Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.     

Bombesin promotes vasculogenesis and angiogenesis in chick chorio-allantoic membrane: A morphometric, structural, and ultrastructural study

Experiments were performed on the chorio-allantoic membrane (CAM) of the chick to evaluate the effects of bombesin (BN) on vascular neoformation. In morphometrical assays, 10(-13)-10(-4) M BN promoted dose-dependent vascular development. Newly formed vessels converged toward the BN release site in a spoked wheel arrangement, suggesting a diffusion gradient mechanism. Structural and ultrastructural analysis of CAM specimens collected near the BN release site showed that both vasculogenetic and angiogenetic processes cooperated in vascular neoformation that involved committed cells from the mesenchyme (angioblasts) as well as endothelial cells. No pattern of vascular development was detected away from the BN release site. Findings from the present study emphasize the role of BN in vascular net development of respiratory organs.     

Hedgehog signaling is essential for endothelial tube formation during vasculogenesis

During embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, we show that endodermally derived sonic hedgehog is both necessary and sufficient for vascular tube formation in avian embryos. We also show that Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation.     

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

Segregation of the embryonic vascular and hemopoietic systems.

The origin of endothelial cells and their subsequent assembly into the primary vascular system have been mostly analyzed in the avian embryo. Following the discovery of specific growth factors and their cognate receptors, the molecular mechanisms underlying these processes have been unraveled in both birds and mammals. In particular, experimental studies of the angiogenic vascular endothelial growth factor (VEGF) and its receptors, carried out in both vertebrate classes, have provided significant insight into the developmental biology of endothelial cells. The VEGF receptor VEGFR2 is the earliest marker known to be expressed by endothelial precursor cells of avian and mouse embryos. Based on the localization of VEGFR2+ cells in the avian embryo and on clonal culture experiments, two types of endothelial precursor cells can be distinguished from gastrulation stages onward: posterior mesodermal VEGFR2+ hemangioblasts, which have the capacity to differentiate into endothelial and hemopoietic cells, and anterior VEGFR2+ angioblasts, which can only give rise to endothelial cells.     

Endoderm is required for vascular endothelial tube formation, but not for angioblast specification

Angioblasts, the precursor cells that comprise the endothelial layer of blood vessels, arise from a purely mesodermal population. Individual angioblasts coalesce to form the primary vascular plexus through a process called vasculogenesis. A number of reports in the literature suggest that signals from the adjacent endoderm are necessary to induce angioblast specification within the mesoderm. We present evidence, using both embryological and molecular techniques, indicating that endoderm is not necessary for the induction of angioblasts. Xenopus embryos that had endoderm physically removed at the onset of gastrulation still express vascular markers. Furthermore, animal caps stimulated with bFGF form angioblasts in the absence of any detectable endodermal markers. These results show that endoderm is not required for the initial formation of angioblasts. While Xenopus embryos lacking endoderm contain aggregates of angioblasts, these angioblasts fail to assemble into endothelial tubes. Endothelial tube formation can be rescued, however, by implantation of endodermal tissue from sibling embryos. Based on these studies in Xenopus, and corroborating experiments using the quail embryo, we conclude that endoderm is not required for angioblast specification, but does play an essential role in the formation of vascular tubes.     

Angioblast differentiation and morphogenesis of the vascular endothelium in the mouse embryo

Bandeiraea simplicifolia B4 isolectin (BSLB4) and polyclonal antisera against von Willebrand factor (VWF) were used to study the origin of endothelial cells and their organization into blood vessels in the postimplantation mouse embryo. Examination of BSLB4-stained whole mounted and sectioned embryos revealed intense staining of the endothelium, highlighting large vessels, capillaries, and many individual cells. Dorsal aorta formation was first obvious at E7 when many lectin-positive cells appeared in paraxial and lateral plate mesoderm. As development proceeded to E8, BSLB4-positive cells became organized into craniocaudal lines destined to become the aorta proper. At E9, BSLB4 stained all vessels of the embryo including the dorsal aorta, the intersomitic arteries, and the endocardium. VWF expression was not detected until E8 when BSLB4/VWF double-stained sections revealed the dorsal aortae as the first VWF-positive vessels, while other endothelium visible with BSLB4 remained negative for VWF immunostaining. By E12 many other vessels became VWF-positive, including the aortic arches, the intersomitic arteries, and the cardinal veins. However, many angioblasts and capillaries remained VWF-negative, reflecting the heterogeneous expression of VWF among endothelium that has been reported in adults of other species. The histochemical data reported here support the conclusions of earlier avian studies by showing distinct vascular patterns in the initial formation of vessels from isolated angioblasts (vasculogenesis), followed by the extension and organization of the initial vascular structures (angiogenesis). Moreover, our data suggest that the endothelium arises from distinct VWF-positive sources associated with the dorsal aorta, as well as VWF-negative sources associated with other vessels in the embryo.     

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.     

Human fetal aorta-derived vascular progenitor cells: identification and potential application in ischemic diseases

Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (i.e., angioblasts), is poorly understood. Here we report a summary of our recent studies on the identification of a population of vascular progenitor cells (VPCs) in human fetal aorta. These undifferentiated mesenchymal cells co-express endothelial and myogenic markers (CD133+, CD34+, KDR+, desmin+) and are localized in outer layer of the aortic stroma of 11–12 weeks old human fetuses. Under stimulation with VEGF-A or PDGF-BB, VPCs give origin to a mixed population of mature endothelial and mural cells, respectively. When embedded in a three-dimensional collagen gel, VPCs organize into cohesive cellular cords that resembled mature vascular structures. The therapeutic efficacy of a small number of VPCs transplanted into ischemic limb muscle was demonstrated in immunodeficient mice. Investigation of the effect of VPCs on experimental heart ischemia and on diabetic ischemic ulcers in mice is in progress and seems to confirm their efficacy. On the whole, fetal aorta represents an important source for the investigation of phenotypic and functional features of human vascular progenitor cells.     

A scanning and transmission electron microscopic study of the bat lung

The lungs of two adult species of bat Epomophorus wahlbergi and Miniopterus minor fixed with 2.3% glutaraldehyde were processed for SEM (scanning electron microscope) and TEM (transmission electron microscope) examination by the standard procedures. The bat lung comprised a blood and air conducting zone (consisting of bronchi, bronchioles and large blood vessels), the intermediate zone (made up of alveolar ducts), and the respiratory zone, which consisted of alveoli and blood capillaries. The interalveolar septa comprised basically granular pneumocytes (type II cells), squamous pneumocytes (type I cells), endothelial cells, and, in the interstitium, collagen and elastic fibres with occasional fibrocytes. Blood capillaries were interposed in the interalveolar septa, thus bulging into adjacent alveoli. It was noted that grossly, architecturally and structurally, the bat lung was similar to that of a terrestrial mammal. However, in previous morphometric and physiological studies it has been found that bats have a large lung, a thin pulmonary blood-gas barrier, a large pulmonary capillary blood volume, and high haematocrit and haemoglobin concentration. The bat lung, while retaining the basic mammalian pulmonary design, is well adapted to provide the large amount of oxygen demanded by flight. The avian pulmonary design (the lung-air sac system) is thus not a prerequisite to flight.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO₂ 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.     

Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells

Apelin, the proposed endogenous peptide ligand of the novel G-protein-coupled receptor APJ, has been shown to possess potent vasodilator and positive inotropic effects in rats and humans in vivo. However, in humans, no endogenous source of apelin has been reported. Therefore, based on the presence of APJ and mRNA encoding apelin in human tissues, we investigated the expression of apelin in fresh-frozen human tissue from right atrium, left ventricle, lung, kidney, adrenal and large conduit vessels using immunocytochemistry. Apelin-like immunoreactivity (apelin-LI) was detected in vascular endothelial cells lining blood vessels in the human heart, kidney, adrenal gland and lung and in endothelial cells of large conduit vessels. Apelin-LI was also present in endocardial endothelial cells lining recesses of the right atrium. Apelin-LI was not present or below the level of detection in cardiomyocytes, Purkinje's cells, pulmonary or renal epithelial cells, secretory cells of the adrenal gland, vascular smooth muscle cells, adipocytes, nerves and connective tissue. The restricted presence of apelin-LI in endothelial cells suggests that endothelial apelin may play a role as a locally secreted cardiovascular mediator acting on APJ receptors present on the vascular smooth muscle and on cardiac myocytes to regulate vascular tone and cardiac contractility.     

Circulating blood island-derived cells contribute to vasculogenesis in the embryo proper

While recent findings have established that cells derived from the bone marrow can contribute to vasculogenesis in the adult, it is unclear whether an analogous population of cells in the embryo can also contribute to vasculogenesis. Using a retroviral labeling strategy, we demonstrate that circulating blood island-derived cells contribute to the genesis of both extra- and intraembryonic blood vessels in the early quail embryo. This finding establishes that vasculogenesis in the embryo is a composite of two processes: the direct in situ formation of blood vessels from mesodermally derived angioblasts and the incorporation and differentiation of circulating endothelial cell progenitors into forming embryonic blood vessels.     

Anatomy of a pulmonary immune response: Kinetics in different leukocyte pools

In pulmonary immune reactions the cells which can be obtained by bronchoalveolar lavage (BAL) are only one part of the picture. In this study the kinetics of an experimental pulmonary immune response were investigated simultaneously in different lung compartments in the same rat. On days 0, 1, 2, 3, 4, 5 and 11 after intratracheal challenge with sheep red blood cells, leukocytes were taken from the bronchoalveolar, the interstitial and the marginal lung vascular pool as well as from the peripheral blood. Total numbers of granulocytes, NK cells, B and T cells, CD4⁺ and CD8⁺ cells were determined. Histology and in vivo labeling of proliferating cells was performed. On day 1 after challenge an increase of granulocytes in the BAL was found. In the BAL the total number of T lymphocytes increased on day 1 and day 2 and the CD4/CD8 ratio increased from day 1 to day 5, indicating an influx of CD4⁺ T cells. Changes in the lung interstitium showed a similar tendency, but were not found in the marginal pool or blood. Histologically cellular infiltrates were seen around the pulmonary small vessels. Little local proliferation occurred in the different lung compartments, indicating mainly immigration of cells. Further studies will focus on the expression of adhesion molecules during an immune response, to learn more about the mechanisms responsible for the increase of lymphocytes.     

Exacerbation of vascular endothelial injury in the generalized Shwartzman reaction by the administration of anti-E-selectin antibody

Previously, we reported that the consecutive administration of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) induced systemic injury of vascular endothelial cells. The aim of this study was to investigate the participation of vascular adhesion molecules in the vascular endothelial injury of GSR. The administration of anti-E-selectin antibody in GSR-induced mice resulted in massive apoptosis of vascular endothelial cells and congestion in blood vessels. Further, marked hemorrhage was found in the pulmonary alveoli of those mice. GSR, especially lung injury, was definitely exacerbated by the administration of anti-E-selectin antibody. On the other hand, the administration of anti-VCAM-1 antibody did not induce such injury of vascular endothelial cells. The possible role of E-selectin in the exacerbation of vascular endothelial injury in GSR is discussed.     

Sphingosine-1-phosphate signaling promotes critical migratory events in vasculogenesis

Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).     

Postnatal vasculogenesis

It is generally accepted that vasculogenesis is limited to early embryogenesis and is believed not to occur in adult, whereas angiogenesis occurs in both the developing embryo and postnatal life. However, the distinction between them is not absolute, because both require endothelial cell proliferation and migration and three-dimensional reorganization of newly formed blood vessels, nor are they mutually exclusive, inasmuch as angioblasts can be incorporated into expanding pre-existing blood vessels. Recent observations indicate that vasculogenesis may not be restricted to early embryogenesis, but may also have a physiological role or contribute to the pathology of vascular diseases in adults. The major evidence in favor of this new view comes from: (i) demonstration of the presence of circulating endothelial cells and endothelial precursor cells; (ii) newly described mechanisms of blood vessel formation in tumor growth. The potential biomedical applications of endothelial precursor cells and the new opportunities for the development of new forms of tumor-targeted treatments are discussed.     

In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells

A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary microvascular permeability. Endotoxin-induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.