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097 atherosclerotic lesions have regions of low p h


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097 atherosclerotic lesions have regions of low p h

  1. 1. Atherosclerotic Lesions Have Regions of Low pH Editorial Slides VP Watch –July 31, 2002 - Volume 2, Issue 30 Provided by: David S. Leake, Ph.D. Cardiovascular Research Group Cell and Molecular Biology Research Division School of Animal and Microbial Sciences The University of Reading United Kingdom
  2. 2. Do Atherosclerotic Lesions Have An Extracellular Acidic pH? • It has been proposed that atherosclerotic lesions may have a localised acidic pH.1 • They may be acidic by analogy to – Inflammatory sites – Ischaemic sites – Tumours • Macrophages release lactic acid and protons and acidify their immediate environment to as low as pH 3.6. 2 • Until now the pH of atherosclerotic lesions has not been measured.
  3. 3. Oxidised LDL and Atherosclerosis • The local oxidation of LDL in atherosclerotic lesions may be important in this disease. 3 • Oxidised LDL is taken up faster by macrophages and may contribute to foam cell formation. • Oxidised LDL activates inflammatory genes, inhibits the activity of nitric oxide and induces apoptosis in cells.
  4. 4. pH and LDL Oxidation • LDL oxidation by macrophages catalysed by iron is much faster at acidic pH4 . • The early stages of LDL oxidation by copper are slower at acidic pH, but the generation of a highly-oxidised form of LDL that is taken up rapidly by macrophages is faster5 .
  5. 5. pH and LDL Oxidation • In the presence of antioxidants, such as serum or histidine, the early stages of LDL oxidation by copper are faster at acidic pH, because the antioxidants are less effective at acidic pH. 6 • LDL oxidation by iron and the iron–carrying protein transferrin is much faster at acidic pH. 7 • LDL oxidation by the copper-containing protein caeruloplasmin is faster after caeruloplasmin has been pre-incubated at acidic pH. 8
  6. 6. Atherosclerotic Lesions Have Regions of Reduced pH • Naghavi and co-workers at the Center for Vulnerable Plaque Research at the University of Texas Houston Health Science Center have for the first time measured the extracellular pH of atherosclerotic lesions. 9
  7. 7. Methods (1) • Human atherosclerotic carotid artery specimens were obtained after endarterectomy. • Atherosclerotic aortas of Watanabe heritable hyperlipidaemic rabbits and (as a control) human undiseased umbilical arteries were also investigated. • They were immediately incubated in culture medium at 37°C and incubated for 30min under 5% CO2. • The pH of the lesions was measured with a glass tipped needle microelectrode of 750µm diameter attached to a micromanipulator and advanced to a depth of 200µm.
  8. 8. Methods (2) • pH-Sensitive fluorescent probes were also used. • BCECF (2’,7’-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein acid) is not permeable to cells and would have measured just the extracellular pH. • SNARF (acetoxymethyl ester derivatve) is permeable to cells and would have measured both the extracellular and intracellular pH. • After incubation with these compounds, the specimens were quickly frozen and sections taken. The fluorescence was then imaged.
  9. 9. Results (1) • The extracellular pH of the lipid-rich areas of the carotid plaques was 7.15 ± 0.01 (S.D.) and that of the calcified areas 7.73 ± 0.01, which were highly significantly different. • Some regions of the human and rabbit lesions had a pH of 7.0 or below. • The umbilical arteries had a mean pH of 7.24, with little variation.
  10. 10. Results (2) Distribution of extracellular pH in human carotid artery plaques
  11. 11. Results (3) Temperature and pH • The temperature (a measurement of metabolic activity) and the extracellular pH were inversely correlated in the human and rabbit lesions. • This suggests that the lowered pH was due to secondary ischaemia, (i.e. to increased metabolism by the macrophages leading to the release pf lactic acid), rather than to primary hypoxia ( a lack of blood supply to the tissue).
  12. 12. Discussion (1) • This is an important study as it shows for the first time that atherosclerotic lesions have a lowered pH. It is especially noteworthy as human lesions, as well animal lesions, were studied.
  13. 13. Discussion (1) • The authors were motivated to measure the extracellular pH of plaques as such a measurement may possibly in the future detect plaques in vivo that are liable to fissure and precipitate thrombosis leading to myocardial infarctions or strokes. The plaques that are liable to fissure contain a lot of macrophages in the shoulder region of the lipid core and these regions would be expected to have a low extracellular pH.
  14. 14. Discussion (2) Very Localised Acidity • A relatively wide microelectrode (750µm diameter) had to be used, due to the fibrous nature of the arterial wall. As the authors point out, this may have led to the acidity in very localised parts of the lesions (i.e. areas rich in macrophages) being underestimated. • The acidity may also have been underestimated if the microelectrode crushed some of the cells and released their contents, thereby raising the pH of the extracellular fluid (cytosol has a pH of about 7.2 usually).
  15. 15. Questions (1) • It will be important to expand on these findings and to try to measure the pH of atherosclerotic lesions in vivo, although this will be technically demanding. • The extracellular pH of tumours has been shown to be acidic by magnetic resonance microscopy, but at present this is not sensitive enough for atherosclerotic lesions10 . • The authors of the paper discussed here are currently working on a near infrared spectroscopy technique to measure the pH of atherosclerotic plaques and the results of this study will be eagerly awaited11 .
  16. 16. Questions (2) • Now that the extracellular pH of atherosclerotic lesions has been shown to be low, it will be important to understand the effects that an acidic pH has on various aspects of atherosclerosis, such as – LDL oxidation – Apoptosis – Metalloproteinase and other protease activity – Cell migration – Gene expression
  17. 17. References (1) • 1 Leake, D. S. (1997) Atherosclerosis 129, 149-157. Does an acidic pH explain why low density lipoprotein is oxidised in atherosclerotic lesions? • 2 Silver, I. A., Murrills, R. J. & Etherington, D. J. (1988) Experimental Cell Research 175, 266-276. Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts • 3 Steinberg, D. (1997) J. Biol. Chem. 272, 20963-20966. Low density lipoprotein oxidation and its pathobiological significance • 4 Morgan, J. & Leake, D. S. (1993) FEBS Lett. 333, 275-279. Acidic pH increases the oxidation of LDL by macrophages • 5 Morgan, J. & Leake, D. S. (1995) J. Lipid Res. 36, 2504-2512. Oxidation of low density lipoprotein by iron or copper at acidic pH • 6 Patterson, R. A. & Leake, D. S. (1998) FEBS Lett. 434, 317-321. Human serum, cysteine and histidine inhibit the oxidation of low density lipoprotein less at acidic pH
  18. 18. References (2) • 7 Lamb, D. J. & Leake, D. S. (1994) FEBS Lett. 352, 15-18. Iron released from transferrin at acidic pH can catalyse the oxidation of low density lipoprotein • 8 Lamb, D. J. & Leake, D. S. (1994) FEBS Lett. 338, 122-126. Acidic pH enables caeruloplasmin to catalyse the modification of low-density lipoprotein • 9 Naghavi, M., John , R., Naguib, S., Siadaty, M. S., Grasu, R., Kurian, K. C., van Winkle, W. B., Soller, B., Litovsky, S., Madjid, M., Willerson, J. T. & Cassells, W. (2002) Atherosclerosis 164, 27-35. pH Heterogeneity of human and rabbit atherosclerotic plaques; a new insight into detection of vulnerable plaque • 10 Griffiths, J. R. (1991) British Journal of Cancer 64, 425-427. Are cancer cells acidic? • 11 Khan, T., Soller, B., Madjid, M., Willerson, J. T., Casscells, S. W. & Naghavi, M. (2002) Arterioscler. Thromb. Vasc. Biol. 22, P134. Progress with the calibration of a 3F near infrared spectroscopy fiber optic catheter for monitoring the pH of atherosclerotic plaque: Introducing a novel approach for detection of vulnerable plaque