Hydrogen molecules (H2) improve perfusion recovery via antioxidant effects in experimental peripheral arterial disease

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.

Keywords: hydrogen molecule; reactive oxygen species; peripheralarterial disease; angiogenesis; arteriogenesis.

Figure 1.

H₂-saturated water improves perfusion recovery following HLI. (A) Laser Doppler imaging indicated a significant increase in perfusion recovery in Balb/c mice treated with H₂-saturated water on days 14 and 21 following HLI (n=10 per group). (B) At day 21 following HLI, the ischemic gastrocnemius muscle from mice treated with H₂-saturated water had a significantly higher capillary density (CD31, red) when compared with those treated with dehydrogenized water (n=8 mice/group). Scale bar, 60 µM. (C) Artery density (α-smooth muscle actin, green) in the abductor muscle from mice treated with H₂-saturated water was higher compared with the control mice (n=8 per group). Scale bar, 100 µM. Data are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. respective control. H₂, hydrogen molecule-saturated water; control, mice with dehydrogenized water. HLI, hindlimb ischemia; I, ischemic; NI, non-ischemic.

Figure 2.

H2-saturated water decreases oxidative stress, increases macrophage M2 polarization in ischemic muscle. H₂-saturated water intake (A) decreases MDA levels, (B) increases cGMP levels and increases M2-like macrophage polarization levels, as indicated by (C) increased expression levels of an M2-like macrophage marker (arg-1) and (D) decreased expression levels of an M1-like macrophage marker (tnf) in the ischemic muscle 7 days after experimental peripheral arterial disease. Data are presented as the mean ± standard error of the mean. H₂, hydrogen molecule-saturated water; control, mice with dehydrogenized water. arg-1, arginase-1; tnf, tumor necrosis factor; MDA, malondialdehyde; cGMP, cyclic guanine monophosphate.

Figure 3.

H₂-saturated medium increases tube formation and cellular cGMP levels and decreases cellular ROS levels in cultured HUVECs. (A) HUVECs were plated on Matrigel with reduced growth factors and incubated for 12 h in HSS conditions with H₂-saturated medium or medium without H₂. H₂ treatment resulted in enhanced tube formation, which was quantified as total length of the cords per visual field, and total loops per visual field as represented by the bar graph. Scale bar, 100 µm. (B) H₂-saturated medium resulted in reduced ROS levels as indicated by 2′,7′-dichlorodihydrofluorescein diacetate staining in cultured HUVECs 1–6 h under HSS. *P<0.05, **P<0.01 vs. respective control. (C) H₂-saturated medium resulted in increased cellular cGMP levels 24 h under HSS. Data are representative of 2–3 separate batches of HUVECs (n=8–12 per group). Data are presented as the mean ± standard error of the mean. H₂, hydrogen molecule-saturated medium; control, medium without H₂. ROS, reactive oxygen species; cGMP, cyclic guanine monophosphate; HUVEC, human umbilical vein endothelial cell; HSS, hypoxia serum starvation.

Figure 4.

H₂ decreases ROS, increases macrophage M2 polarization in vitro. H₂-saturated medium (A) decreases cellular ROS levels, (B) increases the expression of an M2-like macrophage marker (arg-1) and (C) decreases the expression of an M1-like macrophage marker (tnf) in cultured bone marrow-derived macrophages. Data are representative of 2–3 separate batches of macrophages (n=8–12 per group). Data are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. respective control. H₂, hydrogen molecule-saturated medium; control, medium without H₂. ROS, reactive oxygen species; arg-1, arginase-1; tnf, tumor necrosis factor.