Spatial lipidomics of coronary atherosclerotic plaque development in a familial hypercholesterolemia swine model.

Coronary atherosclerosis is caused by plaque build-up, with lipids playing a pivotal role in its progression. However, lipid composition and distribution within coronary atherosclerosis remain unknown. This study aims to characterize lipids and investigate differences in lipid composition across disease stages to aid in the understanding of disease progression. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize lipid distributions in coronary artery sections (n = 17) from hypercholesterolemic swine. We performed histology on consecutive sections to classify the artery segments and to investigate colocalization between lipids and histological regions of interest in advanced plaque, including necrotic core and inflammatory cells. Segments were classified as healthy (n = 6), mild (n = 6), and advanced disease (n = 5) artery segments. Multivariate data analysis was employed to find differences in lipid composition between the segment types, and the lipids' spatial distribution was investigated using non-negative matrix factorization (NMF). Through this process, MALDI-MSI detected 473 lipid-related features. NMF clustering described three components in positive ionization mode: triacylglycerides (TAG), phosphatidylcholines (PC), and cholesterol species. In negative ionization mode, two components were identified: one driven by phosphatidylinositol(PI)(38:4), and one driven by ceramide-phosphoethanolamine(36:1). Multivariate data analysis showed the association between advanced disease and specific lipid signatures like PC(O-40:5) and cholesterylester(CE)(18:2). Ether-linked phospholipids and LysoPC species were found to colocalize with necrotic core, and mostly CE, ceramide, and PI species colocalized with inflammatory cells. This study, therefore, uncovers distinct lipid signatures correlated with plaque development and their colocalization with necrotic core and inflammatory cells, enhancing our understanding of coronary atherosclerosis progression.

Regional aortic wall shear stress increases over time in patients with a bicuspid aortic valve.

BACKGROUND: Aortic wall shear stress (WSS) is a known predictor of ascending aortic growth in patients with a bicuspid aortic valve (BAV). The aim of this study was to study regional WSS and changes over time in BAV patients. METHODS: BAV patients and age-matched healthy controls underwent four-dimensional (4D) flow cardiovascular magnetic resonance (CMR). Regional, peak systolic ascending aortic WSS, aortic valve function, aortic stiffness measures, and aortic dimensions were assessed. In BAV patients, 4D flow CMR was repeated after 3 years of follow-up and both at baseline and follow-up computed tomography angiography (CTA) were acquired. Aortic growth (volume increase of ≥5%) was measured on CTA. Regional WSS differences within patients' aorta and WSS changes over time were analyzed using linear mixed-effect models and were associated with clinical parameters. RESULTS: Thirty BAV patients (aged 34 years [interquartile range (IQR) 25-41]) were included in the follow-up analysis. Additionally, another 16 BAV patients and 32 healthy controls (aged 33 years [IQR 28-48]) were included for other regional analyses. Magnitude, axial, and circumferential WSS increased over time (all p < 0.001) irrespective of aortic growth. The percentage of regions exposed to a magnitude WSS >95th percentile of healthy controls increased from 21% (baseline 506/2400 regions) to 31% (follow-up 734/2400 regions) (p < 0.001). WSS angle, a measure of helicity near the aortic wall, decreased during follow-up. Magnitude WSS changes over time were associated with systolic blood pressure, peak aortic valve velocity, aortic valve regurgitation fraction, aortic stiffness indexes, and normalized flow displacement (all p < 0.05). CONCLUSION: An increase in regional WSS over time was observed in BAV patients, irrespective of aortic growth. The increasing WSSs, comprising a larger area of the aorta, warrant further research to investigate the possible predictive value for aortic dissection.

Aortic geometry and long-term outcome in patients with a repaired coarctation.

OBJECTIVE: This study aims to compare aortic morphology between repaired coarctation patients and controls, and to identify aortic morphological risk factors for hypertension and cardiovascular events (CVEs) in coarctation patients. METHODS: Repaired coarctation patients with computed tomography angiography (CTA) or magnetic resonance angiography (MRA) were included, followed-up and compared with sex-matched and age-matched controls. Three-dimensional aortic shape was reconstructed using patients' CTA or MRA, or four-dimensional flow cardiovascular magnetic resonance in controls, and advanced geometrical characteristics were calculated and visualised using statistical shape modelling. In patients, we examined the association of geometrical characteristics with (1) baseline hypertension, using multivariable logistic regression; and (2) cardiovascular events (CVE, composite of aortic complications, coronary artery disease, ventricular arrhythmias, heart failure hospitalisation, stroke, transient ischaemic attacks and cardiovascular death), using multivariable Cox regression. The least absolute shrinkage and selection operator (LASSO) method selected the most informative multivariable model. RESULTS: Sixty-five repaired coarctation patients (23 years (IQR 19-38)) were included, of which 44 (68%) patients were hypertensive at baseline. After a median follow-up of 8.7 years (IQR 4.8-15.4), 27 CVEs occurred in 20 patients. Aortic arch dimensions were smaller in patients compared with controls (diameter p<0.001, wall surface area p=0.026, volume p=0.007). Patients had more aortic arch torsion (p<0.001) and a higher curvature (p<0.001). No geometrical characteristics were associated with hypertension. LASSO selected left ventricular mass, male sex, tortuosity and age for the multivariable model. Left ventricular mass (p=0.014) was independently associated with CVE, and aortic tortuosity showed a trend towards significance (p=0.070). CONCLUSION: Repaired coarctation patients have a smaller aortic arch and a more tortuous course of the aorta compared with controls. Besides left ventricular mass index, geometrical features might be of importance in long-term risk assessment in coarctation patients.

Shear stress is uncoupled from atheroprotective KLK10 in atherosclerotic plaques.

BACKGROUND AND AIMS: Physiological shear stress promotes vascular homeostasis by inducing protective molecules in endothelial cells (EC). However, physiological shear stress has been linked to atherosclerosis progression in some individuals with heightened cardiovascular risk. To address this apparent paradox, we hypothesized that diseased arteries may exhibit reduced responsiveness to the protective effects of physiological shear stress. Consequently, we compared the transcriptome of EC exposed to physiological shear stress in healthy arteries versus atherosclerotic conditions. METHODS: Employing 3D light sheet imaging and computational fluid dynamics, we identified NOS3 as a marker of physiological shear stress in both healthy and atherosclerotic murine arteries. Single-cell RNA sequencing was performed on EC from healthy (C57BL/6) mice, mildly diseased (Apoe-/- normal diet) mice, and highly diseased (Apoe-/- high fat diet) mice. The transcriptomes of Nos3high cells (exposed to physiological shear stress) were compared among the groups. RESULTS: Nos3high EC were associated with several markers of physiological shear stress in healthy arteries. Clustering of Nos3high EC revealed 8 different EC subsets that varied in proportion between healthy and diseased arteries. Cluster-specific nested functional enrichment of gene ontology terms revealed that Nos3high EC in diseased arteries were enriched for inflammatory and apoptotic gene expression. These alterations were accompanied by changes in several mechanoreceptors, including the atheroprotective factor KLK10, which was enriched in Nos3high EC in healthy arteries but markedly reduced in severely diseased arteries. CONCLUSIONS: Physiological shear stress is uncoupled from atheroprotective KLK10 within atherosclerotic plaques. This sheds light on the complex interplay between shear stress, endothelial function, and the progression of atherosclerosis in individuals at risk of cardiovascular complications.

Mechanical wall stress and wall shear stress are associated with atherosclerosis development in non-calcified coronary segments.

BACKGROUND AND AIMS: Atherosclerotic plaque onset and progression are known to be affected by local biomechanical factors. While the role of wall shear stress (WSS) has been studied, the impact of another biomechanical factor, namely mechanical wall stress (MWS), remains poorly understood. In this study, we investigated the association of MWS, independently and combined with WSS, towards atherosclerosis in coronary arteries. METHODS: Thirty-four human coronary arteries were analyzed using near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) and optical coherence tomography (OCT) at baseline and after 12 months. Baseline WSS and MWS were calculated using computational models, and wall thickness (ΔWT) and lipid-rich necrotic core size (ΔLRNC) change were measured in non-calcified coronary segments. The arteries were further divided into 1.5 mm/45° sectors and categorized as plaque-free or plaque sectors. For each category, associations between biomechanical factors (WSS & MWS) and changes in coronary wall (ΔWT & ΔLRNC) were studied using linear mixed models. RESULTS: In plaque-free sectors, higher MWS (p < 0.001) was associated with greater vessel wall growth. Plaque sectors demonstrated wall thickness reduction over time, likely due to medical therapy, where higher levels of WSS and WMS, individually and combined, (p < 0.05) were associated with a greater reduction. Sectors with low MWS combined with high WSS demonstrated the highest LRNC increase (p < 0.01). CONCLUSIONS: In this study, we investigated the association of the (largely-overlooked) biomechanical factor MWS with coronary atherosclerosis, individually and combined with WSS. Our results demonstrated that both MWS and WSS significantly correlate with atherosclerotic plaque initiation and development.

Identifying lipid traces of atherogenic mechanisms in human carotid plaque.

BACKGROUND AND AIMS: Lipids play an important role in atherosclerotic plaque development and are interesting candidate predictive biomarkers. However, the link between circulating lipids, accumulating lipids in the vessel wall, and plaque destabilization processes in humans remains largely unknown. This study aims to provide new insights into the role of lipids in atherosclerosis using lipidomics and mass spectrometry imaging to investigate lipid signatures in advanced human carotid plaque and plasma samples. METHODS: We used lipidomics and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to investigate lipid signatures of advanced human carotid plaque and plasma obtained from patients who underwent carotid endarterectomy (n = 14 out of 17 whose plaque samples were analyzed by DESI-MSI). Multivariate data analysis and unsupervised clustering were applied to identify lipids that were the most discriminative species between different patterns in plaque and plasma. These patterns were interpreted by quantitative comparison with conventional histology. RESULTS: Lipidomics detected more than 300 lipid species in plasma and plaque, with markedly different relative abundances. DESI-MSI visualized the spatial distribution of 611 lipid-related m/z features in plaques, of which 330 m/z features could be assigned based on exact mass, comparison to the lipidomic data, and high mass resolution MSI. Matching spatial lipid patterns to histological areas of interest revealed several molecular species that were colocalized with pertinent disease processes in plaque including specific sphingomyelin and ceramide species with calcification, phospholipids and free fatty acids with inflammation, and triacylglycerols and phosphatidylinositols with fibrin-rich areas. CONCLUSIONS: By comparing lipid species in plaque and plasma, we identified those circulating species that were also prominently present in plaque. Quantitative comparison of lipid spectral patterns with histology revealed the presence of specific lipid species in destabilized plaque areas, corroborating previous in vitro and animal studies.

Wall shear stress-related plaque growth of lipid-rich plaques in human coronary arteries: an near-infrared spectroscopy and optical coherence tomography study.

AIMS: Low wall shear stress (WSS) is acknowledged to play a role in plaque development through its influence on local endothelial function. Also, lipid-rich plaques (LRPs) are associated with endothelial dysfunction. However, little is known about the interplay between WSS and the presence of lipids with respect to plaque progression. Therefore, we aimed to study the differences in WSS-related plaque progression between LRPs, non-LRPs, or plaque-free regions in human coronary arteries. METHODS AND RESULTS: In the present single-centre, prospective study, 40 patients who presented with an acute coronary syndrome successfully underwent near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) and optical coherence tomography (OCT) of at least one non-culprit vessel at baseline and completed a 1-year follow-up. WSS was computed applying computational fluid dynamics to a three-dimensional reconstruction of the coronary artery based on the fusion of the IVUS-segmented lumen with a CT-derived centreline, using invasive flow measurements as boundary conditions. For data analysis, each artery was divided into 1.5 mm/45° sectors. Plaque growth based on IVUS-derived percentage atheroma volume change was compared between LRPs, non-LRPs, and plaque-free wall segments, as assessed by both OCT and NIRS. Both NIRS- and OCT-detected lipid-rich sectors showed a significantly higher plaque progression than non-LRPs or plaque-free regions. Exposure to low WSS was associated with a higher plaque progression than exposure to mid or high WSS, even in the regions classified as a plaque-free wall. Furthermore, low WSS and the presence of lipids had a synergistic effect on plaque growth, resulting in the highest plaque progression in lipid-rich regions exposed to low shear stress. CONCLUSION: This study demonstrates that NIRS- and OCT-detected lipid-rich regions exposed to low WSS are subject to enhanced plaque growth over a 1-year follow-up. The presence of lipids and low WSS proves to have a synergistic effect on plaque growth.

Wall shear stress angle is associated with aortic growth in bicuspid aortic valve patients.

AIMS: Aortic wall shear stress (WSS) distributions in bicuspid aortic valve (BAV) patients have been associated with aortic dilatation, but prospective, longitudinal data are missing. This study assessed differences in aortic WSS distributions between BAV patients and healthy controls and determined the association of WSS with aortic growth in patients. METHODS AND RESULTS: Sixty subjects underwent four-dimensional (4D) flow cardiovascular magnetic resonance of the thoracic aorta (32 BAV patients and 28 healthy controls). Peak velocity, pulse wave velocity, aortic distensibility, peak systolic WSS (magnitude, axial, and circumferential), and WSS angle were assessed. WSS angle is defined as the angle between the WSSmagnitude and WSSaxial component. In BAV patients, three-year computed tomography angiography-based aortic volumetric growth was determined in the proximal and entire ascending aorta. WSSaxial was significantly lower in BAV patients compared with controls (0.93 vs. 0.72 Pa, P = 0.047) and WSScircumferential and WSS angle were significantly higher (0.29 vs. 0.64 Pa and 18° vs. 40°, both P < 0.001). Significant volumetric growth of the proximal ascending aorta occurred in BAV patients (from 49.1 to 52.5 cm3, P = 0.003). In multivariable analysis corrected for baseline aortic volume and diastolic blood pressure, WSS angle was the only parameter independently associated with proximal aortic growth (P = 0.031). In the entire ascending aorta, besides the WSS angle, the WSSmagnitude was also independently associated with growth. CONCLUSION: Increased WSScircumferential and especially WSS angle are typical in BAV patients. WSS angle was found to predict aortic growth. These findings highlight the potential role of WSS measurements in BAV patients to stratify patients at risk for aortic dilation.

Vascular biomechanics and molecular disease activity in the thoracic aorta: a novel imaging method.

AIMS: The influence haemodynamics have on vessel wall pathobiology in aortic disease is incomplete. This aim of this study was to develop a repeatable method for assessing the relationship between aortic wall shear stress (WSS) and disease activity by fusing 4D flow cardiovascular magnetic resonance (CMR) with hybrid positron emission tomography (PET). METHODS AND RESULTS: As part of an ongoing clinical trial, patients with bicuspid aortic valve (BAV) were prospectively imaged with both 18F-sodium fluoride (18F-NaF) PET, a marker of calcification activity, and 4D flow CMR. We developed novel software allowing accurate 3D co-registration and high-resolution comparison of aortic peak systolic WSS and 18F-NaF PET uptake (maximum tissue-to-background ratio). Intra-observer repeatability of both measurements was determined using Bland-Altman plots and intra-class correlation coefficients (ICCs). The relationship between localized WSS and 18F-NaF uptake was analysed using linear mixed-effect models. Twenty-three patients with BAV (median age 50 [44-55] years, 22% female) were included. Intra-observer repeatability for WSS (ICC = 0.92) and 18F-NaF (ICC = 0.91) measurements obtained within 1.4 ± 0.6 cm2 regions of interest was excellent. On multivariable analysis, 18F-NaF PET uptake was independently and negatively associated with WSS as well as diastolic blood pressure (both P < 0.05), adjusted for age. CONCLUSION: Fused assessment of WSS and 18F-NaF PET uptake is feasible and repeatable, demonstrating a clear association between these two factors. This high spatial resolution approach has major potential to advance our understanding of the relationship between vascular haemodynamics and disease activity.

Micro Spectroscopic Photoacoustic (µsPA) imaging of advanced carotid atherosclerosis.

Atherosclerosis is a lipid-driven and an inflammatory disease of the artery walls. The composition of atherosclerotic plaque stratifies the risk of a specific plaque to cause a cardiovascular event. In an optical resolution photoacoustic microscopy setup, of 45 µm resolution, we extracted plaque lipid photoacoustic (PA) spectral signatures of human endarterectomy samples in the range of 1150-1240 nm, using matrix assisted laser desorption ionization mass spectrometry imaging as a reference. We found plaque PA signals to correlate best with sphingomyelins and cholesteryl esters. PA signal spectral variations within the plaque area were compared to reference molecular patterns and absorption spectra of lipid laboratory standards. Variability in the lipid spectroscopic features extracted by principal component analysis of all samples revealed three distinct components with peaks at: 1164, 1188, 1196 and 1210 nm. This result will guide the development of PA-based atherosclerosis disease staging capitalizing on lipidomics of atherosclerotic tissue.

The definition of low wall shear stress and its effect on plaque progression estimation in human coronary arteries.

Wall shear stress (WSS), the frictional force of the blood on the vessel wall, plays a crucial role in atherosclerotic plaque development. Low WSS has been associated with plaque growth, however previous research used different approaches to define low WSS to investigate its effect on plaque progression. In this study, we used four methodologies to allocate low, mid and high WSS in one dataset of human coronary arteries and investigated the predictive power of low WSS for plaque progression. Coronary reconstructions were based on multimodality imaging, using intravascular ultrasound and CT-imaging. Vessel-specific flow was measured using Doppler wire and computational fluid dynamics was performed to calculate WSS. The absolute WSS range varied greatly between the coronary arteries. On the population level, the established pattern of most plaque progression at low WSS was apparent in all methodologies defining the WSS categories. However, for the individual patient, when using measured flow to determine WSS, the absolute WSS values range so widely, that the use of absolute thresholds to determine low WSS was not appropriate to identify regions at high risk for plaque progression.

Lipid-rich Plaques Detected by Near-infrared Spectroscopy Are More Frequently Exposed to High Shear Stress.

High wall shear stress (WSS) and near-infrared spectroscopy (NIRS) detected lipid-rich plaque (LRP) are both known to be associated with plaque destabilization and future adverse cardiovascular events. However, knowledge of spatial co-localization of LRP and high WSS is lacking. This study investigated the co-localization of LRP based on NIRS and high WSS. Fifty-three patients presenting acute coronary syndrome underwent NIRS-intravascular-ultrasound (NIRS-IVUS) imaging of a non-culprit coronary artery. WSS was obtained using WSS profiling in 3D-reconstructions of the coronary arteries based on fusion of IVUS-segmented lumen and CT-derived 3D-centerline. Thirty-eight vessels were available for final analysis and divided into 0.5 mm/45° sectors. LRP sectors, as identified by NIRS, were more often colocalized with high WSS than sectors without LRP. Moreover, there was a dose-dependent relationship between lipid content and high WSS exposure. This study is a first step in understanding the evolution of LRPs to vulnerable plaques. Graphical Abstract.

The Association Between Time-Varying Wall Shear Stress and the Development of Plaque Ulcerations in Carotid Arteries From the Plaque at Risk Study.

Background and Purpose: Shear stress (WSS) is involved in the pathophysiology of atherosclerotic disease and might affect plaque ulceration. In this case-control study, we compared carotid plaques that developed a new ulcer during follow-up and plaques that remained silent for their exposure to time-dependent oscillatory shear stress parameters at baseline. Materials and Methods: Eighteen patients who underwent CTA and MRI of their carotid arteries at baseline and 2 years follow-up were included. These 18 patients consisted of six patients who demonstrated a new ulcer and 12 control patients selected from a larger cohort with similar MRI-based plaque characteristics as the ulcer group. (Oscillatory) WSS parameters [time average WSS, oscillatory shear index (OSI), and relative residence time (RRT)] were calculated using computational fluid dynamics applying the MRI-based geometry of the carotid arteries and compared among plaques (wall thickness>2 mm) with and without ulceration (Mann-Whitney U test) and ulcer-site vs. non-ulcer-site within the plaque (Wilcoxon signed rank test). More detailed analysis on ulcer cases was performed and the predictive value of oscillatory WSS parameters was calculated using linear and logistic mixed-effect regression models. Results: The ulcer group demonstrated no difference in maximum WSS [9.9 (6.6-18.5) vs. 13.6 (9.7-17.7) Pa, p = 0.349], a lower maximum OSI [0.04 (0.01-0.10) vs. 0.12 (0.06-0.20) p = 0.019] and lower maximum RRT [1.25 (0.78-2.03) Pa-1 vs. 2.93 (2.03-5.28) Pa-1, p = 0.011] compared to controls. The location of the ulcer (ulcer-site) within the plaque was not always at the maximal WSS, but demonstrated higher average WSS, lower average RRT and OSI at the ulcer-site compared to the non-ulcer-sites. High WSS (WSS>4.3 Pa) and low RRT (RRT < 0.25 Pa) were associated with ulceration with an odds ratio of 3.6 [CI 2.1-6.3] and 2.6 [CI 1.54-4.44] respectively, which remained significant after adjustment for wall thickness. Conclusion: In this explorative study, ulcers were not exclusively located at plaque regions exposed to the highest WSS, OSI, or RRT, but high WSS and low RRT regions had a significantly higher odds to present ulceration within the plaque even after adjustment for wall thickness.

Multidirectional wall shear stress promotes advanced coronary plaque development: comparing five shear stress metrics.

AIMS: Atherosclerotic plaque development has been associated with wall shear stress (WSS). However, the multidirectionality of blood flow, and thus of WSS, is rarely taken into account. The purpose of this study was to comprehensively compare five metrics that describe (multidirectional) WSS behaviour and assess how WSS multidirectionality affects coronary plaque initiation and progression. METHODS AND RESULTS: Adult familial hypercholesterolaemic pigs (n = 10) that were fed a high-fat diet, underwent imaging of the three main coronary arteries at three-time points [3 (T1), 9 (T2), and 10-12 (T3) months]. Three-dimensional geometry of the arterial lumen, in combination with local flow velocity measurements, was used to calculate WSS at T1 and T2. For analysis, arteries were divided into 3 mm/45° sectors (n = 3648). Changes in wall thickness and final plaque composition were assessed with near-infrared spectroscopy-intravascular ultrasound, optical coherence tomography imaging, and histology. Both in pigs with advanced and mild disease, the highest plaque progression rate was exclusively found at low time-averaged WSS (TAWSS) or high multidirectional WSS regions at both T1 and T2. However, the eventually largest plaque growth was located in regions with initial low TAWSS or high multidirectional WSS that, over time, became exposed to high TAWSS or low multidirectional WSS at T2. Besides plaque size, also the presence of vulnerable plaque components at the last time point was related to low and multidirectional WSS. Almost all WSS metrics had good predictive values for the development of plaque (47-50%) and advanced fibrous cap atheroma (FCA) development (59-61%). CONCLUSION: This study demonstrates that low and multidirectional WSS promote both initiation and progression of coronary atherosclerotic plaques. The high-predictive values of the multidirectional WSS metrics for FCA development indicate their potential as an additional clinical marker for the vulnerable disease.