Influence of cardiovascular parameters on angiogenesis

The cardiovascular system is the first system to function during vertebrate development. Heart development, vasculogenesis, angiogenesis and hematopoiesis are early processes which are known to be influenced by a complex interaction of gene function and environmental factors. The zebrafish has proved to be an ideal model animal for studying these processes, because the transparency of the embryo and larvae allow an unobstructed view of the heart and of almost every blood vessel in the whole animal. Recently developed non invasive micro techniques allow for the acquisition of numerous cardiovascular parameters like heart rate, cardiac output, red blood cell count and imaging of the vascular bed including blood distribution in these tiny animals (3-4mm). The genetic control of angiogenesis and erythropoiesis are in focus of current research and are well understood for many different species, but above from genetic control many developmental decisions are influenced by physical interaction. One aim of this project is to provide insight into physiological mechanisms in zebrafish (Danio rerio) which drive erythropoiesis and angiogenesis by analyzing the influence of hypoxia on erythropoiesis and angiogenesis as well as the interaction between blood cell density and angiogenesis during early development (from 3-21 day post fertilization). Another aim is to prove the hypothesis that blood pressure is a crucial force for angiogenetic processes in developing zebrafish. By raising animals at low oxygen concentration (hypoxia) we will analyze the effect of hypoxia on angiogenesis and erythropoiesis during early development. The experimental group will be compared to the control group by correlating the pattern of vascular casts with digital image analysis. Blood cell density will be obtained by dividing the number of red blood cells by the blood volume of a vessel segments along the dorsal artery and vein. Digital motion analysis enables us to determine blood distribution in the whole animal. By comparing zebrafish with blood cell densities different from wildtype (mutant, micro surgically or pharmacologically obtained) and wildtype animals we will be able to demonstrate whether changing hematocrit has an effect on the early vascular development. To test the importance of blood pressure for angiogenesis, zebrafish mutants with chronically decreased cardiac output and blood pressure (mutants breakdance, slow motion and pharmacologically obtained) will be raised and compared with wildtype animals for differences in their vascular patterning and.

The zebrafish has developed into a successful and far common model organism in the genetics and developmental biology. The great molecular biological interest in this animal well suitable for such studies has led to an enormous number of publications in this field. The molecular biological studies predominate in zebrafish compared to other model animals, such as mouse, rat and frog, which are already characterized physiologically very well. Reason for it is the small size of this animal which amounts to only few millimetres and refuses access to conventional measurement procedures, e.g. blood analyses. In the context of this project new methods were developed to make such mesurements possible. This is microscopic image analysis which enabled measurement of numerous relevant parameters in the context of this research project. With these methods a biomedical basic research is integrally possible in the field of heart circulation, blood and respiratory illnesses. This will allow the development of drugs and therapies in this field with the help fo the zebrafish model. The international interest in these methods becomes evident by numerous active cooperations with other research laboratories in this field. The results of our examinations demonstrate that zebrafish larvae reared under oxygen deficiency (a global problem) in very early stadia (9 days post hatch) a very flexible in adjusting their blood cell concentration. The cardiovascular as well as the respiratory regulation are already active very early, too. The special it is that none of these regulations are needed at this early stage because due to the animals tiny size oxygen enters the animal by diffusion in a sufficient quantity. Subsequently we have followed the question about the purpose of this early flexibility. With suitable experiments we have examined changes in the development of the animals at malfunctions of the heart or the blood composition (anaemia). A clear connection between blood cell concentration and the mean heart rate exists. The flexibility of the cardiorespiratory system apparently enables a partly compensation of malfunctions due to such syndromes. According to our knowledge this is possible by a complex interaction of gene expression`s and so-called epigenetic factors. Epigenetic factors have influence on the individual development without direct involvement of the genes. Changes in gene and protein expression (genes and proteins which is responsible for blood vessels and the formation of blood were examined) could be quanitified well. The evaluation of the epigenetic influence turned out to be difficult because of the complex interaction of cardiovascular performance, blood parameters and gene express ion did not allow distinguishing between direct or indirect effects. It was striking that temporary development disturbances were damped by the flexibility of the system very well so that malfunctions of the heart circulatory system did not leave any lasting deficiencies in the vascular bed.