Development of lentiviral-based strategies to modulate angiogenesis during post-infarction heart remodeling

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 15-03-2018Esta tesis tiene embargado el acceso al texto completo hasta el 15-09-2019A pesar de la mejora notable por la implementación de la angioplastia en el tratamiento agudo del infarto de miocardio, las consecuencias a largo plazo del infarto de miocardio, incluida la insuficiencia cardíaca, siguen siendo un desafío para la medicina moderna. En las últimas décadas, la necesidad de una terapia eficaz para mejorar o, mejor dicho, evitar una remodelación adversa y, como consecuencia, la insuficiencia cardíaca después de un infarto de miocardio, sigue sin cumplirse. Hasta la fecha, las terapias probadas se han dirigido hacia diversos objetivos, incluida la prevención de la muerte de los cardiomiocitos, la reprogramación de fibroblastos cardíacos en cardiomiocitos o progenitores cardíacos, el ajuste de las respuestas inmune y la modulación de la angiogénesis. Aquí, implementamos una nueva terapia de génica-celular, que combina las acciones de dos factores proangiogénicos VEGF-A y S1P administrados por células de médula ósea infectadas con lentivirus ex vivo. La selección de los factores se basó en su eficacia para inducir angiogénesis en un ensayo de anillo aórtico. En el modelo de isquemia-reperfusión de infarto de miocardio, las células de médula ósea que sobreexpresan hVEGF o hSphK1 se inyectaron secuencialmente en los ratones los días 4 y 7 después del infarto de miocardio. El análisis de los ratones control y los tratados 28 días después de la isquemia-reperfusión mostró una mejoría leve en el volumen del latido sin impacto en la fracción de eyección en ratones tratados con VEGF/S1P. La remodelación adversa del corazón disminuyó en los animales tratados, representada como una mejoría leve en los volúmenes sistólico y diastólico finales del ventrículo izquierdo, la prevención de la hipertrofia de los cardiomiocitos y la mejor preservación de la forma elipsoide del corazón. Además, la terapia secuencial gen-célula VEGF/S1P mitigó la extensión de la cicatriz fibrótica. El análisis de los patrones de la respuesta inmune en ratones tratados y control reveló un perfil distinto de receptores de quimioquinas en los monocitos circulantes de los ratones tratados, lo que sugiere su cambio hacia un fenotipo más reparador. Además, los macrófagos cardíacos fueron menos abundantes en la zona remota de los ratones inyectados con BMVEGF/BMSphK1. Finalmente, y conforme al objetivo primario, la administración secuencial de VEGF y S1P dio como resultado un aumento de la densidad capilar, un menor angioadaptación adversa y 14 una oxigenación tisular mejorada. Además, el índice fibrótico que indica la abundancia de miofibroblastos disminuyó en la zona de infarto de los corazones de los ratones tratados. Nuestros resultados identifican una nueva estrategia basada en el suministro secuencial de células productoras de factores angiogénicos para mejorar función cardíaca después de un infarto de miocardio.The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union´s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement nº 608027 (¨CardioNext¨ Initial Training Networks project

4D imaging of heart vaso-architecture after myocardial infarction

Cardiovascular diseases remain the number one cause of death globally. There is an ongoing desire to study the distribution and structural changes of the vaso-architecture in the diseased heart in cardiovascular research groups all over the world. The ability to acquire high resolution 3D-images of the heart vasculature enables to study heart diseases more in detail and eventually obtain interesting new findings and new treatments. In this work, we introduce a pipeline for high resolution 3D-imaging of the changes in mouse heart vasculature after a myocardial infarction is produced with Single Plane Illumination Microscopy (SPIM). To achieve high resolution 3D-images, protocols for optical tissue clearing (CUBIC tissue clearing technique) were combined with vasculature labelling methods (IHC and intravenous perfused lectin), enabling the visualization for the very first time of the whole heart vasculature. We here also describe the methods used for image pre-processing of the acquired data, mainly for correction of SPIM-image artifacts and for segmentation of the structures of interest. Finally, the analysis of the changes in vasculature between healthy hearts with the different stages of chronic myocardial infarction (7, 14 and 28 days post-infarction) will provide us a tool to know how this disease affects not only to infarcted region but to the whole heart volume.Ingeniería Biomédic

Machine learning and fractal-based analysis for the automated diagnosis of cardiovascular diseases using magnetic resonance

Treballs Finals de Grau d'Enginyeria Informàtica, Facultat de Matemàtiques, Universitat de Barcelona, Any: 2023, Director: Polyxeni Gkontra i Joan Carles Tatjer i Montaña[en] Cardiac magnetic resonance (CMR) is the reference imaging modality for the diagnose of cardiovascular diseases. Traditionally, simple CMR parameters related to the volume and shape of the cardiac structures are calculated by the medical professionals by means of manual or semi-automated approaches. This process is time-consuming and prone to human errors. Moreover, despite the importance of these traditional CMR indexes, they often fail to fully capture the complexity of the cardiac tissue. In this work, we propose a novel approach for automated cardiovascular disease diagnosis, using ischemic heart disease as an example use case. Towards this aim, we will use a state-of-the-art technology, supervised machine learning, and a promising mathematical tool, fractal-based analysis. In order to undertand the potential information that can be derived from fractal-based features, we introduce and explore the concepts of Haussdorff dimension, box-counting dimension and lacunarity. We describe the interrelationships among these concepts and present computational algorithms for calculating box-counting dimension and lacunarity. The study is based on data from a large-cohort study, UK Biobank, to extract box-counting dimension and lacunarity from CMR textures focusing on three cardiac structures of medical interest: the left ventricle, the right ventricle and the myocardium. The extraction of these features allows us to obtain quantitative parameters regarding the complexity and heterogeneity of the tissue. These fractal features, both individually and in conjunction with other vascular risk factors and CMR traditional indexes, are employed as inputs to state-of-the-art machine learning models, including SVM, XGBoost, and random forests. The objective is to determine if the inclusion of fractal features enhances the performance of currently employed parameters. The performance evaluation of our models is based on metrics such as balanced accuracy, F1 score, precision, and recall. The results obtained demonstrate the potential of fractal-based features in improving the accuracy and reliability of cardiovascular diseases diagnosis

Clinical quantitative cardiac imaging for the assessment of myocardial ischaemia

Cardiac imaging has a pivotal role in the prevention, diagnosis and treatment of ischaemic heart disease. SPECT is most commonly used for clinical myocardial perfusion imaging, whereas PET is the clinical reference standard for the quantification of myocardial perfusion. MRI does not involve exposure to ionizing radiation, similar to echocardiography, which can be performed at the bedside. CT perfusion imaging is not frequently used but CT offers coronary angiography data, and invasive catheter-based methods can measure coronary flow and pressure. Technical improvements to the quantification of pathophysiological parameters of myocardial ischaemia can be achieved. Clinical consensus recommendations on the appropriateness of each technique were derived following a European quantitative cardiac imaging meeting and using a real-time Delphi process. SPECT using new detectors allows the quantification of myocardial blood flow and is now also suited to patients with a high BMI. PET is well suited to patients with multivessel disease to confirm or exclude balanced ischaemia. MRI allows the evaluation of patients with complex disease who would benefit from imaging of function and fibrosis in addition to perfusion. Echocardiography remains the preferred technique for assessing ischaemia in bedside situations, whereas CT has the greatest value for combined quantification of stenosis and characterization of atherosclerosis in relation to myocardial ischaemia. In patients with a high probability of needing invasive treatment, invasive coronary flow and pressure measurement is well suited to guide treatment decisions. In this Consensus Statement, we summarize the strengths and weaknesses as well as the future technological potential of each imaging modality

Development of Shear-Thinning and Self-Healing Hydrogels Through Guest-Host Interactions for Biomedical Applications

Hydrogels have emerged as an invaluable class of materials for biomedical applications, owing in part to their utility as structural, bioinstructive, and cell-laden implants that mimic many aspects of native tissues. Despite their many positive attributes, conventional hydrogels face numerous challenges toward translational therapies, including difficulty in delivery (i.e., invasive implantation) as well as limited control over biophysical properties (i.e., porosity, degradation, and strength). To address these challenges, the overall goal of this dissertation was the development of a class of supramolecular hydrogels that can be implanted in vivo by simple injection and that have tunable properties — either innate to the system or achieved through additional modifications. Toward this, we developed guest-host (GH) hydrogels that undergo supramolecular assembly through complexation of hyaluronic acid (HA) separately modified by adamantane (Ad-HA, guest) and β-cyclodextrin (CD-HA, host). Modular modifications were made to GH hydrogels to enable tuning of biophysical properties, including the incorporation of matrix-metalloproteinase cleavable peptides between HA and Ad to form enzymatically degradable assemblies. Additionally, dual-crosslinking (DC) of methacrylated CD-HA (CD-MeHA) and thiolated Ad-HA (Ad-HA-SH) by Michael addition subsequent to GH assembly was explored to stiffen hydrogels in vivo following injection. Finally, injectable and tough double network (DN) hydrogels were fabricated, where GH hydrogels were formed in the presence of an interpenetrating covalent network (methacrylated HA, MeHA) crosslinked by Michael addition with a dithiol under cytocompatible conditions. Both GH and DC hydrogels were further explored in vivo, with application to attenuate the maladaptive left ventricular (LV) remodeling that occurs following myocardial infarction (MI) that can result in heart failure. DC hydrogels reduced stress within the infarct region, prevented early ventricular expansion and thereby ameliorated progressive LV remodeling. Moreover, the preservation of myocardial geometry reduced incidence and severity of ischemic mitral regurgitation — an undesirable and devastating consequence of LV remodeling. Overall, the body of work represents a novel approach to engineer biomaterials with unique properties toward biomedical therapies

AGING, A PATHOLOGICAL FACTOR IN NEUROLOGICAL INJURY

One of the main reasons for CNS drugs to fail in clinical development is not considering age as a risk factor while studying chronic age-related neurological/neurodegenerative diseases in preclinical studies. We first set out to gain a comprehensive understanding of the impact of age on various aspects (anatomical, immunological, and biochemical) in rodents that play a key role in determining the onset, progression, and evolution of disease severity. With advancing age, the vascular structure and function are compromised which is hypothesized to accelerate cognitive decline. The initial step toward developing novel therapeutics is to characterize the age-related vascular modifications. Utilizing a vessel painting technique, we labelled the surface cortical vessels of young and aged Sprague-Dawley rats and analyzed for classical angiographic features (junctions, lengths, end points, density, etc). We found significant decrease in vascular components while vascular complexity and lacunarity were significantly increased in the aged brain compared to young brain. These age-dependent changes were prominent at the level of right and left middle cerebral artery (MCA) as well as on a global scale. Next, we investigated the changes on the peripheral immune response following lipopolysaccharide (LPS) induced acute systemic inflammation in young and aged Sprague Dawley rats. We observed age-related immunosuppression in the splenic leukocytes indicative of reduced ability of the spleen to retain the immune cells. We also found dysregulated cytokine/chemokine expression in the plasma following LPS stimulation in aged and young animals. Interestingly, we noticed significant increase in circulatory neutrophil population in the aged animals compared to young animals in response to LPS at 24h. Taken together, these studies confirm the presence of age-related modifications in the vasculature as well as immune system suggesting altered response to injury/infection and thus emphasizing the need to utilize age-appropriate models when studying diseases of the elderly. Lastly, we wanted to test the therapeutic effect of a novel agent in case of brain injury model in aged rodents. Previous studies by our lab and others have showed that targeting mitoNEET using NL-1 was neuroprotective following brain injury models. We wanted to investigate if administration of NL-1 could improve functional outcomes following stroke in an aged rodent model of cerebral ischemia reperfusion injury. We found significant decrease in infarct volume and edema index at 24h post stroke. We also saw enhanced survival and reduced behavior deficits. Moreover, we showed improved BBB integrity, reduced oxidative stress and apoptosis at 72h post stroke. Interestingly, PLGA encapsulated NL-1 at 0.25mg/kg (which is 40-fold lesser dose than NL-1 at 10mg/kg) produced better therapeutic effects. Future studies should focus on understanding the mechanism underlying the biology of aging thus enabling the development of novel therapeutic targets for neurological disorders/diseases

Magnetic Resonance Imaging Studies of Angiogenesis and Stem Cell Implantations in Rodent Models of Cerebral Lesions

Molecular biology and stem cell research have had an immense impact on our understanding of neurological diseases, for which little or no therapeutic options exist today. Manipulation of the underlying disease-specific molecular and cellular events promises more efficient therapy. Angiogenesis, i.e. the regrowth of new vessels from an existing vascular network, has been identified as a key contributor for the progression of tumor and, more recently, for regeneration after stroke. Donation of stem cells has proved beneficial to treat cerebral lesions. However, before angiogenesis-targeted and stem cell therapies can safely be used in patients, underlying biological processes need to be better understood in animal models. Noninvasive imaging is essential in order to follow biological processes or stem cell fate in both space and time. We optimized steady state contrast enhanced magnetic resonance imaging (SSCE MRI) to monitor vascular changes in rodent models of tumor and stroke. A modification of mathematical modeling of MR signal from the vascular network allowed for the first time simultaneous measurements of relaxation time T2 and SSCE MRI derived blood volume, vessel size, and vessel density. Limitations of SSCE MRI in tissues with high blood volume and non-cylindrically shaped vessels were explored. SSCE MRI detected angiogenesis and response to anti-angiogenic treatment in two rodent tumor models. In both tumor models, reduction of blood volume in small vessels and a shift towards larger vessels was observed upon treatment. After stroke, decreased vessel density and increased vessel size was found, which was most pronounced one week after the infarct. This is in agreement with two initial, recently published clinical studies. Overall, very little signs of angiogenesis were found. Furthermore, superparamagnetic iron oxide (SPIO) labels were used to study neural stem cells (NSCs) in vivo with MRI. SPIO labeling revealed a decrease in volume of intracerebral grafts over 4 months, assessed by T2* weighted MRI. Since SPIO labels are challenging to quantify and their MR contrast can easily be confounded, we explored the potential of in vivo 19F MRI of 19F labeled NSCs. Hardware was developed for in vitro and in vivo 19F MRI. NSCs were labeled with little effect on cell function and in vivo detection limits were determined at ~10,000 cells within 1 h imaging time. A correction for the inhomogeneous magnetic field profile of surface coils was validated in vitro and applied for both sensitive and quantitative in vivo cell imaging. As external MRI labels do not provide information on NSC function we combined 19F MRI with bioluminescence imaging (BLI). The BLI signal allowed quantification of viable cells whereas 19F MRI provided graft location and density in 3D over 4 weeks both in the healthy and stroke brain. A massive decrease in number of viable cells was detected independent of the microenvironment. This indicates that functional recovery reported in many studies of NSC implantation after stroke, is rather due to release of factors by NSCs than direct tissue replacement. In light of these indirect effects, combination of the imaging methods developed in this dissertation with other functional and structural imaging methods is suggested in order to further elucidate interactions of NSCs with the vasculature

Dynamic nanostructured scaffolds as advanced biomaterials

Growing replacement tissues and organs in the laboratory will revolutionise healthcare; however, the maturation of cells into functional tissue constructs requires the controlled presentation of biochemical factors within a mechanically suitable scaffold. In nature, the presentation of such signals is provided through factors and structures existent within the nanoarchitecture of the extracellular matrix (ECM); therefore, in tissue engineering there is significant need to develop dynamic advanced artificial tissue constructs capable of mimicking the complexities of the native ECM. The requirement for bioactive, innervated constructs that contain biologically relevant signals delivered through tuneable mechanisms has yet to be achieved. One approach to address this key-challenge is offered through bioprinting, which allows for the controlled spatial distribution of bioinks containing cells, structures and signals within a single printed construct. However, currently bioprinting applications are severely limited by bioink function - with the majority of bioinks either lacking sufficient mechanical properties or biochemical signalling. Therefore, there is a key need to develop bioinks which adequately mimic the native ECM on a nanostructured, chemical level - particularly in establishing effective control over cell fate and tissue innervation. Tissue composition and extracellular signalling varies substantially between tissue-types, and therefore, advanced approaches that allow for ease of mechanical and biological tuneability through modular mechanisms would provide a practical avenue for bioink development. Self-assembling peptides (SAPs) are a unique class of biomaterials capable of spontaneously forming simple biomimetic structures which entangle to form highly hydrated, bioactive networks with favourable conditions for cell maturation. These biomaterials are easily tuned through modification of amino acid sequence, enabling tailored control over biochemical signalling between cells and scaffold. This provides the ability to artificially replicate natural signalling in a controlled manner - bringing about desired cell behaviour. Using these peptides, a variety of synergistic ECM-protein analogues have been developed, including Fmoc-FRGDF containing fibronectin's attachment motif RGD, and Fmoc-DIKAV, containing laminin's attachment motif IKVAV. Fmoc-SAPs possess the ability to be further functionalised through macromolecule addition, allowing for the presentation of charged, developmentally or structurally-important macromolecules on the surface of peptide fibrils. These macromolecules can integrate with the peptide networks, facilitating additional signalling and allowing for mechanical tunability. Here, we take advantage of these properties to develop an advanced and dynamic bioink for bioprinting applications. Initially, material enhancement is investigated through development of multi-sequence scaffolds. Specifically, Fmoc-FRGDF is combined with a synergistic cell attachment motif PHSRN, either through sequence engineering (Fmoc-FRGSFPHSRN) or through control over assembly properties (Fmoc-FRGDF/Fmoc-PHSRN coassembly). Here, the coassembled (Fmoc-FRGDF/Fmoc-PHSRN) system forms a synergistic network which promotes the attachment, proliferation and migration of muscle cells in vitro. The potential of Fmoc-SAP multi-sequence scaffolds is further investigated through the development of an artificial tumour microenvironment for cancer-cell studies. Here, Fmoc-FRGDF is combined with Fmoc-DIKVAV and used as a spheroid (LLC, NOR-10, LLC + NOR-10) micro-environment. The coassembled Fmoc-FRGDF/Fmoc-DIKVAV microenvironment enhances cancer-cell growth and progression compared to 2D cultures, non-encapsulate spheroids, and spheroids encapsulated in agarose. Agarose was selected as a control owing to the similar physical properties yet lack of biofunctionalisation. Results from this study reinforce the potential of Fmoc-SAPs as advanced microenvironments, and further support the ease of biological functionalisation inherent with this material. Further scaffold functionalisation is investigated through macromolecule addition. Here, one of two macromolecules are coassembled into a Fmoc-FRGDF network. The first macromolecule is fucoidan, a seaweed-derived polysaccharide with known anti-inflammatory properties, while the second is versican, a developmentally important proteoglycan which plays a variety of roles in muscle development. Versican was selected owing to its charge similarity to fucoidan, yet vastly different biological function. Fucoidan addition was found to increase fibre bundling and alter hydrogel mechanical properties, while versican addition had no substantial effect on hydrogel mechanics when compared to an Fmoc-FRGDF empty-vector control. Cell morphology was substantially altered by macromolecule addition, with fucoidan samples resulting in smaller, rounder cells with fewer multinucleated syncytia compared to an Fmoc-FRGDF control, while versican hydrogels showed an initial decrease in cell-size and multinucleation after 24h and a comparable cell-size and multinucleation following 72h. Here, it is possible that macromolecule addition perturbs cells attachment, and therefore, macromolecule selection is a key consideration. Interestingly, the regain of cell morphological characteristics in versican-containing hydrogels following 72h indicates the ability of cells to break-down versican, while the maintenance of small, round cells in the fucoidan hydrogels shows an inability for cells to break down fucoidan. The ability of Fmoc-SAPs to form components in bioinks is investigated through assembly with gelatin methacryloyl (GelMA) macromolecules. Initially, GelMA nanostructure and mechanical properties are investigated in response to increased degree of methacrylation or increased control. Here, structure-function relationships are drawn, and 18% methacryloyl Gelma (LM-GelMA) is selected for further bioink development owing to favourable thermoresponsive viscoelastic properties and improved strain tolerance. LM-GelMA assembly with coassembled Fmoc-FRGDF/Fmoc-PHSRN is investigated as a potential avenue to develop biologically and mechanically tuneable hydrogels. The incorporation of Fmoc-SAPs allows for control over sequence selection, while control over mechanical properties is offered through GelMA inclusion. LM-GelMA/Fmoc-FRGDF/Fmoc-PHSRN (FPG-Hybrid) bioinks demonstrate enhanced printability and are shown to support primary myoblast differentiation. The potential of Fmoc-SAP/GelMA bioinks to act as a modular bioink toolkit is further investigated through Fmoc-FRGDF/Fmoc-PHSRN substitution with Fmoc-DIKVAV, to develop a neural-suitable bioink (DIKVAV-Hybrid). This DIKVAV-Hybrid bioink demonstrated unique mechanical morphological properties and is shown to support rat cortical neurosphere viability. Throughout this project, the networks have been vigorously characterised through various analytical techniques, including micro/nanoimaging (Transmission electron microscopy, Atomic force microscopy, Cryo-scanning electron microscopy), Small-angle X-ray scattering, Small-angle neutron scattering, rheology, and spectroscopy; while the overall effectiveness of these systems have been analysed through in vitro muscle and neural cultures. Work detailed through this thesis aims to vigorously characterise Fmoc-SAP hydrogels and bioinks, providing the foundations for further biological studies and material optimisation