Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia

Abstract

Oxygen supply and diffusion into tissues are necessary for survival. The oxygen partial pressure (pO(2)), which is a key component of the physiological state of an organ, results from the balance between oxygen delivery and its consumption. In mammals, oxygen is transported by red blood cells circulating in a well-organized vasculature. Oxygen delivery is dependent on the metabolic requirements and functional status of each organ. Consequently, in a physiological condition, organ and tissue are characterized by their own unique 'tissue normoxia' or 'physioxia' status. Tissue oxygenation is severely disturbed during pathological conditions such as cancer, diabetes, coronary heart disease, stroke, etc., which are associated with decrease in pO(2), i.e. 'hypoxia'. In this review, we present an array of methods currently used for assessing tissue oxygenation. We show that hypoxia is marked during tumour development and has strong consequences for oxygenation and its influence upon chemotherapy efficiency. Then we compare this to physiological pO(2) values of human organs. Finally we evaluate consequences of physioxia on cell activity and its molecular modulations. More importantly we emphasize the discrepancy between in vivo and in vitro tissue and cells oxygen status which can have detrimental effects on experimental outcome. It appears that the values corresponding to the physioxia are ranging between 11% and 1% O(2) whereas current in vitro experimentations are usually performed in 19.95% O(2), an artificial context as far as oxygen balance is concerned. It is important to realize that most of the experiments performed in so-called normoxia might be dangerously misleading.

Fig 1

Nitroimidazole derivatives used as hypoxia markers. (A) Schematic of the proposed mechanism underlying the binding of pimonidazole to cellular macromolecules, as an example of nitroimidazole tracer. (B) Hypoxic sites detection in B16-melanoma induced tumour by pimonidazole binding, visualized in red. More cells are proliferating [4′,6′-diamidino-2-phenylindole (DAPI)-labelled cells in blue] close to the blood vessel. Endothelial cells lining the blood vessel wall are stained in green after incubation with a fluorescent antibody specific for PECAM-1 (CD31).

Fig 2

Principles of PET. ¹⁸F radiotracer is intravenously injected. The tracer decays by emitting a positron, which annihilates with a nearby electron to produce two γ-rays. The PET scanner can detect the coincident γ-rays, and images can be reconstructed showing the location(s) and concentration of the tracer of interest. Sectional PET image is shown: normal uptake in brain (Br) and myocardium (C), and renal excretion into the urinary bladder (B) are visible. Also seen is a tumour (T) in the lungs that takes up more ¹⁸F radiotracer than the surrounding tissues. Adapted with permission from Macmillan Publishers Ltd: Nature Reviews Cancer, Gambhir, copyright 2002[5].

Fig 3

pO₂ maps of rat tumours obtained using ¹⁹F nuclear magnetic resonance using hexafluorobenzene as the reporter molecule. The H tumour (a well-differentiated and slow-growing tumour) was significantly better oxygenated than the AT1 tumour (an anaplastic and faster-growing subline). In response to oxygen inhalation, pO₂ increased significantly in both the H and the AT1 tumours. Adapted with permission from Neoplasma press, Zhao et al., 2003[14].

Fig 4

B16-melanoma-induced tumour and muscle pO₂ measured using polarographic needle, in mouse. B16 tumour cells are injected subcutaneously in mouse and pO₂ tumour is measured after 14 days, in comparison to the healthy muscle tissue. Experiment representative of 5 (mean ± S.E.M.).

Fig 5

Delivery of oxygen from the atmosphere to the blood, in human, and normal pO₂ in mmHg. Figure was produced using Servier Medical Art. (http://www.servier.com).

Fig 6

Relative HIF-1 and HIF-2 contributions to the hypoxic response over time at 1% (A) and 5% (B) oxygen as demonstrated in human neuroblastoma cells. Model based on a summary of data obtained from protein level variations (Western blot), chromatin immunoprocipitations (ChIP) and activation of target genes ± siRNA treatment. Reproduced with permission from Lofstedt et al., Cell Cycle, 2007[80].

Fig 7

pO₂ dependence of the expression of three CAMs in skin microvascular endothelial cells: ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106. Cells were maintained in normoxia, skin physioxia (26.6 mmHg or 3.5% O₂) or hypoxia (less than 7.6 mmHg or 1% O₂) during 24 hrs. Skin microvascular endothelial cells coming mostly from the dermis, physioxia was considered to be around 3–3.5% O₂[58]. Then they were incubated with specific antibodies and labelled with a fluorescent secondary antibody. Fluorescence was quantified by flow cytometry. Changes in adhesion molecules expression are shown as percentage of fluorescence intensity related to fluorescence intensity of non-treated cells (DIF, mean from three experiments ± S.E.M.; *P < 0.05).

Fig 8

Oxygen dependent modulation of angiogenin secretion by four distinct cell lines constituting the skin. Human skin endothelial microvascular cell line (HSkMEC), fibroblasts MSU 1.1, normal melanocyte cell line and HaCat keratinocytes have been maintained in normoxia, skin physioxia (22.8 mmHg or 3% O₂) or hypoxia (less than 7.6 mmHg or 1% O₂) during 16 hrs. Supernatants have been collected and angiogenin content assessed by the cytometric bead assay (CBA) technique. Results are expressed as picograms of angiogenin secreted by 10 × 6 cells in one hour (mean ± S.E.M.); *P < 0.05.