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Correspondence to: Charles D. Little, Dept of Cell Biology, BSB626, Medical Univ of South Carolina, 173 Ashley Ave., Charleston, SC 29425.
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Summary |
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The avian embryo is well suited for the study of blood vessel morphogenesis. This is especially true of investigations that focus on the de novo formation of blood vessels from mesoderm, a process referred to as vasculogenesis. To examine the cellular and molecular mechanisms regulating vasculogenesis, we developed a bioassay that employs intact avian embryos. Among the many bioactive molecules we have examined, vascular epithelial growth factor (VEGF) stands out for its ability to affect vasculogenesis. Using the whole-embryo assay, we discovered that VEGF induces a vascular malformation we refer to as hyperfusion. Our studies showed that microinjection of recombinant VEGF165 converted the normally discrete network of embryonic blood vessels into enlarged endothelial sinuses. Depending on the amount of VEGF injected and the time of postinjection incubation, the misbehavior of the primordial endothelial cells can become so exaggerated that for all practical purposes the embryo contains a single enormous vascular sinus; all normal vessels are subsumed into a composite vascular structure. This morphology is reminiscent of the abnormal vascular sinuses characteristic of certain neovascular pathologies. (J Histochem Cytochem 47:13511355, 1999)
Key Words: vasculogenesis, vascular fusion, VEGF, vascular hyperfusion
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Introduction |
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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 (
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 (
The first report to demonstrate that hyperfusion is operative in adults is a VEGF gain of function study by Blau and colleagues (
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 (
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 Figure 1A and Figure 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.
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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).
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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.
We know that large-caliber vessels, such as the dorsal aortae and the sinus venosae, form within 1020 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 (
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
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 (
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 (
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
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 (Figure 3D3F). Endothelial cell proliferation and/or recruitment continues unabated while the vascular fusion process is occurring.
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 (Figure 3D3F). This last assertion is strongly supported by the fact that VEGF overstimulation leads to formation of large vascular sinuses (
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.
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Footnotes |
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Presented at the 50th Annual Meeting of the Histochemical Society, Bethesda, MD, April 1617, 1999.
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Acknowledgments |
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Supported by NIH R01 HL57645 and P01 HL52813 to CDL, and R01 HL57375 and the American Heart Association S986515 to CJD.
Received for publication July 6, 1999; accepted July 6, 1999.
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