Abstract
Reactive oxygen species (ROS) impair neovascularization and perfusion recovery following limb ischemia in patients with peripheral arterial disease (PAD). Hydrogen molecules (H2) comprise an antioxidant gas that has been reported to neutralize cytotoxic ROS. The present study investigated whether H2 may serve as a novel therapeutic strategy for PAD. H2‑saturated water or dehydrogenized water was supplied to mice with experimental PAD. Laser Doppler perfusion imaging demonstrated that H2‑saturated water improved perfusion recovery, decreased the rate of necrosis, increased the capillary density in the gastrocnemius muscle and increased the artery density in the abductor muscle in the ischemic limbs, at 14 and 21 days post‑hindlimb ischemia. Ischemic muscle tissue was harvested 7 days after experimental PAD for biochemical testing and H2 was observed to reduce the levels of malondialdehyde and increase the levels of cyclic guanine monophosphate (cGMP). In cultured endothelial cells, H2‑saturated culture medium resulted in reduced ROS levels, increased tube formation and increased cGMP levels. In macrophages, H2 decreased cellular ROS levels and promoted M2 polarization. H2‑saturated water increases angiogenesis and arteriogenesis and subsequently improves perfusion recovery in a mouse PAD model via reduction of ROS levels.