Characterization of hepatic fatty acids using magnetic resonance spectroscopy for the assessment of treatment response to metformin in an eNOS-/- mouse model of metabolic nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Liver biopsy remains the gold standard for diagnosis and staging of disease. There is a clinical need for noninvasive diagnostic tools for risk stratification, follow-up, and monitoring treatment response that are currently lacking, as well as preclinical models that recapitulate the etiology of the human condition. We have characterized the progression of NAFLD in eNOS-/- mice fed a high fat diet (HFD) using noninvasive Dixon-based magnetic resonance imaging and single voxel STEAM spectroscopy-based protocols to measure liver fat fraction at 3 T. After 8 weeks of diet intervention, eNOS-/- mice exhibited significant accumulation of intra-abdominal and liver fat compared with control mice. Liver fat fraction measured by 1 H-MRS in vivo showed a good correlation with the NAFLD activity score measured by histology. Treatment of HFD-fed NOS3-/- mice with metformin showed significantly reduced liver fat fraction and altered hepatic lipidomic profile compared with untreated mice. Our results show the potential of in vivo liver MRI and 1 H-MRS to noninvasively diagnose and stage the progression of NAFLD and to monitor treatment response in an eNOS-/- murine model that represents the classic NAFLD phenotype associated with metabolic syndrome.

Myocardial T1, T2, T2*, and fat fraction quantification via low-rank motion-corrected cardiac MR fingerprinting.

PURPOSE: Develop a novel 2D cardiac MR fingerprinting (MRF) approach to enable simultaneous T1, T2, T2*, and fat fraction (FF) myocardial tissue characterization in a single breath-hold scan. METHODS: Simultaneous, co-registered, multi-parametric mapping of T1, T2, and FF has been recently achieved with cardiac MRF. Here, we further incorporate T2* quantification within this approach, enabling simultaneous T1, T2, T2*, and FF myocardial tissue characterization in a single breath-hold scan. T2* quantification is achieved with an eight-echo readout that requires a long cardiac acquisition window. A novel low-rank motion-corrected (LRMC) reconstruction is exploited to correct for cardiac motion within the long acquisition window. The proposed T1/T2/T2*/FF cardiac MRF was evaluated in phantom and in 10 healthy subjects in comparison to conventional mapping techniques. RESULTS: The proposed approach achieved high quality parametric mapping of T1, T2, T2*, and FF with corresponding normalized RMS error (RMSE) T1 = 5.9%, T2 = 9.6% (T2 values <100 ms), T2* = 3.3% (T2* values <100 ms), and FF = 0.8% observed in phantom scans. In vivo, the proposed approach produced higher left-ventricular myocardial T1 values than MOLLI (1148 vs 1056 ms), lower T2 values than T2-GraSE (42.8 vs 50.6 ms), lower T2* values than eight-echo gradient echo (GRE) (35.0 vs 39.4 ms), and higher FF values than six-echo GRE (0.8 vs 0.3 %) reference techniques. The proposed approach achieved considerable reduction in motion artifacts compared to cardiac MRF without motion correction, improved spatial uniformity, and statistically higher apparent precision relative to conventional mapping for all parameters. CONCLUSION: The proposed cardiac MRF approach enables simultaneous, co-registered mapping of T1, T2, T2*, and FF in a single breath-hold for comprehensive myocardial tissue characterization, achieving higher apparent precision than conventional methods.

Simultaneous comprehensive liver T1 , T2 , T2∗ , T1ρ , and fat fraction characterization with MR fingerprinting.

PURPOSE: To develop a novel simultaneous co-registered T1 , T2 , T2∗ , T1ρ , and fat fraction abdominal MR fingerprinting (MRF) approach for fully comprehensive liver-tissue characterization in a single breath-hold scan. METHODS: A gradient-echo liver MRF sequence with low fixed flip angle, multi-echo radial readout, and varying magnetization preparation pulses for multiparametric encoding is performed at 1.5 T. The T2∗ and fat fraction are estimated from a graph/cut water/fat separation method using a six-peak fat model. Water/fat singular images obtained are then matched to an MRF dictionary, estimating water-specific T1 , T2 , and T1ρ . The proposed approach was tested in phantoms and 10 healthy subjects and compared against conventional sequences. RESULTS: For the phantom studies, linear fits show excellent coefficients of determination (r2 > 0.9) for every parametric map. For in vivo studies, the average values measured within regions of interest drawn on liver, spleen, muscle, and fat are statistically different from the reference scans (p < 0.05) for T1 , T2 , and T1⍴ but not for T2∗ and fat fraction, whereas correlation between MRF and reference scans is excellent for each parameter (r2 > 0.92 for every parameter). CONCLUSION: The proposed multi-echo inversion-recovery, T2 , and T1⍴ prepared liver MRF sequence presented in this work allows for quantitative T1 , T2 , T2∗ , T1⍴ , and fat fraction liver-tissue characterization in a single breath-hold scan of 18 seconds. The approach showed good agreement and correlation with respect to reference clinical maps.

Simultaneous T1 , T2 , and T1ρ cardiac magnetic resonance fingerprinting for contrast agent-free myocardial tissue characterization.

PURPOSE: To develop a simultaneous T1 , T2 , and T1ρ cardiac magnetic resonance fingerprinting (MRF) approach to enable comprehensive contrast agent-free myocardial tissue characterization in a single breath-hold scan. METHODS: A 2D gradient-echo electrocardiogram-triggered cardiac MRF sequence with low flip angles, varying magnetization preparation, and spiral trajectory was acquired at 1.5 T to encode T1 , T2 , and T1⍴ simultaneously. The MRF images were reconstructed using low-rank inversion, regularized with a multicontrast patch-based higher-order reconstruction. Parametric maps were generated and matched in the singular value domain to extended phase graph-based dictionaries. The proposed approach was tested in phantoms and 10 healthy subjects and compared against conventional methods in terms of coefficients of determination and best fits for the phantom study, and in terms of Bland-Altman agreement, average values and coefficient of variation of T1 , T2 , and T1⍴ for the healthy subjects study. RESULTS: The T1 , T2 , and T1⍴ MRF values showed excellent correlation with conventional spin-echo and clinical mapping methods in phantom studies (r2 > 0.97). Measured MRF values in myocardial tissue (mean ± SD) were 1133 ± 33 ms, 38.8 ± 3.5 ms, and 52.0 ± 4.0 ms for T1 , T2 and T1⍴ , respectively, against 1053 ± 47 ms, 50.4 ± 3.9 ms, and 55.9 ± 3.3 ms for T1 modified Look-Locker inversion imaging, T2 gradient and spin echo, and T1⍴ turbo field echo, respectively. CONCLUSION: A cardiac MRF approach for simultaneous quantification of myocardial T1 , T2 , and T1ρ in a single breath-hold MR scan of about 16 seconds has been proposed. The approach has been investigated in phantoms and healthy subjects showing good agreement with reference spin echo measurements and conventional clinical maps.

Imaging of Dysfunctional Elastogenesis in Atherosclerosis Using an Improved Gadolinium-Based Tetrameric MRI Probe Targeted to Tropoelastin.

Dysfunctional elastin turnover plays a major role in the progression of atherosclerotic plaques. Failure of tropoelastin cross-linking into mature elastin leads to the accumulation of tropoelastin within the growing plaque, increasing its instability. Here we present Gd4-TESMA, an MRI contrast agent specifically designed for molecular imaging of tropoelastin within plaques. Gd4-TESMA is a tetrameric probe composed of a tropoelastin-binding peptide (the VVGS-peptide) conjugated with four Gd(III)-DOTA-monoamide chelates. It shows a relaxivity per molecule of 34.0 ± 0.8 mM-1 s-1 (20 MHz, 298 K, pH 7.2), a good binding affinity to tropoelastin (KD = 41 ± 12 µM), and a serum half-life longer than 2 h. Gd4-TESMA accumulates specifically in atherosclerotic plaques in the ApoE-/- murine model of plaque progression, with 2 h persistence of contrast enhancement. As compared to the monomeric counterpart (Gd-TESMA), the tetrameric Gd4-TESMA probe shows a clear advantage regarding both sensitivity and imaging time window, allowing for a better characterization of atherosclerotic plaques.

Assessment of hepatic fatty acids during non-alcoholic steatohepatitis progression using magnetic resonance spectroscopy.

INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver abnormalities including steatosis, steatohepatitis, fibrosis, and cirrhosis. Liver biopsy remains the gold standard method to determine the disease stage in NAFLD but is an invasive and risky procedure. Studies have previously reported that changes in intrahepatic fatty acids (FA) composition are related to the progression of NAFLD, mainly in its early stages. The aim of this study was to characterize the liver FA composition in mice fed a Choline-deficient L-amino-defined (CDAA) diet at different stages of NAFLD using magnetic resonance spectroscopy (MRS). METHODS: We used in-vivo MRS to perform a longitudinal characterization of hepatic FA changes in NAFLD mice for 10 weeks. We validated our findings with ex-vivo MRS, gas chromatography-mass spectrometry and histology. RESULTS: In-vivo and ex-vivo results showed that livers from CDAA-fed mice exhibit a significant increase in liver FA content as well as a change in FA composition compared with control mice. After 4 weeks of CDAA diet, a decrease in polyunsaturated and an increase in monounsaturated FA were observed. These changes were associated with the appearance of early stages of steatohepatitis, confirmed by histology (NAFLD Activity Score (NAS) = 4.5). After 10 weeks of CDAA-diet, the liver FA composition remained stable while the NAS increased further to 6 showing a combination of early and late stages of steatohepatitis. CONCLUSION: Our results suggest that monitoring lipid composition in addition to total water/fat with MRS may yield additional insights that can be translated for non-invasive stratification of high-risk NAFLD patients.

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis.

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of vascular damage and thought to drive atherosclerosis. A method to identify these changes in vivo would be desirable in the clinic. Magnetic resonance imaging (MRI)-based tools and other technologies have enabled a profound understanding of the role of the endothelium in cardiovascular diseases and risk in vivo. There is, however, a need for reproducible and simple approaches for extracting quantifiable data reflective of endothelial damage from a single imaging study. A non-invasive, easy-to-implement, and quantitative MRI workflow was developed to acquire and analyze images that allow the quantification of two imaging biomarkers of arterial endothelial damage (leakiness/permeability and dysfunction). Here, the protocol describes the application of this method in the brachiocephalic artery of atherosclerotic ApoE-/- mice using a clinical MRI scanner. First, late gadolinium enhancement (LGE) and Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping protocols to quantify endothelial leakage using an albumin-binding probe are described. Second, anatomic, and quantitative blood flow sequences to measure endothelial dysfunction, in response to acetylcholine are described. Importantly, the method outlined here allows the acquisition of high-spatial-resolution 3D images with large volumetric coverage enabling accurate segmentation of vessel wall structures to improve inter- and intra-observer variability and to increase reliability and reproducibility. Additionally, it provides quantitative data without the need for high-temporal resolution for complex kinetic modeling, making it model-independent and even allowing for imaging of highly mobile vessels (coronary arteries). Therefore, the approach simplifies and expedites data analysis. Finally, this method can be implemented on different scanners, can be extended to image different arterial beds, and is clinically applicable for use in humans. This method could be used to diagnose and treat patients with atherosclerosis by adopting a precision-medicine approach.

Targeted Molecular Iron Oxide Contrast Agents for Imaging Atherosclerotic Plaque.

Overview: Cardiovascular disease remains a leading cause of death worldwide, with vulnerable plaque rupture the underlying cause of many heart attacks and strokes. Much research is focused on identifying an imaging biomarker to differentiate stable and vulnerable plaque. Magnetic Resonance Imaging (MRI) is a non-ionising and non-invasive imaging modality with excellent soft tissue contrast. However, MRI has relatively low sensitivity (micromolar) for contrast agent detection compared to nuclear imaging techniques. There is also an increasing emphasis on developing MRI probes that are not based on gadolinium chelates because of increasing concerns over associated systemic toxicity and deposits1. To address the sensitivity and safety concerns of gadolinium this project focused on the development of a high relaxivity probe based on superparamagnetic iron oxide nanoparticles for the imaging of atherosclerotic plaque with MRI. With development, this may facilitate differentiating stable and vulnerable plaque in vivo.Aim: To develop a range of MRI contrast agents based on superparamagnetic iron oxide nanoparticles (SPIONs), and test them in a murine model of advanced atherosclerosis. Methods: Nanoparticles of four core sizes were synthesised by thermal decomposition and coated with poly(maleicanhydride-alt-1-octadecene) (PMAO), poly(ethyleneimine) (PEI) or alendronate, then characterised for core size, hydrodynamic size, surface potential and relaxivity. On the basis of these results, one candidate was selected for further studies. In vivo studies using 10 nm PMAO-coated SPIONs were performed in ApoE-/- mice fed a western diet and instrumented with a perivascular cuff on the left carotid artery. Control ApoE-/- mice were fed a normal chow diet and were not instrumented. Mice were scanned on a 3T MR scanner (Philips Achieva) with the novel SPION contrast agent, and an elastin-targeted gadolinium agent that was shown previously to enable visualisation of plaque burden. Histological analysis was undertaken to confirm imaging findings through staining for macrophages, CX3CL1, elastin, tropoelastin, and iron. Results: The lead SPION agent consisted of a 10 nm iron oxide core with poly(maleicanhydride-alt-1-octadecene), (-36.21 mV, r2 18.806 mmol-1/s-1). The irregular faceting of the iron oxide core resulted in high relaxivity and the PMAO provided a foundation for further functionalisation on surface -COOH groups. The properties of the contrast agent, including the negative surface charge and hydrodynamic size, were designed to maximise circulation time and evade rapid clearance through the renal system or phagocytosis. In vitro testing showed that the SPION agent was non-toxic. In vivo results show that the novel contrast agent accumulates in similar vascular regions to a gadolinium-based contrast agent (Gd-ESMA) targeted to elastin, which accumulates in plaque. There was a significant difference in SPION signal between the instrumented and the contralateral non-instrumented vessels in diseased mice (p = 0.0411, student's t-test), and between the instrumented diseased vessel and control vessels (p = 0.0043, 0.0022, student's t-test). There was no significant difference between the uptake of either contrast agent between stable and vulnerable plaques (p = 0.3225, student's t-test). Histological verification was used to identify plaques, and Berlin Blue staining confirmed the presence of nanoparticle deposits within vulnerable plaques and co-localisation with macrophages. Conclusion: This work presents a new MRI contrast agent for atherosclerosis which uses an under-explored surface ligand, demonstrating promising properties for in vivo behaviour, is still in circulation 24 hours post-injection with limited liver uptake, and shows good accumulation in a murine plaque model.

Tropoelastin: an in vivo imaging marker of dysfunctional matrix turnover during abdominal aortic dilation.

AIMS: Dysfunctional matrix turnover is present at sites of abdominal aortic aneurysm (AAA) and leads to the accumulation of monomeric tropoelastin rather than cross-linked elastin. We used a gadolinium-based tropoelastin-specific magnetic resonance contrast agent (Gd-TESMA) to test whether quantifying regional tropoelastin turnover correlates with aortic expansion in a murine model. The binding of Gd-TESMA to excised human AAA was also assessed. METHODS AND RESULTS: We utilized the angiotensin II (Ang II)-infused apolipoprotein E gene knockout (ApoE-/-) murine model of aortic dilation and performed in vivo imaging of tropoelastin by administering Gd-TESMA followed by late gadolinium enhancement (LGE) magnetic resonance imaging (MRI) and T1 mapping at 3 T, with subsequent ex vivo validation. In a cross-sectional study (n = 66; control = 11, infused = 55) we found that Gd-TESMA enhanced MRI was elevated and confined to dilated aortic segments (control: LGE=0.13 ± 0.04 mm2, control R1= 1.1 ± 0.05 s-1 vs. dilated LGE =1.0 ± 0.4 mm2, dilated R1 =2.4 ± 0.9 s-1) and was greater in segments with medium (8.0 ± 3.8 mm3) and large (10.4 ± 4.1 mm3) compared to small (3.6 ± 2.1 mm3) vessel volume. Furthermore, a proof-of-principle longitudinal study (n = 19) using Gd-TESMA enhanced MRI demonstrated a greater proportion of tropoelastin: elastin expression in dilating compared to non-dilating aortas, which correlated with the rate of aortic expansion. Treatment with pravastatin and aspirin (n = 10) did not reduce tropoelastin turnover (0.87 ± 0.3 mm2 vs. 1.0 ± 0.44 mm2) or aortic dilation (4.86 ± 2.44 mm3 vs. 4.0 ± 3.6 mm3). Importantly, Gd-TESMA-enhanced MRI identified accumulation of tropoelastin in excised human aneurysmal tissue (n = 4), which was confirmed histologically. CONCLUSION: Tropoelastin MRI identifies dysfunctional matrix remodelling that is specifically expressed in regions of aortic aneurysm or dissection and correlates with the development and rate of aortic expansion. Thus, it may provide an additive imaging marker to the serial assessment of luminal diameter for surveillance of patients at risk of or with established aortopathy.

Water-fat Dixon cardiac magnetic resonance fingerprinting.

PURPOSE: Cardiac magnetic resonance fingerprinting (cMRF) has been recently introduced to simultaneously provide T1 , T2 , and M0 maps. Here, we develop a 3-point Dixon-cMRF approach to enable simultaneous water specific T1 , T2 , and M0 mapping of the heart and fat fraction (FF) estimation in a single breath-hold scan. METHODS: Dixon-cMRF is achieved by combining cMRF with several innovations that were previously introduced for other applications, including a 3-echo GRE acquisition with golden angle radial readout and a high-dimensional low-rank tensor constrained reconstruction to recover the highly undersampled time series images for each echo. Water-fat separation of the Dixon-cMRF time series is performed to allow for water- and fat-specific T1 , T2 , and M0 estimation, whereas FF estimation is extracted from the M0 maps. Dixon-cMRF was evaluated in a standardized T1 -T2 phantom, in a water-fat phantom, and in healthy subjects in comparison to current clinical standards: MOLLI, SASHA, T2 -GRASE, and 6-point Dixon proton density FF (PDFF) mapping. RESULTS: Dixon-cMRF water T1 and T2 maps showed good agreement with reference T1 and T2 mapping techniques (R2 > 0.99 and maximum normalized RMSE ~5%) in a standardized phantom. Good agreement was also observed between Dixon-cMRF FF and reference PDFF (R2 > 0.99) and between Dixon-cMRF water T1 and T2 and water selective T1 and T2 maps (R2 > 0.99) in a water-fat phantom. In vivo Dixon-cMRF water T1 values were in good agreement with MOLLI and water T2 values were slightly underestimated when compared to T2 -GRASE. Average myocardium septal T1 values were 1129 ± 38 ms, 1026 ± 28 ms, and 1045 ± 32 ms for SASHA, MOLLI, and the proposed water Dixon-cMRF. Average T2 values were 51.7 ± 2.2 ms and 42.8 ± 2.6 ms for T2 -GRASE and water Dixon-cMRF, respectively. Dixon-cMRF FF maps showed good agreement with in vivo PDFF measurements (R2 > 0.98) and average FF in the septum was measured at 1.3%. CONCLUSION: The proposed Dixon-cMRF allows to simultaneously quantify myocardial water T1 , water T2 , and FF in a single breath-hold scan, enabling multi-parametric T1 , T2 , and fat characterization. Moreover, reduced T1 and T2 quantification bias caused by water-fat partial volume was demonstrated in phantom experiments.

Simultaneous Assessment of Cardiac Inflammation and Extracellular Matrix Remodeling after Myocardial Infarction.

Background: Optimal healing of the myocardium following myocardial infarction (MI) requires a suitable degree of inflammation and its timely resolution, together with a well-orchestrated deposition and degradation of extracellular matrix (ECM) proteins. Methods and Results: MI and SHAM-operated animals were imaged at 3,7,14 and 21 days with 3T magnetic resonance imaging (MRI) using a 19F/1H surface coil. Mice were injected with 19F-perfluorocarbon (PFC) nanoparticles to study inflammatory cell recruitment, and with a gadolinium-based elastin-binding contrast agent (Gd-ESMA) to evaluate elastin content. 19F MRI signal co-localized with infarction areas, as confirmed by late-gadolinium enhancement, and was highest 7days post-MI, correlating with macrophage content (MAC-3 immunohistochemistry) (ρ=0.89,P<0.0001). 19F quantification with in vivo (MRI) and ex vivo nuclear magnetic resonance (NMR) spectroscopy correlated linearly (ρ=0.58,P=0.020). T1 mapping after Gd-ESMA injection showed increased relaxation rate (R1) in the infarcted regions and was significantly higher at 21days compared with 7days post-MI (R1[s-1]:21days=2.8 [IQR,2.69-3.30] vs 7days=2.3 [IQR,2.12-2.5], P<0.05), which agreed with an increased tropoelastin content (ρ=0.89, P<0.0001). The predictive value of each contrast agent for beneficial remodeling was evaluated in a longitudinal proof-of-principle study. Neither R1 nor 19F at day 7 were significant predictors for beneficial remodeling (P=0.68;P=0.062). However, the combination of both measurements (R1<2.34Hz and 0.55≤19F≤1.85) resulted in an odds ratio of 30.0 (CI95%:1.41-638.15;P=0.029) for favorable post-MI remodeling. Conclusions: Multinuclear 1H/19F MRI allows the simultaneous assessment of inflammation and elastin remodeling in a murine MI model. The interplay of these biological processes affects cardiac outcome and may have potential for improved diagnosis and personalized treatment.

Tropoelastin: A novel marker for plaque progression and instability.

Background: Elastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques. We developed gadolinium-labeled tropoelastin-specific magnetic resonance contrast agents (Gd-TESMAs) for tropoelastin imaging in animal models. Methods and Results: Two peptides, VVGSPSAQDEASPLS and YPDHVQYTHY were selected to target tropoelastin. In vitro binding, relaxivity, and biodistribution experiments enabled characterization of the probes and selecting the best candidate for in vivo MRI. MRI was performed in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice and New Zealand white rabbits with stable and rupture-prone plaques using Gd-TESMA. Additionally, human carotid endarterectomy specimens were imaged ex vivo. The VVGSPSAQDEASPLS-based probe discriminated between tropoelastin and cross-linked elastin (64±7% vs 1±2%, P=0.001), had high in vitro relaxivity in solution (r1-free=11.7±0.6mM-1s-1, r1-bound to tropoelastin = 44±1mM-1s-1) and favorable pharmacokinetics. In vivo mice vascular enhancement (4wks=0.13±0.007mm2, 8wks=0.22±0.01mm2, 12wks=0.33±0.01mm2, P<0.001) and R1 relaxation rate (4wks=0.90±0.01 s-1, 8wks=1.40±0.03 s-1, 12wks=1.87±0.04s-1, P<0.001) increased with atherosclerosis progression after Gd-TESMA injection. Conversely, statin-treated (0.13±0.01mm2, R1 =1.37±0.03s-1) and control (0.10±0.005mm2, R1 =0.87±0.05s-1) mice showed less enhancement. Rupture-prone rabbit plaques had higher R1 relaxation rate compared with stale plaques (R1=2.26±0.1s-1vs R1=1.43±0.02s-1, P=0.001), after administration of Gd-TESMA that allowed detection of rupture-prone plaques with high sensitivity (84.4%) and specificity (92.3%). Increased vascular R1 relaxation rate was observed in carotid endarterectomy plaques after soaking (R1pre= 1.1±0.26 s-1 vs R1post= 3.0±0.1s-1, P=0.01). Ex vivo analyses confirmed the MRI findings and showed uptake of the contrast agent to be specific for tropoelastin. Conclusions: MRI of tropoelastin provides a novel biomarker for atherosclerotic plaque progression and instability.

MRI with gadofosveset: A potential marker for permeability in myocardial infarction.

BACKGROUND AND AIMS: Acute ischemia is associated with myocardial endothelial damage and microvessel formation, resulting in leakage of plasma albumin into the myocardial extravascular space. In this study, we tested whether an albumin-binding intravascular contrast agent (gadofosveset) allows for improved quantification of myocardial permeability compared to the conventional extracellular contrast agent Gd-DTPA using late gadolinium enhancement (LGE) and T1 mapping in vivo. METHODS: MI was induced in C57BL/6 mice (n = 6) and cardiac magnetic resonance imaging (CMR) was performed at 3, 10 and 21 days post-MI using Gd-DTPA and 24 h later using gadofosveset. Functional, LGE and T1 mapping protocols were performed 45 min post-injection of the contrast agent. RESULTS: LGE images showed that both contrast agents provided similar measurements of infarct area at all time points following MI. Importantly, the myocardial R1 measurements after administration of gadofosveset were higher in the acute phase-day 3 (R1 [s-1] = 6.29 ±â€¯0.29) compared to the maturation phase-days 10 and 21 (R1 [s-1] = 4.76 ±â€¯0.30 and 4.48 ±â€¯0.14), suggesting that the uptake of this agent could be used to stage myocardial remodeling. No differences in myocardial R1 were observed after administration of Gd-DTPA at different time points post-MI (R1 [s-1] = 3d: 3.77 ±â€¯0.37; 10d: 2.74 ±â€¯0.06; 21d: 3.35 ±â€¯0.26). The MRI results were validated by ex vivo histology that showed albumin leakage in the myocardium in the acute phase and microvessel formation at later stages. CONCLUSIONS: We demonstrate the merits of an albumin-binding contrast agent for monitoring changes in myocardial permeability between acute ischemia and chronic post-MI myocardial remodeling.

Increased Vascular Permeability Measured With an Albumin-Binding Magnetic Resonance Contrast Agent Is a Surrogate Marker of Rupture-Prone Atherosclerotic Plaque.

BACKGROUND: Compromised structural integrity of the endothelium and higher microvessel density increase vascular permeability. We investigated whether vascular permeability measured in vivo by magnetic resonance imaging using the albumin-binding contrast agent, gadofosveset, is a surrogate marker of rupture-prone atherosclerotic plaque in a rabbit model. METHODS AND RESULTS: New Zealand white rabbits (n=10) were rendered atherosclerotic by cholesterol-diet and endothelial denudation. Plaque rupture was triggered with Russell's viper venom and histamine. Animals were imaged pre-triggering, at 3 and 12 weeks, to quantify plaque area, vascular permeability, vasodilation, and stiffness and post-triggering to identify thrombus. Plaques identified on the pretrigger scans were classified as stable or rupture-prone based on the absence or presence of thrombus on the corresponding post-trigger magnetic resonance imaging, respectively. All rabbits had developed atherosclerosis, and 60% had ruptured plaques. Rupture-prone plaques had higher vessel wall relaxation rate (R1; 2.30±0.5 versus 1.86±0.3 s-1; P<0.001), measured 30 minutes after gadofosveset administration, and higher R1/plaque area ratio (0.70±0.06 versus 0.47±0.02, P= 0.01) compared with stable plaque at 12 weeks. Rupture-prone plaques had higher percent change in R1 between the 3 and 12 weeks compared with stable plaque (50.80±7.2% versus 14.22±2.2%; P<0.001). Immunohistochemistry revealed increased vessel wall albumin and microvessel density in diseased aortas and especially in ruptured plaque. Electron microscopy showed lack of structural integrity in both luminal and microvascular endothelium in diseased vessels. Functionally, the intrinsic vasodilation of the vessel wall decreased at 12 weeks compared with 3 weeks (18.60±1.0% versus 23.43±0.8%; P<0.001) and in rupture-prone compared with stable lesions (16.40±2.0% versus 21.63±1.2%; P<0.001). Arterial stiffness increased at 12 weeks compared with 3 weeks (5.00±0.1 versus 2.53±0.2 m/s; P<0.001) both in animals with stable and rupture-prone lesions. CONCLUSIONS: T1 mapping using an albumin-binding contrast agent (gadofosveset) could quantify the changes in vascular permeability associated with atherosclerosis progression and rupture-prone plaques.

Assessment of Myocardial Remodeling Using an Elastin/Tropoelastin Specific Agent with High Field Magnetic Resonance Imaging (MRI).

BACKGROUND: Well-defined inflammation, proliferation, and maturation phases orchestrate the remodeling of the injured myocardium after myocardial infarction (MI) by controlling the formation of new extracellular matrix. The extracellular matrix consists mainly of collagen but also fractions of elastin. It is thought that elastin is responsible for maintaining elastic properties of the myocardium, thus reducing the risk of premature rupture. An elastin/tropoelastin-specific contrast agent (Gd-ESMA) was used to image tropoelastin and mature elastin fibers for in vivo assessment of extracellular matrix remodeling post-MI. METHODS AND RESULTS: Gd-ESMA enhancement was studied in a mouse model of myocardial infarction using a 7 T MRI scanner and results were compared to those achieved after injection of a nonspecific control contrast agent, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). In the infarcted tissue, Gd-ESMA uptake (measured as R1 relaxation rate) steadily increased from day 3 to day 21 as a result of the synthesis of elastin/tropoelastin. R1 values were in good agreement with histological findings. A similar R1 behavior was observed in the remote myocardium. No mature cross-linked elastin was found at any time point. In contrast, Gd-DTPA uptake was only observed in the infarct with no changes in R1 values between 3 and 21 days post-MI. CONCLUSIONS: We demonstrate the feasibility of in vivo imaging of extracellular matrix remodeling post-MI using a tropoelastin/elastin binding MR contrast agent, Gd-ESMA. We found that tropoelastin is the main contributor to the increased MRI signal at late stages of MI where its augmentation in areas of infarction was in good agreement with the R1 increase.

Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration.

BACKGROUND AND PURPOSE: There are conflicting data regarding whether netrin-1 retards or accelerates atherosclerosis progression, as it can lead either to monocyte repulsion from or retention within plaques depending on its cellular source. We investigated the effect of aspirin, which is widely used in cardiovascular prophylaxis, on the synthesis of different isoforms of netrin-1 by endothelial cells under pro-inflammatory conditions, and defined the net effect of aspirin-dependent systemic modulation of netrin-1 on atherosclerosis progression. EXPERIMENTAL APPROACH: Netrin-1 synthesis was studied in vitro using human endothelial cells stimulated with TNF-α, with or without aspirin treatment. In vivo experiments were conducted in ApoE(-/-) mice fed with a high-fat diet (HFD), receiving either aspirin or clopidogrel. KEY RESULTS: TNF-α-induced NF-κB activation up-regulated the nuclear isoform of netrin-1, while simultaneously reducing secreted netrin-1. Down-regulation of the secreted isoform compromised the chemorepellent action of the endothelium against monocyte chemotaxis. Aspirin counteracted TNF-α-mediated effects on netrin-1 synthesis by endothelial cells through COX-dependent inhibition of NF-κB and concomitant histone hyperacetylation. Administration of aspirin to ApoE(-/-) mice on HFD increased blood and arterial wall levels of netrin-1 independently of its effects on platelets, accompanied by reduced plaque size and content of monocytes/macrophages, compared with untreated or clopidogrel-treated mice. In vivo blockade of netrin-1 enhanced monocyte plaque infiltration in aspirin-treated ApoE(-/-) mice. CONCLUSIONS AND IMPLICATIONS: Aspirin counteracts down-regulation of secreted netrin-1 induced by pro-inflammatory stimuli in endothelial cells. The aspirin-dependent increase of netrin-1 in ApoE(-/-) mice exerts anti-atherogenic effects by preventing arterial accumulation of monocytes.

Monitoring vascular permeability and remodeling after endothelial injury in a murine model using a magnetic resonance albumin-binding contrast agent.

BACKGROUND: Despite the beneficial effects of vascular interventions, these procedures may damage the endothelium leading to increased vascular permeability and remodeling. Re-endothelialization of the vessel wall, with functionally and structurally intact cells, is controlled by endothelial nitric oxide synthase (NOS3) and is crucial for attenuating adverse effects after injury. We investigated the applicability of the albumin-binding MR contrast agent, gadofosveset, to noninvasively monitor focal changes in vascular permeability and remodeling, after injury, in NOS3-knockout (NOS3(-/-)) and wild-type (WT) mice in vivo. METHODS AND RESULTS: WT and NOS3(-/-) mice were imaged at 7, 15, and 30 days after aortic denudation or sham-surgery. T1 mapping (R1=1/T1, s(-1)) and delayed-enhanced MRI were used as measurements of vascular permeability (R1) and remodeling (vessel wall enhancement, mm(2)) after gadofosveset injection, respectively. Denudation resulted in higher vascular permeability and vessel wall enhancement 7 days after injury in both strains compared with sham-operated animals. However, impaired re-endothelialization and increased neovascularization in NOS3(-/-) mice resulted in significantly higher R1 at 15 and 30 days post injury compared with WT mice that showed re-endothelialization and lack of neovascularization (R1 [s(-1)]=15 days: NOS3 (-/-)4.02 [interquartile range, IQR, 3.77-4.41] versus WT2.39 [IQR, 2.35-2.92]; 30 days: NOS3 (-/-)4.23 [IQR, 3.94-4.68] versus WT2.64 [IQR, 2.33-2.80]). Similarly, vessel wall enhancement was higher in NOS3(-/-) but recovered in WT mice (area [mm(2)]=15 days: NOS3 (-/-)5.20 [IQR, 4.68-6.80] versus WT2.13 [IQR, 0.97-3.31]; 30 days: NOS3 (-/-)7.35 [IQR, 5.66-8.61] versus WT1.60 [IQR, 1.40-3.18]). Ex vivo histological studies corroborated the MRI findings. CONCLUSIONS: We demonstrate that increased vascular permeability and remodeling, after injury, can be assessed noninvasively using an albumin-binding MR contrast agent and may be used as surrogate markers for evaluating the healing response of the vessel wall after injury.

Nitric oxide prevents aortic neointimal hyperplasia by controlling macrophage polarization.

OBJECTIVE: Nitric oxide synthase 3 (NOS3) prevents neointima hyperplasia by still unknown mechanisms. To demonstrate the significance of endothelial nitric oxide in the polarization of infiltrated macrophages through the expression of matrix metalloproteinase (MMP)-13 in neointima formation. APPROACH AND RESULTS: After aortic endothelial denudation, NOS3 null mice show elevated neointima formation, detecting increased mobilization of LSK (lineage-negative [Lin]-stem-cell antigen 1 [SCA1]+KIT+) progenitor cells, and high ratios of M1 (proinflammatory) to M2 (resolving) macrophages, accompanied by high expression of interleukin-5, interleukin-6, MCP-1 (monocyte chemoattractant protein), VEGF (vascular endothelial growth factor), GM-CSF (granulocyte-macrophage colony stimulating factor), interleukin-1ß, and interferon-γ. In conditional c-Myc knockout mice, in which M2 polarization is defective, denuded aortas showed extensive wall thickening as well. Conditioned medium from NOS3-deficient endothelium induced extensive repolarization of M2 macrophages to an M1 phenotype, and vascular smooth muscle cells proliferated and migrated faster in conditioned medium from M1 macrophages. Among the different proteins participating in cell migration, MMP-13 was preferentially expressed by M1 macrophages. M1-mediated vascular smooth muscle cell migration was inhibited when macrophages were isolated from MMP-13-deficient mice, whereas exogenous administration of MMP-13 to vascular smooth muscle cell fully restored migration. Excess vessel wall thickening in mice lacking NOS3 was partially reversed by simultaneous deletion of MMP-13, indicating that NOS3 prevents neointimal hyperplasia by preventing MMP-13 activity. An excess of M1-polarized macrophages that coexpress MMP-13 was also detected in human carotid samples from endarterectomized patients. CONCLUSIONS: These findings indicate that at least M1 macrophage-mediated expression of MMP-13 in NOS3 null mice induces neointima formation after vascular injury, suggesting that MMP-13 may represent a new promising target in vascular disease.

Inhibition of MYC in macrophages: tumor vs inflammation-related diseases.

Inhibition of MYC has been postulated as one of the most promising anti-tumoral therapies. However, if some anti-inflammatory cells express MYC, would an anti-tumoral treatment targeting MYC facilitate subsequent inflammation-related disorders?

NOD1 activation induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis.

The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-ß pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-ß pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.