Branching morphogenesis is a complex developmental program that regulates the formation of the vascular system. A wide variety of extracellular cues, including soluble growth factors, matrix proteins, cell surface integrin receptors, and cell-cell adhesion molecules, influence assembly and patterning of the capillary plexus. These signals are integrated by the cell to regulate the complex changes in endothelial cell shape and motility required to form a network of endothelial tubes.
Study of the morphological and cytoskeletal change during capillary formation has been hampered by the nature of the in vitro assays previously used. Cells in three-dimensional matrix are relatively inaccessible to antibodies and other molecular tools. To address this issue, we have developed an assay in which endothelial cells are overlaid with a thin layer of Matrigel and in which an interconnecting network of endothelial capillary tubes is rapidly formed. These structures resemble capillaries in that they are multicellular and contain lumens. Tube formation occurs through an ordered sequence of events. Cells first elaborate large, highly dynamic cellular protrusions and then form small capillary-like aggregates or cords. These early cord structures do not have lumens. Cells then migrate to form a complex network of tube-like structures and eventually lumen-containing vessels. Lumens appear to arise through the formation of vacuoles by engulfment of dead cells or by simple alignment and structural contact between adjacent cells. Although the exact extracellular stimulus in Matrigel that is responsible for these complex changes is unknown, similar events have been described in other in vitro assays using defined extracellular matrix molecules (Folkman and Haudenschild, 1980
; Montesano and Orci, 1985
; Meyer et al., 1997
). The advantages of the assay described here are the ability to directly visualize the changes in cell morphology and the cytoskeleton during capillary tube formation and the ability to microinject cells before Matrigel overlay. Assays of this nature, when interpreted with caution, provide important information into the mechanisms of capillary assembly.
Cells stimulated to form capillary tubes in the presence of Matrigel disassemble their organized actin stress fibers and form dynamic protrusive structures, leading to migration and capillary assembly. Many protrusions have membrane ruffles or lamellipodia at their leading edge. Surprisingly, however, inhibition of actin polymerization blocked lamellipodia formation but did not affect protrusion formation or the assembly of short primitive capillary-like structures. Inhibition of actin polymerization did block subsequent cell migration and expansion of the capillary network. Similarly, inhibitors of actin-myosin-based contractility also prevented cell migration but not the initial morphological changes. The loss of actin stress fibers and strong adhesion after Matrigel overlay may allow cells to alter their morphology and migrate freely. This likely promotes an increase in cell aggregation and facilitates the cell-cell adhesion needed for capillary assembly. Interestingly, the down-regulation of adhesive proteins such as vinculin has been reported during endothelial tube formation and differentiation (Deroanne et al., 1996
). However, it is clear that adhesion to the ECM and integrin signaling are essential requirements for endothelial cell survival and angiogenesis (Brooks et al., 1994
Inhibition of Rho or of its effector, Rho-kinase, did not block the morphogenetic response to Matrigel or capillary tube formation, although cells retained lamellipodia and capillary structures appeared less stable. Unfortunately, for technical reasons, we have been unable to directly measure Rho activity after Matrigel overlay, but the loss of actin stress fibers seen during morphogenesis suggests that down-regulation of Rho may in fact be required for capillary assembly. In support of this, overexpression of an activated mutant of Rho induces cell contraction and inhibits the morphological response to Matrigel (our unpublished observations). A similar situation exists in neurons where activation of Rho has been shown to inhibit neurite migration and induce growth cone retraction (Jalink et al., 1994
). In this context, it is interesting that many ECM proteins with an antiadhesive function such as tenascin C, SPARC, or thrombospondin have all been shown to play a role in angiogenesis. Tenascin C, for example, induces an elongated morphology in endothelial cells and has been shown to down-regulate Rho activity and stress fibers in fibroblasts (Schenk et al. 1999
; Wenk et al., 2000
Although the Matrigel-induced changes in endothelial cell shape and the formation of primitive capillary-like structures are independent of actin polymerization, they are totally dependent on microtubule dynamics. Relatively little is known of the role of microtubules in endothelial function, although complete disruption of the microtubule network has been reported to inhibit endothelial migration in a wound healing assay (Ettenson and Gotlieb, 1992
). Microtubules are required for migration and shape changes in other cells types such as neurons and astrocytes but not in fibroblasts (Tanaka et al. 1995
; Nobes and Hall, 1999
; Etienne-Manneville and Hall, 2001
The protrusive structures we describe in endothelial cells, like those seen in astrocytes and neurons, are also dependent on Rac function. Because increased microtubule dynamics (for example, after nocodazole treatment) has been shown to result in Rac activation and lamellipodia formation (Waterman-Storer et al., 1999
), we wanted to examine whether the role of microtubules in endothelial cells was simply to activate Rac. However, this was not the case because in cells treated with low-dose taxol, injection of L61 Rac did not restore protrusion formation. As Rac is essential for the earliest changes in cell morphology, we were unable to test directly whether it might also play a role in the later actin-dependent events during capillary morphogenesis. Nevertheless, a dual role for Rac is strongly suggested by the finding that the Rac effector, Pak, is required for endothelial cell motility but not for the formation of microtubule-dependent protrusions or primitive capillary-like structures.
The role of microtubules in capillary tube formation resembles that in neuronal outgrowth where microtubules are required for growth cone extension and guidance (Tanaka et al., 1995
). Recent evidence has uncovered a link between neurogenesis and vascular development, with both processes sharing many of the same patterning cues (Shima and Mailhos, 2000
). Our results suggest that as in neurons, Rho family GTPases integrate the signals from these cues to control endothelial morphogenesis. They control cell shape and movement and are essential for formation of new capillaries in the in vitro assay used here. Further study is needed to define their role in other aspects of angiogenesis such as the establishment of cell polarity, the control of transcription, and the maintenance of capillary architecture.
The potent chemotherapeutic agent taxol, and another antitumor drug, Combresatin A, target microtubules and may also inhibit tumor angiogenesis (Belotti et al., 1996
; Grosios et al. 1999
). It has been assumed that their antitumor effects may be through inhibition of mitosis and cell division. Our results outline a potential alternative mechanism for this effect and suggest that targeting microtubule dynamics may be a useful antiangiogenic strategy. The central role of Rac in the control of microtubule-dependent morphogenesis suggests that the downstream effectors of Rac in this pathway are deserving of further study and this may also be a potential avenue for novel therapeutic research.