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The development of new blood and lymphatic vessels, through the process of angiogenesis and lymphangiogenesis, respectively, is critical to the development and growth of tumors, and integral to the process of metastasis. Lymphatic vessel density can be assessed as a surrogate measure of lymphangiogenesis in human tissue samples. Lymphatic vessel density has been shown to be associated with lymph node metastasis and patient survival in various solid tumor types. Here we describe a method for quantifying the number of lymphatic vessels within tumor tissue that can also be used to assess lymphatic vessel invasion, and compare with blood vessel density and invasion.The development and maturation of the lymphatic vasculature are essential for organ function with disruption leading to severe phenotypes. For example, malfunction of cardiac lymphatics results in myocardial oedema, persistent inflammation and reduced cardiac output. Thus, it is important to study the process of cardiac lymphatic formation and growth from the early stages of fetal development to adulthood. In the murine heart the lymphatics continue to develop and expand postnatally with extensive growth and patterning occurring up to at least 2 weeks after birth. Entinostat nmr Here, we describe a protocol for whole-mount, multi-view imaging and quantification of lymphatic vessel parameters, including vessel junction number (i.e., branching density), vessel length, and number of vessel end points in the murine postnatal heart. This protocol is based on the use of reliable antibodies against key markers of lymphatic endothelial cells (LECs), specifically the glycoprotein lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), the vascular endothelial growth factor receptor 3 (VEGFR3; also known as Fms-related receptor tyrosine kinase 4, FLT4), the mucin-type protein podoplanin (PDPN), and the co-receptor neuropilin 2 (NRP2). For imaging and quantitative analysis of the sub-epicardial network in neonatal hearts, VEGFR3 was selected given its exclusive expression in the lymphatic endothelium. In addition to LECs, LYVE1 expression was detected in tissue-resident macrophages, PDPN in the epicardium, and NRP2 in the autonomic nervous system of the heart. Overall, we characterized the expression patterns of commonly used lymphatic markers in the context of the neonatal heart and provide an image analysis pipeline that can be adapted to study other organs and systems (e.g., blood vasculature and nerve system).Stromal vascular fraction (SVF), isolated from adipose tissue, identifies as a rich cell source comprised of endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, fibroblasts, and immune cells. SVF represents a promising therapeutic heterogonous cell source for growing new blood microvessels due to its rich niche of cells. However, the spatiotemporal dynamics of SVF within living tissues remain largely unknown. The objective of this chapter is to describe a protocol for culturing SVF on mouse mesentery tissues in order to aid in the discovery of SVF dynamics and associated vessel growth over time. SVF was isolated from the inguinal adipose from adult mice and seeded onto mesentery tissues. Tissues were then cultured for up to 5 days and labeled with endothelial cell and pericyte markers. Representative results demonstrate the observation of SVF-derived vasculogenesis characterized by de novo vessel formation and subsequent vessel connection.In the retina EC dysfunction and angiogenesis are driven by an altered microenvironment e.g., diabetes, leading to hypoxia and inflammation in the retinal layers, resulting in excessive vascular leakage and growth. The gold standard for measuring blood-retinal barrier permeability in response to disease and or therapy has been the gold standard Evans blue (EB) assay. However, this technique has limitations in vivo, including nonspecific tissue binding and toxicity. Here we describe a novel imaging methodology combining sodium fluorescein fundus angiography (FFA) with mathematical quantification allowing retinal permeability to be noninvasively and accurately measured at multiple time points in the same animal, minimizing animal use in line with the 3Rs framework. In addition, this technique is a nontoxic, high throughput, sensitive, and cost-effective alternative technique to the Evans blue assay. Moreover, this technique can be translated to other species.Changes in blood vessels and lymphatics in health and disease are easier to understand and interpret when studied microscopically in three dimensions. The mouse trachea is a simple, yet powerful, and versatile model system in which to achieve this. We describe practical immunohistochemical methods for fluorescence and confocal microscopy of wholemounts of the mouse trachea to achieve this purpose in which the entire vasculature can be visualized from the organ level to the cellular and subcellular level. Blood vessels and lymphatics have highly stereotyped vascular architectures that repeat in arcades between the tracheal cartilages. Arterioles, capillaries, and venules can be easily identified for the blood vessels, while the lymphatics consist of initial lymphatics and collecting lymphatics. Even small abnormalities in either blood vessels or lymphatics can be noticed and evaluated in three dimensions. We and others have used the mouse trachea for examining in situ angiogenesis and lymphangiogenesis, vascular development and regression, vessel patency, differences in transgenic mice, and pathological changes, such as increased vascular permeability induced by inflammatory mediators.Peripheral vascular disease is a major cause of morbidity and mortality, and is a consequence of impaired blood flow to the limbs. This arises due to the inability of the tissue to develop sufficiently functional collateral vessel circulation to overcome occluded arteries, or microvascular impairment. The mouse hind limb model of hind limb ischemia can be used to investigate the impact of different treatment modalities, behavioral changes, or genetic knockout. Here we described the model in detail, providing examples of adverse events, and details of ex vivo analysis of blood vessel density.Transmission electron microscopy using resin sections still remains an exceptionally useful tool in evaluating cellular ultrastucture within tissue. For the endothelium the best method for maintaining such structure is perfusion fixation fixing the tissue under physiological pressure. Here the focus is on a method of maintaining the vascular wall structure including the endothelial glycocalyx and extending this with tilt series tomography. Shown are typical histological sections from multiple capillary beds including brain, heart and retina using a lanthanide staining technique (LaDy GAGa) to highlight that the differences in normo-physiology are substantial.It is hoped that users will find the notes useful in deciding which specific staining and imaging method would suit their needs so this technically challenging, and low throughput methodology, is used to its best effect.Whole-mount immunostaining allows intact tissue to be surveyed in three dimensions, avoiding the more restricted fields of view provided by visualizing thin sections. This technique is particularly useful for imaging lymphatic and blood networks by high-resolution confocal microscopy, revealing how such vessels are spatially positioned, the subcellular arrangements of individual antigens, and interactions with individual cells within the interstitium or vessel lumen. The purpose of this chapter is to provide a practical guide for obtaining images of lymphatic vessels following immunofluorescence staining, primarily in mouse skin.Understanding the development of the lymphatic vasculature is essential to the understanding of how these vessels function in health and disease. High-resolution imaging of histological techniques such as immunostaining of sectioned tissue provides a snapshot into lymphatic vessel morphogenesis, patterning, and organization. Whole-mount staining of embryonic dermal vasculature allows for a deeper analysis and characterization of the developing lymphatic vascular network.Angiogenesis refers to the expansion of blood vessels from a preexisting vascular plexus, and it is a fundamental process for organ development, the female reproductive system, and wound healing, but it is also a common denominator in several diseases such as cancer and neovascular eye disease. For these reasons, shedding light on the molecular and cellular mechanisms of angiogenesis has the potential to devise new therapeutic strategies to refrain pathological vessel growth or even promote new vessel formation in ischemic conditions and organ grafts. The mouse postnatal retina provides an excellent and widely adopted model to study physiological angiogenesis in vivo, and this chapter outlines a detailed protocol for its dissection, staining, and analysis of the vasculature.Pharyngeal arch arteries (PAA) are formed early during mouse embryogenesis and remodel soon thereafter into the aortic arch arteries. Failure of these vessels to form or remodel results in congenital heart defects. This protocol is designed to study the formation of the PAA using whole-mount immunofluorescence staining, followed by tissue clearing with benzyl alcohol/benzyl benzoate (BAAB) and imaging by confocal microscopy. The fine cellular resolution obtained with this technique allows the embryonic vasculature of the pharyngeal arch artery endothelium to be visualized by surface rendering and quantitatively analyzed by counting the number of endothelial cells in both the PAA and the vascular plexus surrounding them.Angiogenic vessel remodeling is a critical step in establishing a hierarchical vessel network. Vessel networks rapidly expand through angiogenesis in response to pro-angiogenic factors. This leads to an initially dense vessel network that requires selective regression of vessel branches to establish a hierarchical conduit for blood flow, a process known as pruning. This involves migration of endothelial cells from low-flow vessels to adjacent high-flow vessels and generally occurs independently of cell death. Vessels may also regress in response to other stimuli, including reduced metabolic demand, redundancy, and pathological stimuli. In these contexts, widespread vessel regression typically occurs and involves loss of endothelial cells by apoptotic cell death. Thus, vessel remodeling occurs via both apoptosis independent and dependent vessel regression. In this chapter, we outline a semi-automated method for quantifying vessel regression using the neonatal model of angiogenesis. We further provide instruction on analyzing endothelial apoptosis in this model.Zebrafish allow unrivalled in vivo imaging of vascular development due to their optical translucency and the availability of transgenic lines which fluorescently label cells and tissues of interest. Advances in light sheet fluorescence microscopy allow longer and faster imaging of live embryos at higher resolutions than previously possible, which facilitates study of dynamic cellular and molecular mechanisms underlying vessel formation and function. Here we describe a workflow using lightsheet microscopy to quantify endothelial cell (EC) migration dynamics during vascular development. Tracking movement of EC nuclei and analyzing the properties of EC migration trajectories permit detailed studies of angiogenesis and vascular remodeling in different contexts.
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