Analysis of capillary-like structures formed by endothelial cells in a novel organotypic assay developed from heart tissue

In this work, we present an algorithm to perform automatic segmentation, feature extraction and dosage classification of images derived from a novel organotypic angiogenesis assay developed to assess the effects of ionising radiation in the mouse heart. The images presented very different conditions of illumination and conditions of density and shapes of cells. The algorithm consists of a pipeline of several steps, which were validated against hand-segmented images. The algorithm provided satisfactory results, as all images were correctly dose-classified. The cells exposed to the lowest radiation dose were observed to have the greatest relative feature variability

Mechanisms of cardiac endothelial damage by ionising radiation

Exposure of the heart to ionising radiation even at low to moderate doses can increase the risk of developing cardiovascular disease (CVD) many years later. This is of significance to cancer survivors who were treated with radiotherapy in the thorax. Damage to the heart microvasculature is thought to play a key role in development of radiation-induced CVD. The aim of this project was to investigate the effects of radiation on heart microvascular endothelial function using in vitro and in vivo approaches. In particular effects of radiation on angiogenesis were investigated since angiogenesis is important for heart repair after myocardial injury. The hearts of C57BL/6 and APOE-/- mice were X-ray irradiated with doses of 0.2 to 16 Gray by the group’s collaborators and animals were sacrificed at 20-60 weeks post-irradiation. Angiogenic sprouting was assessed in heart explants embedded in fibrin. A dose dependent reduction in angiogenic sprouting was observed, which was significant after ≥8 Gy at 20 weeks and ≥2 Gy at 60 weeks post-irradiation demonstrating that vascular damage was progressive. Radiation inhibited in vitro endothelial migration at doses of ≥0.2 Gy. Radiation also inhibited endothelial tubule formation in matrigel and in organotypic assays in which endothelial cells were co-cultured with fibroblasts. In addition to damaging cells directly, radiation also induces indirect effects through bystander interactions. Results showed that irradiated fibroblasts inhibited angiogenesis through soluble factors they secreted into their conditioned media. Signaling mechanisms through which radiation alters angiogenic function were studied. Results showed that transforming growth factor beta and Rho-GTPase signaling are involved in the anti-angiogenic activity of radiation. In summary, this study established that low to moderate doses of radiation inhibit endothelial function which could contribute to the development of CVD. The study also identified signaling pathways that may be targeted to protect against radiation-induced heart microvascular damage

The retinoid agonist Tazarotene promotes angiogenesis and wound healing

Therapeutic angiogenesis is a major goal ofregenerative medicine, but no clinically approved small molecule exists that enhancesnew blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitroangiogenesis, promoting branching morphogenesis, and tubule remodeling. The pro-angiogenic phenotype is mediated by Retinoic Acid Receptor (RAR) but not Retinoic X Receptor(RXR) activation, and is characterized by secretion of the pro-angiogenic factors Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGFA), Plasminogen Activator, Urokinase (PLAU) and Placental Growth Factor (PGF), and reduced secretion of the antiangiogenic factor Pentraxin-3 (PTX3) from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing

Quantification of the Effects of Low Dose Radiation and its Impact on Cardiovascular Risks

This work describes an algorithm developed to quantify the effects of low dose radiation on the cardiac endothelial cells with the final objective of inferring how radiation may potentially initiate cardiovascular disease in post radiotherapy-treated patients. The effects are investigated by using an in-vitro co-culture cellular matrix, consisting of endothelial cells on a base of fibroblasts, which in time begin to form capillary (tubular) like structures. A range of radiation doses (0.2-16 Gray (Gy)) was applied to different samples and the effects observed. The automatic segmentation is validated against a set of manual segmented images with satisfactory results presenting a correct classification of 0.93; classification is the measure of comparison between two sets of images, specified as a number from 0 to 1, whereby 1 denotes 100% similarity whilst 0 refers to 0% similarity. Measurements related to geometrical parameters were further obtained. It was found during the course of this project, the largest observable change in endothelial cell structure was found after exposure to 0.2 Grays of radiation

3d Biomimetic Model for Cellular Invasion in Angiogenesis and Cancer

Cell migration is an essential and highly regulated process. Cells migrate to vascularize tissues, to form tissue, and to respond to inflammation. Unfortunately, cell migration is also involved in numerous pathological conditions such as in invasive tumors. Cells can migrate as individual cells or as collective groups of cells. Particularly important in cell migration is the collective migration of cells as it is a hallmark of tissue remodeling events during embryonic morphogenesis, wound repair, and cancer invasion. Perhaps, angiogenesis is one of the most crucial collective migration processes as it is involved in multiple physiological and pathological conditions such as formation of vasculature, wound healing, cancer progression and metastasis. During angiogenesis, endothelial cells migrate collectively from existing vasculature in response to a complex biochemical and mechanical cues to form multicellular structures that eventually develop into new functional blood vessels. Angiogenesis is also a highly dynamic process where multiple cells rearrange and coordinate within a sprout. Such dynamic rearrangement requires different cytoskeletal regulators such as Rho GTPases proteins (RhoA, Rac, and Cdc42). Although the roles of Rho GTPase proteins have been well characterized in 2D cell migration, little is known about their contributions in angiogenic morphogenesis. Here, we engineered a 3D biomimetic microfluidic-based device, called AngioChip, where endothelial cells are induced to migrate collectively from a pre-formed biomimetic cylindrical blood vessel into a 3D interstitial collagen matrix. The sprouts in our AngioChip demonstrate in vivo-like morphogenetic features such as formation of tip-stalk cells, lumen formation, filopodial-like protrusions in leading tip cells, and formation of perfusable neovessels. Using this system, we examine the roles of Cdc42 to regulate many aspects of angiogenic morphogenesis. We find that disturbing Cdc42 activity reduces formation of branches, migration speed, and collective migration. Additionally, Cdc42 also negatively regulate filopodia formation. We also develop the AngioChip into a pancreatic ductal adenocarcinoma (PDAC) on a chip to investigate the interactions between pancreatic cancer cells and blood vessels. Vascular invasion, where PDAC cells invaded towards the vasculature during tumor progression, is a hallmark of metastatic PDAC. Nevertheless, how pancreatic tumor cells interact with the blood vessels remains largely unknown. Using our PDAC-on-a-chip, we reveal a striking observation where PDAC cells invade and de-endothelialize the blood vessels. This de-endothelialization process leads to vascular replacement in the blood vessels and is mediated by proliferation of PDAC through Nodal/Activin-ALK7 signaling

Characterising the in vitro and in vivo function of the RhoG effector DOCK4 during angiogenesis and ischemia.

The RAC1 specific GEF, DOCK4, has been identified as an essential component in the Rho GTPase signalling pathway, imperative for correct vascular patterning and lumenisation during sprouting angiogenesis in vitro. As RAC1 has been previously implicated in the signalling events involved in vascular regrowth within a hypoxic environment, it was hypothesized that DOCK4 may be an important effector in the response to vascular injury and oxygen deprivation. To test this hypothesis, a DOCK4 depleted endothelial co-culture assay was carried out in both hypoxic and normoxic conditions. DOCK4 driven activation of RAC1 has been demonstrated under VEGF signalling, however FGF2 signalling pathways have also been strongly implicated in vascular response to blood vessel injury and hypoxia. Therefore, co-culture assays were carried out to assess sprouting angiogenesis with DOCK4 knockdown in response to FGF2 supplementation. Further, a heterozygous DOCK4 depleted murine model in ischemia studies using a model of HLI was employed together with LDI monitoring of vascular response and regrowth, comparing the response of heterozygous Dock4 KO mice and their WT littermate controls. DOCK4 interacts with the CDC42 GEF DOCK9 but the molecular basis of the interaction is unknown, as is the role of GEF heterodimerization in cell signalling. This study aims to further understand the function of DOCK4 within a pathological sprouting angiogenesis while also investigating the mechanism of interaction between DOCK4 and DOCK9. The two pro-angiogenic growth factors VEGFA and FGF2 drive different phenotypical growth responses during sprouting angiogenesis in vitro. DOCK4 was demonstrated as being an important component of FGF2 stimulated angiogenesis under hypoxia, indicating DOCK4 as important for mechanisms involved in the angiogenic response to ischemia. The specific site of DOCK9 which interacts with the SH3 domain of DOCK4 was not elucidated during this study, however it was determined that DOCK9 proline rich regions identified as PRR 2, 3, 4, and 9 were unlikely to be involved in the interaction. The small molecule inhibitor QL-47 was demonstrated to be a potent anti-angiogenic compound with VEGFA stimulated ECs being particularly sensitive to QL-47. However, it is highly unlikely that the anti-angiogenic effects are due to disruption of the DOCK4-DOCK9 interaction, as the p.C628 cysteine residue was found to not be involved in DOCK4 SH3 domain interaction. Understanding how Rho GTPases are regulated and mechanisms underpinning their activity will progress the understanding of events that drive blood vessel growth while gaining insight into dysregulation during angiogenic pathologies

Development of Biomimetic Models of Human Cardiac Tissue

The leading cause of death worldwide is cardiovascular disease (CVD). Myocardial infarction (MI) (i.e., heart attack) makes up ~8.5% of CVD and is a common cause of heart failure with a 40% five-year mortality after the first MI. This highlights a substantial patient population and an urgent need to develop new therapeutic strategies (e.g., regenerative cell therapies). Moreover, this also indicates that current models may not sufficiently recapitulate human cardiac tissue. To date, drug development strategies have largely depended on high throughput 2D cell models and pre-clinical testing in animal models of MI leading to minimal improvements in the heart failure treatment paradigm over the past 20 years. Relevant human cardiac models would provide insight into human cardiac tissue physiology and maturation while also providing an advanced in vitro screening tool to explore heart failure pathogenesis. Cardiac tissue engineering has allowed for advances in the development of cardiac constructs by combining developments in biomaterials, 3D microtissue culture, and human induced pluripotent stem cells (hiPSC) technology. Notably, approaches that mimic the natural processes in the body (i.e., biomimetic) have led to further insight into cardiac physiology. Here, I have pursued biomimetic strategies to create a biomimetic model of human cardiac tissue using hiPSC-derived cardiomyocytes (hiPSC-CMs). Throughout this development, I explored the role of the matrix microenvironment on cell behavior using functionalized alginate, the influence of pacemaker-like exogenous electrical stimulation on the maturation of hiPSC-CM spheroids with endogenous electrically conductive nanomaterials, and the development of vascularized, functional cardiac organoids by mimicking the coronary vasculogenesis stage of cardiac development. The research established here provided a biomimetic groundwork for future development into in vitro human cardiac tissue models for applications in basic research, drug discovery, and cell therapy