Angiogenesis refers to all processes that result in the formation of new blood vessels from existing ones. Vasculogenesis, on the other hand, defines the process of de novo vessel formation and is based on the specification of endothelial progenitors, known as angioblasts, from the mesoderm. As a result, vasculogenesis establishes the initial vascular network, which includes the dorsal aorta and cardinal vein, but angiogenesis modifies and grows it. Ellertsdottir and colleagues recently summarised the hallmarks of zebrafish vasculogenesis, but many novel aspects of zebrafish angiogenesis have been discovered in recent years.

Angiogenesis is primarily accomplished through vessel sprouting, which can be broken down into four major steps: tip cell formation, tubule morphogenesis and lumen formation, adaptation to tissue needs, and, finally, stabilisation and maturation of the newly formed vessels. A non-sprouting mechanism of microvascular growth has also been described, which involves increasing the vascular surface by inserting a slew of transcapillary pillars in a process known as “intussusception.”

Parallel to the investigation of the angiogenic process, a plethora of in vitro and in vivo assays have been developed to investigate the cellular and molecular mechanisms involved. Each model has advantages and disadvantages, and their proper combination is critical for revealing the impact of the element under analysis within the overall process. In vitro assays have been widely used to answer questions about endothelial cell behaviours such as proliferation, differentiation, structural organisation, cytokine secretion profiling, and chemotaxis, as well as the molecular mechanisms underlying angiogenesis. Furthermore, because they are quantitative, easily monitored, reproducible, and provide the confidence required for the rapid screening of potential pro- or anti-angiogenic compounds, in vitro systems have aided in the identification and validation of promising compounds to therapeutically promote or inhibit angiogenesis.

The development of microvessels from parent microvessels is an essential component of new tissue development. With the exception of tightly regulated cyclical events in female reproductive organs, almost every normal tissue lacks significant physiological angiogenesis in adulthood due to a balance of pro- and anti-angiogenic endogenous factors.

When the balance shifts in the pro-angiogenic direction, microvascular endothelial cells (ECs) switch to an angiogenic phenotype, triggering an angiogenic reaction that can be stopped or continued. There is a great deal of variation among ECs in different tissues and organs. There are also species distinctions that should not be overlooked. Wound healing, inflammation, rheumatoid arthritis, endometriosis, diabetic retinopathy, macular degeneration, and tumour growth all involve angiogenesis.

Tumor angiogenesis-Angiogenesis is required for tumour growth beyond a microscopic volume. Tumor cells are genomically unstable, making them more likely to produce oncogens and mutate. As a result, tumours frequently acquire a variety of phenotypes over time, including various angiogenic phenotypes. Tumors induce the production of angiogenic factors in the following ways: I by converting neoplastic cells to an angiogenic phenotype; (ii) by activating tumour stroma cells, such as fibroblasts, macrophages, mast cells, and leukocytes, some of which are recruited from adjacent or more distant non-tumor tissues; (iii) by releasing angiogenic factors from the extracellular matrix (ECM). Non-tumor cells’ pro-angiogenic contribution results from interactions with neoplastic cells and the altered ECM.VEGF (VEGF-A, particularly VEGF165/164) is a major pro-angiogenic factor in the majority of human and experimental tumours. Furthermore, hypoxic cells produce VEGF and up-regulate VEGF-receptors on pre-existing ECs as a result of robust cell proliferation and increased cell metabolism. VEGF’s primary functions are to promote EC survival, induce EC proliferation, and improve EC migration and invasion, all of which contribute to angiogenesis. These functions are regulated by VEGF by interacting with its tyrosine kinase receptors and transmitting signals to various downstream proteins. Alternatively, or in addition to the increased expression of pro-angiogenic molecules, a local decrease in the expression of endogenous anti-angiogenic factors may occur, resulting in angiogenesis stimulation. Tumor cell products that influence angiogenesis are active not only in the tumour but also in distal tissues that respond to these stimuli differently. The recruitment of circulating endothelial precursors (CEPs) from the bone marrow by VEGF secreted from a distant tumour is an example of this type of distal effect. VEGF’s primary functions are to promote EC survival, induce EC proliferation, and improve EC migration and invasion, all of which contribute to angiogenesis. These functions are regulated by VEGF by interacting with its tyrosine kinase receptors and transmitting signals to various downstream proteins. Alternatively, or in addition to the increased expression of pro-angiogenic molecules, a local decrease in the expression of endogenous anti-angiogenic factors may occur, resulting in angiogenesis stimulation. Tumor cell products that influence angiogenesis are active not only in the tumour but also in distal tissues that respond to these stimuli differently. The recruitment of circulating endothelial precursors (CEPs) from the bone marrow by VEGF secreted from a distant tumour is an example of this type of distal effect. The anti-tumor effects of directly or indirectly acting exogenous anti-angiogenic agents, including chemotherapeutics, on naturally occurring tumours in humans are most likely tumor-specific. This is due to genomic heterogeneity among neoplastic cells, as well as cross-talk between these cells and stromal host immune cells and fibroblasts, ECs, and the ECM within tumour tissue. Furthermore, the effect of exogenous anti-angiogenic agents on angiogenesis may be site-specific.

The zebrafish embryo has grown in popularity as a model for studying organogenesis. Genetic manipulation has become possible and has increased the power of the zebrafish model by using morpholino antisense oligonucleotides to knockdown gene expression and, more recently, TALEN or CRISPR-Cas mediated genome editing.However, zebrafish embryos are particularly well suited for the study of vascular development because their small size allows for the diffusion of oxygen and nutrients, allowing them to survive for up to a week without a vascular system (cloche mutant embryos) or blood circulation (silent heart mutant/tnnt2 deficient embryos). The vascular anatomy of the developing zebrafish .Despite the fact that imaging capabilities have improved, only a few researchers have begun to look beyond individual cells. Markus Affolter’s lab has begun to break new ground in zebrafish angiogenesis by imaging the behaviour of junctional proteins to uncover the details of morphogenetic mechanisms such as lumen formation or anastomosis. To understand the detailed role of signalling components in cellular behaviour, the field will need to move beyond the presence or absence of a signalling molecule to more sophisticated methods of analysis (e.g., protein localization, turnover and recycling, cytoskeletal rearrangements). Although we have a better understanding of the individual signalling molecules involved in regulating sprouting angiogenesis, the complex interactions between the different signalling pathways, including regional differences, remain unknown.

While the zebrafish is an excellent imaging model, advancements in protein-behavior analysis and genetic approaches (such as the generation of conditional gene deletions) will undoubtedly benefit the research community and our understanding of angiogenic mechanisms.