Angiogenesis "in vitro" of the healthy mouse heart under hypoxia : the role of Angiotensin II and Nitric Oxide

Abstract

Angiogenesis is the process by which blood microvessels are formed from existing ones. Angiogenesis is required for development. It is also important for reducing myocardial hypoxia due to coronary and ischemic heart disease; in myocardial infarction or chronic ischemic heart disease angiogenesis responds to tissue hypoxia by new vessel formation (angiogenesis), which diminishes myocardial ischemia. However, physiological angiogenesis is usually insufficient to re-establish an adequate blood supply to the myocardium, which decreases its proper functioning. Therapeutic angiogenesis in the heart aims at increasing new vessel formation in ischemic myocardium and thus improving myocardial function by increasing blood flow (oxygen and nutrient supply). This may contribute to preventing heart failure and sudden cardiac death. Unfortunately no assay is available to investigate questions around angiogenesis in an easy format and in a way that does not use big numbers of animals. Angiogenesis and hypertension are intrinsically linked; angiogenesis is impaired in hypertension, and microvascular rarefaction is a mainstay of hypertension-induced target organ damage. Many metabolic pathways, for example the Renin-Angiotensin- Aldosterone-System (RAAS) or Nitric Oxide (NO), are involved in the development of hypertension, hypertension-induced target organ damage and also angiogenesis. Contrariwise, treatment of hypertension by drugs such as ACE-inhibitors not only reduces blood pressure and hypertension-induced target organ damage but also improves angiogenesis and thus tissue oxygenation. Specifically, accumulation of Bradykinin in response to ACE inhibition may result in angiogenesis. A study in our laboratory lead us to conclude that impaired angiogenesis in hypertension may result from impaired NO biosynthesis and not from elevated blood pressure itself. In addition, activation or RAAS or other factors may affect angiogenesis in hypertension. The general aim of this thesis was to first contribute at developing a new angiogenesis assay of the heart in vitro and to then use it to investigate the role of Angiotensin II and Nitric Oxide on angiogenesis in the heart in vitro, independent of blood pressure. We also aimed at understanding mechanisms involved in these responses. In order to further study questions of angiogenesis and hypertension in a relevant target organ, we developed and validated a new in vitro assay for investigating angiogenesis of the heart. At the time most experiments were being performed in vivo since no appropriate in vitro model was available. In vivo experiments require a large number of animals, are difficult to perform and are often associated with pain to the animals and their death. Our new in vitro model solved or reduced some of these problems. We found that both hypoxia and serum (5%) are required for angiogenesis to occur in the adult mouse heart in vitro. We analyzed the morphology of the different sprouts and found they were always composed by endothelial cells, and that smooth muscle cells or pericytes align along the sprouts. We conclude that angiogenesis of the heart in vitro can be investigated with a simple assay that allows a large series of experiments to be carried out in a relatively short time and with a minimum number of animals. We have shown that our model is suitable to investigate the actions of different substances on angiogenesis of the heart, i.e., both substances that induce angiogenesis and those that may inhibit it. Subsequently we used our newly developed assay to investigate the role of iNOS on angiogenesis of the mouse heart and aortae under hypoxia. We found that the heart is more sensitive to the different inhibitors than aortae. In vitro angiogenesis of the heart in iNOS knock out mice, in hypoxia, was totally absent. We therefore concluded that organ specific pathways must exist for angiogenesis; and that for angiogenesis of the mouse heart, in hypoxia, iNOS is essential. In the last part of the thesis we describe the work done to understand the role of Angiotensin II in the hypoxic mouse heart. By using different pharmacological agonists and antagonists as well as knock out animals we were able to conclude that the AT2 receptor is the one responsible for angiogenesis in response to Angiotensin II in the healthy and hypoxic mouse heart in our in vitro model. Further experiments led us to conclude that the angiogenic effect of Angiotensin II via the AT2 receptor in the hypoxic mouse heart is mediated via a mechanism that involves the Bradykinin receptor 2. In conclusion we have developed a new in vitro model of angiogenesis in vitro of the heart. Using this model we have analyzed angiogenesis of the hypoxic mouse heart and then characterized effects of Ang II and NO on angiogenesis in vitro.