Uncovering the ‘Hidden Fibrosis’ of Pediatric Congenital Aortic Valve Stenosis via Targeted Mass Spectrometry Approaches

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression, particularly in pediatric patients where bioengineered solutions are not ideal. Collagen deregulation is a hallmark of pediatric CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics and imaging mass spectrometry to investigate collagen fiber production with localized collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified with hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories, providing insight to collagen-cell receptor binding. In addition to chromatographic-based proteomic analysis, Matrix Assisted Laser Desorption Ionization imaging mass spectrometry (MALDI-IMS) methods were developed to further address the localized structure-function relationship of the extracellular matrisome in aortic valve tissue. Here, a novel serial enzyme strategy was developed to define the glycosaminoglycome, N-glycome, as well as the collagen and elastin proteome from a single tissue section for MALDI-IMS applications. These multimodal MALDI-IMS techniques could define unique matrisome profiles based off tissue hemodynamics, as well as identify collagen localization unable to be detected by tradition histopathology. Finally, as a proof-of-concept study toward biomaterials applications, MALDI-IMS was used to localize human collagen-based hydrogels within an infarcted mouse heart, as well as analyze its impact on endogenous extracellular matrix (ECM) remodeling. The studies presented in this dissertation are the first of their kind to detail the collagen types and HYP modifications associated with human AV development and pediatric CAVS. Additionally, our findings show evidence for the use of MALDI-IMS in assessing the therapeutic application of collagen-based biomaterials. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and bioengineered options for valve replacement

Doctor of Philosophy

dissertationMyocardial microstructure plays an important role in sustaining the orchestrated beating motion of the heart. Several microstructural components, including myocytes and auxiliary cells, extracellular space, and blood vessels provide the infrastructure for normal heart function, including excitation propagation, myocyte contraction, delivery of oxygen and nutrients, and removing byproduct wastes. Cardiac diseases cause deleterious changes to some or all of these microstructural components in the detrimental process of cardiac remodeling. Since heart failure is among the leading causes of death in the world, new and novel tools to noninvasively characterize heart microstructure are needed for monitoring and staging of cardiac disease. In this regards, diffusion magnetic resonance imaging (MRI) provides a promising framework to probe and quantify tissue microstructure without the need for exogenous contrast agent. As diffusion in 3-dimensional space is characterized by the diffusion tensor, MR diffusion tensor imaging (DTI) is being used to noninvasively measure anisotropic diffusion, and thus the magnitude and spatial orientation of microstructural organization of tissues, including the heart. However, even though in vivo cardiac DTI has become more clinically available, to date the origin and behavior of different microstructural components on the measured DTI signal remain to be explicitly specified. The presented studies in this work demonstrate that DTI can be used as a noninvasive and contrast-free imaging modality to characterize myocyte size and density, extracellular collagen content, and the directional magnitude of blood flow. The identified applications are expected to provide metrics to enable physicians to detect, quantify, and stage different microstructural components during progression of cardiac disease

Characterization and modeling of the human left atrium using optical coherence tomography

With current needs to better understand the interaction between atrial tissue microstructure and atrial fibrillation dynamics, micrometer scale imaging with optical coherence tomography has significant potential to provide further insight on arrhythmia mechanisms and improve treatment guidance. However, optical coherence tomography imaging of cardiac tissue in humans is largely unexplored, and the ability of optical coherence tomography to identify the structural substrate of atrial fibrillation has not yet been investigated. Therefore, the objective of this thesis was to develop an optical coherence tomography imaging atlas of the human heart, study the utility of optical coherence tomography in providing useful features of human left atrial tissues, and develop a framework for optical coherence tomography-informed cardiac modeling that could be used to probe dynamics between electrophysiology and tissue structure. Human left atrial tissues were comprehensively imaged by optical coherence tomography for the first time, providing an imaging atlas that can guide identification of left atrial tissue features from optical coherence tomography imaging. Optical coherence tomography image features corresponding to myofiber and collagen fiber orientation, adipose tissue, endocardial thickness and composition, and venous media were established. Varying collagen fiber distributions in the myocardial sleeves were identified within the pulmonary veins. A scheme for mapping optical coherence tomography data of dissected left atrial tissues to a three-dimensional, anatomical model of the human left atrium was also developed, enabling the mapping of distributions of imaged adipose tissue and fiber orientation to the whole left atrial geometry. These results inform future applications of structural substrate mapping in the human left atrium using optical coherence tomography-integrated catheters, as well as potential directions of ex vivo optical coherence tomography atrial imaging studies. Additionally, we developed a workflow for creating optical mapping models of atrial tissue as informed by optical coherence tomography. Tissue geometry, fiber orientation, ablation lesion geometry, and heterogeneous tissue types were extracted from optical coherence tomography images and incorporated into tissue-specific meshes. Electrophysiological propagation was simulated and combined with photon scattering simulations to evaluate the influence of tissue-specific structure on electrical and optical mapping signals. Through tissue-specific modeling of myofiber orientation, ablation lesions, and heterogeneous tissue types, the influence of myofiber orientation on transmural activation, the relationship between fluorescent signals and lesion geometry, and the blurring of optical mapping signals in the presence of heterogeneous tissue types were investigated. By providing a comprehensive optical coherence tomography image database of the human left atrium and a workflow for developing optical coherence tomography-informed cardiac tissue models, this work establishes the foundation for utilizing optical coherence tomography to improve the structural substrate characterization of atrial fibrillation. Future developments include analysis of optical coherence tomography imaged tissue structure with respect to clinical presentation, development of automated processing to better leverage the large amount of imaging data, enhancements and validation of the modeling scheme, and in vivo evaluation of the left atrial structural substrate through optical coherence tomography-integrated catheter

Investigation of Neonatal Pulmonary Structure and Function via Proton and Hyperpolarized Gas Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is a modality that utilizes the phenomenon of nuclear magnetic resonance (NMR) to yield tomographic images of the body. Proton (1H) MRI has historically been successful in soft tissues but has suffered in the lung due to a variety of technical challenges, such as the low proton-density, rapid T2* relaxation time of the lung parenchymal tissue, and inherent physiological motion in the chest. Recent developments in radial ultrashort echo time (UTE) MRI have in part overcome these issues. In addition, there has been much progress in techniques for hyperpolarization of noble gases (3He and 129Xe) out of thermal equilibrium via spin exchange optical pumping, which can greatly enhance the gas NMR signal such that it is detectable within the airspaces of the lung on MRI. The lung is a unique organ due to its complex structural and functional dynamics, and its early development through the neonatal (newborn) period is not yet well understood in normal or abnormal conditions. Pulmonary morbidities are relatively common in infants and are present in a majority of patients admitted to the neonatal intensive care unit, often stemming from preterm birth and/or congenital defects. Current clinical lung imaging in these patients is typically limited to chest x-ray radiography, which does not provide tomographic information and so has lowered sensitivity. More rarely, x-ray computed tomography (CT) is used but exposes infants to ionizing radiation and typically requires sedation, both of which pose increased risks to pediatric patients. Thus the opportunity is ripe for application of novel pulmonary MRI techniques to the infant population. However, MR imaging of very small pulmonary structure and microstructure requires fundamental changes in the imaging theory of both 1H UTE MRI and hyperpolarized gas diffusion MRI. Furthermore, such young patients are often non-compliant, yielding a need for new and innovative techniques for monitoring respiratory and bulk motion. This dissertation describes methodology development and provides experimental results in both 1H UTE MRI and hyperpolarized 3He and 129Xe gas diffusion MRI, with investigation into the structure and function of infant lungs at both the macrostructural and microstructural level. In particular, anisotropically restricted gas diffusion within infant alveolar microstructure is investigated as a measurement of airspace size and geometry. Additionally, the phenomenon of respiratory and bulk motion-tracking via modulation of the k-space center\u27s magnitude and phase is explored and applied via UTE MRI in various neonatal pulmonary conditions to extract imaging-based metrics of diagnostic value. Further, the proton-density regime of pulmonary UTE MRI is validated in translational applications. These techniques are applied in infants with various pulmonary conditions, including patients diagnosed with bronchopulmonary dysplasia, congenital diaphragmatic hernia, esophageal atresia/tracheoesophageal fistula, tracheomalacia, and no suspected lung disease. In addition, explanted lung specimens from both infants with and without lung disease are examined. Development and implementation of these techniques involves a strong understanding of the physics-based theory of NMR, hyperpolarization, and MR imaging, in addition to foundations in hardware, software, and image analysis techniques. This thesis first outlines the theory and background of NMR, MRI, and pulmonary physiology and development (Part I), then proceeds into the theory, equipment, and imaging experiments for hyperpolarized gas diffusion MRI in infant lung airspaces (Part II), and finally details the theory, data processing methods, and applications of pulmonary UTE MRI in infant patients (Part III). The potential for clinical translation of the neonatal pulmonary MRI methods presented in this dissertation is very high, with the foundations of these techniques firmly rooted in the laws of physics

3D Stem Cell Culture

Recently, stem cells have been drawing increasing interest in basic and translational research that aims to understand stem cell biology and generate new therapies for various disorders. Many stem cells can be cultured in 2D relatively easily using tissue culture plastic. However, many of these cultures do not represent the natural conditions of stem cells in the body. In the body, microenvironments include numerous supporting cells and molecules. Therefore, researchers and clinicians have sought ideal stem cell preparations for basic research and clinical applications, which may be attainable through 3D culture of stem cells. The 3D cultures mimic the conditions of the natural environment of stem cells better, as cells in 3D cultures exhibit many unique and desirable characteristics that could be beneficial for therapeutic interventions. 3D stem cell cultures may employ supporting structures, such as various matrices or scaffolds, in addition to stem cells, to support complex structures. This book brings together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cell culture techniques and also to highlight stem cell preparations for possible novel therapeutic applications

Automated Vascular Smooth Muscle Segmentation, Reconstruction, Classification and Simulation on Whole-Slide Histology

Histology of the microvasculature depicts detailed characteristics relevant to tissue perfusion. One important histologic feature is the smooth muscle component of the microvessel wall, which is responsible for controlling vessel caliber. Abnormalities can cause disease and organ failure, as seen in hypertensive retinopathy, diabetic ischemia, Alzheimer’s disease and improper cardiovascular development. However, assessments of smooth muscle cell content are conventionally performed on selected fields of view on 2D sections, which may lead to measurement bias. We have developed a software platform for automated (1) 3D vascular reconstruction, (2) detection and segmentation of muscularized microvessels, (3) classification of vascular subtypes, and (4) simulation of function through blood flow modeling. Vessels were stained for α-actin using 3,3\u27-Diaminobenzidine, assessing both normal (n=9 mice) and regenerated vasculature (n=5 at day 14, n=4 at day 28). 2D locally adaptive segmentation involved vessel detection, skeletonization, and fragment connection. 3D reconstruction was performed using our novel nucleus landmark-based registration. Arterioles and venules were categorized using supervised machine learning based on texture and morphometry. Simulation of blood flow for the normal and regenerated vasculature was performed at baseline and during demand based on the structural measures obtained from the above tools. Vessel medial area and vessel wall thickness were found to be greater in the normal vasculature as compared to the regenerated vasculature (p\u3c0.001) and a higher density of arterioles was found in the regenerated tissue (p\u3c0.05). Validation showed: a Dice coefficient of 0.88 (compared to manual) for the segmentations, a 3D reconstruction target registration error of 4 μm, and area under the receiver operator curve of 0.89 for vessel classification. We found 89% and 67% decreases in the blood flow through the network for the regenerated vasculature during increased oxygen demand as compared to the normal vasculature, respectively for 14 and 28 days post-ischemia. We developed a software platform for automated vasculature histology analysis involving 3D reconstruction, segmentation, and arteriole vs. venule classification. This advanced the knowledge of conventional histology sampling compared to whole slide analysis, the morphological and density differences in the regenerated vasculature, and the effect of the differences on blood flow and function

Non-invasive imaging of fibrosis with positron emission tomography in a rat model with systemic hypertension and myocardial fibrosis

Heart failure is one of the leading causes of death worldwide. Hypertension can initiate myocardial remodelling processes which, often via fibrotic triggers through the renin-angiotensin-aldosterone system, can lead to the development of heart failure. A main contributor of these pathways is angiotensin II, increased levels of which can induce volume and pressure overload in the cardiovascular system, making it an important factor in both hypertension and associated cardiovascular disease. A main process during cardiac remodelling is fibrosis which can be divided into two types: reactive and replacement fibrosis. The latter refers to the changes via scar formation at an injury site while the former (interstitial or perivascular fibrosis) can happen as a response to changes in the physical or chemical environment within the tissue such as hypertension or inflammation. Fibrillary collagen is an important extracellular matrix component and abundantly deposited during fibrosis. Collagen can have various subtypes based on its structure which can add different characteristics to the tissue. During collagen biosynthesis, cis- or trans-proline containing pro-α chains can be integrated into the protein, where chains containing cis isomer are associated with more distensible and abnormal collagen and those with trans isomer with more rigid triple helix collagen. Other factors can also influence the development of heart failure via the myocardial remodelling processes, such as inflammatory and angiogenic pathways. Heart failure can be diagnosed and assessed in the clinic via blood tests and imaging techniques such as ultrasound, magnetic resonance imaging (MRI), computerised tomography (CT), and single-photon emission computed tomography (SPECT) / positron emission tomography (PET). This thesis aimed to investigate the effect of increased angiotensin II and subsequent hypertension on the levels of myocardial collagen synthesis and to test PET radiotracers cis-4-18F-fluoro-L-proline and trans-4-18F-fluoro-L-proline for the detection of myocardial fibrosis and potential differentiation of the types of collagen fibers. The overarching hypothesis of the project was that myocardial fibrosis can be imaged non-invasively with PET in a rat pressure overload model with via persistent hypertension resulting in end-organ damage. A hypertensive rat model with myocardial remodelling was established via angiotensin II infusion using osmotic mini-pumps. Treatment length and dosage were tested and the optimal protocol was chosen to induce myocardial fibrosis. The model was assessed for myocardial collagen content as well as markers of inflammation and vasculature. On a separate set of experiments, the performance of PET radiotracers, cis-4-18F-fluoro-L-proline and trans-4-18F-fluoro-L-proline, was assessed in naïve rats to understand their in vivo metabolism and kinetics. Then, the optimised animal model of hypertensive heart failure and PET imaging protocols were used to investigate whether the new imaging probes could visualise areas if increased collagen synthesis and whether the uptake was related to the type of collagen involved. Using 500 ng/kg/min angiotensin II dose for 4 weeks duration was adequate to induce myocardial fibrosis and hypertension in the rat model. The fibrosis pattern was mainly perivascular in nature. Immunostaining also showed increased CD68 in the myocardium of rats on 250 ng/kg/min but not with the higher dose. The highest percentage of cells stained positive for all three of CD68, TSPO and isolectin B4 was found in the atria of animals on the higher angiotensin II dose, which area also showed the most fibrosis overall. Both radiotracers were successfully assessed in naïve rats, showing no metabolism and favourable kinetics in vivo, allowing for simplified quantification of radiotracer uptake. Myocardial radiotracer uptake in the angiotensin II treated cohort showed increased myocardial signal with the trans-4-18F-fluoro-L-proline radiotracer but no significant differences with cis-4-18F-fluoro-L-proline compared to vehicle treated animals. Animals undergoing the imaging experiment showed increased myocardial fibrosis similarly to rats in the model development experiments. Myocardial fibrosis develops during hypertension induced via angiotensin II treatment, and this change can be measured with PET imaging targeting collagen biosynthesis. Further investigation into the types of collagens involved and how they contribute to pathology needs to be carried out to characterise the underlying biological processes in more detail. Fluoroproline radiotracer PET imaging can become a valuable tool for the assessment of fibrosis pathology both in terms of early detection and disease progression

The role of myocardial fibrosis in outcome following mitral valve repair in degenerative mitral regurgitation

Primary degenerative mitral regurgitation (MR) is a disease of increasing prevalence. Its optimal management is surgical repair, but surgery timings remain controversial. Current guidelines that suggest ‘watchful-waiting’ have been criticised for promoting rescue surgery after the establishment of symptoms or left ventricular (LV) dysfunction. Conversely, non-selective early surgical approaches result in unnecessary surgery for some patients. Myocardial fibrosis has been hypothesised to accumulate in MR, leading to eventual overt LV dysfunction. This thesis examines this hypothesis, assesses the prognostic impact of myocardial fibrosis, and determines its value as a biomarker for optimising the timing of surgery. In a prospective multicentre study of severe MR patients, I provide definitive histological evidence for the presence of myocardial fibrosis, before the onset of symptoms. Due to its patchy nature, non-invasive quantification of fibrosis on cardiac magnetic resonance (CMR) was a superior marker of preoperative myocardial function and symptom burden. However, neither histology- nor CMR-derived fibrosis correlated with postoperative outcomes. Despite successful surgery, symptomatic patients continued to possess worse cardiopulmonary exercise (CPET) performance and symptom burden quantified via patient-response questionnaires (PROMs) compared to asymptomatic patients, providing additional support for the benefits of early surgery. Further evaluation of surveillance CPET and PROMs is indicated for patients in whom early surgery is clinically inappropriate

Molecular imaging of tissue repair after myocardial infarction : preclinical evaluation of novel 68Ga-labeled PET tracers

Congestive heart failure (HF) develops soon after acute myocardial infarction (AMI) in almost 25% of initial survivors. Modern cardiac imaging methods are useful for HF diagnostics and, possibly, the detection of underlying molecular mechanisms involved in myocardial repair. CD44, a cell-surface glycoprotein, is involved in various cellular functions, including cell proliferation, adhesion, migration and lymphocyte activation. Integrins are transmembrane proteins involved in various signaling pathways related to inflammation, angiogenesis and fibrosis. Expression of proteolytic matrix metalloproteinases 2 and 9 (MMP-2/9) also associates with extracellular matrix remodeling. The purpose of this thesis was to evaluate novel Gallium-68 labeled imaging agents targeting αvβ3 integrin, MMP-2/9, or CD44, for positron emission tomography (PET) imaging of post-MI repair in a surgical rat model. The MMP- 2/9 targeting tracer watarkias also evaluated for imaging of atherosclerotic lesions in a hypercholesterolemic mouse model. In vivo PET imaging, ex vivo biodistribution, ex vivo autoradiography, and immunohistochemistry were utilized to assess tracer stability, uptake in various tissues, as well as uptake correlation with various cellular level processes. Of the studied tracers, αvβ3 integrin targeting tracer showed the most optimal characteristics for imaging of myocardial healing processes. Tracer uptake in the damaged myocardium was clearly visible in vivo, and blood clearance as well as tracer stability were sufficient. The CD44 targeting tracer showed initial potential warranting further development, as the tracer uptake was associated with myocardial inflammation. MMP-2/9 targeted imaging showed significant limitations due to tracer instability and slow clearance. In conclusion, imaging of αvβ3 integrin expression is a potential tool for the purpose of evaluating myocardial repair after MI.Sydänkudoksen infarktinjälkeisen paranemisen molekyylikuvantaminen : uusien 68Ga-leimattujen merkkiaineiden prekliininen arviointi Sydämen vajaatoiminta kehittyy pian akuutin sydäninfarktin jälkeen lähes 25 prosentille eloonjääneistä. Nykyaikaiset sydämen kuvantamismenetelmät ovat hyödyllisiä diagnostiikassa ja mahdollisesti sydänlihaksen muovautumiseen liittyvien molekyylimekanismien havaitsemisessa. Solupinnan glykoproteiini CD44 osallistuu erilaisiin soluvälitteisiin toimintoihin, kuten proliferaatioon, adheesioon, migraatioon ja lymfosyyttien aktivaatioon. Integriinit ovat transmembraaniproteiineja, jotka osallistuvat erilaisiin signalointireitteihin liittyen tulehdukseen, angiogeneesiin ja fibroosiin. Proteolyyttisten matriksin metalloproteinaasi 2:n ja 9:n (MMP-2/9) ilmentyminen liittyy niin ikään solunulkoisen matriksin uudelleenmuovautumiseen. Tämän väitöskirjan tarkoituksena on arvioida uusia Gallium-68-leimattuja koettimia positroniemissiotomografiaa (PET) varten. Tutkitut koettimet kohdistuvat joko αvβ3-integriiniin, MMP-2/9:ään tai CD44:ään. Tutkimus toteutettiin infarktinjälkeisen sydämen vajaatoiminnan kirurgisessa rottamallissa. MMP-2/9-koetinta arvioitiin myös ateroskleroottisten muutosten kuvantamiseen hyperkolesterolemisessa hiirimallissa. αvβ3-integriiniin kohdentuvan merkkiaineen kertymä näkyi selkeästi in vivo, ja veren puhdistuma sekä merkkiaineen stabiilisuus olivat riittävät. CD44 kuvantamiskohteena osoitti alkuvaiheen potentiaalia, joka mahdollistaa jatkokehityksen, sillä merkkiaineen kertymä assosioitui infarktinjälkeiseen tulehdusreaktioon. MMP-2/9- kohdennetulle kuvantamiselle puolestaan ilmeni merkittäviä rajoituksia merkkiaineiden epävakauden ja hitaan veripuhdistuman vuoksi Yhteenvetona voidaan todeta, että αvβ3-integriinifragmentin kuvantaminen on potentiaalinen työkalu sydänlihaksen paranemisprosessien arvioimiseksi akuutin sydäninfarktin jälkeen