Amongst the functions of the nitric oxide radical (NO) are its actions as a cytotoxic/cytostatic agent, and as a vasodilator and as a platelet antiadhesive/antiaggregatory agent. The applicant has shown that NO may maintain low vascular resistance and attenuate the action of vasoconstrictors in the human fetal placental vasculature and has described the presence of a constitutive calcium calmodulin-dependent endothelial isoform of nitric oxide synthase (NOS) in syncytiotrophoblast, but not cytotrophoblast of human villous vascular tissue. NO secreted from syncytiotrophoblast may act as a platelet antiadhesive and antiaggregatory agent in the intervillous space. When cytotrophoblasts differentiate in culture to form syncytiotrophoblast, they express this endothelial NOS isoform. However, regulation of the activity of this isoform has not yet been investigated in trophoblast. Villous trophoblast may also express an inducible isoform of NOS to secrete larger quantities of NO to exert cytotoxic/cytostatic effects and protect the fetal allograft against maternal pathogens. Expression of this induced isoform may be regulated in both a positive and negative manner by cytokines, growth factors and steroids. NO itself may exert a negative feedback effect on NOS activity to prevent potentially deleterious effects of NO on the cells producing it. Preeclampsia and intrauterine growth restriction (IUGR), which are conditions where increased fetal and maternal morbidity and mortality are seen, are associated with abnormalities of trophoblast invasion of the uterus leading to poor perfusion of the placenta, perhaps a relative placental hypoxia and altered trophoblast function. The applicant has preliminary evidence for altered expression of the endothelial NOS isoform in villous trophoblast and villous vessels in preeclampsia and IUGR. Specifically the applicant will determine: (1) the presence of constitutive and/or inducible isoforms of NOS in human villous trophoblast tissue and cell culture using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization; (2) regulation of the expression and activity of the constitutive endothelial isoform in trophoblast cultures and a trophoblast line by changes in oxygen tension; (3) positive and negative regulation of expression of the inducible isoform of NOS in trophoblast by cytokines, growth factors and steroids; (4) the relationship of NOS expression to trophoblast differentiation in an in vitro cell culture system; and (5) the expression of NOS isoforms in trophoblast from pregnancies complicated by preeclampsia and/or intrauterine growth restriction in comparison with gestational age-matched controls.
