Mechanisms Of Blood Vessel Formation Research Articles

Vascular Organization: Lessons from Development and Disease.

Organ formation requires tight coordination with vascular growth. Intricate networks of blood vessels course through all organs and tissues and are composed of both endothelial cells (ECs) and associated mural cells. Despite decades of research into the biology of blood vessel formation and homeostasis, little is known about how the vasculature ensures its properly coordinated growth and intimate development with the cells of different organs. Even more mystifying is how a highly dynamic endothelium quiesces to differentiate into mature vessels, and how disruption of this mature quiescence results in pathological conditions. Interestingly, both intra- and interorgan vascular architecture hold critical importance to the maintenance of blood vessels, as the rate of flow and supply of supportive angiogenic factors can be altered with deleterious effects. In this article, we review the basic mechanisms of blood vessel formation and maintenance with an emphasis on organ-specific vascular development, and we examine the case of transient pulmonary arteriovenous malformations as a case study of vascular homeostatic mechanisms.

Current status and future of anti-angiogenic drugs in lung cancer.

Lung cancer, as a malignant tumor with both high incidence and mortality in China, is one of the major causes of death in our population and one of the major public health problems in China. Effective treatment of lung cancer is a major public health task for all human beings. Angiogenesis plays an important role in the development of tumor, not only as a basic condition for tumor growth, but also as a significant factor to promote tumor metastasis. Therefore, anti-angiogenesis has become a vital means to inhibit tumor development, and anti-angiogenic drugs can rebalance pro- and anti-angiogenic factors to inhibit tumor cells. This article reviews the mechanism of blood vessel formation in tumor tissues and the mechanism of action of different anti-angiogenic drugs, the combination therapy of anti-angiogenic drugs and other anti-tumor drugs, and the mechanism of anti-angiogenic drug resistance.

Generation of specialized blood vessels via lymphatic transdifferentiation.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. Wefurther use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstratingthat LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Isolation of Highly Purified and Viable Retinal Endothelial Cells

The neonatal mouse retinal vascularization model has been widely used in the vascular biology field to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Recent advances in next-generation sequencing can further elucidate mechanisms of blood vessel formation and remodeling in this, as well as other, vascular development models. However, an optimized method for isolating retinal endothelial cells that limits tissue digestion-induced cell damage is required for next-generation sequencing applications. In this study, we established a method for isolating neonatal retinal endothelial cells that optimizes cell viability and purity. The CD31+/CD45− endothelial cell population was fluorescence-activated cell sorting (FACS)-isolated from digested postnatal retinas, found to be highly enriched for endothelial cell gene expression, and exhibited no change in viability for 60 min post-FACS. Thus, this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. Combining this isolation method with next-generation sequencing will enable further delineation of mechanisms underlying vascular development and maturation.

Resistance Mechanisms to Anti-angiogenic Therapies in Cancer.

Tumor growth and metastasis rely on tumor vascular network for the adequate supply of oxygen and nutrients. Tumor angiogenesis relies on a highly complex program of growth factor signaling, endothelial cell (EC) proliferation, extracellular matrix (ECM) remodeling, and stromal cell interactions. Numerous pro-angiogenic drivers have been identified, the most important of which is the vascular endothelial growth factor (VEGF). The importance of pro-angiogenic inducers in tumor growth, invasion and extravasation make them an excellent therapeutic target in several types of cancers. Hence, the number of anti-angiogenic agents developed for cancer treatment has risen over the past decade, with at least eighty drugs being investigated in preclinical studies and phase I-III clinical trials. To date, the most common approaches to the inhibition of the VEGF axis include the blockade of VEGF receptors (VEGFRs) or ligands by neutralizing antibodies, as well as the inhibition of receptor tyrosine kinase (RTK) enzymes. Despite promising preclinical results, anti-angiogenic monotherapies led only to mild clinical benefits. The minimal benefits could be secondary to primary or acquired resistance, through the activation of alternative mechanisms that sustain tumor vascularization and growth. Mechanisms of resistance are categorized into VEGF-dependent alterations, non-VEGF pathways and stromal cell interactions. Thus, complementary approaches such as the combination of these inhibitors with agents targeting alternative mechanisms of blood vessel formation are urgently needed. This review provides an updated overview on the pathophysiology of angiogenesis during tumor growth. It also sheds light on the different pro-angiogenic and anti-angiogenic agents that have been developed to date. Finally, it highlights the preclinical evidence for mechanisms of angiogenic resistance and suggests novel therapeutic approaches that might be exploited with the ultimate aim of overcoming resistance and improving clinical outcomes for patients with cancer.

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

TGF-β Signaling Induces the Expression of OPN in Blood Vessel Endothelial Cells.

The mechanism of blood vessel formation and degeneration still remains unclear. Transforming growth factor-β1 (TGF-β1) signaling is a critical pathway in this progression and can induce multiple biological effects. Osteopontin (OPN) is involved in mineral metabolism and the inflammatory response associated with vascular calcification. To identify the relationship between TGF-β signaling pathway and OPN, we stimulated human vascular endothelial cells (HVECs) and human aortic endothelial cells (HAECs) using various concentration of TGF-β1 in vitro. As assessed by flow cytometry and western blots, apoptosis levels were significantly increased with TGF-β1 treatment. We also demonstrated that OPN increased in vitro with TGF-β signaling by western blot and quantitative real time polymerase chain reaction (qRT-PCR) analyses. The inhibitory phosphorylation of endothelial nitric-oxide synthase (eNOS) (Thr495) was also up-regulated by TGF-β signaling. Meanwhile, the anti-inflammatory factor Nrf2 and the activating phosphorylation of eNOS (Ser1177) were down-regulated. Taken together, our findings demonstrate that TGF-β signaling can induce the expression of OPN, which may play an important role in the dysfunction of the vascular wall.

Downregulation of transgelin blocks interleukin-8 utilization and suppresses vasculogenic mimicry in breast cancer cells.

Vasculogenic mimicry (VM) is a non-classical mechanism recently described in many tumors, whereby cancer cells, rather than endothelial cells, form blood vessels. Transgelin is an actin-binding protein that has been implicated in multiple stages of cancer development. In this study, we investigated the role of transgelin in VM and assessed its effect on the expression of endothelial and angiogenesis-related genes during VM in MDA-MB-231 breast cancer cells. We confirmed the ability of MDA-MB-231 cells to undergo VM through a tube formation assay. Flow cytometry analysis revealed an increase in the expression of the endothelial-related markers VE-cadherin and CD34 in cells that underwent VM, compared with those growing in a monolayer, which was confirmed by immunocytochemistry. We employed siRNA to silence transgelin, and knockdown efficiency was determined by western blot analyses. Downregulation of transgelin suppressed cell proliferation and tube formation, but increased IL-8 levels in Matrigel cultures. RT-PCR analyses revealed that the expression of IL-8, VE-cadherin, and CD34 was unaffected by transgelin knockdown, indicating that increased IL-8 expression was not due to enhanced transcriptional activity. More importantly, the inhibition of IL-8/CXCR2 signaling also resulted in suppression of VM with increased IL-8 levels, confirming that increased IL-8 levels after transgelin knockdown was due to inhibition of IL-8 uptake. Our findings indicate that transgelin regulates VM by enhancing IL uptake. These observations are relevant to the future development of efficient antivascular agents. Impact statement Vasculogenic mimicry (VM) is an angiogenic-independent mechanism of blood vessel formation whereby aggressive tumor cells undergo formation of capillary-like structures. Thus, interventions aimed at angiogenesis might not target the entire tumor vasculature. A more holistic approach is therefore needed in the development of improved antivascular agents. Transgelin, an actin-binding protein, has been associated with multiple stages of cancer development such as proliferation, migration and invasion, but little is known about its role in vasculogenic mimicry. We present here, an additional mechanism by which transgelin promotes malignancy by way of its association with the occurrence of VM. Although transgelin knockdown did not affect the transcript levels of most of the angiogenesis-related genes in this study, it was associated with the inhibition of the uptake of IL-8, accompanied by suppressed VM, indicating that transgelin is required for VM. These observations are relevant to the future development of efficient antivascular agents.

Therapeutic Angiogenesis in Ischemic Tissues by Growth Factors and Bone Marrow Mononuclear Cells Administration: Biological Foundation and Clinical Prospects.

The processes of new vessels formation in tissues are supported by two definite mechanisms: de novo development of blood vessels (vasculogenesis) through the accumulation of progenitor cells during early prenatal stage, and extension of a pre-existing microcirculatory network by endothelial cell germination (angiogenesis), the essential mechanism of blood vessel formation in postnatal period. Angiogenesis is associated with a series of inductive, permissive and restrictive communications that result in the appearance, differentiation, and formation of new vessels. The goal of therapeutic angiogenesis is to improve blood circulation, relay survival factors and regenerative stem cell populations to sites of tissue repair, and ultimately recover function and form of the tissue. Growth factors and bone marrow mononuclear cells represent a very interesting research field for the realization of therapeutic angiogenesis in ischemic tissues. They provide a potential key component in the healing processes of ischemic injured tissues.

Understanding and targeting resistance to anti-angiogenic therapies.

Therapies targeting tumor angiogenesis are used in a variety of malignancies, however not all patients benefit from treatment and impact on tumor control may be transient and modest. Mechanisms of resistance to anti-angiogenic therapies can be broadly categorized into VEGF-axis dependent alterations, non-VEGF pathways, and stromal cell interactions. Complimentary combinations of agents that inhibit alternative mechanisms of blood vessel formation may optimize inhibition of angiogenesis and improve clinical benefit for patients. The purpose of this review is to detail the preclinical evidence for mechanisms of angiogenic resistance and provide an overview of novel therapeutic approaches exploiting these pathways.

Transgenesis and imaging in birds, and available transgenic reporter lines

Avian embryos are important model organism to study higher vertebrate development. Easy accessibility to developing avian embryos enables a variety of experimental applications to understand specific functions of molecules, tissue-tissue interactions, and cell lineages. The whole-mount ex ovo culture technique for avian embryos permits time-lapse imaging analysis for a better understanding of cell behaviors underlying tissue morphogenesis in physiological conditions. To study mechanisms of blood vessel formation and remodeling in developing embryos by using a time-lapse imaging approach, a transgenic quail model, Tg(tie1:H2B-eYFP), was generated. From a cell behavior perspective, Tg(tie1:H2B-eYFP) quail embryos are a suitable model to shed light on how the structure and pattern of blood vessels are established in higher vertebrates. In this manuscript, we give an overview on the biological and technological background of the transgenic quail model and describe procedures for the ex ovo culture of quail embryos and time-lapse imaging analysis.

Vascular Lumen Formation

The vascular system developed early in evolution. It is required in large multicellular organisms for the transport of nutrients, oxygen, and waste products to and from tissues. The vascular system is composed of hollow tubes, which have a high level of complexity in vertebrates. Vasculogenesis describes the de novo formation of blood vessels, e.g., aorta formation in vertebrate embryogenesis. In contrast, angiogenesis is the formation of blood vessels from preexisting ones, e.g., sprouting of intersomitic blood vessels from the aorta. Importantly, the lumen of all blood vessels in vertebrates is lined and formed by endothelial cells. In both vasculogenesis and angiogenesis, lumen formation takes place in a cord of endothelial cells. It involves a complex molecular mechanism composed of endothelial cell repulsion at the cell-cell contacts within the endothelial cell cords, junctional rearrangement, and endothelial cell shape change. As the vascular system also participates in the course of many diseases, such as cancer, stroke, and myocardial infarction, it is important to understand and make use of the molecular mechanisms of blood vessel formation to better understand and manipulate the pathomechanisms involved.

Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

Visualization and experimental analysis of blood vessel formation using transgenic zebrafish

The mechanisms of blood vessel formation have become a subject of enormous scientific and clinical interest. However, it is difficult to visualize the developing vasculature in most living animals due to the ubiquitous and deep localization of vessels within other tissues. The establishment of vascular-specific transgenic zebrafish with fluorescently "tagged" blood vessels has facilitated high-resolution imaging studies of developing blood and lymphatic vessels in vivo. Use of these transgenic lines for genetic and chemical screening, experimental manipulations, and time-lapse imaging has extended our knowledge of how complex networks of vessels assemble in vivo.

Vasculogenesis, angiogenesis, and arteriogenesis: Mechanisms of blood vessel formation and remodeling

In this review, the concept of oxygen homeostasis will be presented as an organizing principle for discussion of the phylogeny, ontogeny, physiology, and pathology of blood vessel formation and remodeling, with a focus on molecular mechanisms and potential therapeutic applications.

Potential role of the Slit/Robo signal pathway in angiogenesis

Intensive investigations on angiogenesis and vasculogenesis have increased our understanding of molecular mechanisms of blood vessel formation during pathologic and developmental conditions. However, endothelial cells (ECs), the main component of vasculature, are heterogeneous, as revealed by our phenotypic and molecular biological studies in the laboratory, and it is still hard to adequately understand the molecular mechanisms of angiogenesis and vasculogenesis. Indeed, there are several major ligand/receptor signal pathways: VEGF/VEGFR, Jagged-1/Notch, Wnt ligand/frizzled receptor, and ephrin/Eph; each of which having distinct and independent roles during vascular formation. In this review, we focus on the angiogenic effect of the Slit and Robo signal pathway that was formally known as neuronal axon guidance. Among the existing vascular signals, this pathway is the most recently found ligand/receptor vascular signal, and may play important physiological roles as other major receptor/ligand signals do. Here, we briefly address: (1) the background of Slit and Robo families; (2) expression patterns of Slit and Robo; (3) functional roles of the Slit/Robo pathway in vascular formation; and (4) confronting tasks of this novel vascular pathway in the near future. Together, a summary of these data suggest the essential role of the Slit/Robo pathway in angiogenesis, and may explain why multiple vascular signals exist in heterogenic endothelial cells.

Induction of lymphatic endothelial cell differentiation in embryoid bodies

AbstractThe molecular mechanisms that regulate the formation of the lymphatic vascular system remain poorly characterized. Whereas studies in embryonic stem (ES) cells have provided major new insights into the mechanisms of blood vessel formation, the development of lymphatic endothelium has not been previously observed. We established embryoid bodies (EBs) from murine ES cells in the presence or absence of lymphangiogenic growth factors. We found that lymphatic endothelial cells develop at day 18 after EB formation. These cells express CD31 and the lymphatic lineage markers Prox-1 and Lyve-1, but not the vascular marker MECA-32, and they frequently sprout from preexisting blood vessels. Lymphatic vessel formation was potently promoted by VEGF-A and VEGF-C but not by bFGF. Our results reveal, for the first time, that ES cells can differentiate into lymphatic endothelial cells, and they identify the EB assay as a powerful new tool to dissect the molecular mechanisms that control lymphatic vessel formation.

Mast cells and eosinophils: A novel link between inflammation and angiogenesis in allergic diseases

Mast cells and eosinophils are the key cells in the early and late stages of allergic inflammation. There is increasing evidence that angiogenesis plays an important role both in the development of inflammation and in the pathophysiology of tissue remodeling during allergic disorders. In this review we provide recent data showing a link between allergy and angiogenesis and some possible mechanisms through which vascular endothelial growth factor and the immune system can interact. We discuss the multifaceted roles of mast cells and eosinophils in tissue remodeling and angiogenesis during allergic diseases and whether these cells can be both source and target cells for pro-angiogenic mediators.