cGMP accumulation is reduced by TNF-α stimulation or PDE2 transfection. HUVECs were incubated with TNF-α (10 ng/mL) for 18 hours followed by PDP incubation (1 μM) for further 20 minutes. SNP (100 μM) was added for 5 minutes with subsequent lysis and measurement of total cellular cGMP levels using an enzyme-linked immunosorbent assay (A). Data presented are mean ± SEM of 5 independent experiments. ^*P < .05; ^**P< .01. PDE2A3 cDNA was cloned into the pcDNA3.1 expression vector. Of the pPDE2A3 expression plasmid, 0.1 μg was transfected into approximately 1 million HUVECs and harvested after 18 hours prior to stimulation and subsequent measurement of total cellular cGMP levels using an enzyme-linked immunosorbent assay (B). PDE2 inhibitor PDP (1 μM) was added 20 minutes prior to stimulation with SNP (100 μM) for 5 minutes with subsequent lysis and measurement of cGMP levels. Green fluorescent protein (GFP)–transfected cells were used as control. Data presented are mean ± SEM of 3 independent experiments. ^*P < .05 versus SNP-treated cells; #P < .05 versus SNP/PDP-treated cells; and §P < .05 versus SNP-treated/PDE2-transfected cells.