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-1β, 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.pre-print262 K

BS43 A new collagen III-specific MRI imaging probe to assess cardiac fibrosis

Heart failure (HF) has reached epidemic proportions, affecting approximately 64 million people globally and is the main cause of death and disability.1 Myocardial fibrosis, characterised by changes in the amount and/or distribution of collagen I and III, impairs cardiac function and relates to adverse outcomes of HF.2 3 Clinically, we rely on indirect or surrogate measurements of collagen in the myocardium and current targeted molecular imaging probes are limited to collagen I. Here, we report the discovery of a peptide selective for collagen III and a strategy to develop an imaging probe with superior properties for in vivo molecular magnetic resonance imaging (MRI) applications. A small peptide was screened and selected from a library of peptides with potential to bind to collagen identified based on protein-protein interaction studies. The peptide was conjugated to a DOTA-chelator and labelled with Europium [Eu(III)] for in vitro binding assays; gallium (68Ga) for in vivo PET/CT biodistribution; and gadolinium [Gd(III)] for in vivo MRI studies. The probe was further modified to increase the number of Gd(III) per peptide (from one to four) to amplify and prolong the MRI signal. The probe was validated using a surgical mouse model of myocardial infarction (MI). In vivo MRI was performed at days 10 and 21 post-MI (n=7). Imaging findings were validated with tissue analysis. A negative control probe, carrying a scrambled peptide sequence was used. All MRI experiments were performed at a 3 Tesla clinical MRI scanner. In vitro binding assays showed that the probe has a good affinity towards collagen III (Kd= 5.2±1.3µM) that is in the ideal range for a molecular imaging probe.4 Lack of binding of the scrambled probe (negative control) proved the specificity our probe (figure 1A). In vivo PET/CT biodistribution showed favourable pharmacokinetics with fast blood clearance and no unspecific binding (figure 1B). In vivo cardiac MRI showed selective late gadolinium enhancement (LGE) of the fibrotic scar at day 10 which decreased by day 21. This observation is expected as collagen III naturally gets replaced by collagen I at the later stages of cardiac fibrosis. The imaging data are validated histologically showing co-localisation of the MRI signal with collagen III (green colour) at day 10 and reduction of collagen III at day 21 (figure 2). Importantly, no enhancement was observed using the negative control probe and a clinically approved non-collagen targeting probe (Gadovist). We have developed a new molecular imaging probe specific for collagen type III. Using this probe, we have successfully imaged - previously undetectable - collagen III in cardiac fibrosis. This approach may enable early detection and characterisation of cardiac fibrosis in vivo allowing staging of disease and monitoring of therapie

Animal Models of Cardiovascular Diseases

Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models have contributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of these pathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiac diseases, and the same pathology have been successfully recreated in different species, including small and big animal models of disease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficult to match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the human complication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specialized personnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selected that best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expanding and occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible to devise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases

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

Extracellular Matrix Targeted MRI Probes

International audienceDysregulated remodeling of the extracellular matrix (ECM) can lead to excessive accumulation of ECM proteins (primarily collagen, elastin/ tropoelastin, fibronectin and fibrin) resulting in tissue fibrosis. In many pathologies, changes in the molecular pattern of ECM components have been related to the progression and severity of fibrosis. Thus, magnetic resonance imaging (MRI) probes sensing specific ECM components hold promise for accurate staging of fibrotic diseases. This paper focuses on gadolinium-based contrast agents (GBCA) targeted to ECM components, including structural proteins and enzymes. According to available examples, they can be grouped into: 1) GBCA conjugated to targeting vectors that recognize and noncovalently bind to specific sites on the molecular target; 2) GBCA carrying a reactive chemical function able to bind covalently to the complementary chemical function of the molecular target; and 3) enzyme-responsive probes, whose relaxivity and pharmacokinetics change after enzymatic processing. Pros and cons of each approach are discussed

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.Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

, and fat fraction characterization with MR fingerprinting

Purpose: To develop a novel simultaneous co-registered T1, T2, (Formula presented.), 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 (Formula presented.) 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 &gt; 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 &lt; 0.05) for T1, T2, and T1⍴ but not for (Formula presented.) and fat fraction, whereas correlation between MRF and reference scans is excellent for each parameter (r2 &gt; 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, (Formula presented.), 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.</p