In vitro Thermoreversible Gel Disc Quantitative Assay of Rat Angiogenesis

Abstract

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.