INTRODUCTION

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CORONARY ARTERY DISEASE (CAD) continues to be the leading cause of mortality in the industrialized world, with over 12 million Americans alive today with a history of angina pectoris, myocardial infarction, or both (3). Despite advances in pharmacological therapies as well as catheter-based and surgical revascularization, a significant number of these patients have diffuse coronary disease, small distal vessels, or other comorbidities that make them poor candidates for traditional methods of treatment. This number may represent as many as 12% of all patients with symptomatic coronary disease (59). In addition, as the population ages, the proportion of patients ineligible for these therapies may increase. Consequently, alternative means of improving blood flow to the heart, i.e., therapeutic angiogenesis, may take on a larger role in the treatment of CAD. Although mechanical means such as transmyocardial laser revascularization (38), angiogenic peptides including the fibroblast growth factors (77) and vascular endothelial growth factors (33), and gene therapy (84) have been used clinically, none has yet proven ideal. Consequently, further preclinical study of existing as well as novel therapies is necessary to continue to advance the field. Such preclinical studies require robust large-animal models that allow assessment of both the safety and efficacy of various therapies with predictive value for clinical study. To date, little has been written comparing the various models of chronic ischemia available for use (89), and no review has focused solely on chronic in vivo large-animal models. This review describes those models most commonly employed in large-animal studies of chronic myocardial ischemia, with a focus specifically on porcine models because of their increasing use in recent years, and critically evaluates the strengths and weaknesses of each in terms of their utility for preclinical trials of angiogenic therapies.

	WHAT ANIMAL MODELS SHOULD REPRODUCE: REFRACTORY ANGINA PECTORIS AND END-STAGE CAD

TOP ABSTRACT INTRODUCTION WHAT ANIMAL MODELS SHOULD... LARGE-ANIMAL MODELS PRESENTLY... ADDITIONAL CONSIDERATIONS REFERENCES

Clinical trials of therapeutic angiogenesis have generally included patients with refractory angina pectoris and so-called "end-stage" CAD (75). This term refers to patients with the persistence of severe anginal symptoms (Canadian Cardiovascular Society Class III and IV) despite maximal conventional antianginal combination therapy and coronary atherosclerosis not amenable to revascularization by percutaneous means or surgical bypass. The overwhelming majority of these patients have multivessel coronary disease and have undergone prior revascularization procedures (75). However, patients eligible for these alternative therapies generally have a relatively large amount of viable myocardium and only moderately impaired left ventricular function. Consequently, heart transplantation is not an option for this patient population (75). Patients with large areas of prior myocardial infarction and its attendant necrosis and scar formation are excluded because these changes are not reversible with improvements in myocardial perfusion (19). Rather, patients should have ischemic yet viable myocardium as demonstrated by positron emission tomography, thallium or technetium-sestamibi scintigraphy, or dobutamine echocardiography. The latter situations are reversible with improvements in myocardial blood flow (19).

Because of the heterogeneity of coronary artery disease and the unpredictability of collateral development, no two patients with symptomatic coronary disease will have the same pathophysiology or clinical features (61). In general, however, patients will manifest with ischemia, defined as a lack of oxygen at the cellular level caused by inadequate coronary flow (61). Patients eligible for proangiogenic therapies are frequently considered to have areas of "hibernating" myocardium (87), although in reality many will have some combination of the various recognized clinical ischemic syndromes such as effort ischemia, chronic myocardial stunning, and chronic hibernation.

Chronically stunned myocardium describes regions of the heart with persistent dysfunction at rest despite normal basal perfusion (88). This condition of persistent postischemic dysfunction is felt to result from repetitive episodes of stress-induced ischemia in myocardial regions with impaired coronary flow reserve. This differs from chronic myocardial hibernation, which refers to myocardial regions with persistent dysfunction but which are hypoperfused at rest (88). Although these ischemic syndromes appear distinct on paper, there is likely much overlap between them in the clinical setting. Common to both conditions are characteristic structural alterations (Fig. 1) affecting both the cardiomyocytes and extracellular matrix, including loss of contractile material, glycogen accumulation, numerous small mitochondria, irregular nuclei, and cytoskeletal alterations, as well as increased collagen, fibronectin, and structural proteins, among others (88).

View larger version (120K): [in this window] [in a new window]   Fig. 1.   A: electron microscopy (original magnification ×900) of biopsy from chronic hibernating myocardium in a miniswine with an experimentally produced high-grade proximal left circumflex coronary artery stenosis. Note loss of contractile elements within viable cardiomyocytes (arrowheads). These changes are most prominent in the perinuclear area. B: on higher power (original magnification ×7,100), large areas of glycogen accumulation (G) are visible within the areas of sarcomere loss along with numerous small mitochondria (M). N, nucleus. C: nonischemic anteroseptal region demonstrating normal ultrastructure (original magnification ×900). Reproduced from Ref. 82 with permission.

A recent study has demonstrated that, in swine with high-grade coronary stenoses and dysfunctional yet viable myocardium, chronic stunning and hibernation appear to coexist, with approximately two-thirds of the dysfunctional segments within the distribution of the stenotic epicardial coronary artery being hypoperfused at rest (hibernating) and one-third normally perfused (stunned) (43). Similar findings have been made in a porcine ameroid constrictor model (65). Fallavollita and Canty (25) have put forth the theory of a transition from stunning to hibernation over time in viable, chronically dysfunctional myocardium. Although the exact contribution of chronic stunning and hibernation to the clinically observed syndrome in any particular patient may not be discernible, differentiation between the two is probably not clinically relevant because the treatment for both conditions involves improving myocardial perfusion, which if done in a timely manner will result in improvement in left ventricular function (37).

One important consideration is that there appears to be a spectrum in terms of recovery of function after revascularization under these conditions (61). As noted above, there are structural changes that occur in chronically dysfunctional myocardium, and these alterations are felt to affect the ability of the myocardium to recover function after revascularization (88). Elsässer and colleagues (22) have described a self-perpetuating "vicious cycle" in hibernating myocardium whereby a regional inflammatory reaction in the ischemic territories leads to progressive fibrosis and cellular degeneration. The importance of the progressive nature of these phenotypic changes is that patients with more advanced cardiomyocyte deterioration have less return of function after revascularization than those with less advanced changes (23, 32, 76). Because the therapeutic angiogenesis trials to date have generally included "no option" patients with severe inoperable CAD, one should not be surprised that improvements in ventricular function seen in these trials have been insignificant.

	LARGE-ANIMAL MODELS PRESENTLY IN USE

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Maxwell et al. (55) in 1987 demonstrated the wide spectrum of collateral flow that exists between various mammalian species (Fig. 2). This study demonstrated that the dog, which for many decades has been the most commonly used species in studies of myocardial ischemia, has a variable and often substantial collateral circulation network. These preformed collaterals are capable of providing up to 40% of normal flow to the perfusion bed of an acutely occluded epicardial coronary artery (31). Consequently, most recent large-animal models of chronic ischemia have utilized swine because their coronary anatomy, with minimal preexisting coronary collateral vessels, is similar to that of humans (31, 55, 74). In addition, their cardiac physiology, with the distribution of the coronary artery blood supply, including a typically right-dominant coronary system, and cardiac conduction system are very similar to humans (83). Likewise, the heart size-to-body weight ratio (0.005) for the typical 30-kg pig used in most laboratory studies is identical to that of humans (36). Finally, the swine heart is similar to that of humans from a metabolic standpoint as well, relying predominantly on nonesterified fatty acids as the preferred substrate under normal conditions, accounting for up to 80% of myocardial energy production (1). During sublethal myocardial ischemia, the -oxidation of fatty acids diminishes concomitant with increased glucose extraction (Randle's cycle) (1), a property we and others have demonstrated using positron emission tomography (PET) imaging of the chronically ischemic porcine heart (43). These anatomic and physiological factors, which have been reviewed in detail elsewhere (36), become critical in selecting an animal model for the study of therapeutic angiogenesis. Consequently, this review will focus on the most commonly used swine models of chronic, reversible myocardial ischemia. These studies have generally utilized one of three methods for producing stenosis or occlusion of an epicardial coronary artery: an ameroid constrictor, fixed stenosis, or hydraulic occluder. Other models, such as the repetitive coronary occlusion model (46, 54a, 91), that have been used exclusively in dogs will not be discussed. Because the ultimate goal of proangiogenic therapies is improved myocardial perfusion and function, special attention will be given to regional myocardial blood flow (MBF) and function in the various porcine models.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Species differences in collateral flow expressed as flow delivered to ischemic zone after epicardial coronary ligation as a percent of that within nonischemic regions of heart. Reproduced from Ref. 31 with permission.

Ameroid constrictor model. The most widely used porcine model of chronic ischemia has been the ameroid constrictor (24, 45, 60, 66), which has been utilized in preclinical studies of numerous proangiogenic therapies including recombinant vascular endothelial growth factor (69) and basic fibroblast growth factor (47, 68), autologous bone marrow (28), and gene therapy (54), as well as transmyocardial laser revascularization (35, 70). Originally described by Litvak and colleagues in 1957 (52), these constrictors are constructed of the hygroscopic material casein encased within a steel sleeve. When the device is implanted around an artery, the constrictor absorbs water and swells, compressing the artery and producing total coronary occlusion over a period of 14-30 or more days (24, 45), a period that may be prolonged by coating the ameroid with petrolatum, thus slowing the absorption of water by the occluder (90). However, as discussed in a recent review (87), although ameroid constrictors cause external arterial compression as their cross-sectional diameter diminishes, they also cause mechanical trauma that may lead to endothelial damage, platelet aggregation, and thrombus formation, as well as potentially inciting a foreign body reaction with local scar formation. Consequently, the common perception that these constrictors cause gradual coronary occlusion may be an oversimplification (87).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Time course of changes in systolic wall thickening (WT) distal to a left circumflex (LCx) ameroid constrictor in conscious pigs during the development of progressive coronary artery stenosis. The data are depicted as percent change from the baseline values obtained the first day after the ameroid constrictor was implanted. Systolic wall thickening distal to the ameroid began to fall after 1 wk, reached a nadir of 56 ± 6% below baseline at 20 ± 3 days (P < 0.05), began to recover, and was no longer significantly depressed at 34 ± 2 days. * Significant change from day 1. Reproduced from Ref. 79 with permission.

View larger version (124K): [in this window] [in a new window]   Fig. 4.   Angiogram of a porcine heart slice after chronic coronary occlusion with an ameroid constrictor on the LCx coronary artery. Note the significant microvascular collateral network that has developed in the entire perfusion territory of the left circumflex bed. The collateral vessels are typically endomural with a subendocardial plexus of anastomoses from the left anterior descending (LAD) and right coronary arteries with the right coronary being the predominant source (93). Reproduced from Ref. 29 with permission.

Fixed stenosis model. Unlike the ameroid constrictor, the fixed stenosis model has not been used for the assessment of proangiogenic therapies to date but rather has been widely utilized in studies investigating the pathophysiology of hibernating myocardium (14, 25, 26, 48). One rationale for using a fixed stenosis rather than a total occlusion model is that with a fixed degree of high-grade narrowing of a coronary artery in swine, unlike the situation with the ameroid constrictor, MBF in the region subtended by the stenotic artery is significantly decreased at rest (14, 26, 48). The reason for this discrepancy appears to be that fewer collaterals are formed with lesser degrees of occlusion (57). This is supported by prior experimental studies (7, 26) suggesting that significant collateral recruitment in swine does not occur in the absence of total coronary occlusion. This is similar to the situation in humans, in whom angiographically evident collaterals generally do not become apparent until epicardial coronary occlusion exceeds 90%, although heterogeneity clearly exists (51).

Hydraulic occluder model. Another large-animal model of chronic ischemia has involved placing an adjustable hydraulic occluder around an epicardial coronary artery to produce a fixed degree of coronary stenosis (9). This model is similar to the fixed stenosis model in that the coronary artery is reduced in diameter by an external device, although with this latter model the occluder is typically placed either proximal or distal to a myocardial flow probe. The proximal end of the occluder is externalized at the time of surgery and then may be inflated with either air or liquid material once the animal is recovered from surgery. In this manner, the degree of coronary stenosis can be finely adjusted to reduce baseline flow through the epicardial coronary artery as measured by the flow probe by a given amount and thus produce a stenosis of the severity desired. Bolukoglu and colleagues (9) were among the first to employ this type of model in an attempt to reproduce the clinical condition of chronic hibernating myocardium. These investigators placed a Doppler flow probe and hydraulic occluder in sequence around the proximal LAD of swine. The occluder was then inflated during the surgical procedure to reduce flow velocity as measured by the flow probe located just proximal to the occluder by 50%. This stenosis was then maintained for 7 days. Perioperative mortality rate in this study was nearly 44% with deaths due to cardiac arrhythmias from coronary spasm after inflation of the occluder. For those animals surviving the 7-day period, systolic shortening in the area at risk was decreased by ~40%. Metabolic studies at 7 days indicated no evidence for acidosis or cell death, preserved contractile reserve during low-dose dobutamine infusion, and no infarction by histological exam, all of which suggest that the stenosis produced chronically ischemic yet viable myocardium in the LAD distribution. Interestingly, cross sections of the LAD at the point of placement of the occluder demonstrated adventitial and medial injury with subendothelial fibroblast proliferation and elevation of overlying endothelial cells, consistent with occluder-induced vessel injury. These structural alterations within the vessel appeared to be functionally significant and potentially permanent, as there was no change in the velocity of blood flow through the region in which the occluder had been placed even after the stenosis was released at 7 days postoperatively. It is likely that these ultrastructural alterations in vessel architecture are common to both the ameroid constrictor and fixed stenosis models as well.

View larger version (151K): [in this window] [in a new window]   Fig. 5.   Coronary angiogram demonstrating experimental preparation. Note location of hydraulic occluder (radiolucent and not visualized) producing a high-grade stenosis of the proximal LCx coronary artery. Normal-diameter LAD coronary artery is labeled for comparison. The ultrasonic flow probe is located downstream from the occluder on the LCx and allows the degree of stenosis to be adjusted to produce the desired reduction in epicardial flow. Reproduced from Ref. 82 with permission.

View larger version (49K): [in this window] [in a new window]   Fig. 6.   A: positron emission tomographic [13N]ammonia perfusion scan of an animal instrumented with a high-grade proximal LCx coronary stenosis demonstrating a flow defect in the lateral and posteroinferior walls of the left ventricle as seen on the short axis view. B: corresponding [18F]fluorodeoxyglucose uptake scan showing a relative increase in glucose utilization in the region of the flow defect consistent with preserved myocardial viability (10). Reproduced from Ref. 82 with permission.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   A: regional wall motion score index (WMSI) (left) by dobutamine stress echocardiography for the LCx (solid lines) and LAD (dashed lines) distributions of the left ventricle in a group of animals 1 mo after creation of a high-grade proximal LCx stenosis. Note severe hypocontractility at rest in the LCx region with significant improvement in wall motion during low-dose dobutamine infusion followed by deterioration at higher doses. This biphasic response of initial improvement followed by deterioration with dobutamine stimulation is characteristic of ischemic, viable myocardium (2, 15). WMSI: 1 = normal, 2 = hypokinetic, 3 = akinetic, 4 = dyskinetic. B: short axis echocardiographic view of the left ventricle at rest (right) demonstrating inferoposterolateral ischemic wall motion abnormality in left circumflex distribution (arrows). PM, papillary muscles.

	ADDITIONAL CONSIDERATIONS

TOP ABSTRACT INTRODUCTION WHAT ANIMAL MODELS SHOULD... LARGE-ANIMAL MODELS PRESENTLY... ADDITIONAL CONSIDERATIONS REFERENCES

Many experimental protocols no longer use farm-bred swine because of numerous problems inherent to these animals, including a high susceptibility to malignant hyperthermia (58) and ventricular fibrillation (36) after periods of excitement, rapid growth (94), and difficulty adapting to laboratory conditions (94). Because of these problems with the use of farm-bred swine, especially in chronic experiments, several species of miniswine have been developed that overcome many of these limitations (62, 83). We have found miniswine to be well suited to long-term survival studies and recommend them over farm-bred swine for any type of chronic study, including translational studies investigating the efficacy of proangiogenic techniques.

A second consideration regarding study design for assessing proangiogenic therapies regards the duration of ischemia before application of the angiogenic therapy. As noted above, chronically ischemic yet viable myocardium is characterized by progressive myocyte apoptosis (14), ventricular remodeling (48), and structural alterations including loss of contractile material within cardiomyocytes and increases in the amount of interstitial connective tissue (88). Presumably, these alterations are related to the diminished ventricular function observed in the ischemic regions, as studies have demonstrated that patients with more advanced cardiomyocyte deterioration have less return of function after revascularization than those with less advanced changes (22, 23, 32, 76). For example, Beanlands and colleagues (6) studied the effects of the timing of surgical revascularization on the recovery of left ventricular function and survival in patients with hibernating myocardium and found that delayed revascularization was associated with increased preoperative mortality and a lack of improvement in left ventricular function compared with those operated on early. Consequently, because of the progressive nature of these phenotypic alterations in the absence of revascularization, the period of experimentally induced ischemia in studies investigating proangiogenic therapies should probably not be excessively prolonged; otherwise, functional recovery may be limited despite improvement in regional perfusion. This would hamper efficacy studies for angiogenic agents.

Unger (87) and Post and colleagues (65) have recently reviewed the various potential experimental endpoints in studies of proangiogenic therapies. At a minimum, studies should include an assessment of regional myocardial perfusion and function pre- and posttreatment as well as histological evaluation of angiogenesis and arteriogenesis. Histological analysis should include immunohistochemical staining for endothelial cell-specific markers to confirm the presence of endothelial cells within suspected blood vessels (44). Regarding serial measurements of myocardial perfusion, numerous studies in both animals and humans (12, 53) have demonstrated the spatial heterogeneity of absolute values of myocardial blood flow between different regions of the left ventricle as well as the inherent variability in serial measurements of absolute flow for a given region of the heart over time (17, 56). Consequently, in assessing potential changes in regional myocardial perfusion, normalization of the data, in which perfusion in the ischemic region of interest is expressed as a percentage of that in the nonischemic areas of the same heart, is preferred for ease of comparison of serial measurements given the temporal and spatial heterogeneity in absolute flow values (42, 67).

A wide variety of angiogenic therapies have been demonstrated effective in various porcine chronic ischemia models, including recombinant fibroblast (8, 47, 68) and vascular endothelial growth factors (40, 69), gene therapy techniques (54, 70), autologous bone marrow (28), and transmyocardial laser revascularization (35, 39, 41, 42, 44, 70), among others. These numerous experimental successes have been seen despite much less impressive results when using many of these same therapies in the clinical arena (33, 49, 50, 80). The reason for this discrepancy is unknown, but potential explanations may relate to differences between the animal models utilized and human subjects (81). Until recently, neovascularization in the chronically ischemic adult heart had been thought to be due to the processes of angiogenesis and arteriogenesis. Angiogenesis refers to the sprouting of new capillaries from preexisting ones and is mainly caused by hypoxia and mediated via activation of hypoxia-inducible factor, which serves to increase transcription of VEGF and its receptors and stabilize VEGF mRNA (72). Arteriogenesis, on the other hand, is the growth of arteries from preexisting arterioles; it is the type of vascular growth responsible for maturation of collateral conduits and produces vessels capable of carrying significant blood flow as well as being visualized with angiography (27, 72). Primary arteriogenic stimuli include shear stress and inflammation in which an invasion of monocytes and other white blood cells leads to the production of growth factors such as the fibroblast growth factors (FGF) with subsequent vascular growth (71, 73). Recent work (4, 5), however, has demonstrated that neovascularization in adults is not restricted to angiogenesis and arteriogenesis but rather involves vasculogenesis as well (27, 63). Vasculogenesis refers to the process of in situ formation of blood vessels from endothelial progenitor cells termed angioblasts (27, 63). Angioblasts migrate and fuse with other endothelial progenitor cells and capillaries to form a primitive network of vessels known as the primary capillary plexus. After this primary capillary plexus is formed, it is remodeled by sprouting and branching via the process of angiogenesis. Thus angiogenesis, vasculogenesis, and arteriogenesis all potentially contribute to neovascularization in the adult heart (27), although some authors (71) have suggested that arteriogenesis is necessary for significant improvements in myocardial blood flow and ultimately represents the desired effect of "therapeutic coronary angiogenesis." However, most studies on arteriogenesis have focused on areas in which preexistent collaterals are prevalent such as in the peripheral extremities, and it remains to be seen whether the same conditions are required in the heart or whether de novo formation of collaterals is the predominant mechanism. Regardless, because by definition arteriogenesis requires a source arteriole from which to grow, human subjects with so-called "end-stage" coronary artery disease may lack the appropriate substrate (81). In addition, the processes of angiogenesis and vasculogenesis may be impaired in these individuals as well (27). As discussed in this review, the experimental models typically utilized in translational studies of proangiogenic agents involve young, healthy animals with single-vessel coronary disease in which the remaining vessels are normal and thus potentially more able to sprout collateral vessels capable of improving myocardial blood flow to the ischemic regions. These differences may explain the disparate results seen to date in animal vs. human studies. In addition, they suggest that animal models of multivessel CAD may need to be developed to provide a more rigorous assessment of therapies appearing promising in single-vessel disease models.

Finally, neovascularization represents a highly ordered physiological mechanism under tight regulation with many factors active at the molecular level to influence the process (63), including numerous soluble polypeptides such as VEGF, angiopoietins, FGF, platelet-derived growth factors, transforming growth factor-, tumor necrosis factor-, and colony-stimulating factors, as well as many others. In addition, several membrane-bound proteins play prominent roles in angiogenesis, including various members of the integrin, cadherin, and ephrin families. Finally, mechanical forces acting on the endothelium also contribute to the regulation of angiogenesis (63). The importance of further investigation into the interactions of these regulatory processes and their potential modification for therapeutic benefit cannot be overemphasized. In addition, genomic and proteomic approaches will take on an ever-important position in the field of angiogenesis research in the future (30, 64). Consequently, the physiological and anatomic benefits of porcine models need to be weighted against smaller animal models such as rats and mice in which there is a far greater ability to perform studies in defined genetic models or apply genomic and/or proteomic techniques.

In summary, the field of therapeutic angiogenesis remains in its infancy, with moderate successes being reported in the clinical arena in some instances and disappointments in others. If the field is to advance, scientifically rigorous translational studies carried out in appropriate animal models will be necessary to develop and assess potential proangiogenic therapies before their use in humans. Although continued work using small animal models such as mice remains important, the ultimate test of therapeutic efficacy in the preclinical setting involves large-animal models of myocardial ischemia. Consequently, we hope the information contained in this review will help investigators make informed decisions regarding the planning of future studies such that these therapies may one day find their way into every day clinical practice.