Elsevier

Atherosclerosis

Volume 164, Issue 1, September 2002, Pages 27-35
Atherosclerosis

pH Heterogeneity of human and rabbit atherosclerotic plaques; a new insight into detection of vulnerable plaque

https://doi.org/10.1016/S0021-9150(02)00018-7Get rights and content

Abstract

Background: Atherosclerotic plaques are heterogeneous with respect to inflammation, calcification, vascularity, oxygen, and temperature. We hypothesized that they also vary in pH and measured pH in living human carotid endarterectomized atherosclerotic plaques (CEA), Watanabe heritable hyperlipidemic (WHHL) rabbit aortas and human umbilical arteries (HUA). Methods and results: We measured pH of CEA of 48 patients, nine WHHL rabbit aortas and 11 HUA specimens (as controls) using a glass type microelectrode mounted on a micromanipulator in a 37 °C incubator. We also used single emission and also dual emission fluorescence ratio imaging microscopy employing pH-sensitive probes to confirm pH heterogeneity. Mean pH measured at 415 points of CEA was 7.55±0.32; at 275 points of WHHL rabbit aortas it was 7.40±0.43; and in 233 points of HUA it was 7.24±0.1. In CEA, pH of yellow (lipid-rich) areas was significantly lower than pH in calcified areas (7.15±0.01 vs. 7.73±0.01, P<0.0001). The coefficients of variation (heterogeneity) of pH in CEA, WHHL rabbit aortas, and HUA were 0.038±0.010, 0.039±0.007, and 0.009±0.003, respectively (P=0.0001). Fluorescence microscopic imaging confirmed pH heterogeneity in both humans and rabbits but not in HUA. In a variance components analysis 82% of the heterogeneity was due to the within-plaque variation and 2% was attributable to between-plaque variation. Conclusions: Our findings support the hypothesis of pH heterogeneity in plaques, and suggest a possible role for detecting low pH in the detection of plaque vulnerability. The source of pH heterogeneity particularly acidic pH, its impact on the stability of plaques and its potential clinical utility in locating vulnerable plaques remain to be evaluated.

Introduction

Despite major advances in cardiovascular science and technology during the past three decades, approximately half of all myocardial infarctions and sudden deaths occur unexpectedly. It is widely accepted that coronary atherosclerotic plaques and thrombotic complications resulting from their rupture or erosion are the underlying causes of this major health problem. The majority of these vulnerable plaques exhibit active inflammation, a large necrotic lipid core, a thin fibrous cap, and confer a stenosis of less than 70%. These lesions are not detectable by stress testing or coronary angiography [1], [2], [3].

Atherosclerotic plaques have generally been classified on the basis of structural properties, such as plaque volume, degree of stenosis, calcification, cap thickness, and lipid core to provide a ‘structural classification’, [4], [5]. In order to supplement the ‘structural classification’ of plaques, our group is exploring the possibility of a ‘functional classification’ based on physiological variables such as plaque temperature, pH, oxygen consumption, lactate production, free radicals formation, and monocyte recruitment rate. Previously, we have reported temperature heterogeneity in human atherosclerotic plaques that have the morphological characteristics of vulnerability, [6] and this has been corroborated by Stefanadis and colleagues [7]. In our previous study, temperature heterogeneity correlated with density and proximity of inflammatory cells, most of which were macrophages [6].

Here we tested the hypotheses that, (1) human and rabbit atherosclerotic plaques exhibit more heterogeneity of pH than normal arterial wall; and (2) that pH and temperature are inversely correlated.

Section snippets

Materials and methods

With the approval of the St. Luke's Episcopal Hospital Institutional Review Board, we obtained carotid plaque specimens from the surgeon operating on patients undergoing carotid endarterectomy and took them immediately to our nearby laboratory in a 37 °C container. In order to develop and test our study protocol and calibrate the microelectrodes and pH meter, the first five plaques were examined and necessary changes in the study protocols were made.

Next, a series of 30 cases was investigated

Statistical analysis

We used SPSS for windows (version 8) software for analysis and graph generation. Unless stated otherwise, data are presented as mean±S.D. Heterogeneity was defined as the CV, which is the standard deviation (S.D.) divided by the mean. The Kolmogorov–Smirnov test of normality with Lilliefors significance correction was used to test whether the distribution of variables had a normal pattern. Mann–Whitney and Kruskal–Wallis tests were used for analyzing the variables with a non-normal distribution.

Results

Results of pH measurement in living human CEA specimen are shown in Fig. 1. There was a non-normal distribution (P<0.0001). As is apparent in Fig. 1, there was a bimodal distribution, which is a rough indicator of the presence of regional clusters in pH measurements.

The coefficient variation (CV) of pH for the first 30 plaques from the first phase of the study was 0.033±0.015 which was very close to the CV obtained from 18 CEA specimens in the main phase of the study 0.038±0.010, (P=0.2).

In the

Discussion

In this study, living atherosclerotic plaques exhibited marked pH heterogeneity. To our knowledge, this is the first report of this finding. The areas showing the lowest pH were yellow in color and presumed to have a lipid core (Fig. 7, panels A and B). The regions with higher pH were almost exclusively in the calcified regions. (Fig. 7, panels C and D). Using a consistent method for collecting and washing CEA specimens, we observed more pH heterogeneity within specimens than between specimens,

Study limitations

This study has several limitations. The pH was measured ex vivo after carotid endarterectomy. This process is presumed to be associated with variable amounts of trauma (traction, ischemia, etc.), which presumably lowers the pH for all specimens. We provided identical and near-physiologic conditions for all the specimens by incubating them in DMEM (pH 7.4) at 37 °C for 30 min prior to measuring the pH, but the actual measurement of pH was done in a 37 °C humidified (>90% H2O saturation) incubator.

Potential mechanisms of pH heterogeneity

Inflammation is known to increase the metabolic activity resulting in an increase in temperature and reduction of pH. An acidic environment in inflammatory foci has been recognized for decades. Some have also speculated that the biology of inflammation is affected by its hydrogen concentration [9]. It is generally accepted that a shift to anaerobic metabolism and lactate formation is the underlying mechanism for increased hydrogen ion concentration in inflamed tissues.

It is now widely accepted

Implications

pH heterogeneity can affect numerous plaque functions. It has been reported that lowering the pH augments the oxidation of LDL by releasing Fe and Cu radicals and decreasing anti-oxidant defense capacity (l-cysteine and l-histidine); [18], [19] on this basis Leake speculated that the plaques should be acidic [20].

It is well established that in contrast to the matrix metalloproteinases that work optimally at a neutral pH, certain proteases such as serine protienases are active in low pH.

Applications

In order to investigate the possible clinical utility of pH and/or lactate in the detection of vulnerable plaques, we are developing a near-infrared spectroscopic catheter for pH and lactate imaging of atherosclerotic plaque in vivo. Magnetic resonance spectroscopy may also be used for the same purpose and has the advantage of being used non-invasively given enough spatial resolution and signal to noise ratio.

In summary, we have found evidence of considerable pH heterogeneity in atherosclerotic

Acknowledgements

The authors wish to acknowledge Khawar Gul, Mark Snuggs, Bujin Guo, David Engler, and Tania Khan for sharing their knowledge and kind assistance. The study was funded through US Army DREAMS Project at the University of Texas-Houston Health Science Center and Texas Heart Institute.

References (28)

  • C. Stefanadis et al.

    Thermal heterogeneity within human atherosclerotic coronary arteries detected in vivo: a new method of detection by application of a special thermography catheter

    Circulation

    (1999)
  • T. Binzoni et al.

    Temperature dependence of human gastrocnemius pH and high-energy phosphate concentration by noninvasive techniques

    Magn. Reson. Med.

    (2000)
  • V. Menkin

    Biology of inflammation

    Science

    (1956)
  • R. Ross

    Atherosclerosis—an inflammatory disease

    New Engl. J. Med.

    (1999)
  • Cited by (168)

    • Assessing the colloidal stability of copper doped ZIF-8 in water and serum

      2023, Colloids and Surfaces A: Physicochemical and Engineering Aspects
    View all citing articles on Scopus
    View full text