In mice, Nhe1-deficiency or anti-IgE antibody reduces atherosclerosis and blocks lesion acidification. probe LS662, followed by coregistered fluorescent molecular tomography-computed tomography imaging, identifies acidic regions in atherosclerotic lesions in live mice, ushering a non-invasive and radiation-free imaging approach to monitor atherosclerotic lesions in live subjects. mice10,11. We previously showed that IgE activates Nhe1, reduces extracellular pH, and induces macrophage apoptosis. Acidic media alone induced macrophage apoptosis12. In this study, we use pHrodo to report that human and mouse atherosclerotic lesions are acidic and colocalize with macrophage clusters, IgE expression, and cell death. Using Nhe1 heterozygous mice, we prove a role of Nhe1 in atherosclerotic lesion acidification and atherogenesis. IgE induces immunocomplex formation between FcR1 and Nhe1 in macrophages. IgE and macrophage expression of FcR1 and Nhe1 all contribute to atherogenesis and lesion acidification. Fluorescent molecular tomography (FMT) Docosahexaenoic Acid methyl ester imaging using a pH-sensitive near-infrared (NIR) probe with or without computed tomography (CT) localizes acidified atherosclerotic lesions in live mice. This concept may lead to the development of non-invasive and non-radiation-imaging method to monitor atherosclerotic lesion Docosahexaenoic Acid methyl ester growth in live mice and humans. Results pHrodo localizes acidic regions in atherosclerotic lesions pHrodo is a cell permeable fluorogenic probe that becomes fluorescent under acidic pH (pH? ?7.0), which allows the examination of intracellular acidic compartments13. The pHrodo used in this study becomes red fluorescent in acidic environment and allows direct visualization under a confocal microscope. When fresh human carotid atherosclerotic segments were incubated in a pHrodo solution followed by OCT embedding and sectioning, this red fluorogenic probe localized acidic regions in human carotid atherosclerotic lesions (Fig. ?(Fig.1a).1a). Immunohistological analysis of adjacent sections revealed CD68-positive macrophage accumulation, IgE expression, and TUNEL-positive cell apoptosis in the same regions (Fig. ?(Fig.1a).1a). Hematoxylin and eosin (H&E) staining characterized the lesion morphology and von Kossa staining showed this region remained free of calcification. A calcified human atherosclerotic lesion was used as von Kossa staining positive control (Fig. ?(Fig.1b).1b). Therefore, lesion acidification did not necessarily occur at the site of calcification. Open in a separate window Fig. 1 Acidic human and murine atherosclerotic lesions are rich in macrophages, IgE expression, and cell apoptosis. a pHrodo-positive acidic area, Compact disc68-positive macrophages, IgE, and TUNEL-positive cells are colocalized in parallel areas from individual atherosclerotic lesions (data are Docosahexaenoic Acid methyl ester consultant from five donors). b Parallel section eosin and hematoxylin staining and von Kossa staining for tissues morphology and calcification. A individual atherosclerotic lesion section with calcification can be used being a von Kossa staining positive control. c In parallel atherosclerotic lesion areas from mice, the pHrodo-positive acidic region (crimson) are abundant with Macintosh-3-positive macrophages. d IgE appearance and TUNEL-positive cells in the same section of -panel c. e pHrodo is normally replaced with drinking water as detrimental control in atherosclerotic lesion from mice. Data are representative from seven mice. Pubs: 500?m, inset pubs: 250?m After incubating the aortic root base from atherosclerotic mice that fed an atherogenic diet plan for three months using the pHrodo probe, we also detected acidic locations in mouse atherosclerotic lesions (Fig. ?(Fig.1c,1c, still left -panel). Immunostaining of adjacent areas showed that pHrodo crimson fluorescent areas also included clusters of Macintosh-3-positive macrophages (Fig. ?(Fig.1c,1c, correct Docosahexaenoic Acid methyl ester -panel), IgE-reactivity (Fig. ?(Fig.1d,1d, still left -panel), and TUNEL-positive apoptotic cells (Fig. ?(Fig.1d,1d, correct -panel). To check the specificity from the pHrodo probe, we repeated the labeling procedure using solvent (drinking water) without pHrodo and discovered just the green autofluorescence in the elastica in aortic root base in the mice (Fig. ?(Fig.1e).1e). Development of such acidic locations links to IgE-induced Nhe1 activation of macrophages and perhaps various other cell types, resulting in proton acidification and extrusion from the extracellular milieu12,14,15. Nhe1-insufficiency protects mice from atherosclerosis Nhe1-mediated proton extrusion, extracellular milieu acidification, and consequent macrophage apoptosis might donate to atherogenesis DEPC-1 directly. To check this hypothesis, we produced Nhe1 heterozygous mice and their littermate control mice, because Nhe1 homozygous-deficient mice develop ataxia and epileptic-like seizures, display postnatal development arrest, and expire within per month after delivery16 frequently,17. After three months on an.