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

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.