Vasculogenesis

Morphogenetic Events

The dorsal aortae are formed by vasculogenesis. The role of VEGF in the assembly of the dorsal aorta was examined by perturbation studies in which VEGF was added or inactivated. VEGF has been inactivated by injection of soluble VEGF receptor-1/Fc hybrid protein in quail embryos [5] and by mouse or zebrafish knockout or mutation. In the quail embryo, VEGF has been added through direct injection of recombinant protein [5] or by surgical implantation of heparin chromatography beads preincubated with recombinant human VEGF165 [6], which serve as a slow-release source of VEGF (Figure 2). VEGF was delivered to somites of quail embryos with six or seven somite pairs, because somites give rise to few angioblasts. This was followed by seven hours of culture, until the embryos had 9 to 12 somite pairs. Embryos were fixed and stained as whole mounts with the QH-1 monoclonal antibody (Figure 2A). At early stages of vasculogenesis, ectopic VEGF delivery resulted in increased vascular densities such that the dorsal aorta (DA) and capillary plexus (CP) just lateral to the embryo appeared fused together, with obliteration of the avascular area (AVA) that normally separates the DA from the CP (Figures 2B and C).

The embryo and its vasculature develop in a rostral-caudal (head-to-tail) fashion, such that the most highly developed vascular pattern is located rostrally. Ectopic VEGF can affect morphogenetic events of vasculogenesis along the entire rostral-caudal axis of the embryo, resulting in hypervascularization [6]. However, qualitative differences in the vascular changes were observed at different levels. Ectopic VEGF at rostral levels resulted in an increased number of vessel branches between the DA and CP, similar to intersomitic arteries branching from the tubular dorsal aortae at this anatomic level. Moving caudal (closer to the tail), VEGF delivery at the level of a middle somite or the most recently formed somite resulted in a more generalized expansion of the vasculature. There was complete loss of the AVA, such that the DA and CP fused together (Figures 2B and C). The effect of ectopic VEGF can be seen by comparing of the side where VEGF was delivered to the opposite side, or to a control embryo (Figure 2A). These data suggest that VEGF influences multiple events of vascular morphogenesis as they occur simultaneously along the rostral-caudal axis. These include cohesion of angioblasts to form a solid cord caudally, further differentiation of the cord as a lumen forms at the middle somite level, and finally sprouting from the tubular DA at rostral levels, likely through angiogenesis (see Figure 1).

Mechanisms

There are several cellular mechanisms by which VEGF can influence endothelial cells as they form networks in vitro or in vivo. These include induction of endothelial cell proliferation or chemotaxis toward a VEGF source, where it can influence the size and pattern of blood vessels. Although it is known that increased or decreased VEGF levels in the developing embryo can profoundly affect vascular morphogenesis, to date the precise mechanism of VEGF activity remains unclear. VEGF does not induce the formation of new angioblasts from uncommitted mesoderm. Rather, it is believed to act on existing populations of angioblasts by guiding them to form a vascular pattern with vessels specifically located and of defined size. VEGF is not believed to influence the developing vasculature in our experiments by inducing endothelial cell or angioblast proliferation. The effects of VEGF were observed in as little as four hours,

PATTERN THE DA

LATERAL MESODERM

Figure 2 The Effect of VEGF and Endoderm on Angioblast Recruitment from Lateral Mesoderm to Pattern the Dorsal Aorta. Quail embryos are shown following experimental methods to study vascular morphogenesis. For each panel, the head of the embryo is up, the scale bar represents 100 mm, and asterisks (*) indicate VEGF delivery locations. (A) A normal quail embryo stained with QH-1. (B) An embryo with VEGF delivered to somite 7. (C) A schematic representation of the effect of ectopic VEGF on the vascular pattern, as seen in cross section. (D) An embryo with VEGF delivery to both sides, followed by a cut (made as indicated approximately 100 mm lateral to the midline) to separate the lateral mesoderm (a source of angioblasts) from the embryo proper, resulting in ablation of the VEGF delivery effect. (E) The effect of lateral mesoderm removal on the ability of ectopic VEGF to alter vascular patterns. VEGF is a chemoattractant for angioblasts from the lateral mesoderm to form the dorsal aortae. (F) An embryo without a VEGF bead, in which the endoderm was removed from one side. On the side without endoderm (-ENDO), the DA failed to form; rather, angioblasts (ANG) remain as single cells or clusters, compared to the unoperated (NORMAL) side of the embryo. (G) An embryo in which endoderm was removed from one side (-ENDO), followed by VEGF delivery on the same side. In this case, VEGF was able to partially restore the normal vascular pattern. (H) The effect of endoderm removal on vascular patterning. VEGF expressed within the endoderm is necessary for the proper localization and patterning of the dorsal aorta. (I) The role of VEGF in vascular morphogenesis. Ectopic VEGF delivery causes hypervascularization (left) by recruiting angioblasts from lateral mesoderm. Endogeneous VEGF within the endoderm recruits angioblasts from lateral mesoderm (arrows) and acts to localize and pattern the DA. In all diagrams: AVA, avascular area; CP, capillary plexus; DA, dorsal aorta; IM, intermediate mesoderm; NC, notochord; NT, neural tube; PD, pronephric duct; SM, somatic mesoderm; SP, splanchnic mesoderm; SOM, somite; VEGF, delivery site.

PATTERN THE DA

LATERAL MESODERM

Figure 2 The Effect of VEGF and Endoderm on Angioblast Recruitment from Lateral Mesoderm to Pattern the Dorsal Aorta. Quail embryos are shown following experimental methods to study vascular morphogenesis. For each panel, the head of the embryo is up, the scale bar represents 100 mm, and asterisks (*) indicate VEGF delivery locations. (A) A normal quail embryo stained with QH-1. (B) An embryo with VEGF delivered to somite 7. (C) A schematic representation of the effect of ectopic VEGF on the vascular pattern, as seen in cross section. (D) An embryo with VEGF delivery to both sides, followed by a cut (made as indicated approximately 100 mm lateral to the midline) to separate the lateral mesoderm (a source of angioblasts) from the embryo proper, resulting in ablation of the VEGF delivery effect. (E) The effect of lateral mesoderm removal on the ability of ectopic VEGF to alter vascular patterns. VEGF is a chemoattractant for angioblasts from the lateral mesoderm to form the dorsal aortae. (F) An embryo without a VEGF bead, in which the endoderm was removed from one side. On the side without endoderm (-ENDO), the DA failed to form; rather, angioblasts (ANG) remain as single cells or clusters, compared to the unoperated (NORMAL) side of the embryo. (G) An embryo in which endoderm was removed from one side (-ENDO), followed by VEGF delivery on the same side. In this case, VEGF was able to partially restore the normal vascular pattern. (H) The effect of endoderm removal on vascular patterning. VEGF expressed within the endoderm is necessary for the proper localization and patterning of the dorsal aorta. (I) The role of VEGF in vascular morphogenesis. Ectopic VEGF delivery causes hypervascularization (left) by recruiting angioblasts from lateral mesoderm. Endogeneous VEGF within the endoderm recruits angioblasts from lateral mesoderm (arrows) and acts to localize and pattern the DA. In all diagrams: AVA, avascular area; CP, capillary plexus; DA, dorsal aorta; IM, intermediate mesoderm; NC, notochord; NT, neural tube; PD, pronephric duct; SM, somatic mesoderm; SP, splanchnic mesoderm; SOM, somite; VEGF, delivery site.

while the proliferation rate of quail embryo cells at the stages examined is approximately ten hours [6]. Rather, VEGF is believed to induce endothelial cells and angioblasts to undergo directed migration (chemotaxis) towards a VEGF source. Evidence for this mechanism in quail is from experiments with VEGF delivered to both sides of the embryo, followed by removal of lateral meso-derm (a tissue rich in angioblasts). After removal of lateral mesoderm, the effect of ectopic VEGF on vascular pattern formation was reduced (Figures 2D and E), suggesting that cells recruited by the VEGF to form the dorsal aorta were removed.

VEGF is known to regulate the behavior of endothelial cells during vasculogenesis, such that adding VEGF increases protrusive activity, while soluble VEGFR1 (a VEGF inhibitor) reduces protrusive activity. Protrusive activity is the extension of filopodia from endothelial cells as they extend and migrate to form a vascular pattern. This activity is necessary for endothelial cell shape changes that take place during vascular morphogenesis [5].

The Role of Endoderm

In the developing quail embryo, high levels of VEGF are expressed within the planar endoderm that underlies the DA. VEGF within the endoderm may provide a chemotactic cue to recruit angioblasts and endothelial cells from the lateral mesoderm to the midline, thereby positioning the DA. Directed cell migration of angioblasts toward an area of VEGF expression was demonstrated during development of the dorsal aorta in the frog, Xenopus laevis [7]. In Xenopus, the hypochord (an endoderm-derived midline structure that lies below the notochord) expresses predominantly the lowest molecular weight isoform of VEGF (VEGF122), a diffusible form of the growth factor. Diffusion of VEGF122 stimulates angioblast migration toward the midline of the embryo. Expression of ectopic VEGF in lateral mesoderm can divert angioblasts away from the hypochord. Vital dye labeling studies confirmed lateral mesoderm as the source of angioblasts destined to form the DA [7]. VEGF is thought to play essentially the same role in dorsal aorta formation in zebrafish [4].

In the quail embryo, removal of endoderm resulted in failure of the dorsal aorta to form (Figures 2F and H). However, unlike the situation in Xenopus, angioblasts were recruited to the midline, where they remained as single cells or clusters in the absence of endoderm, without undergoing cohesion to form the DA. On the contralateral side of the embryo, where the endoderm was left intact, a DA formed (Figure 2F). VEGF delivery to the side where endoderm was removed partially restored the vascular pattern and hyper-vascularized the control side as previously described (Figure 2G). Therefore, VEGF within the endoderm of the quail embryo locates and patterns the DA (Figure 2H). This may differ from the results in Xenopus because of the developmental stage when endoderm was removed in the quail (earlier removal may prevent angioblast migration), or the planar quail endoderm may function differently from the Xenopus hypochord. Taken together, data from lateral meso-derm removal and endoderm removal experiments in quail suggest that VEGF within the endoderm recruits angioblasts to the midline, where they undergo cellular changes under the continued influence of VEGF to form the DA (Figure 2I).

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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