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. 2012 Apr 1;94(1):144-53.
doi: 10.1093/cvr/cvs024. Epub 2012 Jan 27.

Oral Intake of Hydrogen-Rich Water Inhibits Intimal Hyperplasia in Arterialized Vein Grafts in Rats

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Free PMC article

Oral Intake of Hydrogen-Rich Water Inhibits Intimal Hyperplasia in Arterialized Vein Grafts in Rats

Qiang Sun et al. Cardiovasc Res. .
Free PMC article

Abstract

Aims: Arterialized vein grafts often fail due to intimal hyperplasia. Hydrogen potently protects organs and cells from many insults via its anti-inflammatory and antioxidant properties. We investigated the efficacy of oral administration of hydrogen-rich water (HW) for prevention of intimal hyperplasia.

Methods and results: The inferior vena cava was excised, stored in cold Ringer solution for 2 h, and placed as an interposition graft in the abdominal aorta of syngeneic Lewis rats. HW was generated by immersing a magnesium stick in tap water (Mg + 2H(2)O → Mg (OH)(2) + H(2)). Beginning on the day of graft implantation, recipients were given tap water [regular water (RW)], HW or HW that had been subsequently degassed water (DW). Six weeks after grafting, the grafts in the rats given RW or DW had developed intimal hyperplasia, accompanied by increased oxidative injury. HW significantly suppressed intimal hyperplasia. One week after grafting, the grafts in HW-treated rats exhibited improved endothelial integrity with less platelet and white blood cell aggregation. Up-regulation of the mRNAs for intracellular adhesion molecules was attenuated in the vein grafts of the rats receiving HW. Activation of p38 mitogen-activated protein kinase, matrix metalloproteinase (MMP)-2, and MMP-9 was also significantly inhibited in grafts receiving HW. In rat smooth muscle cell (A7r5) cultures, hydrogen treatment for 24 h reduced smooth muscle cell migration.

Conclusion: Drinking HW significantly reduced neointima formation after vein grafting in rats. Drinking HW may have therapeutic value as a novel therapy for intimal hyperplasia and could easily be incorporated into daily life.

Figures

Figure 1
Figure 1
(A) Morphological assessment of arterialized vein grafts 6 weeks after engraftment. Vein grafts from rats that received regular water (RW), degassed water (DW), or hydrogen-rich water (HW). Representative images of tissue autofluorescence, Masson trichrome stain and immunohistochemical staining for α-SMA or ED-1 are shown. (B) Histogram quantifying intimal area/intimal + medial area in the vein grafts. (C) Macrophage infiltration as assessed by ED-1 staining. HPF, high power field (n = 6 for RW, n = 5 for DW and n = 6 for HW; 5 HPF were counted per sample; *P < 0.05 vs. RW, #P < 0.05 vs. DW).
Figure 2
Figure 2
Evaluation of oxidative injury in the vein grafts. (A) Arterialized vein grafts taken 6 weeks after implantation were assessed using immunohistochemical stain for nitrotyrosine, 4HNE and 8-OHdG. Recipients were treated with RW, DW, or HW. Quantitation of staining intensity for (B) nitrotyrosine and (C) 4-HNE. (D) The number of cells positive for 8-OHdG (n = 6 per group; 5 HPF were counted per sample). (E) MDA levels in the vein grafts (n = 6 per group, *P < 0.05 vs. RW, #P < 0.05 vs. DW).
Figure 3
Figure 3
(A) SEM of the luminal surface of vein grafts taken 7 days after engraftment. Arrows indicate microthrombi; arrowheads indicate aggregated platelets. Images are representative of three vein grafts from each treatment group. (upper panels: lower magnification, lower panels: higher magnification) (B) Real-time RT–PCR for ICAM-1 and endothelin receptor-A (ETA). (n = 5 for each group, *P < 0.05 vs. RW, #P < 0.05 vs. DW).
Figure 4
Figure 4
(A) Western blots for p38MAPK and ERK1/2 using the vein graft samples harvested 1 week after engraftment. (B) Real-time RT–PCR for TNF-α and IL-6. (n = 5 for each group, *P < 0.05 vs. RW, #P < 0.05 vs. DW).
Figure 5
Figure 5
(A) Western blots for MMP-2 and MMP-9 using the vein graft samples harvested 1 week after engraftment. Images are representative of three independent experiments. (B) Enzymatic activity of MMP-2 and MMP-9 determined by zymography. (n = 3 for each group, *P < 0.05 vs. RW, #P < 0.05 vs. DW). Histogram of band intensity of the bands for MMP-2 and MMP-9 zymogram of four different samples as measured by ImageJ computer software.
Figure 6
Figure 6
The wound migration assay (A) without FBS and (B) with FBS in the culture medium. Wounds in monolayers of A7r5 SMC cells. Representative images of three independent experiments are shown. Histograms depict average migration index. The original wound edge is marked with broken line (*P < 0.05 vs. control). (C) Cell proliferation assay of cultured A7r5 SMC.

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