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(Circulation. 1997;96:2317-2324.)
© 1997 American Heart Association, Inc.

Articles

Peroxynitrite Reduces Myocardial Infarct Size and Preserves Coronary Endothelium After Ischemia and Reperfusion in Cats

Tareck O. Nossuli, BS; Reid Hayward, PhD; Rosario Scalia, MD, PhD; ; Allan M. Lefer, PhD

From the Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Dr Allan M. Lefer, Department of Physiology, Jefferson Medical College, 1020 Locust St, Philadelphia, PA 19107.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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 µmol/L) in a feline model of MI/R injury.

Methods and Results ONOO^- (1 µmol/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.

Key Words: neutrophils • vasorelaxation • leukocytes • endothelium • endothelium-derived factors • ischemia • reperfusion

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Early reperfusion remains an important means to limit the extension of myocyte damage and necrosis in acute myocardial ischemia. However, recent evidence indicates that reperfusion itself can extend injury to the myocardium and further jeopardize viable myocardial cells.^{1 2} Reperfusion of an ischemic coronary circulation is characterized by endothelial dysfunction occurring within 2.5 minutes of reperfusion, in the absence of ultrastructural damage.³ The initial insult involved in reperfusion injury appears to be mediated by oxygen-derived free radicals produced upon reperfusion by coronary vascular endothelial cells.³ A number of studies have directly demonstrated the involvement of oxygen-derived free radicals through the use of spin trap agents or electron spin resonance spectroscopy.^{4 5} Other groups have reported the involvement of oxygen radicals by using agents such as superoxide dismutase and catalase to block the generation of deleterious free radicals and have observed beneficial effects with these scavengers.^{3 6 7} Recently, a free radical receiving considerable attention as a potential mediator of cell injury is peroxynitrite (ONOO^-).

ONOO^- is a reaction product formed by the interaction between nitric oxide (NO) and superoxide at equimolar concentrations⁸ thought to be formed in vivo.⁹ ONOO^- can be degraded to a radical with hydroxyl-like reactivity,¹⁰ which has been thought to exert cytotoxic effects in several systems. In vitro, ONOO^- is highly bactericidal to Escherichia coli^{11 12} and can cause oxidation of sulfhydryl groups¹³ as well as protein strand breakage¹⁴ and apoptosis¹⁵ at high micromolar concentrations. ONOO^- has also been implicated as a toxic mediator in the pathophysiology of endotoxemia,¹⁶ atherosclerosis,¹⁷ and mesenteric ischemia and reperfusion.¹⁸

In contrast, ONOO^- has also been shown to have several beneficial effects similar to those of NO. Thus, ONOO^-, at low micromolar concentrations, inhibits platelet aggregation^{19 20 21} and produces vasorelaxation in human and dog coronary arteries.^{21 22} Furthermore, ONOO^- can produce S-nitrosothiols,^{19 22 23} which can stimulate guanylyl cyclase and release NO. It has also recently been shown that nanomolar concentrations of ONOO^- maintained contractility in the isolated, buffer-perfused rat heart from cardiac stunning occurring in ischemia and reperfusion.²⁴ These in vitro studies showed that ONOO^- could protect against cardiac contractile dysfunction over a period of 30 minutes. However, there were three important shortcomings of this study: (1) myocardial cellular injury of necrosis was not assessed, (2) ONOO^- was not administered to a blood-perfused heart, and (3) coronary endothelial integrity could not be determined. To our knowledge, there have been no reports on the in vivo effects of exogenously administered ONOO^- in the setting of myocardial ischemia and reperfusion (MI/R).

Therefore, the purposes of this study were to investigate the in vivo effects of a physiologically relevant concentration of ONOO^- in a well-characterized feline model of MI/R, assessing (a) the degree of myocardial cell protection, (b) the preservation of coronary endothelial function, and (c) the extent of leukocyte-endothelium adherence on coronary artery segments in this setting.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Myocardial Ischemia and Reperfusion In Vivo
All procedures used in the present study are in accordance with the guidelines of the American Physiological Society for the Use and Care of Experimental Animals. Adult male cats (weight, 2.4 to 3.2 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV). An intratracheal cannula was inserted through a midline incision, and cats were placed on mechanical ventilation (Harvard small animal respirator). Two methods were used for administration of ONOO^- (1 µmol/L) diluted in 0.9% NaCl titrated to pH 8.4. In the first method, a catheter was inserted into the right jugular vein. In the second method, a catheter was inserted directly into the lumen of the left ventricle through the apical dimple with care taken not to interfere with the mitral valve or the papillary muscles. The right femoral vein was cannulated for administration of additional pentobarbital as needed to maintain a surgical plane of anesthesia. The right femoral artery was cannulated for continuous measurement of mean arterial blood pressure (MABP) and for withdrawing blood samples. After a midsternal thoracotomy, the anterior pericardium was incised and a 3-0 silk ligature was placed around the left anterior descending (LAD) coronary artery 8 to 10 mm from its origin. Standard lead II of the scalar ECG was used to determine heart rate (HR) and ST-segment elevation. The ECG and MABP were continuously monitored on a model 78304A unit oscilloscope (Hewlett Packard) and recorded on an oscillographic recorder (Gould model 2107-4490-00) every 20 minutes. The pressure-rate index (PRI), used as an index of myocardial oxygen demand, was calculated as the product of MABP+HR/1000. Two milliliters of blood was drawn every 2 hours to count circulating white blood cells according to established procedures.²⁵

Experimental Protocol
After completion of all surgical procedures, the cats were allowed to stabilize for 30 minutes before baseline readings of ECG and MABP were recorded. In cats subjected to MI/R, ischemia was induced by tightening the previously placed reversible silk ligature around the LAD so that the vessel was completely occluded. This was designated as time 0. Eighty minutes after coronary occlusion (that is, 10 minutes before reperfusion), intraventricular infusion of ONOO^- (1 µmol/L) in pH 8.4 saline or pH 8.4 saline alone was initiated and maintained throughout the 4.5-hour reperfusion period. Ten minutes later, the LAD ligature was untied and the ischemic myocardium was allowed to reperfuse for 270 minutes. Prior pilot experiments have shown that intravenous administration of ONOO^- to achieve circulating levels of 400 to 1000 nmol/L ONOO^- were ineffective in this model of MI/R.

Cats were randomly divided into three groups: (1) six sham MI/R cats receiving ONOO^- (1 µmol/L) in 0.9% NaCl at pH 8.4, (2) six MI/R cats receiving 0.9% NaCl at pH 8.4 as a vehicle, and (3) six MI/R cats receiving ONOO^- (1 µmol/L) in 0.9% NaCl at pH 8.4. Sham MI/R cats were subjected to the same surgical procedures and observed for the same duration of time as MI/R cats except that the LAD coronary artery was not occluded.

Quantification of Myocardial Area at Risk and Necrotic Area
At the end of the 270-minute reperfusion, the ligature around the LAD was again tightened. Twenty milliliters of 0.5% Evans blue (Sigma Chemical Co) was rapidly injected into the left ventricle to stain the area of myocardium, which was perfused by the patent, nonoccluded coronary arteries (that is, the left circumflex [LCx] and right coronary arteries) according to previously described methods.²⁵ The irreversibly injured or necrotic portion of the myocardium at risk that did not stain was separated from the stained portion of the myocardium. The three portions of the myocardium (nonischemic, ischemic nonnecrotic, and ischemic necrotic tissue) were subsequently weighed.²⁵ Results were expressed as the area at risk indexed to the total left ventricular mass, and the area of necrotic tissue indexed to either the area at risk or the total left ventricular mass.

Autologous Cat PMN Isolation and Labeling
Peripheral blood (20 mL) was collected from the cannulated femoral artery just before thoracotomy, and polymorphonuclear neutrophils (PMNs) were isolated by the method of Lafrado and Olsen.²⁶ In brief, after centrifugation, the pellet was mixed with 8 mL of 6% dextran (MW, 60 000 to 90 000, Sigma) and phosphate-buffered saline (PBS) to allow the red blood cells to settle. The leukocyte-enriched upper fraction was layered onto the Percoll:platelet-poor plasma (PPP) gradient (density gradient of 80%, 62%, and 50%). The gradient was centrifuged to separate the different cell populations. PMNs were collected from the 62% to 80% interface and washed with PBS. PMN preparations obtained by this method were in general >95% pure and >95% viable.

Isolated autologous cat PMNs were subsequently labeled with a fluorescent dye (PKH2 Green Fluorescent Cell Linker Kit, Sigma Immunochemicals) according to the method of Yuan and Fleming.²⁷ One milliliter of the diluent A was added to a cell pellet containing 10 million cells. One milliliter of PKH2-GL dye solution (4 µmol/L) was added to the cell suspension and incubated for 7 minutes at room temperature. Two milliliters of PBS with 10% PPP was then added to stop the labeling reaction. Cells were collected and centrifuged at 400g for 10 minutes and were resuspended in PBS. This labeling procedure does not affect either the normal morphology or function of PMNs.^{27 28}

PMN Adherence to the Cat Coronary Endothelium
PMNs were isolated and fluorescently labeled as described above. Both LAD and LCx coronary segments were isolated from ischemic-reperfused cat hearts and placed into warmed Krebs-Henseleit (KH) solution. These ex vivo segments were cut 2 to 3 mm in length. The segments were placed endothelial surface up into a cell culture dish filled with 3 mL of oxygenated KH solution and incubated in culture dishes with autologous labeled PMNs for 20 minutes at 37°C. After incubation, the coronary segments were rinsed lightly to remove nonadherent PMNs, removed, and placed onto microscope slides. Adherent PMNs were counted with the use of epifluorescence microscopy (Nikon) on five separate fields from each vessel segment and expressed as PMNs/mm² of coronary vascular endothelial surface area as previously described.²⁵

Additionally, LAD and LCx segments were isolated from cats that had not undergone ischemia-reperfusion. These in vitro segments underwent the same procedure as above but were stimulated for 10 minutes with 2 U/mL thrombin, with or without ONOO^- (100 nmol/L or 1 µmol/L). The ONOO^- was added 3 minutes before thrombin stimulation, and adherence of autologous PMNs were evaluated as above.

Isolated Coronary Artery and Aortic Ring Vasoactivity
Both LAD and LCx coronary arteries were isolated and placed into warmed KH solution as described above. Arteries were cut into rings of 2- to 3-mm length. The rings were then mounted on stainless steel hooks, transferred to tissue baths, and connected to FT-03 force transducers (model 7, Grass Instrument Co) as described previously.²⁶ Relaxation of isolated cat LAD and LCx coronary artery rings to the endothelium-dependent dilators (100 nmol/L acetylcholine [ACh] and 1 µmol/L calcium ionophore A23187) and to the endothelium-independent dilator NaNO₂ at 100 µmol/L was calculated as the percent decrease from the peak U-46619–induced (100 nmol/L) precontraction value as previously described.²⁸ In several additional studies, rat aortic rings were isolated and set up as described for cat coronary arteries above. However, the aortic rings were deendothelialized by gentle application of a cotton swab. These rings relaxed fully to NaNO₂ but relaxed <10% to acetylcholine. Solutions of ONOO^- at pH 8.4 were added to the aortic ring baths at the same time as the beginning of the infusion in MI/R cats and 4.5 hours later, coinciding with the end of the reperfusion protocol. The percent vasorelaxation of the aortic rings was recorded.

Immunohistochemical Localization of P-Selectin in the Cat Myocardium
To determine the effect of ONOO^- on expression of P-selectin after ischemia and reperfusion, two cats were exposed to 90 minutes of ischemia and 30 minutes of reperfusion and received ONOO^- (1 µmol/L) 10 minutes before reperfusion. Two additional ischemic-reperfused cats received the vehicle for ONOO^- and two other cats were sham-operated control cats not subject to ischemia. After 30 minutes of reperfusion the hearts were removed and the aorta was cannulated and perfused with KH buffer for 3 minutes, followed by ice-cold 4% paraformaldehyde in PBS for 3 minutes. Slices of cardiac tissue were dehydrated with graded acetone washes at 4°C. Tissue sections were imbedded in plastic (Immunobed, Polysciences Inc), and 4-mm-thick sections were cut and transferred to Vectabond-coated slides (Vector Laboratories). Immunohistochemical localization of P-selectin was accomplished with the use of the avidin-biotin immunoperoxidase technique (Vectastain ABC Reagent, Vector Laboratories). Positive staining was defined as a venule displaying brown reaction product on >50% of the circumference of its endothelium as previously described.²⁹ Ten sections from each heart and 50 venules per tissue section were examined, and the percentage of positive staining venules was then calculated.

Peroxynitrite
ONOO^- (obtained from the Alexis Corporation), which was freshly prepared, was delivered via express mail in dry ice and stored at -80°C. The ONOO^- was synthesized from acidified nitrite and hydrogen peroxide according to the method of Beckman et al.³⁰ The concentration of ONOO^- was monitored before use in each experiment by measuring the extinction coefficient at 302 nm after the addition of 5 mL of ONOO^- in 3 mL of 1N sodium hydroxide at pH 12. Only ONOO^- aliquots that exhibited activity >95% of the stipulated concentrations were used. At the end of each experiment, the extinction coefficient of the ONOO^- that was used was again measured. It was determined that the ONOO^- used during the experiment was still >90% active at the end of reperfusion, thus confirming that ONOO^- itself was delivered to each cat. Aliquots of ONOO^- were diluted in an appropriate volume of freshly prepared, ice-cold, pH 8.4 saline. The pH of this saline solution was adjusted by addition of an appropriate volume of 0.1N NaOH directly to the normal saline. The ONOO^-–pH 8.4 saline solution was then infused at a rate of 1 mL/h to achieve a concentration of 1 µmol/L in the coronary circulation. A decomposed form of ONOO^- prepared from the same stock of ONOO^- was found in preliminary tests to be inactive in all aspects of this study, thus eliminating the effects of nitrite and hydrogen peroxide.

Statistical Analysis
All values in the text and figures are presented as mean±SEM of n independent experiments. All data were subjected to ANOVA followed by post hoc analyses with Fisher's t test. All data on ST elevation, white cell counts, and PRI were analyzed by ANOVA incorporating repeated measures. Probability values of <.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of ONOO^- on the Extent of Myocardial Necrosis
To determine the ability of ONOO^- to protect against myocardial necrosis, we measured the amount of necrotic tissue both as a percent of the area at risk and as a percent of the total left ventricular mass (Fig 1). There was no significant difference in the wet weights of the areas at risk as expressed as a percent of the total left ventricular mass, indicating that a comparable degree of jeopardy occurred in both ischemic groups. With the intraventricular infusion method, the extent of necrosis, whether expressed as a percent of the area at risk or as a percent of the total left ventricular mass, was reduced by >50% in the ONOO^--treated group compared with cats receiving pH 8.4 normal saline alone (P<.001). Thus ONOO^- infused intraventricularly exerted a clear cardioprotective effect compared with ischemic-reperfused cats receiving only vehicle. However, with the intravenous method of infusion, the extent of necrosis expressed as a percentage of the total area at risk was not significantly different between the vehicle and ONOO^--treated groups (1 µmol/L) (33.4±3 versus 34.8±4.3, for three cats in each group, respectively). The data obtained with the different infusion methods suggest that ONOO^- could be a local-action rather than a systemic-action agent because the intraventricular infusion directly into the arterial side of the circulation showed cardioprotection but the intravenous infusion demonstrated no such effects.

View larger version (33K): [in this window] [in a new window]   Figure 1. Tissue wet weights of area at risk as a percentage of the total left ventricular wet weight (left), necrotic tissue as a percent of the area at risk (AAR) (middle), and necrotic tissue as a percent of the total left ventricle (LV) wet weight (right) for the two groups of cats that underwent myocardial ischemia and reperfusion (MI/R). Heights of bars are means and brackets are ±SEM. Cats receiving peroxynitrite (ONOO-) 1 µmol/L exhibited significantly attenuated cardiac necrosis. Numbers at the bottom of the bars indicate the numbers of cats studied.

Cardiac Electrophysiological, Hematologic, and Hemodynamic Variables
Before ischemia, all hemodynamic and electrophysiological variables were similar among the three groups of cats. However, during ischemia, in the MI/R groups, a significant and comparable peak elevation of the ST segment occurred (between 0.25 and 0.34 mV), indicating that the early ischemic insult was equivalent between the two ischemic groups. Moreover, the PRI (Table) was also similar during ischemia, indicating that myocardial oxygen demand was comparable between the two ischemic groups. After reperfusion, the ST segment returned to near control values (that is, zero) in all cats, indicating a successful degree of reperfusion. Furthermore, we observed no significant effects of ONOO^- on circulating white blood cell counts over the course of the study (Table). Thus the cardioprotective effects of ONOO^- were not due to a decreased neutrophil count in the MI/R cats. This demonstrates that changes in hemodynamic, electrophysiological, and hematologic variables during the MI/R protocol are not likely to have been the cause of the observed cardioprotection. In addition, we determined the biological effectiveness of the ONOO^- over the 4.5 hours of the infusion by assessing the vasodilator action of the ONOO^- at the start and the end of the ONOO^- infusion. One milliliter of the ONOO^- infusion solution relaxed 12 rat aortic rings 63±7% initially and 61±4% after the 4.5 hours. These values are not significantly different and indicate that the ONOO^- retained full biological activity throughout the duration of the infusion. However, at 1 µmol/L, ONOO^- did not exert any significant vasorelaxant effect.

View this table: [in this window] [in a new window]   Table 1. Circulating White Blood Cell Count and PRI in Cats Subjected to MI/R

Effects of ONOO^- on Post–Reperfusion-Induced Endothelial Dysfunction
Because endothelial dysfunction is an early and critical event in neutrophil-mediated myocardial injury, we examined the response of isolated coronary vascular rings to the endothelium-dependent vasodilators ACh and the calcium ionophore A23187 compared with the endothelium-independent vasodilator NaNO_2. These results are summarized in Fig 2. The coronary artery rings from the nonischemic control group demonstrated nearly total relaxation to all vasodilators, indicating a functionally normal endothelium and vascular smooth muscle. Although coronary rings isolated from MI/R cats receiving only vehicle exhibited full relaxation to NaNO₂, relaxation to both endothelium-dependent dilators (ACh and A23187) was markedly attenuated compared with nonischemic, operated control cats (P<.001), signifying a significant degree of endothelial dysfunction. In contrast, coronary rings isolated from MI/R cats receiving ONOO^- developed only a small decline in endothelium-dependent relaxation compared with those isolated from control cats. Coronary artery rings isolated from cats treated with ONOO^- exhibited a significantly greater relaxation than those of the MI/R group receiving only vehicle. These results indicate that ONOO^- exerts a vasculoprotective effect on the coronary vascular endothelium.

View larger version (51K): [in this window] [in a new window]   Figure 2. Summary of the vasorelaxant responses of coronary artery rings isolated from sham-operated control cats and cats subject to myocardial ischemia and reperfusion (MI/R). Left anterior descending (LAD) coronary arteries (left) and nonischemic left circumflex (LCx) coronary arteries (right) were challenged with 100 nmol/L acetylcholine (ACh), 1 µmol/L A23187, and 100 µmol/L NaNO2. Bar heights are means, brackets are ±SEM, and numbers at the bottom of the bars indicate the numbers of rings studied. *P<.01 vs MI/R + peroxynitrite–treated group (ONOO-, 1 µmol/L). **P<.02 vs vehicle-treated group.

Effect of In Vivo ONOO^- on Neutrophil Adherence to Ex Vivo Coronary Endothelium
Firm adhesion of neutrophils to the vascular endothelium is an important step in the neutrophil-endothelium interaction occurring in inflammatory states such as ischemia-reperfusion. Therefore, we assessed the extent of neutrophil adherence to the LCx and the LAD ex vivo vascular segments obtained at the conclusion of each protocol (Fig 3). Few neutrophils adhered to LCx artery segments (that is, artery segments not exposed to ischemia or reperfusion) in all three groups of cats. Also, few neutrophils adhered to the endothelium of LAD coronary artery segments isolated from control nonischemic cats. In contrast, LAD segments isolated from ischemic-reperfused cats receiving only vehicle exhibited a marked increase (threefold to fourfold) in PMN adherence compared with nonischemic controls (P<.001). However, LAD segments isolated from ischemic-reperfused cats receiving the ONOO^- infusion exhibited significantly lower PMN adherence than that of ischemic-reperfused cats receiving only vehicle (P<.01), although the degree of adherence was significantly above that observed in control cats. These data suggest that ONOO^- can significantly modulate PMN-endothelium interactions in an in vivo setting of MI/R injury.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effects of peroxynitrite (ONOO-, 1 µmol/L) or pH 8.4 saline on adherence of unstimulated neutrophils (PMNs) to coronary endothelium. Sham-operated controls (Sham) as well as rings from cats subjected to myocardial ischemia/reperfusion (MI/R) were studied. In the two MI/R groups, ischemic-reperfused left anterior descending (LAD) and nonischemic-reperfused left circumflex (LCx) coronary endothelium were studied. Data are expressed as numbers of neutrophils adhering per square millimeter of coronary artery endothelial surface area. Bar heights are means, brackets are SEM, and numbers at the bottom of the bars indicate the numbers of coronary artery segments studied.

Effect of ONOO^- on Immunohistochemical Localization of P-Selectin
The percentage of coronary venules staining positive for P-selectin in sham MI/R cats, as well as in the area not at risk in MI/R groups, was similarly low-in the range of 10% (Fig 4). Ninety minutes of ischemia followed by 30 minutes of reperfusion resulted in a significant increase in the percentage of venules staining positive for P-selectin in untreated ischemic-reperfused cats (Fig 4). This represents a fivefold to sixfold increase in the surface expression of P-selectin under these conditions. This increased expression of P-selectin on the coronary microvasculature was significantly attenuated by intraventricular infusion of ONOO^- (P<.01) to values not significantly different from those observed in control nonischemic cats.

View larger version (23K): [in this window] [in a new window]   Figure 4. Percentage of coronary venules staining positive for P-selectin in the three experimental groups of cats. Sham-operated controls (Sham) as well as cats subjected to myocardial ischemia and reperfusion (MI/R) were studied. Cats received either peroxynitrite (ONOO-, 1 µmol/L) or vehicle (saline at pH 8.4). Bar heights represent mean values and brackets indicate ±SEM. Two cats were studied in each group, 10 sections were studied in each cat heart, and 50 venules were counted in each section. ANAR indicates area not at risk; AAR, area at risk.

Effect of Exogenously Administered ONOO^- on PMN Adherence to Nonischemic Coronary Artery Segments In Vitro
We also studied the ability of ONOO^- to alter adherence of autologous unstimulated PMNs to coronary vascular segments not exposed to ischemia-reperfusion but stimulated with 2 U/mL thrombin. Stimulation of the isolated cat coronary endothelium resulted in a sixfold increase in PMN adherence to the endothelium (Fig 5). ONOO^- (100 nmol/L) resulted in a 48% reduction of PMN adherence to the thrombin-stimulated cat coronary endothelium (P<.01 versus thrombin alone). Similarly, ONOO^- (1 µmol/L) decreased PMN adherence to the coronary endothelium by 51% (P<.01 versus thrombin alone). These data demonstrate that ONOO^- significantly reduces cat PMN adherence to thrombin-stimulated, nonischemic cat coronary artery segments (Fig 5).

View larger version (26K): [in this window] [in a new window]   Figure 5. In vitro adherence of autologous cat neutrophils (PMNs) to thrombin-stimulated nonischemic cat coronary segments. Peroxynitrite (ONOO-) at (100 nmol/L and 1 µmol/L) significantly attenuated PMN adherence compared with thrombin alone (P<.01). All values are mean±SEM. Numbers at the bottom of bars indicate the numbers of segments studied. KH indicates Krebs-Henseleit buffer control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data presented in this study provide clear evidence that physiologically relevant doses of ONOO^- infused directly into the left ventricle, but not intravenously, protect both the myocardium and the coronary endothelium in feline MI/R. ONOO^- (1 µmol/L) infusion, starting just before reperfusion, decreased the necrotic area indexed to the area at risk by 50% compared with MI/R cats receiving only pH 8.4 saline. This significant cardioprotective effect was accompanied by an endothelial preservation, as evidenced by (a) a marked decrease in PMN adhesion to both in vitro and ex vivo coronary vascular endothelium segments, (b) maintenance of the vascular responsiveness to endothelium-dependent vasodilators, and (c) attenuated P-selectin expression on the coronary microvasculature. The observation that intraventricular infusion is beneficial whereas intravenous infusion is not effective is probably due to the short half-life of ONOO^- (that is, 1 second at pH 7.4). Therefore, most of the ONOO^- given intravenously would be cleared by the lungs and metabolized before it reaches the heart.

In the present report, there were no significant differences in ST-segment elevation or the areas at risk between ischemic cats receiving ONOO^- or its vehicle, indicating that the severity of the ischemic insult was similar in both groups of ischemic cats. In addition, neither the PRI, an index of myocardial oxygen demand, nor the circulating white cell counts were significantly different at any time between the two ischemic groups. Taken together, these results support the position that the observed cardioprotective effects of ONOO^- could not be attributed to the differences in severity of ischemia, the myocardial oxygen demand, or the number of circulating leukocytes.

Many previous studies have reported that high micromolar to millimolar concentrations of ONOO^- exert toxic effects in several cell systems.^{31 32} However, some of these reports did not take into account the physiological buffering systems that normally occur in vivo. It is now clear that the reactivity and toxicity of ONOO^- significantly depend on its chemical environment.^{12 33} For example, Denicola et al³⁴ reported that ONOO^- interacts with the normal bicarbonate buffer system in human plasma and becomes much less toxic even at high micromolar concentrations. Thus when evaluating in vitro studies involving ONOO^-, it is essential to determine whether normal levels of buffers or antioxidants that mimic the in vivo situation are present.

Of even greater concern is the question of whether the in vivo concentration of ONOO^- could ever reach the high micromolar to millimolar concentrations used in most in vitro studies. This is because the circulating physiological concentration of NO is 1 to 20 nmol/L³⁵ but can increase two to three orders of magnitude in certain disease states as the result of the action of inducible NO synthase (iNOS).³⁶ Because NO and superoxide must be in equimolar ratio for ONOO^- to be formed,⁸ and because the half-life of ONOO^- is only 1 second at physiological pH, the effective concentration of ONOO^- would remain in the nanomolar range or very low micromolar range even if ONOO^- is produced in situations in which iNOS is activated. Thus the supraphysiological concentrations of ONOO^- used to achieve the toxic effects observed in vitro are unlikely to occur in vivo. In fact, Wang et al³⁷ have shown that ischemic-reperfused rat hearts produce <100 nmol/L ONOO^-. Thus even if one adds the exogenous 100 to 1000 nmol/L ONOO^- to endogenous levels, one would still have high nanomolar concentrations of ONOO^-.

There are three different potential mechanisms that could account for the beneficial cardioprotective and endothelial protective effects of ONOO^- infusion observed in this study. First, ONOO^- can S-nitrosylate glutathione or other thiol-containing substances in tissues, causing the formation of S-nitrosothiols.^{19 23} S-nitrosothiols can directly activate guanylyl cyclase in vascular smooth muscle, thus causing vascular relaxation,³⁸ and these S-nitrosothiols can also release NO over sustained periods of time.²³ Second, ONOO^- can complex with substances containing alcohol groups such as sugars to form NO donors, which inhibit platelet aggregation and produce vasorelaxation in response to the NO release.³⁹ These effects are unlikely to exert a marked cardioprotective effect by themselves. However, if platelets form complexes with PMNs, as has been shown in humans during unstable angina,⁴⁰ then the antiplatelet effect of ONOO^- may be of significance in this setting. Third, ONOO^- can directly activate guanylyl cyclase, leading to cGMP accumulation in vascular smooth muscle, causing relaxation.⁴¹ At present, we cannot differentiate among these possibilities.

The hallmark of ischemia-reperfusion injury is early endothelial dysfunction caused by reduced NO release.^{42 43} The decreased NO level promotes the subsequent upregulation of cell adhesion molecules, which can increase leukocyte-endothelium interactions.^{42 43} This allows activated neutrophils to migrate across the endothelium to the cardiac myocytes and release their toxic mediators, promoting further endothelial dysfunction and cardiac myocyte injury.⁴⁴ The initial increase in PMN-endothelium interaction is primarily mediated by P-selectin, which has been observed to be upregulated in such disease states as MI/R and hypercholesterolemia.^{45 46} Thus basal NO release appears to be essential for maintaining a normal endothelial surface free of adherent leukocytes. Consistent with this hypothesis is the finding that authentic NO or organic NO donors protect against MI/R injury.^{47 48 49} Moreover, NO has been shown to inhibit the expression of ICAM-1,⁵⁰ which is another important adhesion molecule involved in ischemia and reperfusion.⁵¹ Thus, inhibition of adhesion molecules could be an important mechanism for the cardioprotective actions of NO.

In contrast to these reports, others^{52 53 54} have shown that inhibition of NO synthase (NOS) with an NOS inhibitor can protect against myocardial injury. Although ONOO^- levels were not measured, these authors speculated that the cardioprotective effect was due to inhibition of ONOO^- allegedly formed during ischemia and reperfusion. In this regard, Matheis et al⁵² studied neonatal piglets subjected to total body hypoxia, conditions far removed from adult ischemia-reperfusion injury. The others used the setting of isolated, buffer-perfused hearts subjected to ischemia and reperfusion without leukocytes.^{53 54} Under these conditions, NO has little chance of exerting any relevant beneficial effect. Moreover, the NOS inhibitor may have augmented oxygen delivery to these maximally dilated hearts. In contrast, in neutrophil-perfused rat hearts, L-NAME aggravated reperfusion injury, whereas an NO donor protected.⁴⁷ In the present study, ONOO^- significantly attenuated PMN adhesion to coronary endothelium both under ex vivo and in vitro conditions, supporting the hypothesis that ONOO^- can modulate the expression of cell adhesion molecules. This concept is further supported by the fact that ONOO^- infusion significantly attenuated P-selectin expression in the cat coronary microvasculature, as shown by immunohistochemical analysis. This could contribute to the preservation of endothelial function and to the observed cardioprotective effects in ischemia and reperfusion.

Summary
We have demonstrated that low concentrations of ONOO^- infused directly into the left ventricle significantly protect the feline myocardium against MI/R injury. This study also shows that ONOO^- can modulate PMN-endothelium interaction and preserve endothelial function in the ischemic-reperfused coronary vasculature, thus promoting vascular homeostasis. To our knowledge, this is the first report to show cardioprotective and vasculoprotective effects of ONOO^- in vivo. Although physiologically achievable concentrations of ONOO^- can be cardioprotective in the setting of MI/R, one must be cautious in interpreting these findings because there are several reports showing ONOO^- to be cytotoxic in vitro. Nevertheless, our data suggest that it is time to reassess the relevance of high concentrations of ONOO^- and thus reexamine the role of physiologically relevant concentrations of ONOO^- in cardiovascular disease states.

	Acknowledgments

 
This study was supported by Research Grant No. GM-45434 from the National Institute of General Medical Sciences of the National Institutes of Health. T. Nossuli was a Predoctoral Fellow; Dr Hayward was an NIH Postdoctoral Trainee (HL-07599).

Received April 7, 1997; accepted May 3, 1997.

	References

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