Biological and Molecular Biological Aspects of Angiogenesis in Coronary Collateral Development

Summary

We have studied over several years the development of coronary collateral vessels in pigs and dogs following progressive coronary artery occlusion by ameroid-, hydraulic-and (intermittent) pneumatic occlusion. Preexistent collateral vessels respond with growth by DNA-synthesis, mitosis and proliferation of endothelium and smooth muscle cells. Experiments with chronically-implanted, remote-controlled, hydraulic-occluders in canine hearts showed that when the time between onset of stenosis and complete occlusion was 3 days, mostly endothelial cells entered the S-phase of the reproductive cell cycle. When it took 5 days, mostly smooth muscle cells were undergoing DNA-synthesis. In the dog heart, this growth occurs preferentially in epicardial vessels. Preexistent arterioles and small arteries are completely remodeled during the growth process, and the internal elastic lamina is digested first (probably by invading leucocytes), to allow expansion of the growing vessel. After the expansion phase is over, a new elastic lamina is formed. Monocyte invasion is a typical occurrence in growing collaterals of dog. Monocytes are known to produce a host of growth factors that may stimulate endothelium and smooth muscle to divide. The chemotactic stimulus for monocytes to adhere to endothelium may be ammonia from the deamination of endothelium. Adenosine is a metabolite of ATP which is broken down in ischemia. Monocyte involvement is less well observed in pig collaterals. In the pig heart, numerous small vessels, located throughout the entire LV-wall thickness with a slight preference for the subendocardium, respond to progressive coronary occlusion and ischemia. The entire microvasculature of the risk region in the pig heart responds with enlargement by growth. This is a useful response because of the paucity of the preexistent collateral network in the pig heart: when a critical degree of stenosis is reached, the minimum capillary resistance falls, thereby ensuring nearly adequate tissue perfusion for only a small increase in collateral flow.

Monocytes were detected in dog collaterals by direct inspection (scanning electron microscopy of opened whole mounted collaterals), a method not applicable to the much smaller pig vessels. Monoclonal antibodies against human monocytes did not cross-react with pig monocytes.

Dog and pig hearts also differ after the completion of vascular growth. After chronic left circumflex occlusion the peripheral coronary pressure equals the pressure at the origin of the collateral (= stem pressure = 0.8 × aortic pressure). Although this is also true for the pig, i.e. PCP = Pstem, stem pressure is much lower, indicating that pig collaterals originate from much smaller vessels.

Mature dog collaterals can increase their initial tissue mass by a factor of 50.

Another approach to the study of collateral growth is intermittent coronary occlusion as introduced by Franklin and Tomoike. We occluded the left circumflex coronary artery in the pig (using a chronically-implanted pneumatic-occluder) for 2 min every 30 min, using the Tomoike-scheme. More than 400 occlusions were necessary to cause a significant increase in collateral flow, and to avoid systolic bulging (2D and M-mode echo). Unfortunately, a control group receiving only the implant achieved the same result with only two test occlusions after 4 weeks. Extracardiac collaterals (adhesions) complicate this experiment in pigs.

Growth by DNA-synthesis and mitosis usually occurs following stimulation by peptide-mitogens. We have searched for a heart-derived growth factor, and we have isolated two heparin-binding growth factors from normal pig, canine and bovine heart. These have a very high sequence homology with beta-ECGF which is involved in the growth of collaterals; the stored tissue hormone is released by proteolysis of the basement membrane, and new beta-ECGF is produced by upregulation of its gene. In situ hybridization with a heterologous beta-ECGF cRNA-probe showed upregulated gene expression only in growing pig blood vessels.

We are currently searching for a myocardial ischemia-derived myocyte factor that upregulates genes for peptide growth factors. The search strategy is based not on knowledge on the protein level, but rather on expected differences in transcription of genes in the collateralized versus non-collateralized parts of the heart, using a subtractive cloning approach.

The successful identification of new and known peptide growth factors and competence factors necessitates studies designed to unravel their interaction. It is not presently known how the six factors that are known to be angiogenic interact in the heart following the onset of ischemia. Preliminary data suggest an interaction between growth factors and coagulation factors. Stimulated endothelial cells produce urokinase and tissue plasminogen activator, which pave the way for new endothelial cells. Growth factors like TGF-beta, PDGF and PDECGF can also be carried into the potentially ischemias region via platelets which adhere at damaged collateral endothelium, and are the richest known source of these factors.