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TRANSLATIONAL PHYSIOLOGY
1Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, Japan; 2Department of Medicine and Center for Vascular Biology, Indiana University Medical School, Indianapolis, Indiana; and 3Department of Physiology, Louisiana State University Health Science Center, New Orleans, Louisiana
Submitted 6 July 2004 ; accepted in final form 14 December 2004
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ABSTRACT |
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vascular endothelial growth factor; matrix metalloproteinase; plasminogen
Angiostatin inhibits tumor angiogenesis and metastases (11, 12) and is produced via degradation of plasminogen (1, 12) by matrix metalloproteinases (MMP)-2, -7, and -9 (12, 13). MMP-2 and -9 activities are upregulated in atherosclerotic plaques and failing heart in humans (4, 23), cellular environments under oxidant stress and with reduced nitric oxide (NO) bioavailability. Recently, we reported that angiostatin levels are increased in a model of inadequate coronary collateral growth and angiogenesis in response to ischemia under NO synthesis inhibition, despite high levels of VEGF (8, 9). These results indicate that coronary collateral growth and development may be dependent on a balance between levels of growth inhibitors and growth factors. Therefore, we proposed that the impairment of coronary collateral growth is contributed to by increased production of angiostatin in humans. To test this hypothesis, we measured the expression of angiostatin, VEGF, plasminogen/plasmin, and activities of MMP-2 and -9 in pericardial fluid taken from patients with CAD who underwent coronary bypass surgery.
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METHODS |
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Sampling pericardial fluid during surgery. Immediately after a small incision of the pericardium was made, undiluted pericardial fluid was obtained. The samples were collected in sterile tubes and immediately placed on ice. The average amount of pericardial fluid obtained from each patient was 3.2 ± 0.3 ml. After clarification of the cellular components by centrifugation at 3,000 g for 10 min at 4°C, supernatants were rapidly frozen and stored at –80°C until use.
Western analyses. Pericardial fluid (15 µl) was collected and diluted 1:1 in SDS sample buffer. Each sample (30 µl) was separated in a 10% SDS-PAGE gel. We used mouse monoclonal angiostatin primary antibody (Upstate Biotechnology) and VEGF primary antibody, and the secondary antibody (IgG) was labeled with horseradish peroxidase (HRP). After incubation with HRP substrate (Pierce SuperSignal), membranes were exposed on film, and signals were visualized, digitized, and analyzed using NIH Image software, in units of density x band area, termed optical density units.
SDS-PAGE zymography. We performed quantitative gelatin zymography as previously described (9). Pericardial fluid samples (3 µl) were separated by dilution into zymography sample buffer. The samples were loaded into the wells of a 7.5% gelatin gel and electrophoresed. The gel was then removed and incubated for 1 h at room temperature in 100 ml of renaturing buffer (2.5% Triton X-100) on a rotary shaker. The buffer was decanted and replaced with 100 ml of development buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2, and 0.02% Brij 35). The gel was incubated at 37°C for 18 h. Each gel was stained with 100 ml of 0.5% Coomassie blue G-250 in 30% methanol and 10% acetic acid for 3 h and then destained with three changes of 30% methanol and 10% acetic acid. Gels were digitized using a scanning digitizing system and analyzed using NIH Image software (product of density and band area). In the preliminary study, we identified the 72-kDa band as MMP-2 and the 92-kDa band as MMP-9 by performing Western blotting.
Statistical analysis.
All results are expressed as means ± SE. To ascertain whether there was any relationship to risk factors, a comparison of the variables between the two patient groups, with no apparent and well-developed collaterals, respectively, was performed using ANOVA and Pearson's 
2 test, for the analysis of continuous and categorical variables, respectively. A P value <0.05 was regarded as significant.
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RESULTS |
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-adrenergic and calcium channel antagonists was higher in the groups with collateral development (P < 0.05). Notably, the other medications [lipid-lowering agents and angiotensin-converting enzyme (ACE) inhibitors] were not different between the two groups.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Coronary collateral growth in CAD. The growth or recruitment of coronary collateral circulation ameliorates adverse outcomes of CAD such as myocardial infarction, angina, and sudden death (5, 7). Myocardial viability after acute infarction correlates with the extent of collateral blood flow of the affected vascular segment. Therefore, the extent of increase of collateral conductance is pivotal in determining the preservation of myocardium during ischemia. Recent clinical trials evaluated the administration of growth factors such as VEGF or bFGF for therapeutic angiogenic effects in CAD (17, 21). However, these clinical trials have not shown significantly positive therapeutic outcomes, suggesting endogenous impediments to coronary collateralization in humans that are not sufficiently complemented by the provision of exogenous growth factors.
A complicating factor in this study is the lack of recruitment of pericardial fluid samples from a control group of normal volunteers. Thus we do not know how the levels of angiostatin we measured in either group compare with those from normal human subjects without CAD. This caveat should be considered in the interpretation of our results, in which we proffer that the conclusions should be confined to the groups of patients with CAD and not extrapolated to normal patients. Another limitation is that our study represents only a relatively small group of patients with varying degrees of collateral development, and as such, our results should be interpreted with caution.
Another caveat to our results pertains to the characteristics and clinical data shown in Table 1. Although the age, gender balance, number of bypasses, and proportion of subjects with diabetes are equivalent in the two groups, and the proportion of patients taking ACE inhibitors or lipid-lowering agents is not different between the groups, the proportion of those on calcium channel antagonists or -adrenergic antagonists is greater in the group with Rentrop grade 2 or 3 collaterals. We also do not have a complete understanding about the duration of the pathology in the different groups, which also may complicate the interpretations. Despite these caveats, our study shows a difference in the levels of angiostatin in patients with CAD who develop or do not develop coronary collaterals; namely, angiostatin is negatively correlated with collateral development.
We previously reported that NO is important for coronary collateral development in the context of a repetitive coronary occlusion model designed to mimic the clinical circumstances of intermittent ischemia (8). Upon NO synthase (NOS) inhibition in this canine model, the expected increase in collateral blood flow was not abrogated during repetitive myocardial ischemia despite an increase in VEGF. Many clinical studies have indicated impaired bioavailability of NO in patients with CAD (3, 16). Accordingly, our previous results supported a role of reduced local NO levels in reduced coronary collateral development in patients with risks contributing to endothelial dysfunction.
Collateral growth and angiostatin.
Angiostatin activity as a potent inhibitor of tumor angiogenesis and the growth of tumor cell metastases (1, 12) is indicated by proteolytic fragments of plasminogen including kringles 1–4 as well as kringles 1–3, with the latter representing the original angiostatin of 
38 kDa reported by O'Reilly et al. (11, 12). The fragment including kringles 1–4, with a molecular weight of 45–50 kDa, also inhibits angiogenesis (18, 22). It has been reported that MMP-2, -7, and -9 are important enzymes for the generation of angiostatin (13, 15), which are increased in atherosclerotic plaques as well as failing hearts in humans (4, 23). Recently, we reported that angiostatin plays a role in mediating the reduced coronary angiogenesis in the context of inhibition of NO synthesis, despite the presence of high-level expression of VEGF (9). In the present study, pericardial fluid level of angiostatin was found to be increased in patients with absent collateralization compared with that in patients with well-developed collaterals, whereas MMP activity was not different between these two groups. We propose that the higher level of angiostatin is, in turn, due to a locally elevated level of substrate plasmin/plasminogen. One possibility to describe the mechanism of increased level of angiostatin could be myocardial ischemia-mediated increase in vascular leak, partially induced by VEGF (19). According to the increase in permeability, the level of plasminogen in myocardial interstitial fluid increases. Increased plasminogen is then processed by local MMP activity to angiostatin, which restrains both angiogenesis and collateral development. However, one unresolved issue related to this hypothesis is that we did not measure activities of all enzymes capable of generating angiostatin, e.g., macrophage-derived metalloelastase (2), which also could contribute a basis for elevated angiostatin. Nonetheless, our results show elevated angiostatin levels in pericardial fluid of patients with poor coronary collateral development.
It is interesting to note that the extent of increase in the level of angiostatin noted to be correlated with absent collaterals in this study is not as prominent as that seen in the prior study, which showed NOS inhibition in dogs to result in both abrogation of collateral formation and increased angiostatin in myocardial interstitial fluid. This may reflect a difference between the relatively acute imposition of NOS inhibition in the latter study and the chronic endothelial dysfunction that presumably characterized the individuals in the present study. Our recent study demonstrating a similar extent of elevation of intrapericardial endostatin in patients in the absence of collateral response to ischemia complements this finding (14). The extent of both angiostatin and endostatin elevation in these patients with absent visualized collaterals also may emphasize the importance of considering the influence of multiple factors acting in aggregate to determine the overall level of "collateral-forming tone" existing within a region such as the coronary system. It is tempting to hypothesize that the nonischemic adult heart possesses a local bias against new blood vessel formation, which must be overcome by a coordinated downregulation of inhibitors and expression of stimuli to achieve increased collateral vessel supply.
Another point we are compelled to make is that we did not study whether the concentrations of angiostatin in pericardial fluid were high enough to confer a biological effect. Thus we must offer the caveat that our study does not provide a definitive cause-and-effect conclusion; rather, it offers an association of increased levels of angiostatin with decreased coronary collateral development.
In conclusion, locally elevated levels of angiostatin are correlated with reduced coronary collateral development in lumens. These results suggest that therapies attempting to augment coronary collateral growth should incorporate approaches to limit or neutralize the effects of growth inhibitor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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