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Imaging mass spectrometry reveals heterogeneity of proliferation and metabolism in atherosclerosis
Christelle Guillermier, … , Matthew L. Steinhauser, Jonathan D. Brown
Christelle Guillermier, … , Matthew L. Steinhauser, Jonathan D. Brown
Published June 6, 2019
Citation Information: JCI Insight. 2019;4(11):e128528. https://doi.org/10.1172/jci.insight.128528.
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Technical Advance Vascular biology

Imaging mass spectrometry reveals heterogeneity of proliferation and metabolism in atherosclerosis

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Abstract

Atherosclerotic plaques feature local proliferation of leukocytes and vascular smooth muscle cells (VSMCs) and changes in cellular metabolism. Yet the relationship between glucose utilization and proliferation has been technically impossible to study directly in cells of atherosclerotic plaques in vivo. We used multi-isotope imaging mass spectrometry (MIMS), a quantitative imaging platform, to measure coincident cell division and glucose utilization at suborganelle resolution in atherosclerotic plaques. In established plaques, 65% of intimal foam cells and only 4% of medial VSMCs were labeled with 15N-thymidine after 1 week of isotope treatment. Dividing cells demonstrated heightened glucose labeling. MIMS detected 2H-glucose label in multiple subcellular compartments within foam cells, including lipid droplets, the cytosol, and chromatin. Unexpectedly, we identified an intensely focal region of 2H-label in VSMCs underlying plaques. This signal diminished in regions of aorta without atherosclerosis. In advanced plaques, 15N-thymidine and 2H-glucose labeling in foam cells and VSMCs significantly decreased. These data demonstrate marked heterogeneity in VSMC glucose metabolism that was dependent on both proliferative status and proximity of VSMCs to plaques. Furthermore, these results reveal how quantitative mass spectrometry coupled with isotope imaging can complement other methods used to study cell biology directly in the growing atherosclerotic plaque in vivo.

Authors

Christelle Guillermier, Sean P. Doherty, Adam G. Whitney, Vladimir R. Babaev, MacRae F. Linton, Matthew L. Steinhauser, Jonathan D. Brown

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Figure 1

Multi-isotope imaging mass spectrometry maps proliferation and glucose utilization by key cell types within the complex architecture of atherosclerotic plaque.

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Multi-isotope imaging mass spectrometry maps proliferation and glucose u...
(A) Schematic depicting the application of multi-isotope imaging mass spectrometry (MIMS) to atherosclerosis (left). Ldlr–/– mice were fed a high-fat/high-cholesterol diet. Stable isotope labeling by intraperitoneal injections of 15N-thymidine (1 week) and 2H-glucose (3 days) was initiated before sacrifice. The fixation protocol included osmium tetroxide to preserve lipids (black stain). Toluidine blue staining (middle) revealed atherosclerotic plaques in the aortic roots and guided coordinate selection for MIMS analysis (right). The image shown represents quantification of 12C14N– and provides histological detail. White arrow, endothelial cell (EC); top white asterisk, VSMC; red line, Foam cell core; L, lumen. Scale bar: 20 μm. (B) Quantitative mass images reveal features of cells in atherosclerotic plaque intima: EC (white arrows) and numerous foam cells with lipid droplets (dark spaces) in 12C14N– images. Phosphorus (31P–) images reveal nuclei due to high phosphorus content of chromatin, including foam cell nucleus (2-headed arrow). Hue saturation intensity images are used to display the isotope ratio measurements and map the incorporation of 15N-thymidine (12C15N–/12C14N–) and 2H-glucose (12C22H/12C21H). The lower bound of the scale is set to the background ratio (0%), and the upper bound is set to reveal differences in labeling (150% and 300% above background, respectively). Scale bar: 5 μm. (C) MIMS images of vascular smooth muscle cells (VSMCs) underlying atherosclerotic plaques. Elastin bands (white arrows) appear bright in 12C14N– images. Scale bar: 5 μm.
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