In vitro Thermoreversible Gel Disc Quantitative Assay of Rat Angiogenesis

Shin Wakui1,2, Tomoko Muto3, Naohiko Anzai4, Hiroyuki Takahashi2, Promsuk Jutabha4, Akihiko Sakata2, Michael F. Wempe5, Hiroshi Hano2 and Hitoshi Endou6

1Department of Toxicology, Azabu University School of Veterinary Medicine, Kanagawa, Japan,
2Department of Pathology, The Jikei University School of Medicine, Tokyo, Japan,
3Drug Safety Testing Center, Saitama, Japan, 4Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan,
5School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, USA,
6J-Pharma Co., Ltd., Tokyo, Japan.

AATEX 16(2):59-65, 2011

Appropriate preclinical angiogenic screening is a preeminent consideration for the development of agents that target the tumor microvessels coordination among endothelial cells and pericytes. We previously investigated implantation of a thermoreversible gelation polymer (TGP) disc in an in vivo rat angiogenesis model (Wakui et al., 2006), but this model required many animals and long-term experiments. To reduce and replace animal usage, the present study developed an in vitro TGP disc rat angiogenesis model using a TGP disc pretreated to induce microvessels within five days after implantation in subcutaneous tissue of the rat back. Electron microscopic analysis revealed a low number of small-sized immature capillaries after 24 hours in vitro incubation. After 48 hours incubation, a number of more developed capillaries with narrow lumens were dominantly observed. After 72 hours incubation, the capillary density had decreased, and mature and also degenerated capillaries were observed. Quantitative RT-PCR analysis revealed that the level of Ang-1 mRNA was low after 24-48 hours incubation, and it had increased at 72 hours incubation, while a high level of Ang-2 mRNA was observed at 24-72 hours incubation. In the present in vitro angiogenesis model, the microvessel growth stage was 24-48 hours and the microvessel maturation stage was after 72 hours incubation.

key words: thermoreversible gel, rat, angiogenesis, in vitro, alternative


(AATEX: Altern. Animal Test. EXperiment.: Alternatives to Animal Testing and EXperimentation)