Myeloperoxidase PET Imaging Tracks Intracellular and Extracellular Treatment Changes in Experimental Myocardial Infarction.

Myeloperoxidase (MPO) is a highly oxidative, pro-inflammatory enzyme involved in post-myocardial infarction (MI) injury and is a potential therapeutic target. While multiple MPO inhibitors have been developed, the lack of an imaging reporter to select appropriate patients and assess therapeutic efficacy has hampered clinical development. Thus, a translational imaging method to detect MPO activity non-invasively would help to better understand the role MPO plays in MI and facilitate novel therapy development and clinical validation. Interestingly, many MPO inhibitors affect both intracellular and extracellular MPO, but previous MPO imaging methods can only report extracellular MPO activity. In this study, we found that an MPO-specific PET imaging agent (18F-MAPP) can cross cell membranes to report intracellular MPO activity. We showed that 18F-MAPP can track the treatment effect of an MPO inhibitor (PF-2999) at different doses in experimental MI. The imaging results were corroborated by ex vivo autoradiography and gamma counting data. Furthermore, extracellular and intracellular MPO activity assays revealed that 18F-MAPP imaging can report the changes induced by PF-2999 on both intracellular and extracellular MPO activities. These findings support 18F-MAPP as a translational candidate to noninvasively report MPO activity and accelerate drug development against MPO and other related inflammatory targets.

An activatable PET imaging radioprobe is a dynamic reporter of myeloperoxidase activity in vivo.

Myeloperoxidase (MPO) is a critical proinflammatory enzyme implicated in cardiovascular, neurological, and rheumatological diseases. Emerging therapies targeting inflammation have raised interest in tracking MPO activity in patients. We describe 18F-MAPP, an activatable MPO activity radioprobe for positron emission tomography (PET) imaging. The activated radioprobe binds to proteins and accumulates at sites of MPO activity. The radioprobe 18F-MAPP has a short blood half-life, remains stable in plasma, does not demonstrate cytotoxicity, and crosses the intact blood-brain barrier. The 18F-MAPP imaging detected sites of elevated MPO activity in living mice embedded with human MPO and in mice induced with chemical inflammation or myocardial infarction. The 18F-MAPP PET imaging noninvasively differentiated varying amounts of MPO activity, competitive inhibition, and MPO deficiency in living animals, confirming specificity and showing that the radioprobe can quantify changes in in vivo MPO activity. The radiosynthesis has been optimized and automated, an important step in translation. These data indicate that 18F-MAPP is a promising translational candidate to noninvasively monitor MPO activity and inflammation in patients.

In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis.

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Molecular MR Imaging of Myeloperoxidase Distinguishes Steatosis from Steatohepatitis in Nonalcoholic Fatty Liver Disease.

Purpose To test whether MPO-Gd, an activatable molecular magnetic resonance (MR) imaging agent specific for myeloperoxidase (MPO) activity, could detect MPO activity in nonalcoholic steatohepatitis (NASH) mouse models and human liver biopsy samples. Materials and Methods In this study, 20 leptin receptor-deficient and three MPO knockout mice were injected with endotoxin (lipopolysaccharide) or fed a methionine and choline-deficient (MCD) diet to induce experimental NASH and underwent MR imaging with MPO-Gd. Saline-injected and control diet-fed leptin receptor-deficient mice were used as respective controls. MPO protein and activity measurements and histologic analyses were performed. Eleven human liver biopsy samples underwent MPO-Gd-enhanced MR imaging ex vivo and subsequent histologic evaluation. Results were compared with Student t test or Mann-Whitney U test. Results With endotoxin, a significantly increased contrast-to-noise ratio (CNR) was found compared with sham (mean CNR, 1.81 [95% confidence interval {CI}: 1.53, 2.10] vs 1.02 [95% CI: 0.89, 1.14]; P = .03) at MPO-Gd MR imaging. In the diet-induced NASH model, an increased CNR was also found compared with sham mice (mean CNR, 1.33 [95% CI: 1.27, 1.40] vs 0.98 [95% CI: 0.83, 1.12]; P = .008). Conversely, CNR remained at baseline in NASH mice imaged with gadopentetate dimeglumine and in MPO knockout NASH mice with MPO-Gd, which proves specificity of MPO-Gd. Ex vivo molecular MR imaging of liver biopsy samples from NASH and control patients confirmed results from animal studies (mean CNR for NASH vs control patients, 2.61 [95% CI: 1.48, 3.74] vs 1.29 [95% CI: 1.06, 1.52]; P = .004). Conclusion MPO-Gd showed elevated MPO activity in NAFLD mouse models and human liver biopsy samples. © RSNA, 2017 Online supplemental material is available for this article. An earlier incorrect version of this article appeared online. This article was corrected on April 6, 2017.

Endothelial receptor proteins in acute venous thrombosis and delayed thrombus resolution in cerebral sinus vein thrombosis.

BACKGROUND AND PURPOSE: Cerebral sinus venous thrombosis (CSVT) is a rare but life-threatening disease and its diagnosis remains challenging. Blood biomarkers, including D-Dimer are currently not recommended in guidelines. Soluble endothelial receptor proteins (sICAM-1, sPECAM-1 and sVCAM-1) have been shown to be promising diagnostic biomarkers in deep vein thrombosis (DVT) and pulmonary embolism (PE). Therefore, we examined endothelial receptor proteins as potential biomarkers for detecting CSVT. METHODS: In this bi-centre, prospective study, we quantified D-Dimer as well as sICAM-1, sPECAM-1 and sVCAM-1 in plasma of patients with clinically suspected CSVT managed in the neurological emergency department (ED) of a tertiary care hospital. All patients underwent cerebral magnetic resonance imaging (MRI) and were followed up after 3, 6 and 12 months to detect thrombus resolution. RESULTS: Twenty-four out of 75 (32%) patients with clinically suspected CSVT presenting with headache to the ED were diagnosed with acute CSVT. These patients had a mean age of 45 ± 16 years and 78% were female. In patients with CSVT, mean baseline D-dimer (p < 0.001) and sPECAM-1 (p < 0.001) were significantly higher compared to patients without CSVT. The combination of D-Dimer and sPECAM-1 yielded the best ROC-AUC (0.994; < 0.001) with a negative predictive value of 95.7% and a positive predictive value of 95.5%. In addition, higher baseline sPECAM-1 levels (> 198 ng/ml) on admission were associated with delayed venous thrombus resolution at 3 months (AUC = 0.83). CONCLUSION: sPECAM-1 in combination with D-Dimer should be used to improve the diagnostic accuracy of acute CSVT and sPECAM-1 may predict long-term outcome of CSVT. Confirmatory results are needed in other settings in order to show their value in the management concept of CSVT patients.

Dabigatran in Cerebral Sinus Vein Thrombosis and Thrombophilia.

BACKGROUND AND PURPOSE: Thrombophilic gene alterations are a major risk factor for cerebral sinus vein thrombosis (CSVT). Up to 30% of all patients with cerebral sinus vein thrombosis (CSVT) are found to have thrombophilic defects such as prothrombin mutation (PTM) or factor V Leiden (FVL). Their repercussions on the plasma levels of dabigatran etexilate are unclear. In this prospective case-control study, we aimed to investigate whether thrombophilia in CSVT has an influence on dabigatran peak-plasma levels. METHODS: We monitored 10 patients over 12 months with acute CSVT, genetic thrombophilia with off-label use of dabigatran etexilate 150 mg twice a day and measured dabigatran peak-plasma levels and radiological outcome. We also monitored patients without genetic thrombophilia with dabigatran etexilate 150 mg twice a day and compared the efficiency and dabigatran peak-plasma levels. RESULTS: Patients with homozygote PTM had significantly lower dabigatran peak concentration compared to patients with FVL or the control group (23 ± 4.2 vs. 152.3 ± 27.5 and 159.6 ± 63.08; p-value ≤ 0.05) There was no significant difference in dabigatran etexilate plasma levels between the heterozygote PTM group compared to patients with FVL or the control group (p = 0.29). There was no correlation between dabigatran peak concentration and delayed thrombus dissolution. CONCLUSIONS: Dabigatran peak concentration was stable in patients with heterozygote FVL and heterozygote PTM, but not in homozygote PTM, compared to controls. Genetic screening for thrombophilia in patients after CSVT may be useful to make patient tailored therapeutic decisions regarding oral anticoagulation and may decrease thrombotic events.

Mouse model of anti-NMDA receptor post-herpes simplex encephalitis.

Objective: To develop an endogenous rodent model of postinfectious anti-NMDA receptor (NMDAR) encephalitis. Methods: Six mice were inoculated intranasally with herpes simplex virus (HSV) 1 and subsequently treated with acyclovir for 2 weeks. Serum was collected at 3, 6, and 8 weeks postinoculation and tested for NMDAR antibodies through a cell-based assay. Eight weeks postinoculation, mice were killed and their brains were sectioned and immunostained with antibodies to postsynaptic density (PSD)-95 and NMDARs. Colocalization of hippocampal PSD-95 and NMDAR clusters, representing postsynaptic membrane NMDARs, was quantified via confocal imaging. Hippocampi were additionally analyzed for NMDAR and PSD-95 protein using Western blot analysis. Results: Four of 6 mice (67%) developed serum antibodies to NMDARs: 1 at 3 weeks, 1 at 6 weeks, and 2 at 8 weeks postinoculation. As compared to inoculated mice that did not develop NMDAR antibodies, immunofluorescence staining revealed decreased hippocampal postsynaptic membrane NMDARs in mice with serum antibodies at 8 weeks postinoculation. Western blot analysis showed that mice that had NMDAR antibodies at 8 weeks had decreased total NMDAR but not PSD-95 protein in hippocampal extracts (p < 0.05). Conclusions: Mice inoculated intranasally with HSV-1 developed serum NMDAR antibodies. These antibodies were associated with reduced hippocampal NMDARs, as has been shown in previous models where antibodies from patients with anti-NMDAR encephalitis were infused into mice, paving the way for future studies into the pathophysiology of autoimmune encephalitides.

A versatile imaging platform with fluorescence and CT imaging capabilities that detects myeloperoxidase activity and inflammation at different scales.

Aberrant innate immune response drives the pathophysiology of many diseases. Myeloperoxidase (MPO) is a highly oxidative enzyme secreted by activated myeloid pro-inflammatory immune cells such as neutrophils and macrophages, and is a key mediator of the damaging innate immune response. Current technologies for detecting MPO activity in living organisms are sparse and suffer from any combination of low specificity, low tissue penetration, or low spatial resolution. We describe a versatile imaging platform to detect MPO activity using an activatable construct conjugated to a biotin moiety (MPO-activatable biotinylated sensor, MABS) that allows monitoring the innate immune response and its modulation at different scales and settings. Methods: We designed and synthesized MABS that contains MPO-specific and biotin moieties, and validated its specificity and sensitivity combining with streptavidin-labeled fluorescent agent and gold nanoparticles imaging in vitro and in vivo in multiple mouse models of inflammation and infection, including Matrigel implant, dermatitis, cellulitis, cerebritis and complete Fraud's adjuvant (CFA)-induced inflammation. Results: MABS MPO imaging non-invasively detected varying MPO concentrations, MPO inhibition, and MPO deficiency in vivo with high sensitivity and specificity. MABS can be used to obtain not only a fluorescence imaging agent, but also a CT imaging agent, conferring molecular activity information to a structural imaging modality. Importantly, using this method on tissue-sections, we found that MPO enzymatic activity does not always co-localize with MPO protein detected with conventional techniques (e.g., immunohistochemistry), underscoring the importance of monitoring enzymatic activity. Conclusion: By choosing from different available secondary probes, MABS can be used to create systems suitable to investigate and image MPO activity at different scales and settings.

Myeloperoxidase-Hepatocyte-Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis.

AIMS: Myeloperoxidase (MPO), a highly oxidative enzyme secreted by leukocytes has been implicated in human and experimental nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unknown. In this study, we investigated how MPO contributes to progression from steatosis to NASH. RESULTS: In C57Bl/6J mice fed a diet deficient in methionine and choline to induce NASH, neutrophils and to a lesser extent inflammatory monocytes are markedly increased compared with sham mice and secrete abundant amounts of MPO. Through generation of HOCl, MPO directly causes hepatocyte death in vivo. In vitro experiments demonstrate mitochondrial permeability transition pore induction via activation of SAPK/JNK and PARP. MPO also contributes to activation of hepatic stellate cells (HSCs), the most important source of collagen in the liver. In vitro MPO-activated HSCs have an activation signature (MAPK and PI3K-AKT phosphorylation) and upregulate COL1A1, α-SMA, and CXCL1. MPO-derived oxidative stress also activates transforming growth factor ß (TGF-ß) in vitro, and TGF-ß signaling inhibition with SB-431542 decreased steatosis and fibrosis in vivo. Conversely, congenital absence of MPO results in reduced hepatocyte injury, decreased levels of TGF-ß, fewer activated HSCs, and less severe fibrosis in vivo. INNOVATION AND CONCLUSION: Cumulatively, these findings demonstrate important cross talk between inflammatory myeloid cells, hepatocytes, and HSCs via MPO and establish MPO as part of a proapoptotic and profibrotic pathway of progression in NASH, as well as a potential therapeutic target to ameliorate this disease.

Gelsolin decreases actin toxicity and inflammation in murine multiple sclerosis.

Gelsolin is the fourth most abundant protein in the body and its depletion in the blood has been found in multiple sclerosis (MS) patients. How gelsolin affects the MS brain has not been studied. We found that while the secreted form of gelsolin (pGSN) decreased in the blood of experimental autoimmune encephalomyelitis (EAE) mice, pGSN concentration increased in the EAE brain. Recombinant human pGSN (rhp-GSN) decreased extracellular actin and myeloperoxidase activity in the brain, resulting in reduced disease activity and less severe clinical disease, suggesting that gelsolin could be a potential therapeutic target for MS.