Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane

1 In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9-12 of chick embryo development. After that period, vascular density remains constant. 2 Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT-PCR), increased from the 8th day reaching a maximum (70% increase) at days 10-11. 3 NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. 4 Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. 5 Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process.