Heme oxygenase isoforms (HO-1/HO-2) catalyze the conversion of heme to carbon monoxide (CO) and bilirubin. In this study, HO-1-deficient endothelial cells were transduced with HO-1 in the antisense orientation to determine whether supplementation with CO or bilirubin would regulate cell proliferation and angiogenesis. Western blotting, enzyme activity, CO and prostaglandin E(2) (PGE(2)) production, and cell-cycle analysis were used to assess transgenic expression and functionality of the recombinant protein. A Matrigel matrix was used for assessment of in vitro capillary formation. Transduction with HO-1 antisense resulted in decreased capillary formation, cell proliferation, and cell-cycle progression, and increased PGE(2) production compared with control. HO-1 deficiency was also associated with increased expression of p21 and p27, but had no significant effect on p16 and p53. We also compared two different CO donors for their ability to rescue angiogenesis. Compared with control, HO-1-deficient endothelial cells showed increased angiogenesis following tricarbonyldichlororuthenium( II) dimer ([Ru(CO)(3)Cl(2)](2)) (CORM-1) starting at 50 microM, whereas tricarbonylchloro(glycinato) ruthenium(II) (CORM-3), starting at 25 microM, was a potent enhancer of angiogenesis. The addition of bilirubin did not restore angiogenesis. These data suggest that HO-mediated angiogenesis and cell proliferation were dependent on HO-1- and not HO-2-derived CO.