Multicellular Sprouting during Vasculogenesis

Living organisms, from bacteria to vertebrates, are well known to generate sophisticated multicellular patterns. Numerous recent interdisciplinary studies have focused on the formation and regulation of these structures. Advances in automatized microscopy allow the time-resolved tracking of embryonic development at cellular resolution over an extended area covering most of the embryo. The resulting images yield simultaneous information on the motion of multiple tissue components—both cells and extracellular matrix (ECM) fibers. Recent studies on ECM displacements in bird embryos resulted in a method to distinguish tissue deformation and cell-autonomous motion. Patterning of the primary vascular plexus results from a collective action of primordial endothelial cells. The emerging “polygonal” vascular structure is shown to be formed by cell–cell and cell–ECM interactions: adhesion and protrusive activity (sprouting). Utilizing avb3 integrins, multicellular sprouts invade rapidly into avascular areas. Sprout elongation, in turn, depends on a continuous supply of endothelial cells. Endothelial cells migrate along the sprout, towards its tip, in a vascular endothelial (VE) cadherin-dependent process. The observed abundance of multicellular sprout formation in various in vitro and in vivo systems can be explained by a general mechanism based on preferential attraction to elongated structures. Our interacting particle model exhibits robust sprouting dynamics and results in patterns with morphometry similar to native primordial vascular plexuses—without ancillary assumptions involving chemotaxis or mechanochemical signaling.

Introduction

It is widely assumed that adhesion-based activities such as exertion of traction and compressional forces, shape-change and motility are the physical means by which tissues and organs are formed (Trinkaus, 1984, Forgacs and Newman, 2005). However, our knowledge is limited about how collective cell behavior creates a specific physical tissue or organ (Keller et al., 2003).

Vasculogenesis of warm blooded vertebrates, the formation of a primary vascular pattern from mesodermal-derived precursors (angioblasts), is an excellent system in which to address tissue pattern emergence. In bird embryos, well before the onset of circulation, hundreds of essentially identical vascular endothelial cells create a polygonal network within a relatively uncomplicated, sheet-like anatomical environment (Risau and Flamme, 1995). Each link in the polygonal network is a cord consisting of 3–10 endothelial cells (Drake et al., 1997).

It has been known for nearly a century that endothelial cells differentiate from solitary angioblasts within the avian embryo (Area Pellucida) (Reagan, 1915, Sabin, 1920). Committed angioblasts display a random spatial distribution within the mesoderm, without an observable preexisting pattern at length scales comparable to that of the future primary vascular polygons (Drake et al., 1997).

Vasculogenic processes may also occur in certain pathophysiologies. Recent data provided evidence that vascular endothelial cell progenitors exist in the adult and may become bloodborne, enter extravascular tissues, and promote de novo vessel formation (Zammaretti and Zisch, 2005, Rumpold). For that reason, endothelial progenitors, mobilized in situ or transplanted, are a major target of therapeutic vascularization approaches to prevent ischemic disease and control endothelial injury. Moreover, endothelial progenitors represent a potential target for strategies to block tumor growth, and a requisite mechanism for tissue engineering (Wu et al., 2004). The capacity of less than fully differentiated cells to assemble into vascular-like tubes is also manifested in various tumors. Best characterized are highly malignant melanomas in which tumor cells are assembling into tubes to secure blood supply (Hendrix et al., 2003). Thus, arguably, the least understood and most important question facing vascular developmental biologists and tissue engineers today is—what are the general principles guiding morphogenesis of an endothelial tube network?

Conventional models of vasculogenesis often assume that endothelial cells migrate to pre- and well-defined positions following extracellular guidance cues or chemoattractants (Ambler, Cleaver and Krieg, 1998, Poole and Coffin, 1989). However, the capacity of endothelial cells to form a polygonal pattern is preserved in various in vitro systems, where the presence of a genetic prepattern is unlikely. The mouse allantois, when explanted, forms a vascular network very similar to the primary vascular pattern (LaRue et al., 2003)—instead of a pair of umbilical vessels. Similarly, a polygonal vascular network emerges when endothelial cells are placed in three-dimensional collagen gels (Montesano and Orci, 1985, Davis). We argue that the ubiquitous polygonal networks are self-organized in the sense that we do not expect a direct correspondence between gene expression patterns and the position of individual segments within the primary vascular network.

This concept of self-organization is markedly different from that of genetic hard-wiring. During insect segmentation the position and identity of body segments is directly correlated with a gene expression pattern. Similarly, genetic prepatterning is clearly involved in the determination of major vessels in the developing vascular network. In fish, where the major vessels assemble directly, without forming an intermediate vascular plexus, specific vascular malformations are correlated with genetic defects (Weinstein, 1999). Thus, endothelial progenitors presumably respond to various environmental cues, including the presence of other endothelial cells as well as genetically preprogrammed “zip codes.”

Various hypotheses are proposed to explain the self-organized aspect of vasculogenesis. The mechanochemical mechanism assumes cells to exert mechanical stress on the underlying substrate, and the resulting stress to guide cell motility (Murray, Murray, 2003). A variant of the mechanochemical model proposes that angioblasts first segregate into compact clusters and engage the surrounding ECM fibers. As a result of traction forces, ECM bundles develop, which in turn later route the motile primordial endothelial cells between clusters (Drake, Manoussaki, Vernon).

A recent body of research focused on pattern emergence based on autocrine chemotactic signaling (Gamba, Serini). The suggested chemoattractant, VEGF₁₆₅, is unlikely to fit the model assumptions in embryonic vasculogenesis as VEGF₁₆₅ is expressed throughout the embryo except in endothelial cells (Flamme, Poole). However, patterning guided by an unspecified chemoattractant continues to serve as the basis of biologically plausible models resolving individual cells (Merks et al., 2006) as well as freely diffusive or matrix-bound chemoattractants (Bauer et al., 2007).

Recently we proposed that mechanical effects may also modulate cell migration guided by adjacent cells, a view we will further explore in Chapter III.