Endothelial progenitor cells (EPCs) play a critical role in the repair of damaged blood vessels and/or in the growth of new ones in ischemic tissues. Elevated levels of
oxygen radicals, which accumulate in the ischemic tissue, could compromise the angiogenic potential of EPCs. To determine if oxidative stress alters the angiogenic response of EPCs and to identify possible cellular targets that protect EPCs from the damaging effects of oxidative stress, we have investigated vascular development in embryonic bodies (EBs) under hyperoxic conditions. Murine EBs at differentiaton day 2 were cultured for 3 days under normoxic (21% O(2)) or hyperoxic (60% O(2)) conditions. Hyperoxic EBs showed a moderate reduction in
Pecam-1,
Vegfr-2, eNOS and Tie2
mRNA levels compared to normoxic EBs. However, immunostaining of hyperoxic EBs with
antibodies against
PECAM-1 after 1 week recovery at room air revealed a defective vasculature completely deficient in branches, while normoxic EBs developed a normal vascular plexus.
Oxygen-induced defective vascular development correlated with a dramatic decrease in
soluble guanylyl cyclase,
phosphodiesterase (Pde) 4B and Pde4C mRNAs. Oxidative stress did not affect the expression of
adenylyl cyclase 6 and Pde5. The abnormal vascular development caused by
hyperoxia was reverted by pharmacological treatments that increased cGMP levels, such as
8-bromo-cGMP or 4-{[3',4'-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline, a specific inhibitor of PDE5. These results indicated that oxidative stress inhibits vascular development from EPCs through its effects on levels of
cyclic nucleotides and suggested that
therapies that target
cyclic nucleotide turnover may be useful in protecting vascular repair under oxidative conditions.