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Ann Thorac Surg 1995;60:778-788
© 1995 The Society of Thoracic Surgeons
Cardiac Surgical Research and Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
Abstract
Background. The consequences of ischemia-reperfusion injury on myocytes has been studied intensely, and previous investigations of methods of myocardial protection during global and regional ischemia have focused on resultant alterations in myocardial function. However, the coronary artery endothelium is also vulnerable to damage, and only recently have investigators been able to assess coronary endothelial function.
Methods. This review examines some aspects of coronary flow abnormalities that occur after ischemia and reperfusion. In addition, we summarize recent data that address the hypothesis that injury to the coronary artery endothelium may contribute to the pathophysiology of global (and regional) cardiac ischemia and reperfusion.
Results. It appears that ischemia and reperfusion selectively injure a component in the receptor/G-protein complex linking receptor-stimulus coupling to the activation of nitric oxide synthase. Further, oxygen radicals may contribute to this injury. Recent investigations demonstrate that oxygen radicals impair the receptor/G-protein complex specific to the nitric oxide signal transduction pathway rather than causing global receptor/G-protein dysfunction.
Conclusions. The understanding of endothelial cell function and the elucidation of the nitric oxide pathway should further clarify our understanding of the pathogenesis of endothelial reperfusion injury and coronary vasospasm and contribute to the development of effective therapeutic interventions.
Braunwald and Kloner [1] characterized myocardial reperfusion as ``a double-edged sword,'' and indeed, although the benefit of early reperfusion after acute myocardial ischemia is indisputable, reestablishment of coronary flow paradoxically triggers a cascade of events that injures the coronary endothelium and accelerates ischemically induced myocardial injury [2]. Jennings and associates [3] were among the first to suggest that reperfusion may in fact hasten necrosis in myocytes subjected to prior ischemia. Much has been written regarding the functional, histologic, and biochemical consequences of ischemia and reperfusion on the myocardium; here we will examine the effects of ischemia and reperfusion on the coronary artery.
The ``No-Reflow'' Phenomenon
In 1974, Kloner and colleagues [4] reported that myocardial blood flow in dogs was reduced after release of coronary occlusion, an event they referred to as the ``no-reflow'' phenomenon. Historically, four theories have been advanced to explain this phenomenon. First, interstitial edema, intracellular edema, or both could cause extravascular compression of the coronary arteries and arterioles, thereby preventing normal blood flow [5]. Second, the decrease in flow could be the result of impaired autoregulatory responses, or alternatively, this might represent ``appropriate autoregulation'' caused by decreased metabolic demands of the postischemic heart. Third, coronary artery smooth muscle damage might augment vascular tone and impair the ability of the vascular smooth muscle to relax in response to normal mechanisms. Finally, impaired release of endothelium-derived relaxing factors, prostacyclin, or both could decrease coronary blood flow by increasing arterial tone [6].
Each of these hypotheses has been tested in isolated perfused rabbit hearts subjected to ischemia and reperfusion [7]. To assess the contribution of myocardial edema to the no-reflow phenomenon, ischemic hearts were reperfused with either a control or hypertonic (mannitol) solution. Although hearts reperfused with mannitol developed significantly less myocardial edema, there was no improvement in coronary flow compared with the more edematous hearts, which had been reperfused with isotonic medium (Table 1
).
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), but this could not be attributed to an impairment in autoregulation. In fact, plots of myocardial oxygen consumption versus coronary flow demonstrated a steeper slope in postischemic hearts than in control hearts (Table 2). This suggests that reperfused hearts may actually be more sensitive to metabolic regulation than control hearts.
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). Thus, it appears that reduced coronary flow after a global ischemic insult is due to coronary artery endothelial injury, and this is distinct from myocyte injury.
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Nitric oxide, which causes vascular relaxation through a direct action on vascular smooth muscle, produced comparable relaxation in the control left circumflex and the reperfused left anterior descending coronary arteries (Fig 3). Similarly, reperfusion had no effect on arterial contractions to either potassium chloride or prostaglandin F2
, both of which act directly on coronary vascular smooth muscle to produce vasoconstriction.
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). Importantly, ischemia alone does significantly alter endothelium-dependent relaxation; it is only on reperfusion that these reductions are clearly evidenced.
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Reperfusion and Endothelial Injury
Characterizing the Nature of Injury
In response to aggregating platelets, normal endothelium releases both prostacyclin and nitric oxide. Nitric oxide acts directly on the vascular smooth muscle to produce relaxation through a cyclic guanosine monophosphate-dependent pathway, whereas prostacyclin acts through cyclic adenosine monophosphate-dependent mechanisms [10, 11]. Although the actions of nitric oxide and prostacyclin are synergistic [12], nitric oxide is primarily responsible for the vasodilation that occurs in response to aggregating platelets [13, 14].
It has been possible to more precisely characterize the impairment in endothelium-dependent relaxations after myocardial reperfusion using a canine model of global myocardial ischemia [15]. Hearts supported by cardiopulmonary bypass were exposed to 45 minutes of aortic cross-clamping followed by 60 minutes of reperfusion; coronary arteries were then studied in organ chamber experiments. Again, endothelium-dependent relaxations to aggregating platelets, serotonin, and ADP were reduced in reperfused coronary arteries but not in ischemic, nonreperfused vessels compared with control vessels exposed to cardiopulmonary bypass alone.
Studies performed in the presence and absence of indomethacin, a cyclooxygenase inhibitor, and N-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthase, demonstrated that the reperfusion-mediated decrease in endothelium-dependent relaxation resulted from decreased release of nitric oxide, not prostacyclin.
Endothelial cells produce nitric oxide in response to a wide range of agonists including norepinephrine, histamine, ADP, thrombin [16], sodium fluoride, phospholipase C, calcium ionophore, serotonin, angiotensin, prostaglandin F2, potassium ions [17], endothelin [18], and electric stimulation. There appear to be two distinct classes of agonists: receptor-mediated agonists-acetylcholine, ADP, serotonin, norepinephrine, and histamine-and receptor-independent agonists-sodium fluoride, phospholipase C, potassium ions, and the calcium ionophore A23187. The activity of these various agents reflects their capacity to stimulate various steps in the signal transduction pathway linking endothelial cell receptor-ligand binding to the ultimate synthesis of nitric oxide from its substrate L-arginine (Fig 5).
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), caused similar endothelium-dependent relaxations in control and reperfused vessels (Fig 6). In contrast, sodium fluoride, which selectively activates the pertussis toxin-sensitive G-protein (Fig 7), failed to elicit normal endothelium-dependent relaxations after reperfusion. This impairment was specifically due to global cardiac ischemia and reperfusion; coronary arteries from animals exposed to global ischemia without reperfusion exhibited normal endothelium-dependent relaxation to sodium fluoride.
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Defining the Mechanism of Injury
Reports implicating oxygen radicals in the pathogenesis of reperfusion injury are prevalent. Using a canine model of regional coronary ischemia and reperfusion, Lawson and co-workers [23] were able to demonstrate significant reduction in infarct size in animals treated with superoxide dismutase, a scavenger of superoxide anion. This and numerous similar observations [24] lead quite naturally to the question of whether oxygen radicals might selectively injure the receptor/G-protein complex component of the nitric oxide synthase signal transduction pathway.
To test this hypothesis, coronary arteries were incubated in vitro with oxygen radicals generated by the xanthine/xanthine oxidase enzyme system [25]. As seen in intact animal studies of coronary reperfusion, coronary arteries exposed to oxygen radicals in vitro failed to relax to sodium fluoride (Fig 8A). Indeed, in vitro exposure to oxygen radicals impaired coronary artery relaxation to the endothelium-dependent, receptor-dependent agonist ADP (Fig 8B) but had no effect on endothelium-dependent, receptor-independent relaxation to the calcium ionophore A23187 (Fig 8C). Thus, the pattern of vascular reactivity in coronary artery segments exposed to oxygen-derived free radicals in vitro paralleled exactly that observed after global myocardial ischemia and reperfusion in vivo.
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) suggests that oxidative injury may selectively injure the receptor/G-protein complex specific to the nitric oxide signal transduction pathway rather than causing global receptor/G-protein dysfunction.
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Endothelium-Derived Contracting Factors
Enhanced vascular tone after coronary reperfusion is not the exclusive result of decreased production of relaxing factors. The release of an endothelium-derived contracting factor (EDCF) has been demonstrated in reperfused vessels in response to hypoxia, but the identity of this factor is unknown [18, 28]. The factor can diffuse [29] but has not been quantified in bioassay. Catecholamines, serotonin, histamine, adenine nucleotides, and cyclooxygenase products have been excluded as the contracting factor or factors induced by hypoxia [30, 31].
The discovery that blockers of nitric oxide synthesis inhibit endothelium-dependent hypoxic contraction in reperfused vessels was somewhat unexpected. This finding, at first glance, appeared to contradict the well-known actions of nitric oxide as a potent vasodilator. It is likely that these observations reflect the interaction of nitric oxide (NO) with superoxide anion (O2-) to form the peroxynitrite anion (ONOO-): NO + O2- 
ONOO-.
Peroxynitrite anion is a toxic, unstable, and highly reactive compound [32]. It is possible that superoxide anion produced during hypoxia may combine with nitric oxide produced by the endothelial cell to form peroxynitrite anion [33]. Peroxynitrite anion might then be metabolized to the endothelium-dependent contracting factor or alternatively, may act to induce its synthesis (Fig 10). This hypothesis is supported by the finding that hypoxic contractions are augmented in arteries after reperfusion injury, an environment rich in oxygen-derived free radicals [34].
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) [38]. Similar increases in plasma endothelin levels have been demonstrated in the isolated perfused rat heart subjected to ischemia and reperfusion and in humans after acute myocardial infarction [39, 40].
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Neutrophil-Endothelial Cell Interactions in Reperfusion Injury
The neutrophil has been implicated as a central mediator of tissue damage in virtually every organ system susceptible to reperfusion injury [43]. Once bound to the endothelial surface, neutrophils accelerate endothelial cell dysfunction and tissue destruction through a variety of cytotoxic mechanisms. In addition to physically plugging capillaries and small arterioles, activated neutrophils release a variety of harmful substances, including reactive oxygen radicals, cytotoxic enzymes, and cytokines [44]. Many of these substances also recruit additional leukocytes and enhance their adhesiveness to the vascular endothelium. These events are not restricted to the endothelial cell surface. With more severe injury, neutrophils infiltrate the involved tissues where enzymes and oxygen radicals may also cause tissue damage.
Further, neutrophils are a rich source of both hydrogen peroxide and superoxide anion, which inactivate nitric oxide and contribute to formation of peroxynitrite; this may lead to peroxidation of membrane phospholipids and inactivation of a variety of enzymes [45].
Neutrophil elastase and, to a lesser extent, collagenase and cathepsin G are capable of degrading the basement membrane that anchors the endothelial cell to the underlying smooth muscle. One consequence of endothelial cell detachment is that the underlying smooth muscle is exposed to the direct vasoconstricting effects of a number of luminal factors, in particular, those released from activated platelets. Elastase also disrupts the bonds linking adjacent cells and may contribute to the increase in capillary permeability that attends ischemia and reperfusion [44].
The factors governing the interaction between neutrophils and endothelial cells are complex and only partially understood. Attachment of neutrophils to the vascular endothelium is mediated by adhesion glycoprotein complexes expressed both on the surface of the neutrophil and on the surface of the endothelial cell. Many factors influence the expression of these glycoprotein complexes and thereby regulate the binding of neutrophils to endothelial cells.
The neutrophil adhesion glycoprotein complex, or CD11/CD18 complex, appears to mediate distinct neutrophil interactions [46, 47]. The expression of the CD11/CD18 complex on the neutrophil cell surface is upregulated by superoxide radicals [48] and opposed by nitric oxide [49]. After ischemia and reperfusion, the abundance of superoxide anions combined with diminished production of nitric oxide increases neutrophil cell surface expression of the CD11/CD18 adhesion complexes, thereby increasing affinity of the neutrophil for the endothelial cell surface.
The two most extensively studied forms of the endothelial cell adherence glycoproteins are the endothelial-leukocyte adhesion molecule 1 (ELAM-1) [50] and the intercellular adhesion molecule 1 (ICAM-1) [51, 52]. Under normal conditions, low levels of ICAM-1 are expressed on the surface of endothelial cells, but ELAM-1 is not. Several factors, including interleukin-1, tumor necrosis factor-, lymphotoxin, and bacterial endotoxin, promote the production and expression of ELAM-1 and ICAM-1 and favor neutrophil adherence to endothelial cells.
Whereas neutrophils appear to contribute importantly to the initiation of myocardial reperfusion injury, the same cannot be said of endothelial reperfusion injury. Indeed it has been difficult to demonstrate a direct causal role for the neutrophil in reperfusion-mediated endothelial injury. The most persuasive evidence that neutrophils are not an absolute requirement in reperfusion injury is the simple observation that reperfusion injury occurs in the absence of blood products. In the isolated perfused rabbit heart model discussed previously, significant reperfusion injury was induced with a reperfusate devoid of blood components.
Similarly, Tsao and co-workers [53] measured myeloperoxidase activity in the myocardium as an estimation of neutrophil accumulation and found significantly higher myocardial myeloperoxidase activity after 180 to 270 minutes of reperfusion. However, after short periods of reperfusion (20 minutes or less), when endothelial function has already been compromised, no significant accumulation of neutrophils or disruption of the endothelium was observed.
Indeed, neutrophil-endothelial cell interactions early in reperfusion injury may be the result of endothelial injury and diminished nitric oxide production rather than endothelial injury being the consequence of neutrophil-endothelial interactions [54].
Endothelial Injury and Coronary Artery Vasospasm
The protective role of the coronary endothelium against thrombosis and vasospasm is dynamic and requires constant response to a host of hematologic factors (Table 3). Just as the blood coagulation system is in a dynamic equilibrium with cascade activation being balanced by inactivation, so too is the thrombotic system. Low levels of platelet adhesion and aggregation are balanced by higher activity of disaggregation and disadhesion. When platelet aggregation occurs in the normal coronary artery, ADP and serotonin released by the aggregating platelets cause the endothelium to release nitric oxide [55]. Nitric oxide release then impairs platelet adhesion, prevents further aggregation, and promotes platelet disaggregation in the blood vessel. As a result of this process, vasodilation and thrombolysis occur, and coronary flow is maintained.
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) [9, 15].
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). However, if the left anterior descending coronary artery is occluded for 45 minutes followed by 60 minutes of reperfusion, subsequent exposure to hypoxia abruptly triggers vasospasm of the reperfused segment (Fig 14) [28]. Organ chamber experiments have confirmed that these hypoxia-induced contractions result from production of an EDCF by the vascular endothelium. These studies suggest that local tissue ischemia, as might occur after ischemia and reperfusion, might stimulate the endothelium to produce EDCF, thereby inducing or exacerbating coronary vasospasm.
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Conclusion
Endothelial regulation of vascular tone is achieved through the balanced production of vasodilators, such as nitric oxide and prostacyclin, and vasoconstrictors, such as endothelin-1 and EDCF. At present, no factor produced by the endothelium appears to be more important in the preservation of vascular patency than nitric oxide. In addition to dilating vascular smooth muscle, nitric oxide suppresses the production of the potent vasoconstrictor endothelin-1, resists the adhesion and aggregation of platelets and neutrophils, and promotes the dissolution of platelet aggregates.
Clearly, most of the events in the evolution of coronary vasospasm could originate from a reduction in nitric oxide production by the vascular endothelium. In the absence of this potent vasodilator, coronary artery tone would increase, narrowing the vessel lumen and limiting myocardial perfusion. In the absence of the suppressive influence of nitric oxide, endothelin-1 production would increase, upregulation of CD18/CD11 complexes would favor the adhesion of neutrophils to the endothelin, and platelet adhesion, aggregation, and activation would proceed unopposed. Together, the vasoconstricting influence of endothelin-1 and vasoactive platelet products would further reduce perfusion of the dependent myocardium. The progression of platelet aggregation to thrombus formation would ultimately occlude the already narrowed vessel. Finally, the hypoxic environment distal to the occlusion would serve as a stimulus for EDCF release and the propagation of coronary vasospasm.
The explosion of interest in endothelial cell function will no doubt further clarify our understanding of the pathogenesis of endothelial reperfusion injury and coronary vasospasm, and contribute to the development of effective therapeutic interventions.
Footnotes
Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 25-28, 1994.
Address reprint requests to Dr Schaff, Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, 200 First St SW, Rochester, MN 55905.
References
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), continuous occlusion (•), or no occlusion (control [•]). Plasma endothelin-1 levels were significantly increased during myocardial ischemia and reperfusion compared with either control or continuous occlusion. (Reprinted from Life Sciences, 48, Tsuji S, Sawamura A, Watanabe H, Takihara K, Park SE, Azuma J, Plasma endothelin levels during myocardial ischemia and reperfusion, 1745-9, Copyright 1991, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.)
