Macrophages in atherosclerotic lesions release lactic acid, lowering the extracellular pH. This study directly measured the pH of human and rabbit atherosclerotic lesions for the first time, finding lipid-rich areas had pH as low as 7.0. Regions with more macrophages had lower pH, inversely correlated with temperature, suggesting acidity arose from macrophage metabolism rather than hypoxia. These findings have implications for understanding how acidic pH affects processes like LDL oxidation and proteolysis in atherosclerosis.

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. 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. 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. 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. 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. 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. 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. 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. 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. Results (2)
Distribution of extracellular pH in human
carotid artery plaques

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 (i.e.to a lack of blood supply to the tissue).

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. 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. 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. 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. 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. 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. 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