in cytokine studies.

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.

Mechanisms of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury.

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 microM ONOO- provided significant cardioprotection, whereas either 0.2 or 20 microM ONOO- did not protect. ONOO- at 2 microM 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.

Cardioprotective effect of interleukin-10 in murine myocardial ischemia-reperfusion.

We investigated the cardioprotective effects of rat interleukin-10 in a murine model of myocardial ischemia-reperfusion (20 min ischemia, 24 h reperfusion). Interleukin-10 (100 microg/rat) administered 15 min prior to reperfusion, significantly (P < 0.01) attenuated myocardial injury compared to rats receiving only 0.9% saline as a vehicle, as indicated by a reduced loss of myocardial creatine kinase from the ischemic-reperfused myocardium. Cardiac myeloperoxidase activity was also significantly (P < 0.01) attenuated by interleukin-10 within the ischemic-reperfused region compared to vehicle treated rats. To further investigate the mechanism of interleukin-10 we observed the in vitro adherence of neutrophil to rat vascular endothelium. Interleukin-10 treatment significantly (P < 0.05) attenuated neutrophil adherence to rat superior mesenteric artery endothelium stimulated with interleukin-1beta. Thus, interleukin-10 demonstrated significant cardioprotective effects as evidenced by a decrease in myocardial creatine kinase loss as well as an inhibition of neutrophil accumulation within the myocardium. It appears as though interleukin-10 mediates its effects, at least in part, by inhibiting leukocyte-endothelial interactions.

Peroxynitrite reduces myocardial infarct size and preserves coronary endothelium after ischemia and reperfusion in cats.

BACKGROUND: Peroxynitrite (ONOO-) is purported to exert cytotoxic effects at high doses. However, physiologically relevant concentrations of ONOO- inhibit polymorphonuclear neutrophil (PMN) adhesion to the endothelium and attenuate PMN-mediated contractile dysfunction in isolated perfused rat hearts. We are unaware of any reports in vivo showing effects of peroxynitrite in myocardial ischemia and reperfusion (MI/R). Thus, the purpose of this study was to examine the in vivo effects of a physiologically relevant concentration of ONOO- (1 micromol/L) in a feline model of MI/R injury. METHODS AND RESULTS: ONOO- (1 micromol/L) or its vehicle (0.9% NaCl at pH 8.4) was infused intraventricularly, starting 10 minutes before reperfusion in cats subjected to 90 minutes of myocardial ischemia and 4.5 hours of reperfusion. ONOO(-)-treated cats demonstrated marked attenuation of cardiac necrosis after MI/R compared with cats receiving only vehicle (P<.001). Moreover, vasorelaxation of ischemic-reperfused left anterior descending (LAD) coronary artery rings in response to the endothelium-dependent dilators acetylcholine and A23187 was greater in rings isolated from ONOO(-)-treated MI/R cats compared with MI/R cats receiving only vehicle, indicating that postreperfusion coronary vascular endothelial function was preserved by ONOO-. ONOO- also significantly reduced adherence of neutrophils to the ischemic-reperfused LAD coronary endothelium. Immunohistochemical localization of P-selectin was also significantly attenuated in hearts from ONOO(-)-infused MI/R cats. CONCLUSIONS: 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.

Regulation of P-selectin expression in human endothelial cells by nitric oxide.

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.

Heparinase III exerts endothelial and cardioprotective effects in feline myocardial ischemia-reperfusion injury.

The initial phase of neutrophil (PMN) adherence in the pathophysiology of myocardial ischemia-reperfusion (MI/R) injury depends on the selectins, particularly P- and L-selectin. Several ligands for these selectins have been identified, one of which may be a heparan sulfate proteoglycan (HSPG). Cats subjected to 90 min of MI and 270 min of R were given either heparinase III (0.033, 0.33 or 3.33 IU/kg/min) or its vehicle beginning 10 min before R and continuing throughout the 270-min R period. Heparinase III at 3.33 IU/kg/min provided a marked cardioprotective effect compared with cats receiving only vehicle as evidenced by a significant attenuation in myocardial necrosis (P < .01). In addition, endothelium-dependent vasorelaxation to acetylcholine in coronary artery rings isolated from MI/R cats treated with heparinase III was significantly preserved (P < .01). Adherence of PMNs to the coronary vascular endothelium after 270 min of R was also significantly attenuated in heparinase III-treated cats compared with vehicle (P < .01). At 0.33 IU/kg/min, heparinase III exerted modest, significant cardioprotective effects, whereas at 0.033 IU/kg/min, no significant beneficial effects were observed. Our results indicate that heparinase III is cardioprotective in a dose-dependent manner, preserves endothelial function and attenuates PMN adherence to the coronary vascular endothelium.

S-nitrosylated tissue-type plasminogen activator protects against myocardial ischemia/reperfusion injury in cats: role of the endothelium.

S-Nitrosylated tissue plasminogen activator (tPA) is formed by S-nitrosylation of the clinically important agent tPA by nitric oxide, thus conferring nitric oxide donor properties to the molecule. Cats were subjected to 90 min of myocardial ischemia and 270 min of reperfusion and were treated with either tPA or S-nitrosylated tPA 10 min before reperfusion. S-Nitrosylated tPA-treated cats demonstrated marked attenuation of cardiac necrosis after myocardial ischemia/reperfusion, compared with cats receiving only tPA (13 +/- 3% vs. 28 +/- 3%, P < .01). Relaxation of ischemic/reperfused left anterior descending coronary artery rings in response to the endothelium-dependent dilators acetylcholine and A23187 was greater in the S-nitrosylated tPA-treated group, compared with the cats receiving only tPA, indicating that coronary vascular endothelial function was preserved by S-nitrosylated tPA. S-Nitrosylated tPA also resulted in markedly reduced adherence of neutrophils to the coronary vascular endothelium, compared with nonnitrosylated tPA (P < .01). Immunohistochemical localization of P-selectin in the ischemic region was also significantly reduced by S-nitrosylated tPA, compared with the control group (P < .01). These data indicate that S-nitrosylated tPA is a cardioprotective agent, likely exerting its effect by site-specific nitric oxide donation resulting in inhibition of neutrophil-endothelium interaction via a P-selectin-dependent mechanism.

Protection from myocardial reperfusion injury by acute administration of 17 beta-estradiol.

Although several studies have demonstrated that chronic exposure to estrogen appears to be cardioprotective, acute circulatory effects of estrogen are largely unknown. Therefore, we studied the effects of acute administration of 17 beta-estradiol in myocardial ischemia/reperfusion. Cats were subjected to 90 min of left anterior descending coronary artery (LAD) occlusion and 270 min of reperfusion (MI/R). Either the estrogenic steroid, 17 beta-estradiol or its non-estrogenic isomer, 17 alpha-estradiol was administered (i.v.) 30 min prior to reperfusion at 1 microgram/kg bolus followed by a constant infusion lasting the remaining duration of the protocol at 1 microgram/kg/h. Control cats were subjected to sham MI/R. Cats treated with 17 beta-estradiol demonstrated a marked reduction in cardiac necrosis following MI/R compared to cats receiving 17 alpha-estradiol or phosphate buffered saline (17 +/- 2% v 33 +/- 1% or 34 +/- 4% area of necrosis indexed to the area-at-risk, P < 0.01). In addition, cats receiving 17 beta-estradiol exhibited reduced myocardial PMN infiltration in necrotic tissue as compared to 17 alpha-estradiol treated cats. Moreover, 17 beta-estradiol administration attenuated neutrophil adherence to ex vivo coronary vascular endothelium compared to the two controls (44 +/- 8 PMNs/mm2 v 79 +/- 7 PMNs/mm2 or 86 +/- 7 PMNs/mm2 P < 0.01). These data indicate that 17 beta-estradiol protects against myocardial ischemia/reperfusion, in part, by attenuating PMN infiltration and subsequent injury due to PMN mediator release.

Myocardial protection by N,N,N-trimethylsphingosine in ischemia reperfusion injury is mediated by inhibition of P-selectin.

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. We examined the cardioprotective effects of N,N,N-trimethylsphingosine (TMS) in a murine model of MI (20 min) and R (24 h) injury in vivo, focusing on leukocyte-endothelial interactions. TMS is a synthetic N-methylated sphingosine derivative that has protein kinase C inhibitory activity and has been shown to prevent leukocyte activation. TMS (18 microgram/kg), administered intravenously 1 min prior to reperfusion, significantly attenuated myocardial necrotic injury assessed by myocardial creatine kinase loss compared with MI/R rats receiving only vehicle (P<0.001). Cardiac myeloperoxidase activity, an index of PMN accumulation in the ischemic myocardium, was also significantly attenuated by TMS compared with rats receiving vehicle (P<0.001). We further examined whether TMS can attenuate leukocyte-endothelial interaction by intravital microscopy. TMS significantly attenuated NG-nitro-L-arginine-methyl ester (L-NAME)-stimulated PMN rolling and adherence to the rat microvascular endothelium. This action of TMS appears to be mediated by reduction of P-selectin expression because immunohistochemical analysis demonstrated that TMS significantly attenuated endothelial P-selectin expression in the L-NAME-superfused rat mesenteric microvasculature. Similarly, TMS markedly attenuated rapid P-selectin expression in rat platelets stimulated with either thrombin or L-NAME assessed by flow cytometry. In conclusion, TMS seems to be an effective cardioprotective agent by inhibiting early leukocyte-endothelial interaction, thus preventing leukocyte accumulation in the ischemic reperfused myocardium.