Mass Spectrometry Imaging of atherosclerosis-affine Gadofluorine following Magnetic Resonance Imaging.

Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr-/- mice were investigated by high-field MRI (7 T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis.

Molecular imaging of myocardial infarction with Gadofluorine P - A combined magnetic resonance and mass spectrometry imaging approach.

BACKGROUND: Molecular MRI is becoming increasingly important for preclinical research. Validation of targeted gadolinium probes in tissue however has been cumbersome up to now. Novel methodology to assess gadolinium distribution in tissue after in vivo application is therefore needed. PURPOSE: To establish combined Magnetic Resonance Imaging (MRI) and Mass Spectrometry Imaging (MSI) for improved detection and quantification of Gadofluorine P deposition in scar formation and myocardial remodeling. MATERIALS AND METHODS: Animal studies were performed according to institutionally approved protocols. Myocardial infarction was induced by permanent ligation of the left ascending artery (LAD) in C57BL/6J mice. MRI was performed at 7T at 1 week and 6 weeks after myocardial infarction. Gadofluorine P was used for dynamic T1 mapping of extracellular matrix synthesis during myocardial healing and compared to Gd-DTPA. After in vivo imaging contrast agent concentration as well as distribution in tissue were validated and quantified by spatially resolved Matrix-Assisted Laser Desorption Ionization (MALDI) MSI and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. RESULTS: Both Gadofluorine P enhancement as well as local tissue content in the myocardial scar were highest at 15 minutes post injection. R1 values increased from 1 to 6 weeks after MI (1.62 s-1 vs 2.68 s-1, p = 0.059) paralleled by an increase in Gadofluorine P concentration in the infarct from 0.019 mM at 1 week to 0.028 mM at 6 weeks (p = 0.048), whereas Gd-DTPA enhancement showed no differences (3.95 s-1 vs 3.47 s-1, p = 0.701). MALDI-MSI results were corroborated by elemental LA-ICP-MS of Gadolinium in healthy and infarcted myocardium. Histology confirmed increased extracellular matrix synthesis at 6 weeks compared to 1 week. CONCLUSION: Adding quantitative MSI to MR imaging enables a quantitative validation of Gadofluorine P distribution in the heart after MI for molecular imaging.

Lipase inhibitor orlistat prevents hepatitis B virus infection by targeting an early step in the virus life cycle.

Hepatitis B Virus (HBV) is a strictly hepatotropic pathogen which is very efficiently targeted to the liver and into its host cell, the hepatocyte. The sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a key virus entry receptor, but the early steps in the virus life cycle are still only barely understood. Here, we investigated the effect of lipase inhibition and lipoprotein uptake on HBV infection using differentiated HepaRG cells and primary human hepatocytes. We found that an excess of triglyceride rich lipoprotein particles in vitro diminished HBV infection and a reduced hepatic virus uptake in vivo if apolipoprotein E is lacking indicating virus transport along with lipoproteins to target hepatocytes. Moreover, we showed that HBV infection of hepatocytes was inhibited by the broadly active lipase inhibitor orlistat, approved as a therapeutic agent which blocks neutral lipid hydrolysis activity. Orlistat treatment targets HBV infection at a post-entry step and inhibited HBV infection during virus inoculation strongly in a dose-dependent manner. In contrast, orlistat had no effect on HBV gene expression or replication or when added after HBV infection. Taken together, our data indicate that HBV connects to the hepatotropic lipoprotein metabolism and that inhibition of cellular hepatic lipase(s) may allow to target early steps of HBV infection.

Targeting Elastase for Molecular Imaging of Early Atherosclerotic Lesions.

OBJECTIVE: Neutrophils accumulate in early atherosclerotic lesions and promote lesion growth. In this study, we evaluated an elastase-specific near-infrared imaging agent for molecular imaging using hybrid fluorescence molecular tomography/x-ray computed tomography. APPROACH AND RESULTS: Murine neutrophils were isolated from bone marrow and incubated with the neutrophil-targeted near-infrared imaging agent Neutrophil Elastase 680 FAST for proof of principle experiments, verifying that the elastase-targeted fluorescent agent is specifically cleaved and activated by neutrophil content after lysis or cell stimulation. For in vivo experiments, low-density lipoprotein receptor-deficient mice were placed on a Western-type diet and imaged after 4, 8, and 12 weeks by fluorescence molecular tomography/x-ray computed tomography. Although this agent remains silent on injection, it produces fluorescent signal after cleavage by neutrophil elastase. After hybrid fluorescence molecular tomography/x-ray computed tomography imaging, mice were euthanized for whole-body cryosectioning and histological analyses. The in vivo fluorescent signal in the area of the aortic arch was highest after 4 weeks of high-fat diet feeding and decreased at 8 and 12 weeks. Ex vivo whole-body cryoslicing confirmed the fluorescent signal to locate to the aortic arch and to originate from the atherosclerotic arterial wall. Histological analysis demonstrated the presence of neutrophils in atherosclerotic lesions. CONCLUSIONS: This study provides evidence that elastase-targeted imaging can be used for in vivo detection of early atherosclerosis. This imaging approach may harbor potential in the clinical setting for earlier diagnosis and treatment of atherosclerosis.

The "Intermediate" CD14++CD16+ monocyte subset increases in severe peripheral artery disease in humans.

Monocytes are key players in atherosclerotic. Human monocytes display a considerable heterogeneity and at least three subsets can be distinguished. While the role of monocyte subset heterogeneity has already been well investigated in coronary artery disease (CAD), the knowledge about monocytes and their heterogeneity in peripheral artery occlusive disease (PAOD) still is limited. Therefore, we aimed to investigate monocyte subset heterogeneity in patients with PAOD. Peripheral blood was obtained from 143 patients suffering from PAOD (Rutherford stage I to VI) and three monocyte subsets were identified by flow cytometry: CD14++CD16- classical monocytes, CD14+CD16++ non-classical monocytes and CD14++CD16+ intermediate monocytes. Additionally the expression of distinct surface markers (CD106, CD162 and myeloperoxidase MPO) was analyzed. Proportions of CD14++CD16+ intermediate monocyte levels were significantly increased in advanced stages of PAOD, while classical and non-classical monocytes displayed no such trend. Moreover, CD162 and MPO expression increased significantly in intermediate monocyte subsets in advanced disease stages. Likewise, increased CD162 and MPO expression was noted in CD14++CD16- classical monocytes. These data suggest substantial dynamics in monocyte subset distributions and phenotypes in different stages of PAOD, which can either serve as biomarkers or as potential therapeutic targets to decrease the inflammatory burden in advanced stages of atherosclerosis.

Increased intermediate CD14++CD16++ monocyte subset levels associate with restenosis after peripheral percutaneous transluminal angioplasty.

BACKGROUND AND AIMS: We aimed at studying the association of three major human monocyte subsets after percutaneous transluminal angioplasty (PTA) in patients with femoropopliteal disease. METHODS: We prospectively studied 67 sequential patients (40 male, 27 female; mean age 71 ± 11 years) treated with femoropopliteal angioplasty. Multi-color flow cytometry characterized monocyte subsets from venous blood for expression of CD14 and CD16 and intracellular myeloperoxidase (MPO) prior to, and 3, 6 and 12 months post PTA. Analyses tested associations between monocyte subsets and risk for restenosis. RESULTS: 16/67 patients (24%) developed restenosis within 12 months after PTA. Patients with hyperlipidemia had increased risk for restenosis (HR = 1.7, 95% CI 0.7-2.9, p = 0.001). Increased baseline monocytes associated with an increased risk of late restenosis (HR = 4.9, 95% CI: 1.3-18.6, p = 0.047). CD14++CD16++ 'intermediate' monocytes assessed at baseline, and after 3, 6, and 12 months significantly associated with the risk for subsequent restenosis: HR = 3.9 (95% CI: 2.4-6.5, p = 0.029), HR = 5.7 (95% CI = 0.7-44.7, p = 0.013), HR = 6.5 (95% CI: 2.5-16.9, p = 0.001) and HR = 1.5 (95% CI = 1.4-15.5 p = 0.001), respectively. Moreover, the probability for freedom of restenosis decreased with increased levels of intermediate subsets at 12 months after PTA. Additionally, intracellular MPO expression in CD14++CD16++ measured at 3, 6 and 12 months associated with an increased restenosis risk (HR = 1.5, 95% CI: 0.8-2.1, p = 0.214, HR = 1.9, 95% CI: 1.0-2.3 p = 0.051 and HR = 1.4, 95% CI: 1.0-1.8, p = 0.052). CONCLUSIONS: Our results imply altered innate immunity after angioplasty. Elevated CD14++CD16++ intermediate monocyte frequencies and increased MPO expression may identify individuals at heightened risk for restenosis.