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

Department of Biology, MedicalSchool, University of Patras, Patras 261 10, Greece1 In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenicmediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity andproduction of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo modelof angiogenesis. In this model, maximum angiogenesis is reached between days 9–12 of chickembryo development. After that period, vascular density remains constant.2 Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptasepolymerase 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, alsoincreased from day 8 reaching a maximum around day 10 (100% increase). Similar results wereobtained 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 maximumaround day 10 (64% increase) and remaining stable at day 13.5 Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibiteddose dependently angiogenesis in the CAM. These results in connection with previous findings fromthis laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOSexpression, enzyme activity and NO production closely parallel the progression of angiogenesis inthe CAM, thus providing an endogenous brake to control this process.British Journal of Pharmacology (2000) 129, 207–213Keywords: Nitric oxide synthase; nitric oxide; angiogenesis; chick chorioallantoic membraneAbbreviations: CAM, chick chorioallantoic membrane;