After the developing embryo has formed a primary vascular plexus by a process termed vasculogenesis, further blood vessels are generated by both sprouting and non-sprouting angiogenesis, which are progressively pruned and remodelled into a functional adult circulatory system. Recent results, particularly from the study of mice lacking some of the signalling systems involved, have greatly improved our understanding of the molecular basis underlying these events, and may suggest new approaches for treating conditions such as cancer that depend on angiogenesis.

Mechanisms of angiogenesis

Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.

3D bioprinting of tissues and organs

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

https://science.sciencemag.org/content/sci/276/5317/1425.full.pdf

Geometric control of cell life and death.

Structure and function of the blood–brain barrier

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular mass and in biological properties such as their ability to bind to cell-surface heparan-sulfate proteoglycans. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo VEGF induces angiogenesis as well as permeabilization of blood vessels, and plays a central role in the regulation of vasculogenesis. Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors. The various VEGF forms bind to two tyrosine-kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1), which are expressed almost exclusively in endothelial cells. Endothelial cells express in addition the neuropilin-1 and neuropilin-2 coreceptors, which bind selectively to the 165 amino acid form of VEGF (VEGF165). This review focuses on recent developments that have widened considerably our understanding of the mechanisms that control VEGF production and VEGF signal transduction and on recent studies that have shed light on the mechanisms by which VEGF regulates angiogenesis.

Vascular endothelial growth factor (VEGF) and its receptors

Organ printing: Tissue spheroids as building blocks☆

Vasculogenesis in the day 6.5 to 9.5 mouse embryo.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial mitogen that is able to induce angiogenesis in vivo [Leung, D. W., Cachianes, G., Kuang, W.-J., Goeddel, D. V. & Ferrara, N. (1989) Science 246 1306-1309]. To determine if VEGF also influences the behavior of primordial endothelial cells, we used an in vivo vascular assay based on the de novo formation of vessels. Japanese quail embryos injected with nanomolar quantities of the 165-residue form of VEGF at the onset of vasculogenesis exhibited profoundly altered vessel development. In fact, the overall patterning of the vascular network was abnormal in all VEGF-injected embryos. The malformations were attributable to two specific endothelial cell activities: (i) inappropriate neovascularization in normally avascular areas and (ii) the unregulated, excessive fusion of vessels. In the first instance, supernumerary vessels directly linked the inflow channel of the heart to the aortic outflow channel. The second aberrant activity led to the formation of vessels with abnormally large lumens. Ultimately, unregulated vessel fusion generated massive vascular sacs that obliterated the identity of individual vessels. These observations show that exogenous VEGF has an impact on the behavior of primordial endothelial cells engaged in vasculogenesis, and they strongly suggest that endogenous VEGF is important in vascular patterning and regulation of vessel size (lumen formation).

Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization

Experimental data in this study demonstrate that integrin alpha v beta 3 is fundamentally involved in the maturation of blood vessels during embryonic neovascularization (vasculogenesis). Integrin alpha v beta 3 was specifically expressed on the surface of angioblasts during vessel development in quail embryos and vitronectin, a ligand for alpha v beta 3, localized to the basal surface of these cells. More importantly, microinjection of the anti-alpha v beta 3 monoclonal antibody, LM609, disrupted the normal pattern of vascular development. After exposure to LM609 the angioblasts in experimental embryos appeared as clusters of rounded cells lacking normal cellular protrusions. This led to disruption of lumen formation and abnormal vessel patterning. These findings demonstrate that during vasculogenesis ligation of integrin alpha v beta 3 on the surface of primordial endothelial cells is critical for the differentiation and maturation of blood vessels. Similar studies on chicken chorioallantoic membrane showed that LM609 blocks angiogenesis. Together the two studies suggest that integrin alpha v beta 3 plays a role in neovascularization of tissues.

/pdf/an-antagonist-of-integrin-alpha-v-beta-3-prevents-maturation-s9mnbcj9me.pdf

Christopher J. Drake

Papers

Organ printing: Tissue spheroids as building blocks☆

Vasculogenesis in the day 6.5 to 9.5 mouse embryo.

Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization

An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embryonic neovascularization.

Hematopoietic origin of microglial and perivascular cells in brain