Hyperbaric Oxygen Therapy Improved Neovascularisation Following Limb Ischaemia-The Role of ROS Mitigation

Abstract

Hyperbaric oxygen (HBO) therapy has emerged as a potential treatment, shown to enhance blood flow and angiogenesis. However, specific effects and mechanisms of HBO on limb ischaemia responding to a hypoxic environment remain largely unknown. We aimed to investigate the therapeutic potential of HBO in the treatment of limb ischaemia. Following limb ischaemia surgery, we evaluated the angiogenic capacity in wild-type C57BL/6J mice subjected to HBO treatment (100% oxygen at 3 ATA for 1 h/day for five consecutive days) compared to untreated controls. Notably, through laser Doppler perfusion imaging and CD31 staining mice receiving HBO postlimb ischaemia surgery exhibited significantly enhanced angiogenic capability and reduced ROS expression compared to nontreated counterparts. Additionally, in vitro experiments were conducted to investigate whether HBO could mitigate endothelial cell dysfunction and reactive oxygen species (ROS) production triggered by oxygen-glucose deprivation (OGD). HBO treatment rescued the impaired proliferation, migration and tube formation of endothelial cells following OGD. Mechanistically, HBO upregulated the expression of proangiogenic proteins, including vascular endothelial growth factor (VEGF), haem oxygenase-1 (HO-1), hypoxia-inducible factor 1 (HIF-1) and nuclear factor erythroid 2-related factor 2 (Nrf2). Collectively, HBO treatment shows promise in augmenting the endogenous angiogenic potential and suppressing ROS levels in limb ischaemia.

Keywords: HBO; ROS; angiogenesis; limb ischaemia.

FIGURE 1

Hyperbaric Oxygen (HBO) enhances angiogenesis in mice postlimb ischaemia. (A) The study design illustrating limb ischaemia mice with or without HBO treatment including three groups: (1) Sham group (n = 8 biologically independent animals), (2) limb ischaemia group (n = 10 biologically independent animals) and (3) HBO + limb ischaemia group (n = 10 biologically independent animals). In the HBO + limb ischaemia group, mice were placed in a HBO chamber and exposed to 100% oxygen at 3 ATA for 1 h per day over five consecutive days, beginning on the first day postsurgery. (B) Representative images and quantification of laser Doppler perfusion flow in limb ischaemia mice treated with or without HBO treatment. (C) Representative images and weight quantifications of harvested ischaemic (L) limbs compared with nonischaemic (R) limbs. (D) Representative images and quantification of CD31 immunostaining representing capillary density. Data are presented as mean ± SEM. One‐way ANOVA followed by Tukey's test was used to compare groups. **p < 0.01, ***p < 0.005 and ****p < 0.001 compared with the indicated groups.

FIGURE 2

Hyperbaric Oxygen (HBO) alleviates ROS accumulation and boosts HIF‐1α and VEGF activities in mice with limb ischaemia. Double immunostaining with endothelial marker CD31, ROS marker 8‐OHdG, HIF‐1α and VEGF in the left calf muscle mice following limb ischaemia surgery, with or without HBO therapy. Representative images and quantification of (A) 8‐OHdG (B) VEGF and (C) HIF‐1α expressions in endothelial cells (red; CD 31 positive). Cell nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data are presented as mean ± SEM. One‐way ANOVA followed by Tukey's test was used to compare groups. *p < 0.05, *** p < 0.005 and ****p < 0.001 compared with the indicated groups.

FIGURE 3

Hyperbaric Oxygen (HBO) augments angiogenesis‐associated protein in mice with limb ischaemia surgery. Representative blots and quantification of VEGFR, VEGF, Erk, Nrf2, HO‐1 and HIF‐1α in the left limbs of mice treated with or without HBO therapy. Data are presented as mean ± SEM. One‐way ANOVA followed by Tukey's test was used to compare groups. *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.001 compared with the indicated groups.

FIGURE 4

Hyperbaric Oxygen (HBO) enhances proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs) under oxygen–glucose deprivation (OGD) injury. Following OGD injury (95% N₂/5% CO₂, no glucose, no serum) for 3 h, the HUVECs were treated with HBO (100% oxygen at 3 ATA) for 1 h. (A) Cell proliferation evaluated by measuring BrdU incorporation into cells for 24 h, (B) cell migration for 6 h and (C) tube formation for 16 h evaluated by the branched points in HUVECs. The experiment was conducted in triplicate. Data are presented as mean ± SEM. One‐way ANOVA followed by Tukey's test was used to compare groups. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the indicated groups.

FIGURE 5

Hyperbaric Oxygen (HBO) attenuates reactive oxygen species (ROS) generation in the human umbilical vein endothelial cells (HUVECs) under oxygen–glucose deprivation (OGD) injury. After HUVECs were subjected to OGD injury for 3 h, HUVECs were treated with or without HBO therapy (100% oxygen at 3 ATA) for 1 h. The intracellular expression of ROS was measured by dihydroethidium (DHE) immunohistological staining. The experiment was conducted in triplicate. Data are presented as mean ± SEM. One‐way ANOVA followed by Tukey's test was used to compare groups. **p < 0.0001 and ****p < 0.0001 compared with the indicated groups.

FIGURE 6

Hyperbaric Oxygen (HBO) improves angiogenesis‐associated mRNA and protein expressions in human umbilical vein endothelial cells (HUVECs) under oxygen–glucose deprivation (OGD) injury. Under OGD conditions for 3 h, HUVECs were treated with or without HBO (100% oxygen at 3 ATA) for 1 h. (A) Representative blots and quantification of VEGFR, VEGF, Erk, Nrf2, HO‐1 and HIF‐1α in HUVECs treated with or without HBO therapy. Transcriptions of (B) VEGF and (C) HIF‐1α detected by reverse transcription‐PCR (RT‐PCR). The experiment was repeated in triplicate. Data are presented as mean ± SEM. One‐way ANOVA followed by Tukey's test was used to compare groups. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the indicated groups.

FIGURE 7

Summary of HBO preventing limb ischemia‐induced endothelial cell dysfunction.