VEGF and Vascular Fusion: Implications for Normal and Pathological Vessels

THIS ARTICLE SUMMARIZES evidence supporting the concept of vascular fusion. In addition, we speculate on the implications of how vascular fusion influences caliber size during morphogenesis of normal and abnormal blood vessels. This general topic is of considerable interest because vascular pathologies often exhibit enlarged and/or sinusoidal vessels. Assuming that vascular fusion is a normal embryonic activity, it follows that vascular endothelial growth factor (VEGF) gain of function should induce formation of abnormally large vessels. Fortunately, several recent studies have addressed this possibility. The work has been accomplished in chicken and mouse embryos and also in adult mouse muscle tissue. We were particularly interested in whether vascular hyperfusion occurs at developmental stages beyond the very early embryos that we study. To date, we are aware of four studies that substantiate our concept of hyperfusion (Drake and Little, 1995).

Three of these studies were conducted using avian and mouse embryos. Soon after our initial report, Flamme and colleagues injected genetically engineered COS cells, which express VEGF, into later-gestation chicken embryos. Their work showed enlarged vessels in response to the elevated VEGF levels (Flamme et al. 1995). According to our view, their observations are consistent with VEGF-induced vascular hyperfusion. Similar studies were conducted in mouse embryos using the keratin promoter in skin and the surfactant promoter in the lung to drive the expression of VEGF. In both studies, enlarged sinusoidal vessels were observed (Detmar et al. 1998; Zeng et al. 1998).

The first report to demonstrate that hyperfusion is operative in adults is a VEGF gain of function study by Blau and colleagues (Springer et al. 1998). These workers prepared skeletal myoblasts that constitutively express VEGF and the Lac Z gene as a marker. The transfected myoblasts were introduced into adult mouse muscle. After 11 days, the presence of abnormal sinusoidal vessels, including angioma-like structures, was observed. Therefore, their data are consistent with the possibility that a vascular fusion-like activity operates in adult organisms.

Together, these gain of function studies strongly suggest that hyperfusion is a universal response of endothelial cells to increased VEGF stimulation. Although the above articles all reported the presence of abnormally large vessels, either the various authors did not comment on the exact cellular mechanisms leading to formation of the enlarged vessels or they attributed VEGF's affects to vascular “dilatation.”

For vascular fusion to operate in adult tissues, we must also consider the possibility that vasculogenesis operates in adults. Interestingly, the work by Springer et al. and two earlier studies strongly suggest that vasculogenesis occurs in adult organisms (Asahara et al. 1997; Takahashi et al. 1999). Assuming for the moment that adult vasculogenesis exists, it follows that this process would probably be subject to the same mechanisms as primary vasculogenesis. These assumptions are entirely consistent with the vascular malformations described by Blau and colleagues (Springer et al. 1998).

It is important to stress that we envision vascular fusion to be a normal morphogenic mechanism. We believe that vascular fusion is required for de novo morphogenesis of large-caliber vessels (i.e., the endocardium, the dorsal aortae, and the sinus venosae). Stated simply, vascular fusion is an endothelial cell-based mechanism by which small-caliber endothelial tubes fuse so as to enclose a common lumen. When overstimulated, this morphogenic pathway produces enlarged vascular malformations.

Although there are data demonstrating that elevated VEGF levels result in the formation of abnormally large vessels, this work leaves open the question of which mechanisms endothelial cells use to achieve normal vessel caliber. Several possibilities are summarized in Figure 1. It is intuitive that any model of endothelial tube morphogenesis must take into account the replication of endothelial cells (Figure 1B). Other relevant cell biological mechanisms are hypertrophy and an increased degree of cell spreading behavior (see Figures 1A and 1C). The remaining text will focus on two related cellular behaviors. The first is how newly differentiated endothelial cells are incorporated into vessels, and the second is normal vascular fusion.

First, in the case of large-vessel morphogenesis, we propose that “newly induced” primordial endothelial cells initially form a small-caliber vessel. This nascent vessel then fuses with a nearby existing vessel(s) (Figure 2). This iterative process culminates in formation of a large-caliber vessel (see Figure 3).

Second, normal vascular fusion requires sufficient numbers of endothelial precursor cells/angioblasts. Our recent observations suggest that increased angioblast density correlates with sites of large-vessel morphogenesis (unpublished observations). Therefore, we propose a model in which the “correct” angioblast density and the ability of primordial endothelial cells to engage in vascular fusion are the key cellular events that influence vessel size. Our published work and unpublished observations support this model and also suggest a cellular mechanism underlying vascular fusion.

Specifically, we recently noted that within hours after quail embryos are injected with recombinant VEGF the primordial endothelial cells respond in a wholly unexpected fashion. There is a striking increase in protrusive activity that immediately proceeds vascular hyperfusion (unpublished data). On the basis of these observations, we propose that VEGF regulates the degree to which endothelial cells engage in protrusive activity. Furthermore, we speculate that regulation of extension and protrusion behavior is the normal checkpoint that governs whether small-caliber endothelial tubes fuse to form larger vessels.

OPEN IN VIEWER

Figure 1

Three mechanisms for increasing the caliber of a vessel lumen. (A) cell spreading; (B) increased cell replication; (C) endothelial cell hypertrophy.

We know that large-caliber vessels, such as the dorsal aortae and the sinus venosae, form within 10–20 μm of small-caliber polygonal vessels. Unfortunately, how such exquisite local control is accomplished is not understood at this time. Nevertheless, we speculate that VEGF is the vascular morphogen that governs vessel caliber. If the foregoing is correct, it follows logically that, when overstimulated, this morphogenic pathway would give rise to abnormally enlarged vessels or hyperfused vascular sinuses. This is precisely the behavior summarized above.

Moreover, we contend that normal and abnormal vascular fusion is an autonomous activity of endothelial cells. Stated differently, vascular smooth muscle cells are not required for vascular fusion. This contention is obvious in the early embryo when such cells are absent. Whether it holds true for adult neovascular processes remains to be determined. However, it is interesting to speculate that vascular smooth muscle cells might inhibit or curtail vascular fusion.

At present there are no published time-lapse video images that definitively establish the cellular events leading to formation of large vessels or abnormal vascular sinuses. Regardless of the exact cellular mechanisms, all VEGF gain of function studies appear to converge on a reproducible phenotype: enlarged, poorly formed vascular channels that are distinct from the normal vasculature in embryos and adult tissues (see references above). Moreover, these experimental data are consistent with earlier literature showing that the blood vessels of certain malignant tumors contain hyperfused, sinusoidal vascular channels (Bugajski et al. 1989; Konerding et al. 1999).

Although it is convenient to draw analogies between primary vasculogenesis and postembryonic vasculogenesis, very little is known about the behavior of endothelial cells during the latter process (see Springer et al. 1998). However, the basic morphogenetic steps of primary vasculogenesis are now understood (Drake and Little 1998; Drake et al. 1998). Therefore, to consider whether a vasculogenesis-like process occurs in adult organisms, it is useful to briefly describe this activity as it occurs in embryos.

We begin with the assumption that molecules such as basic FGF induce splanchnic mesoderm to form a randomly distributed population of angioblasts. This assumption is based mainly on the work of Flamme and colleagues (Flamme and Risau 1992; Krah et al. 1994). In the model that follows, we also assume that VEGF is responsible for stimulating proliferation of primordial endothelial cells. Whether or not VEGF is a mitogen for angioblasts remains to be determined. However recent evidence suggests that angioblasts express Flk-1 or VEGFR2, thus it is possible that VEGF may stimulate their replication (unpublished data).

On the basis of work in quail embryos, we contend that endothelial tube assembly begins immediately after specification of primary mesoderm to an endothelial fate (Figure 3). We know that angioblasts are born within the plane of the splanchnic mesoderm. We have also demonstrated that vasculogenesis does not occur in the plane of the mesoderm but within a ventral layer of ECM (Drake et al. 1997). Therefore, in response to VEGF signaling, angioblasts extend processes into the ventrally positioned plane of ECM lying between the mesoderm and the endoderm. Cells quickly enter the ECM and reposition themselves 10–20 μm away from the splanchnic mesoderm. Static images suggest that small clusters containing two or three cells accumulate in a polygonal pattern (Figure 3A). Concomitantly, the cells begin to assume an extended spindle-shaped morphology (Figure 3B) (unpublished observations).

At the same time that endothelial cells enter the splanchnopleural ECM and begin protrusive activity, they also begin to express surface markers specific for vascular endothelial cells, such as QH1. We assume that the initial protrusions are randomly oriented but always lie within the plane of ECM in which the endothelial cells reside. Quickly, on the order of minutes, the primordial endothelial cells probably become elongated and extend in a nonrandom manner such that the clusters are interconnected by long cell processes (Figure 3B).

Next, we propose that the endothelial cell protrusions exert tractional forces on the ECM fibers that separate the mesoderm from the endoderm. This assertion is based on in vitro work by Vernon and colleagues (for review see Vernon and Sage 1995). The resultant tension is sensed by nearby primordial endothelial cells, which respond by further elongation so as to form a two-cell cord that will eventually become one side of a nascent vascular polygon. Neighboring cells behave in a similar manner, which leads to the formation of a complete polygon. This progressive activity results in a system of repeating planar vascular polygons (Drake and Little 1998; Drake et al. 1998). During this time, lumens begin to appear in the nascent vessels by means of an unknown mechanism(s) that may involve endocytosis (Figure 3C) (Davis and Camarillo 1996). The primary network formed by this activity represents the most basic vascular pattern in vertebrates: a series of polygons lying in a single plane.

OPEN IN VIEWER

Figure 2

Our proposed mechanism for increasing the caliber of the lumen by means of fusing two smaller endothelial tubes. This mechanism is referred to as vascular fusion.

Vascular morphogenetic events that occur after the establishment of the primary network fall under the broad category of vascular remodeling. Here we restrict our comments to the concept of vascular fusion as a means of forming large-caliber vessels, such as the endocardium or the dorsal aortae. We envision a process that employs protrusive activity under the control of VEGF. This is accomplished when cells that comprise small vascular polygons extend processes from one side of a polygon to another, thus subdividing the preexisting polygon (arrow, Figure 3D). This subdivision occurs in such a manner that a continuous lumen is established within the new connection. In regions at which large endothelial tubes form under VEGF induction, this process of subdivision and coalescence continues until the former polygonal network is replaced by a large-caliber vessel (Figures 3D-3F). Endothelial cell proliferation and/or recruitment continues unabated while the vascular fusion process is occurring.

OPEN IN VIEWER

Figure 3

Several progressive steps in the de novo morphogenesis of endothelial tubes. (A) Primordial endothelial cells are initially observed as small clusters separated by relatively uniform intervals. (B) The endothelial cells extend and protrude, eventually forming cord-like assemblies of spindle-shaped cells that connect clusters. (C) By means of poorly understood mechanisms, lumens form within the cords. This process results in primary vascular polygons. (D) To increase the size of lumens at specific locations such as the aortae, the primary vascular polygons undergo vascular fusion (see Figure 2). This results in previously separate microvessels forming a common lumen. Cell protrusions (arrow) bridge between individual lumens. (E) This fusion process continues in elements destined to be large-caliber vessels. (F) The end-point of normal vascular-fusion-mediated morphogenesis is a large-caliber endothelial tube such as the dorsal aorta. It is possible that an identical mechanism (A-F), if overstimulated, could result in the formation of the abnormal vascular sinuses characteristic of some pathologies.

In vascular fields that are destined to form large vessels, VEGF signaling is controlled such that the correct vessel caliber is obtained. Ultimately, this fills in unvascularized spaces between vascular polygons to yield large-caliber vessels such as the dorsal aortae (Figures 3D-3F). This last assertion is strongly supported by the fact that VEGF overstimulation leads to formation of large vascular sinuses (Drake and Little 1995).

Vascular endothelial growth factor has received considerable attention as a means of inducing or stimulating neovascular processes in human patients. Here we propose that VEGF is a vascular morphogen and that overstimultation of this morphogenetic pathway yields abnormally enlarged vessels. If this proposition is correct, it raises questions about the use of VEGF mimetics or recombinant ligands. Therefore, the challenge will be to modulate VEGF signaling therapy so as to achieve a desired vessel size. It is possible that studies in vasculogenetic-stage avian embryos will contribute to achieving this goal.