How blood vessel networks are made and measured

Abstract

Tissue and organ viability depends on the proper systemic distribution of cells, nutrients, and oxygen through blood vessel networks. These networks arise in part via angiogenic sprouting. Vessel sprouting involves the precise coordination of several endothelial cell processes including cell-cell communication, cell migration, and proliferation. In this review, we discuss zebrafish and mammalian models of blood vessel sprouting and the quantification methods used to assess vessel sprouting and network formation in these models. We also review the mechanisms involved in angiogenic sprouting, and we propose that the process consists of distinct stages. Sprout initiation involves endothelial cell interactions with neighboring cells and the environment to establish a specialized tip cell responsible for leading the emerging sprout. Furthermore, local sprout guidance cues that spatially regulate this outward migration are discussed. We also examine subsequent events, such as sprout fusion and lumenization, that lead to maturation of a nascent sprout into a patent blood vessel.

Fig. 1

Stages of blood vessel sprouting. We propose that blood vessel sprouting occurs in distinct stages characterized by specific cellular processes and specific (although partially overlapping) molecular requirements. Stage I is tip cell specification and sprout initiation that occurs by poorly understood processes; stage II is sprout elongation and local guidance that features VEGF/Flt-1 near-field interactions; stage III is sprout elongation in response to extrinsic cues; stage IV is lumen formation, and stage V is sprout fusion and completion of lumenization to produce a new vessel connection capable of blood flow.

Fig. 2

Mammalian models of blood vessel sprouting. Summary of strengths and limitations for each model discussed, as well as a general schematic of each model and reference to a publication with detailed methods for utilizing the model, although multiple publications describe and use most models. Green cells represent endothelial cells, and tan cells represent other cell types present in each model. The gray sphere in the fibrin bead assay category represents the microcarrier bead to which the endothelial cells attach, and the gray area in the rat mesentery assay represents the connective tissue within a mesenteric loop.

Fig. 3

Molecular regulation of blood vessel sprouting. The VEGF-A ligand (green diamonds) binds the Flk-1 receptor (orange Y) on the tip cell, increasing Dll4 expression. The Dll4 ligand engages the Notch receptor on the neighboring lateral base cells and promotes cleavage of the NICD. Translocation of the NICD into the nucleus increases expression of downstream genes including Hey and Hes family genes, which may then decrease Flk-1 and Nrp-1 expression and increase expression of Flt-1 (blue Ys).