In Situ Mapping of the Glucose Metabolism Heterogeneity in Atherosclerosis: Correlation With 2-Deoxyglucose Uptake

Figures

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  Figure 1.. In situ metabolic mapping of atherosclerotic plaques. Representative low-magnification (top) and high-magnification (bottom) in situ enzyme histochemistry images from aortic root plaques of apoE−/− mice ∼7 months after western diet feeding. (A) A heterogeneously high LDH activity is present within the media and intima. (B) G6PD activity is mostly confined to the media and to a lesser extent along the fibrous cap (blue arrows). G6PD activity is substantially lower in the myocardium (white “m”) compared to the vessel wall. (C) SDH activity is minimal in both intima and media in contrast to intense activity of the myocardium. Insets represent negative controls performed in the presence of chromogenic substrate and other reaction constituents except for the relevant metabolic substrates (ie, lactate, glucose-6-phosphate, and succinate).

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  Figure 2.. Quantification of LDH activity of plaque macrophages and smooth muscle cells. (A) Representative CD68 and α-SMA immunostaining and coregistered LDH in situ histochemistry images. Quantification of background-corrected LDH activity versus the intensity of CD68 (B) and α-SMA (C) expression in 13 × 13 µm grid boxes. (D) Representative histograms and (E) the average ± SEM bar charts of LDH activity in CD68 versus intimal and medial α-SMA positive regions. Empty and filled bars in panel E represent early (∼3 months) and advanced (∼7 months) plaques. There is an overall ∼50% higher LDH activity within medial α-SMA+ regions compared to intimal α-SMA+ and CD68+ regions, though significant heterogeneity and overlaps exist in LDH activity among these regions both in the intima and media. Additionally, early plaques had a higher LDH activity in CD68+ and α-SMA+ (both intimal and medial) regions compared to the advanced plaques. N = 8-9 plaques from 3 mice at ∼3 months and 3 mice at ∼7 months after western diet. *: P < .05, **: P < .01 by one-way analysis of variance (ANOVA) followed by Fisher's LSD post hoc test. Asterisks within the bars denote P values from a comparison of enzymatic…

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  Figure 3.. Quantification of G6PD activity of plaque macrophages and smooth muscle cells. (A) Representative CD68 and α-SMA immunostaining and coregistered G6PD in situ histochemistry images. Quantification of background-corrected G6PD activity versus the intensity of α-SMA (B) and CD68 (C) expression. In contrast to the LDH activity, G6PD activity was mostly confined to medial α-SMA+ regions with substantially less overlap between the activity of media and intima except for modest G6PD activities along the fibrous cap as demonstrated by the representative histograms (D) and the bar chart (E). G6PD activity of α-SMA+ and CD68+ cells was comparable between the early and advanced plaques. Empty and filled bars in panel E represent early (∼3 months) and advanced (∼7 months) plaques. N = 8-9 plaques from 3 mice at ∼3 months and 3 mice at ∼7 months after western diet. ***: P < .001 by one-way analysis of variance (ANOVA) followed by Fisher's LSD post hoc test.

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  Figure 4.. In situ metabolic mapping of atherosclerotic plaques. Consistent with the observed patterns in murine atherosclerotic plaques, human carotid endarterectomy specimens demonstrated a high, but markedly heterogeneous, level of LDH activity (A) within both media and intima, as opposed to G6PD activity (B) which was mostly restricted to the media and fibrous cap (N = 3).

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  Figure 5.. Combined high-resolution [3H]2-deoxyglucose autoradiography and enzyme histochemistry of murine brachiocephalic plaques. Combined immunostaining, enzyme histochemistry, and high-resolution autoradiography (Fire Look-Up Table and smoothing algorithm, Image J) of advanced murine brachiocephalic artery plaques was performed to determine if glucose uptake correlates with LDH (A-B) and G6PD (C-D) activities of plaques. Representative imaging (A and C) and quantification of superimposed images using overlaid grids (B and D) demonstrate a progressive, but modest, increase in LDH activity in regions with higher [3H]2-deoxyglucose uptake (B) (N = 18 plaques from 3 mice). However, G6PD activity was not statistically different across different quartiles of [3H]2-deoxyglucose uptake (D) (N = 14 plaques from 3 mice). Q1 to Q4: lowest to highest quartiles of [3H]2-deoxyglucose uptake.