These results suggest that at low concentration, simvastatin exhibits positive effect on proliferation and osteoblastic differentiation of human PDL cells, and these effects may be caused by the inhibition of the mevalonate pathway.

The anti-angiogenic properties of thalidomide have led to the use of the agent as a remedy for multiple myeloma. Nevertheless, the anti-angiogenic moiety of thalidomide remains unidentified. In this study we examined the anti-angiogenic effects of thalidomide in an in vitro model using a three-dimensional collagen gel culture. Angiogenesis was significantly inhibited when the culture was treated with thalidomide plus cytochrome P-450 (CYP2B4), and the migrating cells and tubules were positive for active-caspase-3 in an accompanying immunohistochemical investigation. Transmission electron microscopic observation also confirmed that active-caspase-3-positive cells demonstrated apoptotic characteristics. This study is the first to morphologically demonstrate the effect of thalidomide in directly inducing the apoptosis of new tubules and migrating cells on a three-dimensional collagen gel culture of aorta. Taken together with earlier findings, our new results indicate that the thalidomide-induced inhibition of angiogenesis involves apoptosis in addition to the suppression of TNF-alpha and inhibition of cell migration from aorta explants, i.e., the factors important for capillarogenesis.

The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.

We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.

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