Vascular smooth muscle cells in low SYNTAX scores coronary artery disease exhibit proinflammatory transcripts and proteins correlated with IL1B activation.

BACKGROUND AND AIMS: The SYNTAX score is clinically validated to stratify number of lesions and pattern of CAD. A better understanding of the underlying molecular mechanisms influencing the pattern and complexity of coronary arteries lesions among CAD patients is needed. METHODS: Human arterial biopsies from 49 patients (16 low-SYNTAX-score (LSS, <23), 16 intermediate-SYNTAX-score (ISS, 23 to 32) and 17 high-SYNTAX-score (HSS, >32)) were evaluated using Affymetrix GeneChip® Human Genome U133 Plus 2.0 microarray. The data were validated by Next-Generation Sequencing (NGS). Primary VSMC from patients with low and high SYNTAX scores were isolated and compared using immunohistochemistry, qPCR and immunoblotting to confirm mRNA and proteomic results. RESULTS: The IL1B was verified as the top upstream regulator of 47 inflammatory DEGs in LSS patients and validated by another sets of patient samples using NGS analysis. The upregulated expression of IL1B was translated to increased level of IL1ß protein in the LSS tissue based on immunohistochemical quantitative analysis. Plausibility of idea that IL1B in the arterial wall could be originated from VSMC was checked by exposing culture to proinflammatory conditions where IL1B came out as the top DEG (logFC = 7.083, FDR = 1.38 × 10-114). The LSS patient-derived primary VSMCs confirmed higher levels of IL1B mRNA and protein. CONCLUSIONS: LSS patients could represent a group of patients where IL1B could play a substantial role in disease pathogenesis. The LSS group could represent a plausible cohort of patients for whom anti-inflammatory therapy could be considered.

Development of Molecular Magnetic Resonance Imaging Tools for Longitudinal Tracking of Carotid Atherosclerotic Disease Using Fast Imaging with Steady-State Precession.

Identification of patients with high-risk asymptomatic carotid plaques remains a challenging but essential step in stroke prevention. Current selection criteria for intervention in carotid disease are still determined by symptomatology and degree of luminal stenosis. This strategy has been less effective in identifying the high-risk asymptomatic individual patients. Inflammation is the key factor that drives plaque instability causing clinical sequelae. Currently, there is no imaging tool in routine clinical practice to assess the inflammatory status within atherosclerotic plaques. Herein we describe the development of a novel molecular magnetic resonance imaging (MRI) strategy to interrogate plaque inflammation, and hence its vulnerability in vivo, using dual-targeted iron particle-based probes and fast imaging with steady-state precession (FISP) sequence, adding further prognostic information to luminal stenosis alone. A periarterial cuff was used to generate high-risk plaques at specific timepoints and location of the carotid artery in an apolipoprotein-E-deficient mouse model. Using this platform, we demonstrated that in vivo dual-targeted iron particles with enhanced FISP can (i) target and characterise high-risk vulnerable plaques and (ii) quantitatively report and track the inflammatory activity within carotid plaques longitudinally. This molecular imaging tool may permit (i) accurate monitoring of the risk of carotid plaques and (ii) timely identification of high-risk asymptomatic patients for prophylactic carotid intervention, achieving early stroke prevention.

Translational Molecular Imaging Tool of Vulnerable Carotid Plaque: Evaluate Effects of Statin Therapy on Plaque Inflammation and American Heart Association-Defined Risk Levels in Cuff-Implanted Apolipoprotein E-Deficient Mice.

Identification of high-risk carotid plaques in asymptomatic patients remains a challenging but crucial step in stroke prevention. The challenge is to accurately monitor the development of high-risk carotid plaques and promptly identify patients, who are unresponsive to best medical therapy, and hence targeted for carotid surgical interventions to prevent stroke. Inflammation is a key operator in destabilisation of plaques prior to clinical sequelae. Currently, there is a lack of imaging tool in routine clinical practice, which allows assessment of inflammatory activity within the atherosclerotic plaque. Herein, we have used a periarterial cuff to generate a progressive carotid atherosclerosis model in apolipoprotein E-deficient mice. This model produced clinically relevant plaques with different levels of risk, fulfilling American Heart Association (AHA) classification, at specific timepoints and locations, along the same carotid artery. Exploiting this platform, we have developed smart molecular magnetic resonance imaging (MRI) probes consisting of dual-targeted microparticles of iron oxide (DT-MPIO) against VCAM-1 and P-selectin, to evaluate the anti-inflammatory effect of statin therapy on progressive carotid atherosclerosis. We demonstrated that in vivo DT-MPIO-enhanced MRI can (i) quantitatively track plaque inflammation from early to advanced stage; (ii) identify and characterise high-risk inflamed, vulnerable plaques; and (iii) monitor the response to statin therapy longitudinally. Moreover, this molecular imaging-defined therapeutic response was validated using AHA classification of human plaques, a clinically relevant parameter, approximating the clinical translation of this tool. Further development and translation of this molecular imaging tool into the clinical arena may potentially facilitate more accurate risk stratification, permitting timely identification of the high-risk patients for prophylactic carotid intervention, affording early opportunities for stroke prevention in the future.

Development of Molecular Magnetic Resonance Imaging Tools for Risk Stratification of Carotid Atherosclerotic Disease Using Dual-Targeted Microparticles of Iron Oxide.

Identification of patients with high-risk asymptomatic carotid plaques remains a challenging but crucial step in stroke prevention. Inflammation is the key factor that drives plaque instability. Currently, there is no imaging tool in routine clinical practice to assess the inflammatory status within atherosclerotic plaques. We have developed a molecular magnetic resonance imaging (MRI) tool to quantitatively report the inflammatory activity in atherosclerosis using dual-targeted microparticles of iron oxide (DT-MPIO) against P-selectin and VCAM-1 as a smart MRI probe. A periarterial cuff was used to generate plaques with varying degree of phenotypes, inflammation and risk levels at specific locations along the same single carotid artery in an Apolipoprotein-E-deficient mouse model. Using this platform, we demonstrated that in vivo DT-MPIO-enhanced MRI can (i) target high-risk vulnerable plaques, (ii) differentiate the heterogeneity (i.e. high vs intermediate vs low-risk plaques) within the asymptomatic plaque population and (iii) quantitatively report the inflammatory activity of local plaques in carotid artery. This novel molecular MRI tool may allow characterisation of plaque vulnerability and quantitative reporting of inflammatory status in atherosclerosis. This would permit accurate risk stratification by identifying high-risk asymptomatic individual patients for prophylactic carotid intervention, expediting early stroke prevention and paving the way for personalised management of carotid atherosclerotic disease.

Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.

OBJECTIVE: Identification of patients with high-risk asymptomatic carotid plaques remains an elusive but essential step in stroke prevention. Inflammation is a key process in plaque destabilization and a prelude to clinical sequelae. There are currently no clinical imaging tools to assess the inflammatory activity within plaques. This study characterized inflammation in atherosclerosis using dual-targeted microparticles of iron oxide (DT-MPIO) as a magnetic resonance imaging (MRI) probe. METHODS: DT-MPIO were used to detect and characterize inflammatory markers, vascular cell adhesion molecule 1 (VCAM-1). and P-selectin on (1) tumor necrosis factor-α-treated cells by immunocytochemistry and (2) aortic root plaques of apolipoprotein-E deficient mice by in vivo MRI. Furthermore, apolipoprotein E-deficient mice with focal carotid plaques of different phenotypes were developed by means of periarterial cuff placement to allow in vivo molecular MRI using these probes. The association between biomarkers and the magnetic resonance signal in different contrast groups was assessed longitudinally in these models. RESULTS: Immunocytochemistry confirmed specificity and efficacy of DT-MPIO to VCAM-1 and P-selectin. Using this in vivo molecular MRI strategy, we demonstrated (1) the DT-MPIO-induced magnetic resonance signal tracked with VCAM-1 (r = 0.69; P = .014), P-selectin (r = 0.65; P = .022), and macrophage content (r = 0.59; P = .045) within aortic root plaques and (2) high-risk inflamed plaques were distinguished from noninflamed plaques in the murine carotid artery within a practical clinical imaging time frame. CONCLUSIONS: These molecular MRI probes constitute a novel imaging tool for in vivo characterization of plaque vulnerability and inflammatory activity in atherosclerosis. Further development and translation into the clinical arena will facilitate more accurate risk stratification in carotid atherosclerotic disease in the future.

MRI detection of endothelial cell inflammation using targeted superparamagnetic particles of iron oxide (SPIO).

BACKGROUND: There is currently no clinical imaging technique available to assess the degree of inflammation associated with atherosclerotic plaques. This study aims to develop targeted superparamagnetic particles of iron oxide (SPIO) as a magnetic resonance imaging (MRI) probe for detecting inflamed endothelial cells. METHODS: The in vitro study consists of the characterisation and detection of inflammatory markers on activated endothelial cells by immunocytochemistry and MRI using biotinylated anti-P-selectin and anti-VCAM-1 (vascular cell adhesion molecule 1) antibody and streptavidin conjugated SPIO. RESULTS: Established an in vitro cellular model of endothelial inflammation induced with TNF-α (tumor necrosis factor alpha). Inflammation of endothelial cells was confirmed with both immunocytochemistry and MRI. These results revealed both a temporal and dose dependent expression of the inflammatory markers, P-selectin and VCAM-1, on exposure to TNF-α. CONCLUSION: This study has demonstrated the development of an in vitro model to characterise and detect inflamed endothelial cells by immunocytochemistry and MRI. This will allow the future development of contrast agents and protocols for imaging vascular inflammation in atherosclerosis. This work may form the basis for a translational study to provide clinicians with a novel tool for the in vivo assessment of atherosclerosis.