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Angiopoietins are well known as modulators of vascular growth and remodeling in a complex modality via Tie receptors. Therefore, amongst others angiopoietins were used for further validation and standardization of a previously established quantitative in vitro model of angiogenesis which based on dividing the angiogenic cascade into 6 different stages. The investigations were carried out in cultures derived from neonatal foreskin and adult heart.
Due to the fact, that the angiopoietins Ang-1 and Ang-2 are involved in control of vessel quiescence as well as in regulation of angiogenesis, these factors were added to both, a basic (non-angiogenic) medium and a growth (angiogenic) medium. For quantitation of angiogenesis both factors were used alone or in combination and supplemented to the particular medium from the beginning and continuously throughout the entire investigation time of 60 days.
In the dermal endothelial cell cultures Ang-1 as well as Ang-2 or the combination of both factors effected in the development of an endothelial network (stage 4) within 14 days persisting until the end of investigation (day 60) independently of its application to the basic or growth medium. Indeed, cell density was higher in cultures incubated with angiopoietins in the growth medium, as expected. While addition of Ang-1 led to a distinct endothelial network, incubation with Ang-2 or the combination of both factors resulted in a fragmentary net of endothelial cell. Interestingly all samples remained in stage 4, i.e. no capillary-like structures were developed.
In the cardiac endothelial cell cultures application of Ang-1 and Ang-2 as well as their combination lead to an almost quiescent monolayer during the entire investigation period of 60 days in both, the basic and the growth medium, whereas the cell density was also higher in cultures in which the factors were added to the basic medium. In all samples only a temporary elongation and linear arrangement of endothelial cells (stage 2 to 3) was observable which regressed consistently within a few days.
Thus, while the foreskin-derived endothelial cells reached at least stage 4 of angiogenesis, no characteristic increase of stages in terms of angiogenesis was observable in the endothelial cell cultures derived from the heart. This phenomenon might depend on the organ or age dependent expression of the receptor TIE2. Therefore, the quantity of the receptor TIE2 is currently determined on transcript and protein level in the different cultures. Additionally, investigations should be carried out in further endothelial cell cultures of different charges (different ages of donors) and organs. Furthermore, a new setup of investigation tests the angiopoietins in different stages of angiogenesis with regard on their influence particularly in the later phases of angiogenesis on capillary-like structure development.
(Supported by ?Ministerium für Umwelt, Forsten und Verbraucherschutz, Rheinland-Pfalz?, Germany)