News Physiol Sci 15: 303-308, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 6, 303-308,
December 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
Myocardial Reperfusion Injury: Insights Gained from Gene-Targeted Mice
Steven P. Jones and
David J. Lefer
S. P. Jones and D. J. Lefer are in the Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130.
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Abstract
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Myocardial ischemia-reperfusion injury involves activation of multiple cell types, including leukocytes and endothelial cells. The pursuant inflammation involves diminished nitric oxide production, influx of neutrophils, and myocardial cell injury. Gene-targeted animals provide important clues about the progression of this inflammatory cascade.
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Introduction
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During the last decade, the scientific community witnessed the union of genetically manipulated animals with modern technologies aimed at assessing the physiological or pathological effects of target genes (3). In no area of research has this fusion been more evident than in the realm of cardiovascular diseases. Myocardial ischemia-reperfusion (MI-R) induces a profound inflammatory condition in the myocardium that results in tissue destruction and compromised pump function (1). Understanding the pathobiological mechanisms that alter this response is paramount to ultimately developing therapeutic interventions. MI-R injury results from an ischemic event (in humans usually a result of coronary artery atherosclerosis and subsequent thrombosis). Although the ischemic insult initiates myocardial injury, additional damage occurs as a result of the reinstatement of coronary blood flow. Specifically, activation of multiple cell types, including endothelial cells (ECs) and leukocytes, is a key step in the propagation of the inflammatory sequelae of reperfusion injury. Furthermore, MI-R attenuates the production of certain cardioprotective factors, such as nitric oxide (NO; Fig. 1
). Although much experimental evidence exists in support of the reperfusion component of injury, ischemia without reperfusion will cause the destruction of most of the ischemic myocardium. This leads to an apparent paradox: the need for reestablishing blood flow at the expense of a profound inflammatory response (1). In the present review, we highlight the utility of selected transgenic mice and the lessons learned in the arena of MI-R injury.
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FIGURE 1. A: graphic depiction of a normal coronary vessel free of any disease processes. Nitric oxide (NO) is constitutively liberated from the endothelial cells via endothelial NO synthase (eNOS). Under normal conditions, there are virtually no interactions between the endothelium and neutrophils. Note the lack of constitutive P-selectin expression. However, P-selectin is stored in preformed pools in Weibel-Palade bodies (W-P). VSMC, vascular smooth muscle cell; EC, endothelial cell; RBC, red blood cell; PMN, polymorphonuclear leukocyte (neutrophil); ICAM-1, intercellular adhesion molecule-1; PECAM-1, platelet EC adhesion molecule-1; CM, cardiac myocyte. B: after coronary ischemia-reperfusion, the blood vessel's ability to form NO is severely curtailed. With decreased supplies of NO, the coronary vessel may constrict. Additionally, P-selectin is expressed on the endothelium and PMNs begin the first step in PMN sequestration: rolling. As the PMN slows, tighter interactions between CD18 (on PMN) and ICAM-1 (on endothelium) signify the second phase of PMN-EC interactions: firm adhesion. The final step, diapedesis (transmigration) involves the coordination of many factors, including the adhesion of the neutrophil to endothelial PECAM-1. PSGL-1, P-selectin glycoprotein ligand.
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Cell adhesion molecule-deficient mice in MI-R injury
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Much of this inflammatory response results from intensified leukocyte-EC interactions via enhanced EC adhesion molecule (ECAM) expression subsequent to MI-R. ECAMs aid in the trafficking of leukocytes along a chemotactic gradient. Typically, ECAMs are upregulated following stimulation (e.g., infection by a bacterium). However, in the setting of MI-R, an inappropriate immune signal causes polymorphonuclear leukocytes (PMNs) to localize to the ischemic-reperfused myocardium. Neutrophil-mediated injury is dependent on the interaction of adhesion glycoproteins expressed on the surface of circulating neutrophils [L-selectin, Sialyl Lewisx, P-selectin glycoprotein ligand-1 (PSGL-1), and CD11/CD18] with adhesion molecules expressed on the surface of the coronary endothelium [P-selectin, E-selectin, and intracellular adhesion molecule (ICAM)-1].
PMN-mediated myocardial reperfusion injury is a sequential process involving three interdependent steps: neutrophil rolling, firm adhesion, and transmigration (or diapedesis). Although there are a variety of ECAMs involved in PMN-mediated injury, P-selectin expression initiates and is necessary for neutrophil sequestration (Fig. 2). PMN rolling on the endothelium involves P-selectin (CD62P) expressed on the activated endothelium and PSGL-1 expressed on the stimulated PMN. P-selectin tethers circulating PMNs to the endothelium via low-affinity interactions and consequently regulates PMN rolling. Intracellular Weibel-Palade bodies store preformed pools of P-selectin. On activation, the Weibel-Palade bodies quickly fuse with the luminal surface of the vascular EC and express P-selectin. As the neutrophils decelerate, interactions between neutrophil CD18 and endothelial ICAM-1 promote firm adhesion of the neutrophil to the vessel wall (Fig. 3). ICAM-1 is constitutively expressed at relatively high levels on the endothelium. Additionally, ICAM-1 can be upregulated following an ischemic event. The last step during neutrophil sequestration involves diapedesis through EC-cell junctions via platelet EC adhesion molecule-1 (PECAM-1). Once at the site of inflammation, the activated neutrophils elaborate reactive oxygen species and proteolytic enzymes. Usually, the ultimate goal of this sequence of events is the destruction of the invading pathogen. However, during MI-R injury this cascade becomes activated with deleterious results.
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FIGURE 2. Schematic representation of selectin-mediated PMN-EC interactions. The normal (unstimulated) endothelium is depicted at left. Preformed pools of W-P exist in ECs. On stimulation (inflammation; right), the W-P undergo exocytosis and quickly express P-selectin. Additionally, transcriptional activation [via interleukin (IL)-1ß and tumor necrosis factor (TNF)- release] of E-selectin and P-selectin begins. Endothelial-bound E-selectin and P-selectin form low-affinity interactions (rolling) with PMN-bound PSGL-1, Sialyl Lewisx (SLex), and L-selectin. The coordinated expression of these adhesion molecules slow the PMN and potentiate the next phase of leukocyte sequestration.
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FIGURE 3. Subsequent to rolling, PMN begin to firmly adhere to the activated endothelium. Left: ICAM-1 is constitutively expressed on the endothelium. After an inflammatory stimulus, additional ICAM-1 is produced (cytokine-induced transcriptional activation). Endothelial-bound ICAM-1 interacts with PMN-bound CD18. CD18 serves as the ß-chain of the dimers of LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18). These three molecules differ only in their -chains. Consequently, a CD18-deficient animal will be unable to express functional versions of these 3 molecules.
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Early studies demonstrated that animals rendered neutropenic suffered less myocardial injury following ischemia and reperfusion. Many subsequent studies have addressed the importance of neutrophil adhesion molecules and ECAMs in the pathogenesis of neutrophil-mediated MI-R injury. Studies have shown cardioprotective effects of anti-P-selectin, anti-CD18, or anti-ICAM-1 pharmacotherapy following MI-R. The mechanism seemed clear: a decrease in the number of neutrophils in the ischemic-reperfused myocardium decreased myocardial injury and the degree of cardiac dysfunction. Although many important studies using ECAM inhibitors deserve acknowledgement, this review focuses mainly on studies using mutant mice to identify the contribution of adhesion molecules to MI-R injury. Gene-targeted mice offer the distinct advantage of not being plagued by issues such as species cross-reactivity, drug dosing, and route of administration.
In 1993, the first report of a P-selectin deficient (–/–) mouse emerged (6) and spawned many investigations into the role of P-selectin in various pathological states. Studies of wild-type mice performed in our laboratory (8) demonstrated profound increases in P-selectin expression within the coronary circulation following ischemia and reperfusion. To determine whether this P-selectin expressed in the coronary circulation was important in the pathogenesis of MI-R injury, we developed an in vivo murine model of coronary artery ligation. We found that genetic absence of P-selectin confers cardioprotection in mice (8). In association with this reduction of myocardial necrosis, we also found significant attenuation of the number of neutrophils that accumulated in the ischemic-reperfused myocardium (8). In addition, mice genetically deficient in CD18 (10) and ICAM-1 (9) were also produced and the phenotype was characterized. Subsequently, we examined the role of ICAM-1 in MI-R injury using ICAM-1 –/– mice. ICAM-1 –/– mice demonstrated attenuation of myocardial necrosis compared to wild-type mice (7). In addition, mice deficient in neutrophil CD18 were also subjected to MI-R, and we determined that these mice were significantly protected against myocardial cell injury. These findings serve to corroborate previous experimental studies of pharmacological blockade of PMN-ECAMs in MI-R. Interestingly, when P-selectin –/–, ICAM-1 –/–, and CD18 –/– mice were subjected to longer periods of ischemia, no differences in infarct size were observed compared with wild-type mice (7, 8). This suggests that, despite impaired leukocyte-EC interactions, extended periods of ischemia will yield little salvageable myocardium. In summary, P-selectin, CD18, and ICAM-1 contribute to the propagation of MI-R injury in experimental animal models, as demonstrated in genetically deficient mice.
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NO synthase knockout mice and myocardial reperfusion injury
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Endothelial NO synthase (eNOS) catalyzes the five-electron oxidation of L-arginine to L-citrulline and yields NO. Continuous release of NO from the endothelium is crucial in the maintenance of vascular homeostasis throughout the circulation. It is well accepted that EC-derived NO modulates vasodilation, leukocyte-EC interactions, platelet adhesion, platelet aggregation, microvascular permeability, and smooth muscle cell proliferation (2). As mentioned above, neutrophil-mediated myocardial injury is dependent on the interaction of adhesion glycoproteins expressed on neutrophils, with counterreceptors expressed on the coronary endothelium (Fig. 1
). NO released by the endothelium has been shown to inhibit the surface expression of many ECAMs, including P-selectin, E-selectin, vascular cell adhesion molecule-1, and ICAM-1. Many studies suggest an important capacity for NO-mediated cardioprotection in MI-R injury. Previous studies demonstrated that NO donors and L-arginine administration attenuate myocardial necrosis following MI-R. Furthermore, studies have revealed that the benefits of L-arginine and NO donors in MI-R injury are likely related to attenuation of leukocyte-EC interactions and the antioxidant actions of NO. Despite this wealth of data, controversy still surrounds the role of endothelial-derived NO in the pathogenesis of MI-R injury. However, this issue has become clear following the completion of experiments using the eNOS –/– mouse in studies of MI-R injury.
Mice deficient of all the three NOS isoforms have been produced. However, alterations in the generation of NO from eNOS appears to be more closely associated with cardiovascular disease states. Our laboratory (5) has recently demonstrated a nearly sevenfold enhancement of both leukocyte rolling and firm adhesion in eNOS –/– mice compared with control mice. Furthermore, eNOS deficiency was associated with significantly elevated microvascular P-selectin expression. Treatment with P-selectin blocking agents (i.e., P-selectin monoclonal antibody and soluble PSGL-1) ameliorated the exaggerated PMN-EC interactions in the eNOS –/– mice. The magnitude of PMN-EC interactions in eNOS –/– mice suggests that eNOS is extremely important in the maintenance of normal EC function. This study (5) also assessed the extent of neutrophil transmigration in response to intraperitoneal injection of a proinflammatory stimulus. Again, the eNOS –/– mice exhibited markedly augmented neutrophil sequestration into the peritoneal cavity. These data confirm and extend the idea of constitutive, endothelial-derived NO as a critical determinant of PMN-EC interactions.
Certain risk factors, such as hypertension, diabetes, and atherosclerosis, are associated with impaired function of eNOS. Consequently, elucidating the specific functions of eNOS in cardiovascular diseases may provide insight into the development of treatment strategies for a number of cardiovascular diseases, including myocardial infarction. Recently, we also examined the effect of eNOS deficiency in MI-R injury (4). Our studies suggest a vital role for constitutive eNOS expression in attenuating the extent of myocardial necrosis in vivo. Coronary P-selectin expression was markedly elevated in the eNOS –/– mice following MI-R. Furthermore, myocardial infarct size was significantly larger (twofold) in the complete absence of eNOS than in wild-type animals (Fig. 4). Finally, the eNOS –/– animals showed a significant augmentation in the number of neutrophils in the ischemic-reperfused myocardium. These data further reinforce the idea that endothelial-derived NO plays a key role in moderating neutrophil-EC interactions. Thus eNOS deficiency promotes leukocyte-EC interactions and exposes the animal to a greater risk of PMN-mediated injury. This injury process may also contribute to the cardiovascular abnormalities associated with hypercholesterolemia, diabetes, and/or atherosclerosis.
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FIGURE 4. Photomicrographic examples of midventricular sections from a wild-type heart and an eNOS –/– heart following in vivo myocardial ischemia and reperfusion. The dark blue (Evans blue dye) region signifies the nonischemic zone of the heart. The rest of the myocardium comprises the ischemic zone. Within the ischemic zone, viable cells were stained red by 2,3,5-triphenyltetrazolium chloride and the necrotic area was left unstained. Despite similar-sized ischemic zones, genetic deficiency of eNOS caused ~2-fold as much injury (white area) as that seen in the wild-type heart.
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Future directions
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This review provides an initial survey and assessment of the literature on gene-targeted and transgenic mice in MI-R inflammatory injury. However, there is an ever-expanding list of knockout mice and transgenic mice available for cardiovascular research. Of particular interest to cardiovascular researchers are diabetic mice [db/db, insulin receptor substrate (IRS)-1 –/–, IRS-2 –/–], hypercholesterolemic/dyslipidemic mice (LDL receptor –/–, apolipoprotein E –/–, cholesteryl ester transfer protein transgenic), and mice with cardiac failure/hypertrophy (-adrenergic receptor or ß-adrenergic receptor kinase overexpression). Many other human risk factors, such as obesity and hypertension, also have their murine counterparts. Using these mutant mice, investigators have and will continue to garner important information about the pathobiology of human diseases.
As the number of gene-targeted mice continues to grow, the available techniques to evaluate cardiovascular functional parameters in mice also expand. Laboratories are beginning to employ high-frequency transthoracic echocardiography in mice. This allows for the assessment of global cardiac function and regional wall motion in gene-targeted animals with heart rates of 300-700 beats/minute. Specifically, this noninvasive technique allows serial determinations of variables such as ventricular dimensions, ejection fraction, fractional shortening, and flow velocities. Furthermore, 1.4-Fr high-fidelity pressure transducers can be employed to measure cardiovascular function in mice. These catheters are used for measuring left ventricular systolic and diastolic pressures and offer the ability of determining left ventricular change in pressure/change in time. Surprisingly, most cardiovascular parameters can be assessed in these diminutive creatures.
Current efforts in the field of gene-targeted mice are geared toward models of conditional deficiency or transgenesis. Most mice in use either overexpress or are deficient of a certain gene beginning in utero. However, some genetic deletions are lethal at the embryonic stage or at least cause developmental abnormalities. Although this alone is important information, it precludes the use of these knockout mice in many cardiovascular studies. Fortunately, newer techniques (e.g., Cre-lox) allow the mice to develop to adulthood with essentially a wild-type phenotype. At the discretion of the investigator, the mouse can be rendered deficient of or can overexpress a gene of interest by administration of a particular agent that stimulates the transcriptional activation (or inhibition) of the target portion of DNA (gene). These so-called "knockons" and "knockoffs" may be particularly advantageous over the widely used knockout mice.
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Summary
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Data from studies using pharmacological blockers of ECAMs in MI-R injury laid the foundation for recent studies of leukocyte-ECAM-deficient mice. In experimental animal studies, the preponderance of evidence supports a definite role for neutrophils in MI-R injury. Mice with targeted deficiency of ECAMs (P-selectin or ICAM-1) or leukocyte adhesion molecules (CD18) exhibit decreased neutrophil sequestration and smaller myocardial infarcts. Furthermore, certain cardioprotective factors such as endothelial-derived NO attenuate interactions between cell adhesion molecules on the endothelium and on leukocytes. The absence of eNOS in mice results in enhanced P-selectin expression, neutrophil sequestration, and myocardial injury. Preservation of the constitutive activity of eNOS is vital to the survival of the myocardium subsequent to ischemia.
Gene-targeted mice are serving and will continue to serve as important adjuncts in the study of myocardial diseases. Mice with genetic modifications provide important information when assessed alone and in complement studies using a wide variety of approaches. Genetically modified mice are not intended to replace any particular technique or approach; instead, they should supply the investigator with a powerful tool to enhance our understanding of cardiovascular disease states.
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Acknowledgments
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D. J. Lefer is supported by grants from the National Institutes of Health (RO1-HL-60849 and PO1-DK-43785).
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References
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Introduction
Cell adhesion molecule-deficient...
