Techniques of assessing hypoxia at the bench and bedside.

Tissues require an adequate supply of oxygen in order to maintain normal cell function. Low oxygen tension (hypoxia) is characteristic of a number of conditions, including cancer, atherosclerosis, rheumatoid arthritis, critical limb ischaemia, peripheral vascular disease, and ischaemic heart disease. Tissue hypoxia is found in tumours, atherosclerotic plaque, and ischaemic myocardium. There is a growing interest in methods to detect and assess hypoxia, given that hypoxia is important in the progression of these diseases. Hypoxia can be assessed at the level of the whole organ, tissue, or cell, using both invasive and non-invasive methods, and by a range of immunohistochemical, biochemical, or imaging techniques. This review describes and critiques current methods of assessing hypoxia that are used at the bench and in clinical practice.

Legumain and cathepsin-L expression in human unstable carotid plaque.

OBJECTIVE: The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain over-expression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location. METHODS AND RESULTS: Legumain was measured in human carotid plaque extracts (n=17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P<0.001) and activity (P<0.03) compared with stable regions of the same plaque. Over-expression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30kDa to its 25kDa form compared with controls. CONCLUSION: Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25kDa form. This may be a means by which legumain could contribute to plaque instability.