Morteza Naghavi MD,

Tania Khan, Babs Soller PhD, Peter Melling PhD,

Mohammad Madjid MD, Ward Casscells MD,

James T. Willerson MD

The University of Texas-Houston Texas Heart Institute

Functional Evaluation of Atherosclerotic Functional Evaluation of Atherosclerotic Plaque, Temperature, pH and Lactate Plaque, Temperature, pH and Lactate

Casscells et al have previously discovered that plaque temperature correlates with other markers of vulnerability and that hot plaque may be a vulnerable plaque

(The Lancet 1996)

Introduction: Introduction:

Plaque temperature correlates with number of macrophages

but not with smooth muscle cells

Our ex-vivo findings have been recently corroborated by others in-vivo, suggesting that increased plaque temperature compared to adjacent arterial wall correlates with patients’ clinical presentation. Stefanadis et al

1999

Why should vulnerable plaque be Why should vulnerable plaque be hothot??

Vulnerable plaque is inflamed and inflammation generates heat

What else can be found in a site of What else can be found in a site of inflammation?inflammation?

Increased metabolic rate namely increased oxygen and glucose consumption Free radical formation (ROS and RNS)Hypoxia?

pHpHExtra-cellular low pH or acidic environment is

characteristic of an inflamed site

This is mainly because of massive lactate formation resulted from increased metabolic activity of inflammatory cells. These cells normally do not find enough oxygen to burn glucose therefore, go through anaerobic pathway and generate a large a mount of lactate

Atherosclerotic plaques are known to be hypoxic due to: - Increased oxygen consumption by macrophages - Decreased oxygen diffusion through plaque fibrous cap Both result in anaerobic metabolism.

(Bjornheden et al 1987)

Macrophages are also known to be metabolically very active. For example the ATP consumption of every 2 activated macrophages equals to one isolated maximally loaded cardiomyocyte. (Newsholme et al 1989)

Back to old literature:Back to old literature:

……why plaque pH or lactate? why plaque pH or lactate?

When incubated with Ox-LDL, macrophages are seen to reduce their environmental pH to as low as 5.5. (De Vries et al 1998)

Macrophages oxidize LDL in both neutral and acidic pH, but the LDL oxidation is more pronounced in an acidic environment.

(Leake et al 1995)

What could be the significance of What could be the significance of an acidic plaque?an acidic plaque?

Low pH can activate or increase activity of acidic matrix digesting enzymes resulting in plaque softening, weakening and rupture.

Others recently reported presence of acidic enzymes in human atherosclerotic plaques (Libby et all, 1998)

pH

9.008.88

8.758.63

8.508.38

8.258.13

8.007.88

7.757.63

7.507.38

7.257.13

7.006.88

6.756.63

No

of p

oint

s w

ith th

e sa

me

ph

140

120

100

80

60

40

20

0

pH Distribution in 48 Human Carotid Plaques

This histogram demonstrates distribution of pH measured in 858 points in 48 carotid plaques of 48 patients. A marked variation

ranging from 6.5 to 8.9 is seen.

PH

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

pH Heterogeneity Within and Between Plaques in 48 Human Carotid Specimens

Red box shows the middle quartiles, solid lines shows median, cross-bars show pH range and circles represents extreme values. This graph shows a significant pH variation within and between plaques. The variation within the plaque holds

the majority of the total variation.

Watanabe Rabbit Aorta

pH

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

pH Heterogeneity in 9 Watanabe Rabbit Aortas

2220231717191026252428N =

11 human umbilical artery

pH

9.59.39.18.98.78.58.38.17.97.77.57.37.16.96.76.56.36.15.95.75.5

pH Heterogeneity in 11 Human Umbilical Arteries

calcified&thrombosedLipid Rich

pH

9.0

8.5

8.0

7.5

7.0

6.5

pH in Lipid Rich Yellow Areas vs. Calcified areas

Areas with large lipid core exhibit higher temperature and lower pH

p < 0.01

Lipid Rich Yellow Areas (A and B) vs. Calcified Areas (C and D)

Lipid core

Calcification

H&E Movat

Correlation of pH and Temperature in Human Carotid Plaques Varied by the Areas

We see a marked inverse correlation between temperature and pH of plaques that varies by macroscopic characteristics of plaques.

p < 0.01

Correlation of ph and Temperature in 9 Watanabe Rabbit Aorta

A significant inverse correlation between temperature and pH is seen these rabbits similar to human carotid plaques

A significant inverse correlation between temperature and pH is seen these rabbits similar to human carotid plaques

p < 0.01

Correlation of pH and Temperature in 11 Human Umbilical Arteries

In contrast to human and rabbit plaques in human umbilical there is no relationship seen between temperature and pH

p = 0.2

Fluorescence Ratio (dual emission) Imaging Microscopy of A Predominantly Calcified Plaque Shows Significant Microscopic

pH Heterogeneity with Higher pH in Calcified Areas

Fluorescence Single-Emission Imaging Microscopy of a Predominantly Lipid Rich Plaque Shows

Significant Microscopic pH Heterogeneity Mostly Acidic

What else, besides Temp and pH?What else, besides Temp and pH?Oxidized lipid and proteins

– Ox-LDL– Ox-collagen

Nitrated proteins – Nitrosyl hemoglobin – Nitrosyl tyrosine

?

How to measure Temperature, pH and the other factors in plaque invivo?