Anti-Angiogenic Effects of Resveratrol on Cerebral Angiogenesis

Abstract

Angiogenesis is implicated in diseases of the central nervous system, and its modulation represents an attractive therapeutic strategy. This is the first report of the effects of resveratrol (trans-3,4',5-trihydroxystilbene) on cerebral angiogenesis, using an in vitro model. Processes associated with angiogenesis were studied in cerebral vascular endothelial cells, using the human brain endothelial cell line hCMEC/D3 and primary bovine brain microvessel EC (BBMEC). Comparisons were made to human umbilical vein EC (HUVEC). In cerebral cultures, resveratrol (24h) induced a dose dependent reduction in BrdU incorporation and cell numbers (MTT assay) between 10-100 µM, while lower doses (100 nM-5 µM) had no effect. Cell migration (scratch assay) was inhibited between 10-100 µM depending on cell type. Doses between 10-100 µM reduced average tubule length on Matrigel, while higher doses (50,100 µM) also inhibited process formation around explanted rat aortic rings (ex vivo assay). Cell cycle analysis in hCMEC/D3 (propidium iodide) showed reduced progression to the G2/M phase, with a maximal effect at 25 µM. Resveratrol did not induce apoptosis in hCMEC/D3, based on caspase-3 activity. Cytotoxicity (LDH release) was induced by resveratrol (50,100 µM) in hCMEC/D3 and HUVEC, peaking at ~20% in hCMEC/D3 and ~35% in HUVEC. Cytotoxic effects were not detected in BBMEC. Resveratrol (10-50 µM) inhibited phosphorylation of the serine/threonine kinase Akt, by Western blot (15 min, 1h) with a prolonged inhibition (24h) for 25 µM. In conclusion, this study shows inhibitory effects of resveratrol on cerebral angiogenesis, using an in vitro model. This is discussed in terms of dosage, in vivo equivalence and therapeutic potential.