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An investigation into the molecular mechanisms of hyperbaric oxygen using a human dermal microvascular endothelial cell line and a porcine retinal explant model.

Iwuagwu, Benedicta Ugochi


Benedicta Ugochi Iwuagwu


Rachel Knott

Stuart Cruickshank

Iain Rowe


Diabetes is a debilitating metabolic condition with associated vascular complications that are a major cause of morbidity and mortality. Endothelial dysfunction is central to microvascular complications such as diabetic retinopathy (DR) and impaired wound healing in diabetes. Hyperbaric oxygen therapy (HBOT) is the administration of hyperbaric oxygen (HBO) which involves breathing ≥ 95% oxygen at elevated pressures. It is used for the treatment of recalcitrant ulcers in diabetes. However, the exact molecular mechanism is unclear, and there are questions about its safety, and what contribution the single components of hyperoxia and elevated pressure provide. The effect of HBO on human dermal microvascular endothelial cells (HDMEC) and porcine retinal vasculature is presented with three mechanistic pathways of transcriptional factors regulating the redox (nuclear factor type-2 (Nrf2)), pro-inflammation (Nuclear Factor kappa B; (NFᴋB)), and oxygen signalling (hypoxia inducible factor type 1 (HIF-1)). In this study, HDMEC and porcine retinal explants (a novel explant model) were exposed to treatments simulating HBOT using a bespoke HBO chamber (≥95% oxygen at 2.2 absolute pressures (2.2ATA) for 105 mins), or hyperoxia alone (HYP) (≥95% oxygen) or hyperbaric pressure alone (HYB) (2.2ATA) for 90 mins in low (LG) or high glucose (HG) concentration. HDMEC and explants were exposed to 20 mM and 25 mM D-glucose concentrations respectively for HG treatment. Post-treatments, samples were incubated for 2 h (explants), 4 h (HDMEC) and up to 24 h (explants and HDMEC). HDMEC morphology and metabolic activities were determined using image analysis and the Resazurin assay respectively. Targets of the aforementioned pathways - nrf2 (HO-1), NFĸB (IL-6 mRNA), HIF-1α (VEGF) and PECAM-1 - were examined. HIF-1α, nrf2, HO-1, PECAM-1, VEGF, and NFᴋB levels were detected by immunocytochemical/immunohistochemistry and/or by Western blotting (WB). Total RNA was isolated from HDMEC and cDNA prepared and amplified for each treatment with IL-6 mRNA specific primers with β2m as reference. The study found that porcine retinal explants were established as a viable explant model. HIF-1α immunoreactivity was increased in response to HG relative to LG in retinal explants. In addition, HIF-1α reactivity was augmented post treatments; HBO, HYP and HYB, and further exaggerated in the presence of HG relative to control, with a corresponding increase in PECAM-1. In retinal explants, HIF-1α expression post HBO was increased, including increased nuclear associated HIF-1α reactivity which was sustained for up to 24 h across all retinal layers relative to control. Whilst HG was associated with profound HIF-1α reactivity in retinal explants, HIF-1α expression in HDMEC appeared suppressed in response to HG, which may indicate cell type differences between retinal cells and HDMEC. Mean HDMEC size was significantly varied between samples (p < 0.0001, n = 8), and HDMEC in HYP were significantly larger relative to control or HBO (p < 0.05) and hyperbaric pressure (p < 0.01), but HBO was not associated with HDMEC morphology or size alteration relative to control (p > 0.05). Nrf2 was basal in control sample which was consistent with a homeostatic condition but, ironically, nrf2 was acutely lower in HG relative to LG for HDMEC in control condition (p < 0.05). Increased nrf2 stabilisation and accumulation was seen following HBO, HYP and HYB relative to control. In addition, nrf2 distribution post HBO and to a lesser extent in HYB were nuclear and plasma membrane associated, whilst predominantly perinuclear associated post hyperoxia relative to control. Total HO-1 protein in HDMEC appeared elevated in response to treatments; HBO and HYP relative to control possibly via nrf2 mediated mechanism(s). In addition, HO-1 appeared more elevated in HG relative to LG in control and HBO conditions but HO-1 elevation post HYP seemed independent of glucose concentration, alluding to a predominant hyperoxia mediated effect. More so, VEGF accumulation possibly linked to HO-1 increase appeared imminent post HYP relative to control. In HDMEC, concomitant HYP and HG at 24 h were associated with downregulation of PECAM-1 relative to control, although this response appeared lacking in the retinal explants. HBO, HYP and HYB, but more so HBO were associated with significant IL-6 mRNA downregulation relative to control (p < 0.0001). IL-6 mRNA was significantly less suppressed post HBO (p < 0.01), whilst more suppressed post HYP (p < 0.0001) in HG relative to LG control. This shows a significantly higher level of IL-6 mRNA was induced post HBO, whilst decreased post HYP in response to high glucose. NFĸB (p65) appeared basal post HBO, whilst elevated post HYP and HYB but more so post HYP relative to control which is suggestive of an acute pro-inflammatory response or endothelial cell activation. In conclusion, HDMEC and retinal cells appear to have different responses to HG, which highlights pertinent cell-type differences that may be fundamental in understanding dysfunctions in endothelial cells of retinal or dermal origin. Also, differential responses are evident in the expression of HIF-1α, nrf2 stabilisation and possibly its target protein HO-1, and IL-6 post treatments alone and in concert with HG. Fundamentally, this demonstrates the pivotal role of high glucose on HBO mediated effects. Taken together, HBO effects in HDMEC and retinal explants are distinct and may generate a greater protective environment in relation to redox, immune-response and HIF-1α expression. Further studies are needed to identify the exact mechanisms of redox (nrf2), inflammatory (NFĸB), and HIF-1α expression in HBO particularly in in-vivo setting.

Thesis Type Thesis
Institution Citation IWUAGWU, B.U. 2019. An investigation into the molecular mechanisms of hyperbaric oxygen using a human dermal microvascular endothelial cell line and a porcine retinal explant model. Robert Gordon University [online], PhD thesis. Available from:
Keywords Diabetes; Hyperglycaemia; Endothelial dysfunction; Retinal explants; Hyperbaric oxygen; Diabetic retinopathy


IWUAGWU 2019 An investigation into the molecular (7.3 Mb)

Copyright Statement
Copyright: the author and Robert Gordon University

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