3R-Project 76-01

Development of a model of heart angiogenesis in vitro

present address: Department of Chemistry, Molecular Modeling, University of Basel
Pharmacenter, 4056 Basel, Switzerland
edouard.battegay@unibas.ch, rok.humar@unibas.ch

Keywords: mice; rat; heart; angiogenesis; angiogenesis; ischemia; explants; reduction; replacement

Duration: 2 years Project Completion: 2003

Background and Aim
Angiogenesis is a process of creating new arteries, veins and capillaries in order to increase the blood supply within an organ or tissue. It occurs for example in response to signals from an organ or tissue that is insufficient oxygenised. In the heart, angiogenesis occurs in response to acute or chronic blockages (occlusions) of coronary arteries. These occlusions induce a state of hypoxia known as myocardial ischemia.
Patients with coronary heart disease often have a better clinical outcome if angiogenesis can be stimulated and the collateral blood circulation within the heart can be improved. Because coronary heart disease is very common in humans, substantial research resources are currently being invested into studying the therapeutic stimulation of angiogenesis in the heart.
A growing number of experiments dealing with angiogenesis in the heart are performed in vivo. Current animal models of angiogenesis in vivo often use complete blockage by ligation or a gradual reduction of blood flow in a major coronary artery to induce hypoxia-driven angiogenesis. Many animals die due to myocardial infarction or cardiac rhythm disturbances during these sometimes cumbersome surgical procedures. Furthermore, myocardial ischemia is often quite a painful condition for the animals. Currently, no appropriate assay is available to assess angiogenesis in the heart in vitro. The aim of this study is to develop an in vitro assay of angiogenesis in the heart.

Method and Results
The present in vitro model uses small explants of hearts from laboratory animals killed for other purposes (mice, rats). The explants (~ 1 mm³) are cultivated in fibrin gels for 10 days under different physiological conditions (e.g. normoxia and hypoxia) in standard 48-well cell culture plates. One single heart from an adult rat or mouse suffices to perform 24 different experiments in octuplicates. Evaluation of in vitro angiogenesis of the heart occurs after 10 day of cultivation. Angiogenesis is represented by sprout formation by endothelial cells growing out of the piece of heart in different shapes and forms. Sprout formation is analyzed morphometrically and cells forming the sprouts can be characterized. Characterization of outgrowing cells is performed by different methods: (i) by subcultivation of outgrowing cells with subsequent immunohistochemistry, (ii) by direct immunohistochemistry in the fibrin gel, (iii) by cryostat sections of the tissue explant embedded into the fibrin gel, or (iiii) by electron microscopy.
For validation of the in vitro assay we have investigated different physiological conditions (e.g. normoxia and hypoxia) and frequently used angiogenic stimuli (vascular endothelial growth factor: VEGF, basic fibroblast growth factor: bFGF and platelet-derived growth factor: PDGF). We found that hypoxia (3% O₂) is a prerequisite for angiogenesis in vitro to occur in adult heart and that the growth factors amplify and modify basal in vitro angiogenesis induced by hypoxia. Amongst the tested growth factor PDGF-BB displayed the strongest potential to induce in vitro sprouting, albeit in a disorganized form. VEGF, on the other hand, induces elongated unbranched capillary-like sprouts. By the use of pharmaceutical inhibitors we were able to investigate mechanisms leading to PDGF-BB-mediated heart angiogenesis. Thereby we found that PDGF-mediated in vitro heart angiogenesis under hypoxia is mediated via a mTOR dependent process.
In further validation of the assay, we also found that in vitro angiogenesis of the heart is more restricted with increasing age. In hearts of adolescent mice younger than 8 weeks, angiogenesis occurs faster and requires less nutrients in the microenvironment. Adolescent mice show in vitro angiogenesis under serum free and normoxic (21% O₂) culture conditions. In contrast, adult mice hearts (older than 12 weeks) require serum-enriched medium and hypoxic growth conditions (3% O₂). However, in vitro angiogenesis in adolescent and adult 12-week-old mice develops to similar degrees if adequate culture conditions are chosen. As age increases further (hearts of 20-week-old mice) the potential for sprout formation decreases compared to 12-week-old mice. Thus, angiogenesis appears to be more restricted with aging in our in vitro assay of the heart, very similar to corresponding observations in vivo.

Conclusions and Relevance for 3R
The heart angiogenesis in vitro model allows to assess angiogenesis in hearts of mice and rats. Both, induction and repression of angiogenesis can be investigated. The model allows rapid screening of all sorts of pharmacological compounds by using a small number of animals only. Thus, this model will reduce and partially replace the use of animals in the investigation of angiogenesis of the heart. The flexibility in the selection of experimental conditions and protocols will allow new compounds to be screened for their effects on angiogenesis in the heart.

References
Abstract:
Kiefer, F. N., Dieterle, T., Humar, R., Neysari, S., Li, W., and Battegay E. J. (2002) Development of an assay of angiogenesis in vitro of the heart. J Vasc Res, 39 (S1): 64 (PAN 17)