Depth-Resolved Assessment of Atherosclerosis by Intravascular Photoacoustic-Ultrasound Imaging

Coronary heart disease is the leading cause of death in the United States and the incidence is projected to increase by 18% by 2030. Yet, there remains a pressing clinical need for tools to detect vulnerable atherosclerotic plaques that can rupture and lead to major adverse cardiac events. Plaques that are considered most vulnerable for rupture are thin-capped fibroatheromas, which are grossly defined by hallmarks of a thin fibrous cap, a large lipid-rich necrotic core, inflammatory infiltrate, and positive remodeling. These plaques are often structurally non-obstructive to moderately obstructive, thus asymptomatic and clinically unidentifiable with routine angiography and stress testing. Rather, their vulnerability is a product of their chemical composition. We have developed a dual-mode intravascular catheter which is capable of producing co-registered cross-sectional images of arterial wall morphology and lipid content, via ultrasound and photoacoustic modes, respectively. Validation of this capability will rely on interrogation of atherosclerotic coronary arteries from humans and peripheral arteries from swine, with comparison to gold-standard histopathology and competing technologies. Here, we present ex vivo validation of a novel intravascular photoacoustic-ultrasound (IVPA-US) imaging catheter and the first systematic in vivo IVPA-US imaging study in a preclinical swine model with native disease, necessary benchmarks before proceeding with translation to clinic. We aim to ultimately demonstrate predictive utility to detect plaques that are vulnerable to rupture and trigger adverse cardiac events. In addition, this will be instrumental in elucidating the mechanism of plaque rupture, the development of preventive and therapeutic interventions, and reducing coronary heart disease-related mortality

Carotid plaque vulnerability assessment by microscopic morphology analysis, ultrasound and 3D model reconstruction

This thesis was submitted for the degree of Docter of Philosophy and awarded by Brunel University.Research suggests that plaque morphology plays a crucial role in determining plaque vulnerability. However the relationship between plaque morphology and rupture is still not clearly understood due to the limited information of plaque morphology. The aim of this study is to improve our understanding of the relationship between plaque morphology and rupture, and to use this to predict the risk of plaque rupture from the morphology at the molecular level. This can enable the identification of culprit lesions in clinical situations for assessing plaque rupture risk. Histological assessments were carried out on 18 carotid plaque specimens. The 3-D collagen, lipid and macrophage distributions along the entire length of the plaque were analysed in both ruptured and non-ruptured symptomatic plaques. In addition, plaque morphology on the rupture sites were examined and compared with the surrounding regions. It was found that ruptured plaques had thinner fibrous caps and larger lipid cores compared to non-ruptured plaques. Also, ruptured plaques had lower collagen content compared to non-ruptured plaques, and higher collagen contents upstream compared to downstream region from the plaque throat. At the rupture site there was lower collagen content, and a larger lipid core thickness behind a thin fibrous cap compared with the mean for the longitudinally adjacent and circumferential regions. Macrophage cells were located nearer to the boundary of the luminal wall in ruptured plaques. For both groups, the area occupied by macrophages is greater at the upstream shoulder of the plaque. There is a positive correlation between macrophage area and lipid core area, a negative correlation between macrophage area and collagen content, and between lipid core size and collagen content for both plaque groups. 3D reconstruction of ex-vivo specimens of carotid plaques were carried out by a combined analysis of US imaging and histology. To reconstruct accurate 3D plaque morphology, the non-linear tissue distortion in histological images caused by specimen preparation was corrected by a finite element (FE) based deformable registration procedure. This study shows that it is possible to generate a 3D patient specific plaque model using this method. In addition, the study also quantitatively assesses the tissue distortion caused by histological procedures. It shows that at least 30% tissue shrinkage is expected for plaque tissues. The histology analysis result was also used to evaluate ultrasound (US) tissue characterization accuracy. An ex-vivo 2D ultrasound scan set-up was used to obtain serial transverse images through an atherosclerotic plaque. The different plaque component region obtained from ultrasound images was compared with the associated histology result and photograph of the sections. Plaque tissue characterisation using ex-vivo US can be performed qualitatively, whereas lipid core assessment from ultrasound scan can be semi-quantitative. This finding combined with the negative correlation between lipid core size and collagen content, suggests the ability of US to indirectly quantify plaque collagen content. This study may serve as a platform for future studies on improving ultrasound tissue characterization, and may also potentially be used in risk assessment of plaque rupture

Factors Influencing The Collagen Fiber Angle Distribution in The Mouse Aorta

The aortic extracellular matrix (ECM) consists of microstructural proteins, collagen and elastin, together with proteoglycans and other components. The matrix metalloproteinases (MMPs) are proteolytic enzymes that influence morphological and structural changes in the ECM and can degrade the matrix as it responds to cellular behaviors such as angiogenesis, apoptosis, proliferation, and migration. Collagen is the most important component among the extracellular proteins because it provides strength and stability to the tissue. Changes in collagen content play a major role in the development of atherosclerosis. These changes can be induced by increased or decreased proteinase activity. Therefore, we studied the collagen fiber angle distribution in mouse models of atherosclerosis with or without a deficiency of a selected MMP. Quantification of the collagen fiber data is meaningful in providing insight into the mechanical behavior of the artery and leads to an understanding of how the diseased aorta maintains homeostasis. Furthermore, such data can be utilized to increase the understanding of disease progression, including but not limited to atherosclerosis and aortic aneurysm development. We characterized collagen fiber angles in mouse models of atherosclerosis which were fed a control chow diet or a high-fat Western diet for 6 months. To visualize collagen, we imaged the mouse thoracic and abdominal aorta using second-harmonic generation (SHG) microscopy. Angle measurements were acquired using a well-established computer software program, Continuity 6.4b. The angle measurements were exported into bivariate histograms. We then designed a multiple regression analysis to compare the distributions of absolute angles between two diet groups, controlling for diet, mouse strain, anatomical location, and radial position in the aortic wall. Data extracted from bivariate histograms were analyzed in R, a programming language and statistical software. In trying to understand the changes seen between chow diet and Western diet fed mice, we began to study physiological variables such as blood pressure and blood flow velocity. While we did not find any differences in the hemodynamics, we were able to determine that factors beyond atherogenesis, for example aging, influenced aortic collagen fiber angle distributions. With aging and atherosclerosis, the extracellular matrix may experience an increase in collagen content and fibrous tissue. We evaluated changes in fractional collagen amount, in particular, collagen type I. To understand if changes in collagen fibers are initiated by endothelial dysfunction (a pathological condition that often accompanies atherosclerosis progression), we performed immunohistochemical studies of two endothelial cell-derived factors, intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS). This study yielded data of collagen fiber angle distributions throughout the vessel wall in the aortas of mice with atheroprone phenotypes at different ages and on different diets. Endothelial dysfunction as a stimulus for vascular remodeling remains inconclusive. However, we conclude that the aorta displays a distinct remodeling response in the presence of atherogenic stimuli, even in non-lesioned areas, as observed by a shift in collagen fiber orientation

Assessment of the nanomechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture.Open Acces

Carotid plaque vulnerability assessment by microscopic morphology analysis, ultrasound and 3D model reconstruction

Research suggests that plaque morphology plays a crucial role in determining plaque vulnerability. However the relationship between plaque morphology and rupture is still not clearly understood due to the limited information of plaque morphology. The aim of this study is to improve our understanding of the relationship between plaque morphology and rupture, and to use this to predict the risk of plaque rupture from the morphology at the molecular level. This can enable the identification of culprit lesions in clinical situations for assessing plaque rupture risk. Histological assessments were carried out on 18 carotid plaque specimens. The 3-D collagen, lipid and macrophage distributions along the entire length of the plaque were analysed in both ruptured and non-ruptured symptomatic plaques. In addition, plaque morphology on the rupture sites were examined and compared with the surrounding regions. It was found that ruptured plaques had thinner fibrous caps and larger lipid cores compared to non-ruptured plaques. Also, ruptured plaques had lower collagen content compared to non-ruptured plaques, and higher collagen contents upstream compared to downstream region from the plaque throat. At the rupture site there was lower collagen content, and a larger lipid core thickness behind a thin fibrous cap compared with the mean for the longitudinally adjacent and circumferential regions. Macrophage cells were located nearer to the boundary of the luminal wall in ruptured plaques. For both groups, the area occupied by macrophages is greater at the upstream shoulder of the plaque. There is a positive correlation between macrophage area and lipid core area, a negative correlation between macrophage area and collagen content, and between lipid core size and collagen content for both plaque groups. 3D reconstruction of ex-vivo specimens of carotid plaques were carried out by a combined analysis of US imaging and histology. To reconstruct accurate 3D plaque morphology, the non-linear tissue distortion in histological images caused by specimen preparation was corrected by a finite element (FE) based deformable registration procedure. This study shows that it is possible to generate a 3D patient specific plaque model using this method. In addition, the study also quantitatively assesses the tissue distortion caused by histological procedures. It shows that at least 30% tissue shrinkage is expected for plaque tissues. The histology analysis result was also used to evaluate ultrasound (US) tissue characterization accuracy. An ex-vivo 2D ultrasound scan set-up was used to obtain serial transverse images through an atherosclerotic plaque. The different plaque component region obtained from ultrasound images was compared with the associated histology result and photograph of the sections. Plaque tissue characterisation using ex-vivo US can be performed qualitatively, whereas lipid core assessment from ultrasound scan can be semi-quantitative. This finding combined with the negative correlation between lipid core size and collagen content, suggests the ability of US to indirectly quantify plaque collagen content. This study may serve as a platform for future studies on improving ultrasound tissue characterization, and may also potentially be used in risk assessment of plaque rupture.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Optical coherence tomography for the assessment of coronary atherosclerosis and vessel response after stent implantation

Optical Coherence Tomography (OCT) is a light-based imaging modality that can provide in vivo high-resolution images of the coronary artery with a level of resolution (axial 10-20 µm) ten times higher than intravascular ultrasound. The technique, uses low-coherent near infrarred light to create high-resolution cross sectional images of the vessel. The technology refinement achieved in the last years has made this imaging modality less procedurally demanding opening its possibilities for clinical use. The present thesis provides im

The Use of Intracoronary Optical Coherence Tomography in Interventional Cardiology: Safety, Feasibility and Clinical Applications

Interventional cardiology has witnessed tremendous change since 1977 when Andreas Gruentzig successfully performed the first balloon angioplasty. Whereas initial concerns revolved around maintaining vessel patency with issues of recoil and restenosis, the introduction of stents changed the landscape forever. Inherent with their use, stents, and, more specifically, drugeluting stents (DES), have become central to improved patient outcomes but, at some cost. Catastrophic, yet fortunately still rare complications such as stent thrombosis have re-ignited an intense need for greater scrutiny when developing and, subsequently implanting DES into our patients. The demand for detailed information regarding coronary artery disease has seen intravascular imaging become pivotal at delineating atherosclerosis and tissue responses following stent implantation. In fact, the strategy that relied on angiography alone is evolving to include better confirmation of disease severity and stenting technique. With this, optical coherence tomography (OCT) has grown exponentially with a broad diffusion amongst catheterisation laboratories worldwide. Optical coherence tomography is a procedurally demanding technique. Individual experience is often frustrated initially with disappointing images as a result of inadequate blood clearance. With perseverance and adequate proctorship however, one cannot help but be impressed by the clarity and resolution afforded by this imaging modality. It is these images that have attracted considerable attention at cardiology conferences internationally and have helped instil OCT as the most sensitive intravascular imaging technique available today. The aim of this thesis was to evaluate the role of OCT in contemporary coronary intervention. Part 1 embraces the principles of the technique and the physical properties of OCT (chapter 2) and gives an insight into where OCT is placed compared to other intravascular imaging modalities (chapter 3). Despite the adoption of OCT in more and more catheterisation laboratories, little has been documented as to its safety, so, in chapter 4, we review the procedural safety of intracoronary OCT in a large group of patients across six leading European centres

Intravascular OCT tissue type imaging by automated optical attenuation analysis

We developed attenuation imaging in OCT for atherosclerotic tissue characterization and validated the method ex and in-vivo. We introduced an en-face map of attenuation in the whole artery for plaque visualization. We quantified the attenuation derived from OCT and derived an index for the plaques. A single centre clinical study (OC3T study) was conducted to validate the index to identify thin cap fibroatheromas. We also demonstrated the utility of the attenuation maps and the index in clinical studies as corresponding well with a visual assessment of LCP in the OCT data by expert readers

Quality Assessment of Cardiovascular Cells and Tissues by Raman Microspectroscopy and Imaging

The increasing lifespan of the human population has been accompanied by a higher prevalence of cardiovascular diseases. It has been more than 50 years since the first heart valve was transplanted in a human patient and many new approaches in cardiovascular transplantation and tissue engineering (TE) have been evolving ever since. However, the availability of human donor tissues is limited. Ideal, vital, durable, nonimmunogenic heart valve or cardiovascular replacements are not yet commercially available. Thus, a better understanding of developmental and regulating mechanisms of cardiovascular tissues is essential to develop new implant materials. Moreover, cardiovascular tissue transplants or tissue-engineered grafts need to be monitored before transplantation. This thesis aimed to establish Raman microspectroscopy and Raman imaging as marker-independent, non-destructive technique for quality assessment of cardiovascular transplants and tissue-engineered products. Towards this aim, the influence of an ice-free cryopreservation technique (IFC) on tissue integrity and immunogenicity of heart valves was analyzed. The extracellular matrix (ECM) structures of standard cryopreserved (FC) and IFC allograft leaflets were compared to native leaflets after longterm implantation in sheep. Moreover, the mid-term immunogenic effects on IFC treated xenografts were assessed. Quantitative monitoring of interstitial cryoprotectant (CPA) concentrations was performed for quality control of cryopreserved heart valves. Furthermore, phenotype and tissue origin of human smooth muscle cells (SMCs) that are applied in cardiovascular TE, were analyzed. The ECM remodeling of SMC ring constructs under different culture conditions was monitored. In addition to Raman measurements, routine techniques such as immunocytochemistry, quantitative polymerase chain reaction and histological staining were performed. The results demonstrate the superiority of Raman microspectroscopy and Raman imaging as marker-independent, non-destructive and sensitive method, which is also time- and cost efficient when compared to routine techniques. Raman analysis combined with multivariate data analysis tools allowed for the determination and characterization of structural ECM changes in FC heart valves and real-time quantification of residual CPAs. These techniques enabled the identification and discrimination of single human SMCs based on their tissue origin and phenotype. Moreover, ECM remodeling in tissue-engineered SMC rings was non-invasively monitored. This work affirms the potential of Raman techniques for future applications in in situ quality assessment in cardiovascular research

Targeted EDTA Chelation Therapy with Albumin Nanoparticles to Reverse Arterial Calcification and Restore Vascular Health in Chronic Kidney Disease

Cardiovascular diseases (CVDs) are the leading cause of death globally. An estimated 17.9 million people died from CVDs in 2016, with ~840,000 of them in the United States alone. Traditional risk factors, such as smoking, hypertension, and diabetes, are well discussed. In recent years, chronic kidney disease (CKD) has emerged as a risk factor of equal importance. Patients with mild-to-moderate CKD are much more likely to develop and die from CVDs than progress to end-stage renal failure. Vascular calcification (VC), typical in aging, several genetic and metabolic disorders, is now recognized as a strong and independent predictor of cardiovascular events and mortality, not only in diabetic and CKD patients, even in the general population. VC is classified into two distinct types based on location in the vessel wall; intimal and medial. Elastin-associated medial arterial calcification (CKD) is more specific to CKD and contributes significantly to cardiovascular mortality in these patients. It is responsible for loss of vessel elasticity, increased arterial stiffness, increased pulse pressures and systolic blood pressure, and left ventricular hypertrophy ultimately causing arrhythmias and heart failure. Current clinical practice is mostly focused on prevention and retardation of VC progression. Unfortunately, most patients with CKD remain underdiagnosed, and those diagnosed have already heavily calcified vessels. As such, they are undertreated since preventative strategies no longer work at this stage. Unfortunately, there is no FDA-approved treatment available that reverses calcification in countless CKD patients. A treatment strategy which promotes resorption of calcified lesions, while simultaneously avoiding demineralization from normally calcified tissues (i.e., bones and teeth) remains an urgent health care need. Chelating agents bind to metal cations, can dissolve and wash away calcium deposits if delivered in close proximity to the calcification sites. This work was undertaken to see if we can develop targeted therapies to deliver chelating agents to vascular calcification sites. Amongst chelating agents known for their affinity to Calcium ions (Ca2+), we found that EDTA chelates Ca2+ from hydroxyapatite better than others. In our laboratory, we have developed a unique targeting mechanism by using nanoparticles to deliver chelating agents and other drugs to degraded elastin, a characteristic feature of VC. We take this approach forward in clinically relevant animal models of CKD. First, we tested the targeted nanoparticle-based EDTA chelation therapy in a rat model of adenine-induced renal failure. The targeted nanoparticles delivered EDTA at the sites of vascular calcification and reversed mineral deposition without any side effects. Furthermore, we validated the adenine-CKD model in mice to monitor MAC in vivo and explore the phenotypic and functional alterations associated with it. We were able to target our nanoparticles to calcified arteries in these mice. The mouse model will help us to test whether our EDTA chelation therapy tangibly improves arterial function by restoring vascular health. Lastly, we investigated the possibility of using an ex vivo organ culture model of VC as a simpler, and relatively easier model to assess if EDTA chelation therapy promotes vessel homeostasis. The work presented here represents another major step forward towards the development of targeted EDTA chelation therapy as an unconventional therapeutic approach to reverse pathological calcifications in CKD patients