Review
Tubulogenesis during blood vessel formation

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Abstract

The ability to form and maintain a functional system of contiguous hollow tubes is a critical feature of vascular endothelial cells (ECs). Lumen formation, or tubulogenesis, occurs in blood vessels during both vasculogenesis and angiogenesis in the embryo. Formation of vascular lumens takes place prior to the establishment of blood flow and to vascular remodeling which results in a characteristic hierarchical vessel organization. While epithelial lumen formation has received intense attention in past decades, more recent work has only just begun to elucidate the mechanisms controlling the initiation and morphogenesis of endothelial lumens. Studies using in vitro and in vivo models, including zebrafish and mammals, are beginning to paint an emerging picture of how blood vessels establish their characteristic morphology and become patent. In this article, we review and discuss the molecular and cellular mechanisms driving the formation of vascular tubes, primarily in vivo, and we compare and contrast proposed models for blood vessel lumen formation.

Highlights

► Endothelial lumens are required for proper formation and function of the circulatory system. ► Diverse cellular mechanisms for lumen formation are used in different vascular beds. ► Control of cell adhesion, deadhesion, polarity and internal contractility is critical to lumen formation. ► Rasip1 and Rho family GTPases are essential modulators of lumen formation.

Introduction

The cardiovascular system is the first functional organ system to form in developing embryos across all vertebrate species, providing tissues with nutrient and gas exchange required for life. Formation of a cohesive, seamless and contiguous network of blood vessels to carry blood is therefore essential for proper cardiovascular function. While a growing understanding is emerging regarding the specification, patterning and sprouting of blood vessels, data still lag regarding the cellular morphogenesis and molecular pathways that direct formation of vascular ‘tubes’.

The initial, fundamental building block of the vascular system is termed the angioblast, or endothelial progenitor cell, and the first blood vessels to form in the embryo arise via vasculogenesis. Angioblasts are mesodermally derived cells that emerge following gastrulation as scattered cells in both extraembryonic yolk sac blood islands [1] and within embryonic tissues [2]. During vasculogenesis, the initial vascular plexus is formed as angioblasts rapidly organize and coalesce to form solid cords, at distinct embryonic locations. These lumenless, linear aggregates must then transform into tubes so that they may carry blood.

During ‘tubulogenesis’, angioblasts transition from a cuboidal to a flattened shape, and they rearrange their junctional contact points to the cord periphery, as they mature into endothelial cells (ECs) which line central lumens [3]. Later, as embryonic tissues develop and become increasingly complex, new blood vessels sprout from pre-existing vessels of the primary plexus via sprouting angiogenesis [4]. Sprouting extends existing lumens within these new angiogenic vessels and expands the tubular network. In addition, when vessel networks acquire their recognizable hierarchical arrangement of large and small vessels via remodeling angiogenesis, lumens either expand or shrink in concert with the changing vasculature. The final cardiovascular system in the adult thus emerges from a rather astounding array of varied and divergent cellular mechanisms.

Not surprisingly, vasculogenesis in mammalian embryos precedes both the initiation of heart beating and the formation of all other organs. It represents a first and critical step in a coordinated series of events that together drive vascular morphogenesis, ensuring that all organs acquire their proper vasculature as they form and grow. Initially, angioblasts emerge in the mesoderm throughout embryonic tissues. These specialized progenitor cells migrate and coalesce to form a primitive plexus of solid vascular “cords”, sandwiched at the endodermal–mesodermal interface. This initial vascular network resembles a fisherman's net, with rope-like vessels that are for the most part relatively homogeneous in shape and size. Shortly thereafter, endothelial lumens appear at the heart of each solid cord, transforming them into functional tubes (Fig. 1A). This morphogenetic process is rapid and remains poorly understood.

Strictly speaking, vasculogenic lumen formation and ‘tubulogenesis’ can actually be considered separable events. While lumen formation is a local event, involving a small number of cells opening up a space between them, tubulogenesis is the formation of a continuous lumen along the entire length of a vessel. Formation of continuous lumens is inherently required for blood circulation and cardiovascular function, both critical for tissue growth and viability.

The transformation of solid EC cords into ‘canalized’ vascular tubes has been the focus of growing interest. As different aspects of blood vessel formation become unraveled, new questions arise and preoccupy vascular developmental biologists. How are cells changing to form vascular lumens de novo at the heart of newly formed cords? Where do the forces that generate a central lumen come from, during this process? From within cells, or from surrounding tissues? Is anything pumped into the growing cavities prior to blood flow to keep them from collapsing? How and why do angioblasts ‘thin’ out as they become ECs, and contribute to the cylindrical vascular structure? These questions regarding fundamental cellular behaviors during vasculogenic and angiogenic lumen formation, as well as their molecular underpinnings, remain largely unexplored but are beginning to attract attention [5], [6], [7].

Similar to de novo lumen and tube formation in initial embryonic vessels, lumens must also form in new angiogenic sprouts. However, the mechanisms are likely to differ to some extent, as initial vessels start as solid cords with no connections to existing lumens, while angiogenic vessels grow from pre-existing tubes, with pre-existing lumens (Fig. 1B). Much work has recently focused on sprout formation and the role of VEGF and Notch signaling during this process. The distal growing end of new angiogenic vessels consists of endothelial ‘tip’ cells, which extend long filopodia and migrate in a manner similar to axonal growth cones, sensing their microenvironment. The proximal end of these vessels, by contrast, consists of ‘stalk’ cells that initially form a trailing cord but often quickly becomes lumenized [8], [9]. The process that controls tip cell growth is dependent on a remarkable interplay of selective Notch ligand activation, as well as the establishment of well-controlled VEGF gradients [10]. To date, however, relatively little attention has been focused on the timing, mechanics or molecular regulation of lumen formation within stalk cells. Comparing and contrasting lumen formation in different types of vessels will reveal whether similar mechanisms apply to all vessels or whether locally distinct modes of lumen formation reflect inherent heterogeneity of vessels.

Section snippets

Cellular mechanisms of vascular tubulogenesis

As a tubular organ, the endothelial vasculature shares a lot of common features with epithelial tubes found in a number of other organs, such as lung, kidney, salivary glands and pancreas. A considerable amount of work has been carried out to elucidate epithelial tubulogenesis during the past several decades. Epithelial tubes are generally composed of a sheath of cuboidal or columnar epithelial cells, with defined apical membranes facing a central lumen, lateral edges interfacing with each

Cell–cell junctional molecules

Like epithelial cells, ECs interconnect to form cell-lined networks of tubes. In both tissues, cells adhere via adherens junctions (AJs) and tight junctions (TJs). These junctional molecules are specialized transmembrane and intracellular protein complexes that promote homophilic interactions and tether cells to each other in a zipper-like manner [40]. Proper regulation of cell junctions, including adhesion and de-adhesion, is critical for proper vascular lumen formation. One principle

Summary

Vascular tubulogenesis is critical for blood vessel development and subsequent cardiovascular function. Elegant in vitro and in vivo studies during the past decade, as well as important concepts borrowed from epithelial studies, have together contributed to a growing understanding of this complicated process. Using different models of EC lumen formation has provided valuable insights as to how vascular lumens take shape and mature in different places and at different times. Given the intrinsic

Acknowledgements

We thank George Davis and Stryder Meadows for critical reading of the manuscript and essential discussions. This work was supported by AHA Predoctoral fellowship 09PRE2070035 (KX) and NIH DK079862, AHA Grant-in-Aid 0755054Y and the Basil O’Connor March of Dimes Award (OC).

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