Analysis of gene expression during the macrophage to foam cell transformation using cDNA arrays

Coronary heart disease (CHD) is the leading cause of death in most industrialised countries. Atherosclerosis is the major underlying cause of CHD. Atherosclerosis is a disease process in which monocyte derived macrophages enter the subendothelial space, accumulate excess amounts of cholesterol to form lipid filled foam cells. These foam cells have been found contribute significantly to the aetiology of the fatty streak and subsequent lesions involved in atherosclerosis. The aim of this study was to gain a further understanding of the foam cell formation process by studying gene expression during the macrophage to foam cell transformation. Human THP-1 monocytic cells were differentiated into macrophages using a phorbol ester. Macrophages were subsequently exposed to either acetylated LDL or oxidised LDL to induce foam cell formation. Foam cell formation was assessed using the techniques of flow cytometry, Oil Red о staining, fluorescence microscopy and cholesteryl oleate loading. Gene expression was examined using high density cDNA array technology. RNA was isolated from cells exposed to native or modified LDL. First strand cDNA probes were subsequently generated and applied to high-density cDNA arrays. Two different arrays were probed; an array representative of the Human I M A G E collection and a custom array containing known genes thought to be involved in the cardiovascular disease process. The results of this study showed increased expression in CD36, a known receptor for OxLDL. In addition other genes were also identified including IL Iß and fíbronectin. Cluster analysis of a time series experiment showed the existence of nine distinct clusters of genes with different expression patterns. In particular two genes IL-1 β and TIMP-1 were shown to have a highly correlated pattern of expression

Microglia Control Vascular Architecture via a TGFβ1 Dependent Paracrine Mechanism Linked to Tissue Mechanics

© 2020, The Author(s). Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFβ1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFβ1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFβ1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFβ1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFβ1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS

CELL SURFACE COATINGS FOR MAMMALIAN CELL-BASED THERAPEUTIC DELIVERY

The cell plasma membrane is an interactive interface playing an important role in regulating cell-to-cell, cell-to-tissue contact, and cell-to-environment responses. This environment-responsive phospholipid layer consisting of multiple dynamically balanced macromolecules, such as membrane proteins, carbohydrate and lipids, is regarded as a promising platform for various surface engineering strategies. Through different chemical modification routes, we are able to incorporate various artificial materials into the cell surface for biomedical applications in small molecule and cellular therapeutics. In this dissertation, we establish two different cell coating techniques for applications of cell-mediated drug delivery and the localization of cell-based therapies to specific tissues. The first part of this dissertation establishes a membrane-associated hydrogel patch for drug delivery. The crosslinking of a grafted polymeric patch from a mammalian cell membrane is achieved through surface-mediated photolithographic polymerization. With the use of photomask, the formation of nanoparticle-loaded PEGDA hydrogel is controlled to deposit various geometric features on photoinitiator-immobilized surfaces. Through microarray patch patterning, we analyzed the influence of processing parameters on the accuracy of polymer patterning on a microarray. We then optimized the patterning approach for the formation of PEGDA patches on live A549 cells. In the second part of this dissertation, we study the use of tissue-adhesive coatings to improve the retention of therapeutic mesenchymal stem cells (MSCs) in the heart following intramyocardial or intravenous injection. MSCs were coated with antibodies against ICAM1 to adhere to CAM-overexpressed endothelium present in the heart following MI. Through intramyocardial or intravenous delivery, we observe higher number of coated cells retained in the heart over uncoated ones, supporting enhanced affinity for the inflamed endothelium near the infarct. We correlate the detachment force of antigen-interacted MSCs by a parallel laminar flow assay with the density of ICAM on the substrate and the density of anti-ICAM on the MSC surface. MSC retention on CAMmodified surfaces or activated HUVECs was significantly increased on antibody-coated groups (~90%) under physiologically hemodynamic forces (\u3c 30dyne/cm2), compared to uncoated MSCs (~20%). Moreover, a dramatic reduction of immune cell quantity was observed after intravenous injection, indicating the enhanced immunoregulatory efficacy by systemically delivering ICAM-adhesive MSCs to the site of inflammation

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model

Intravascular ultrasound and magnetic resonance imaging of the pulmonary arteries in pulmonary hypertension

Two relatively new techniques by which the pulmonary arteries can be imaged in life are intravascular ultrasound and magnetic resonance imaging. The main aim of this thesis is to describe the changes which are detectable on intravascular ultrasound and magnetic resonance imaging in patients with pulmonary hypertension and to determine whether these imaging modalities could be of use for the clinical assessment of the condition.Intravascular ultrasound was performed in 10 young adults with Eisenmenger's Syndrome and 4 infants with pulmonary hypertension secondary to a left to right shunt. Vasodilator studies were performed in 5 of the patients with Eisenmenger's. The vessel wall appeared as a single echogenic layer in all patients, making it difficult to define or measure medial thickness with certainty. Morphological changes of intimal hypertrophy and atherosclerosis were evident in patients with Eisenmenger's whereas in the infants the intima appeared thin and smooth, typical of normal artery. The technique gave excellent definition of the vessel lumen allowing continuous measurement of changes in luminal dimensions in response to vasodilators.MRI of the pulmonary arteries was performed in 11 patients with Eisenmenger's and 6 normal controls. In patients with pulmonary hypertension the pulmonary arteries were found to be dilated with reduced distensibility 4 when compared with normals. Calculations of Qp:Qs by MRI in patients with systemic to pulmonary shunts and pulmonary hypertension did not correlate well with values from cardiac catheterisation in all patients.As intravascular and magnetic resonance imaging are confined to the elastic pulmonary arteries, quantitative morphological studies were peformed on 24 post mortem specimens of lungs from patients who had died with pulmonary hypertension to determine whether there was any correlation between changes in the elastic pulmonary arteries and severity of pulmonary vascular disease. When compared with normals there was medial thickening in those with pulmonary hypertension but this was of an insufficient degree to be detectable by current ultrasound catheters. There was no correlation between degree of medial thickening in the elastic pulmonary arteries and severity of pulmonary vascular disease but intimal thickening and atherosclerosis were evident in those with more advanced disease.In conclusion, magnetic resonance imaging was found to have limited role in the assesssment of pulmonary hypertension but with new technical developments could become a non-invasive method of studying pulmonary hypertension in the future. The morphological changes detectable by intravascular ultrasound tend to be in severe disease only but the technique provides a unique method of studying pulmonary vascular reactivity in life

Computerized Analysis of Magnetic Resonance Images to Study Cerebral Anatomy in Developing Neonates

The study of cerebral anatomy in developing neonates is of great importance for the understanding of brain development during the early period of life. This dissertation therefore focuses on three challenges in the modelling of cerebral anatomy in neonates during brain development. The methods that have been developed all use Magnetic Resonance Images (MRI) as source data. To facilitate study of vascular development in the neonatal period, a set of image analysis algorithms are developed to automatically extract and model cerebral vessel trees. The whole process consists of cerebral vessel tracking from automatically placed seed points, vessel tree generation, and vasculature registration and matching. These algorithms have been tested on clinical Time-of- Flight (TOF) MR angiographic datasets. To facilitate study of the neonatal cortex a complete cerebral cortex segmentation and reconstruction pipeline has been developed. Segmentation of the neonatal cortex is not effectively done by existing algorithms designed for the adult brain because the contrast between grey and white matter is reversed. This causes pixels containing tissue mixtures to be incorrectly labelled by conventional methods. The neonatal cortical segmentation method that has been developed is based on a novel expectation-maximization (EM) method with explicit correction for mislabelled partial volume voxels. Based on the resulting cortical segmentation, an implicit surface evolution technique is adopted for the reconstruction of the cortex in neonates. The performance of the method is investigated by performing a detailed landmark study. To facilitate study of cortical development, a cortical surface registration algorithm for aligning the cortical surface is developed. The method first inflates extracted cortical surfaces and then performs a non-rigid surface registration using free-form deformations (FFDs) to remove residual alignment. Validation experiments using data labelled by an expert observer demonstrate that the method can capture local changes and follow the growth of specific sulcus

Multi-constraints based deep learning model for automated segmentation and diagnosis of coronary artery disease in X-ray angiographic images

Background: The detection of coronary artery disease (CAD) from the X-ray coronary angiography is a crucial process which is hindered by various issues such as presence of noise, insufficient contrast of the input images along with the uncertainties caused by the motion due to respiration and variation of angles of vessels. Methods: In this article, an Automated Segmentation and Diagnosis of Coronary Artery Disease (ASCARIS) model is proposed in order to overcome the prevailing challenges in detection of CAD from the X-ray images. Initially, the preprocessing of the input images was carried out by using the modified wiener filter for the removal of both internal and external noise pixels from the images. Then, the enhancement of contrast was carried out by utilizing the optimized maximum principal curvature to preserve the edge information thereby contributing to increasing the segmentation accuracy. Further, the binarization of enhanced images was executed by the means of OTSU thresholding. The segmentation of coronary arteries was performed by implementing the Attention-based Nested U-Net, in which the attention estimator was incorporated to overcome the difficulties caused by intersections and overlapped arteries. The increased segmentation accuracy was achieved by performing angle estimation. Finally, the VGG-16 based architecture was implemented to extract threefold features from the segmented image to perform classification of X-ray images into normal and abnormal classes. Results: The experimentation of the proposed ASCARIS model was carried out in the MATLAB R2020a simulation tool and the evaluation of the proposed model was compared with several existing approaches in terms of accuracy, sensitivity, specificity, revised contrast to noise ratio, mean square error, dice coefficient, Jaccard similarity, Hausdorff distance, Peak signal-to-noise ratio (PSNR), segmentation accuracy and ROC curve. Discussion: The results obtained conclude that the proposed model outperforms the existing approaches in all the evaluation metrics thereby achieving optimized classification of CAD. The proposed method removes the large number of background artifacts and obtains a better vascular structure

Developing Novel Platelet-Based Targeting Strategies for Thrombolytics

Use of plasminogen activators (PAs) as thrombolytic drugs is restricted to life threatening thrombotic settings because these therapies are associated with a high risk of bleeding. We hypothesize that platelet-delivered PAs would preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing pre-formed hemostatic clots. Two potential approaches were pursued: 1) PA-loaded platelets that release the thrombolytic from its granular stores upon activation, and 2) a thrombolytic chimeric protein that specifically binds to human platelets and activated when the platelets are incorporated into a growing thrombus. In our first approach, we desired to develop a strategy for producing platelets ex-vivo from cultured megakaryocytes that ectopically expressed urokinase-PA (uPA). No group had successfully produced sufficient ex-vivo generated platelets before, to side step this issue we infused ex-vivo generated megakaryocytes and showed that we can achieve a significant number of donor-derived platelets from these infused megakaryocytes in a murine model. The resulting platelets were normal in size, surface markers, circulating half-life, and were functional. Infused megakaryocytes localized to the pulmonary vasculature to shed platelets. We demonstrated, beginning with megakaryocytes derived from a transgenic mouse that ectopically express and store uPA in their alpha-granules that we can interfere with thrombosis by platelets generated from these megakaryocytes. In the second approach we produced a chimeric protein by fusing a single chain variable fragment (scFv) directed to the human-αIIb (hαIIb) platelet receptor subunit, with a human thrombin activatable pro-urokinase (uPA-T). The fusion protein (anti-PLT scFv/uPA-T) bound specifically to human and to transgenic mice platelets that expressed hαIIb, termed hαIIb+ mice, but did not bind to wildtype (WT) mouse platelets. Anti-PLT scFv/uPA-T retained its zymogenic properties until activated by thrombin. HαIIb+ mice were protected from forming occlusive thrombi for at least 10 hrs post anti-PLT/uPA-T treatment in contrast to the short functional half-life of soluble uPA-T. Thus this dissertation presents two distinct strategies that in proof of principle studies are each promising as approaches for effective and targeted platelet directed thrombolytic, which merit further study to test clinical applicability

Zeb1 controls neuron differentiation and germinal zone exit by a mesenchymal-epithelial-like transition

In the developing mammalian brain, differentiating neurons mature morphologically via neuronal polarity programs. Despite discovery of polarity pathways acting concurrently with differentiation, it's unclear how neurons traverse complex polarity transitions or how neuronal progenitors delay polarization during development. We report that zinc finger and homeobox transcription factor-1 (Zeb1), a master regulator of epithelial polarity, controls neuronal differentiation by transcriptionally repressing polarity genes in neuronal progenitors. Necessity-sufficiency testing and functional target screening in cerebellar granule neuron progenitors (GNPs) reveal that Zeb1 inhibits polarization and retains progenitors in their germinal zone (GZ). Zeb1 expression is elevated in the Sonic Hedgehog (SHH) medulloblastoma subgroup originating from GNPs with persistent SHH activation. Restored polarity signaling promotes differentiation and rescues GZ exit, suggesting a model for future differentiative therapies. These results reveal unexpected parallels between neuronal differentiation and mesenchymal-to-epithelial transition and suggest that active polarity inhibition contributes to altered GZ exit in pediatric brain cancers.National Institute of Neurological Disorders and Stroke grant: (1R01NS066936); March of Dimes Foundation grant: (#1-FY12-455).info:eu-repo/semantics/publishedVersio

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals