Yoichi Shimizu

Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains

Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.

Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.

Characterization of the role of sphingomyelin synthase 2 in glucose metabolism in whole-body and peripheral tissues in mice.

Sphingomyelin synthase 2 (SMS2) is a proposed potential therapeutic target for obesity and insulin resistance. However, the contributions of SMS2 to glucose metabolism in tissues and its possible therapeutic mechanisms remain unclear. Thus, to determine whole-body glucose utilization and the contributions of each insulin-targeted tissue to glucose uptake, we performed a glucose kinetics study, using the radiolabeled glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG), in wild-type (WT) and SMS2 knockout (KO) mice. Insulin signaling was enhanced in the liver, white adipose tissue and skeletal muscle of SMS2 KO mice compared with those of WT mice. In addition, compared with in WT mice, blood clearance of (18)F-FDG was accelerated in SMS2 KO mice when they were fed either a normal or a high fat diet. (18)F-FDG uptake was also increased in insulin-targeted tissues such as skeletal muscle in the SMS2 KO mice. Whereas skeletal muscle sphingolipid content was not clearly affected, plasma levels of very long-chain fatty acid (VLCFA)-containing ceramides were markedly increased in SMS2 KO mice, compared with in WT mice. We also generated liver-conditional SMS2 KO mice and performed glucose and insulin tolerance tests on mice with a high fat diet. However, no significant effect was observed. Thus, our study provided evidence that genetic inhibition of SMS2 elevated glucose clearance through activation of glucose uptake into insulin-targeted tissues such as skeletal muscle by a mechanism independent of hepatic SMS2. Our findings further indicate that this occurs, at least in part, via indirect mechanisms such as elevation of VLCFA-containing ceramides.

Recent Advances in the Development of PET/SPECT Probes for Atherosclerosis Imaging

The rupture of vulnerable atherosclerotic plaques and subsequent thrombus formation are the major causes of myocardial and cerebral infarction. Accordingly, the detection of vulnerable plaques is important for risk stratification and to provide appropriate treatment. Inflammation imaging using 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) has been most extensively studied for detecting vulnerable atherosclerotic plaques. It is of great importance to develop PET/SPECT probes capable of specifically visualizing the biological molecules involved in atherosclerotic plaque formation and/or progression. In this article, we review recent advances in the development of PET/SPECT probes for visualizing atherosclerotic plaques and their application to therapy monitoring, mainly focusing on experimental studies.

Irbesartan Attenuates Atherosclerosis in Watanabe Heritable Hyperlipidemic Rabbits: Noninvasive Imaging of Inflammation by 18F-Fluorodeoxyglucose Positron Emission Tomography

The purpose of this study was to assess the usefulness of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) in evaluating the antiatherogenic effects of irbesartan, an angiotensin II type 1 receptor blocker. Watanabe heritable hyperlipidemic rabbits were divided into the irbesartan-treated group (75 mg/kg/d; n = 14) and the control group (n = 14). After a 9-month treatment, rabbits underwent 18F-FDG PET. Using the aortic lesions, autoradiography and histologic examinations were performed. PET imaging clearly visualized the thoracic lesions of control rabbits and showed a significant decrease in the 18F-FDG uptake level of irbesartan-treated rabbits (78.8% of controls; p < .05). Irbesartan treatment significantly reduced the plaque size (43.1% of controls) and intraplaque macrophage infiltration level (48.1% of controls). The 18F-FDG uptake level in plaques positively correlated with the plaque size (r = .65, p < .05) and macrophage infiltration level (r = .57, p < .05). Noninvasive imaging by 18F-FDG PET is useful for evaluating the therapeutic effects of irbesartan and reflects inflammation, a key factor involved in the therapeutic effects.The purpose of this study was to assess the usefulness of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) in evaluating the antiatherogenic effects of irbesartan, an angiotensin II type 1 receptor blocker. Watanabe heritable hyperlipidemic rabbits were divided into the irbesartan-treated group (75 mg/kg/d; n = 14) and the control group ( n = 14). After a 9-month treatment, rabbits underwent 18F-FDG PET. Using the aortic lesions, autoradiography and histologic examinations were performed. PET imaging clearly visualized the thoracic lesions of control rabbits and showed a significant decrease in the 18F-FDG uptake level of irbesartan-treated rabbits (78.8% of controls; p < .05). Irbesartan treatment significantly reduced the plaque size (43.1% of controls) and intraplaque macrophage infiltration level (48.1% of controls). The 18F-FDG uptake level in plaques positively correlated with the plaque size ( r = .65, p < .05) and macrophage infiltration level ( r = .57, p < .05). Noninvasive imaging by 18F-FDG PET is useful for evaluating the therapeutic effects of irbesartan and reflects inflammation, a key factor involved in the therapeutic effects.

Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent “Pimonidazole” in Hypoxia

Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.

Dynamic PET evaluation of elevated FLT level after sorafenib treatment in mice bearing human renal cell carcinoma xenograft

BackgroundSorafenib, an oral multikinase inhibitor, has anti-proliferative and anti-angiogenic activities and is therapeutically effective against renal cell carcinoma (RCC). Recently, we have evaluated the tumor responses to sorafenib treatment in a RCC xenograft using [Methyl-3H(N)]-3′-fluoro-3′-deoxythythymidine ([3H]FLT). Contrary to our expectation, the FLT level in the tumor significantly increased after the treatment. In this study, to clarify the reason for the elevated FLT level, dynamic 3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT) positron emission tomography (PET) and kinetic studies were performed in mice bearing a RCC xenograft (A498).The A498 xenograft was established in nude mice, and the mice were assigned to the control (nu2009=u20095) and treatment (nu2009=u20095) groups. The mice in the treatment group were orally given sorafenib (20xa0mg/kg/day p.o.) once daily for 3xa0days. Twenty-four hours after the treatment, dynamic [18F]FLT PET was performed by small-animal PET. Three-dimensional regions of interest (ROIs) were manually defined for the tumors. A three-compartment model fitting was carried out to estimate four rate constants using the time activity curve (TAC) in the tumor and the blood clearance rate of [18F]FLT.ResultsThe dynamic pattern of [18F]FLT levels in the tumor significantly changed after the treatment. The rate constant of [18F]FLT phosphorylation (k3) was significantly higher in the treatment group (0.111u2009±u20090.027 [1/min]) than in the control group (0.082u2009±u20090.009 [1/min]). No significant changes were observed in the distribution volume, the ratio of [18F]FLT forward transport (K1) to reverse transport (k2), between the two groups (0.556u2009±u20090.073 and 0.641u2009±u20090.052 [mL/g] in the control group).ConclusionsOur dynamic PET studies indicated that the increase in FLT level may be caused by the phosphorylation of FLT in the tumor after the sorafenib treatment in the mice bearing a RCC xenograft. Dynamic PET studies with kinetic modeling could provide improved understanding of the biochemical processes involved in tumor responses to therapy.

Discovery and Evaluation of Biomarkers for Atherosclerosis

The usage of biomarkers reflecting atherosclerosis progression is important for preventing serious incidence of cardiovascular events. To elucidate clinically relevant molecular determinants in atherosclerosis, we have taken a comprehensive approach to combine mass spectrometry-based differential proteomics using both clinical and animal model specimens. Clinical plasma samples were collected from patients with acute myocardial infarction (AMI), stable angina (SA), and healthy/low-risk individuals (H-LR). We also obtained plasma and arterial tissue samples from apolipoprotein E-deficient and wild-type mice at various pathognomonic points of age. Cleavable isotope-coded affinity tags were used for differential mass spectrometry. Differential proteomics of clinical plasma samples revealed that more than 10 proteins appeared to be upregulated (relative abundance AMI/H-LR or SA/H-LR >1.5) and 5 proteins downregulated (AMI/H-LR or SA/H-LR <1/1.5). These trends associated with the disease progression are not always coincident with those of mouse ortholog proteins, suggesting a pathophysiological difference between humans and the mouse model. Among the downregulated proteins, the complement factor D (CFD) showed monotonic decrease that was in good agreement with the enzyme-linked immunosorbent assay. These results suggest that the comprehensive and systematic proteomic approach may be promising in terms of the selection and evaluation of biomarker candidates.

Radioimmunodetection of Atherosclerotic Lesions Focusing on the Accumulation Mechanism of Immunoglobulin G

In the diagnosis of atherosclerosis, detailed evaluation of biomarkers related to its lesion formation is desired for estimation of its progression rate. In our previous proteomic studies of atherosclerosis mice, the protein level of thrombospondin-4 (TSP4) in the aorta, but not in plasma, elevated relatively with atherosclerotic plaque formation. Therefore, we supposed that TSP4 would be a potential biomarker for diagnostic imaging of atherosclerotic progression. Immunoglobulin G (IgG) has been widely used as a basic molecule of imaging probes providing images specific to their target biomolecules, owing to the antigen-antibody reaction. Therefore, we first developed anti-TSP4 monoclonal IgG radiolabeled with 99mTc (99mTc-TSP4-mAb). 99mTc-TSP4-mAb showed higher accumulation in atherosclerotic aortas of apoE−/− mice (atherosclerotic model mice); however, we found that the non-targeted monoclonal IgG radiolabeled with 99mTc also showed similar distribution in atherosclerotic aortas of apoE−/− mice. IgG has also known to accumulate nonspecifically in the immunological disease such as inflammatory arthritis. However, the accumulation mechanism of IgG has still been unclear in detail. In this chapter, we would like to introduce recent topics on atherosclerotic imaging, focused on our work exploring the accumulation mechanisms of IgG in atherosclerotic lesions, and elucidating the usefulness of radiolabeled IgG images in the diagnosis of atherosclerosis.

Relationship between biodistribution of a novel thymidine phosphorylase (TP) imaging probe and TP expression levels in normal mice

ObjectiveThymidine phosphorylase (TP) is a key enzyme in the pyrimidine nucleoside salvage pathway and its expression is upregulated in a wide variety of solid tumors. In mice, we previously observed high and specific accumulation levels of our TP imaging probe, radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) not only in high-TP-expressing tumors, but also in the liver and small intestine. To clarify the reason for the high accumulation levels of radioiodinated IIMU in the liver and small intestine, we investigated the expression levels of TP in mice in comparison with the biodistribution of radioiodinated IIMU (123I-IIMU).MethodsBALB/cCrSlc mice were injected with 123I-IIMU, and the radioactivity levels [%ID/g (normalized to a mouse of 25xa0g body weight)] in the tissues of interest were determined 0.5, 1, 3 and 24xa0h after the injection (nxa0=xa05, each time point). To determine the expression levels of TP, BALB/cCrSlc and ddy mice (nxa0=xa03/each strain) were euthanized, and the heart, liver, lung, spleen, kidney, stomach, small intestine, large intestine and brain were collected. The mRNA and protein expression levels of TP in these organs were examined by quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively.ResultsIn BALB/cCrSlc mice administered 123I-IIMU, markedly high radioactivity levels were observed in the liver [1.568xa0±xa00.237 (%ID/g)] and small intestine [0.506xa0±xa00.082 (%ID/g)], whereas those in the other tissues were fairly low [<0.010xa0±xa00.003 (%ID/g)] 30xa0min after the injection. The highest expression levels of TP mRNA were also observed in the liver and small intestine among the tissues tested. Immunoblotting showed intense immunoreactive bands of the TP protein for the liver and small intestine, whereas no notable bands were detected for other tissues. Similar expression profiles of TP mRNA and protein were observed in ddy mice.ConclusionWe confirmed TP expression in various tissues of mice at the mRNA and protein levels: high TP expression levels were observed in the liver and small intestine. These high TP expression levels are consistent with the high accumulation levels of 123I-IIMU in these tissues. Our results may provide important information about the physiological accumulation of 123I-IIMU, which may be useful for the clinical diagnostic imaging of TP.