Role of vascular endothelial-cadherin in vascular morphogenesis

Vascular endothelial (VE)-cadherin is an adhesive transmembrane protein specifically expressed at interendothelial junctions. Its extracellular domain exhibits Ca2+-dependent homophilic reactivity, promoting cell-cell recognition. Mice deficient in VE-cadherin die at mid-gestation resulting from severe vascular defects. At the early phases of vascular development (E8.5) of VE-cadherin-deficient embryos, in situ differentiation of endothelial cells was delayed although their differentiation program appeared normal. Vascularization was defective in the anterior part of the embryo, while dorsal aortae and vitelline and umbilical arteries formed normally in the caudal part. At E9.25, organization of endothelial cells into large vessels was incomplete and angiogenesis was impaired in mutant embryos. Defects were more severe in extraembryonic vasculature. Blood islands of the yolk sac and clusters of angioblasts in allantois failed to establish a capillary plexus and remained isolated. This was not due to defective cell-cell recognition as endothelial cells formed intercellular junctions, as shown by electron microscopy. These data indicate that VE-cadherin is dispensable for endothelial homophilic adhesion but is required for vascular morphogenesis.     

Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.     

Comparative analysis of the circulatory system in Amphipoda (Malacostraca, Crustacea)

We present data on the haemolymph vascular system (HVS) in four representatives of the major amphipod lineages Gammaridea, Hyperiidea and Caprellidea based on corrosion casting and three‐dimensional reconstructions of histological semi‐thin sections. In all these species the HVS comprises a dorsal pulsatile heart, which is continued in the body axis by the anterior and posterior aortae. The heart is equipped with three pairs of incurrent ostia. The number of cardiac arteries that lead off the heart varies among species: in the studied Gammaridea four pairs occur, in Hyperia galba only the three posterior pairs of cardiac arteries occur, while in Caprella mutica cardiac arteries are absent. In all the studied species the posterior aorta leads as a simple tube into the pleon attached to the dorsal diaphragm. The anterior aorta runs from its origin in the anterior part of the second thoracic segment into the cephalothorax. Both pairs of antennae have an arterial supply off the anterior aorta. An overview of previously studied species including our present findings shows the amphipod HVS to be relatively uniform and the gammarid form is discussed as being closest to the ground pattern of Amphipoda.     

Essential role of endothelial Smad4 in vascular remodeling and integrity

New blood vessels are formed through the assembly or sprouting of endothelial cells (ECs) and become stabilized by the formation of perivascular matrix and the association with supporting mural cells. To investigate the role of endothelial Smad4 in vascular development, we deleted the Smad4 gene specifically in ECs using the Cre-LoxP system. EC-specific Smad4 mutant mice died at embryonic day 10.5 due to cardiovascular defects, including attenuated vessels sprouting and remodeling, collapsed dorsal aortas, enlarged hearts with reduced trabeculae, and failed endocardial cushion formation. Noticeably, Smad4-deficient ECs demonstrated an intrinsic defect in tube formation in vitro. Furthermore, the mutant vascular ECs dissociated away from the surrounding cells and suffered from impaired development of vascular smooth muscle cells. The disturbed vascular integrity and maturation was associated with aberrant expression of angiopoietins and a gap junction component, connexin43. Collectively, we have provided direct functional evidence that Smad4 activity in the developing ECs is essential for blood vessel remodeling, maturation, and integrity.     

Vascular abnormalities due to hyperthermia in chick embryos

Intraembryonic vascular abnormalities were studied in chick embryos exposed to temperatures 3 degrees C and 4 degrees C above normal temperature (38 degrees C) from the beginning of incubation. The average duration of hyperthermia was 54 and 53 hours, respectively. Immediately after exposure, the embryos were examined with FITC-Dextran microangiography in vivo. Following hyperthermia various abnormalities in the heart, ventral aortae, aortic arches, omphalomesenteric arteries, and the distal dorsal aortae frequently occurred. There were also significant microvascular changes in the head, in the lateral and caudal parts of the embryos, and in the pellucid area of the yolk sac. In another series incubation at 41 degrees C, hyperthermia of 3 degrees C during the first 3 days of development produced several extraembryonic vascular abnormalities. These included duplication and abnormal branching of the cranial vitelline vein, absence or abnormal course of the omphalomesenteric vessels, aneurysmatic dilatation or abnormal course of the caudal vitelline vein, and aneurysmatic dilatation or occlusion of the abdominal venous sinus. Most frequent findings were blind, congested, and dilated microvascular segments in the pellucid area, commonly associated with an irregular microvascular pattern and perivascular swelling. The abnormalities described are assumed to be caused by the direct effects of hyperthermia upon the developing vessels resulting in microvascular insufficiencies, pathological leakage, and perivascular oedema. Such disturbances may have serious consequences for embryonic vascular development and microcirculation, which in turn may have adverse effects on further embryonic growth and development.     

On the evolution of arterial vascular patterns of tetrapods

The factors that explain the diverse arrangement of the major arteries of tetrapods are not known. Here, I aim to illuminate some of the underpinnings of these patterns. I review the variation in the sauropsid left, right, and dorsal aortae regarding the origin of the gastrointestinal blood vessels and the relative diameters of left and right aortae where they join together to form the dorsal aorta. I focus on these features because the quality of blood that flows through these aortae can vary depending on the state of cardiac shunting and the size of the vessel can provide insight into the quantity of blood borne by the vessels. I then place the information in a phyletic, historical, and ecological context. The plesiomorphic pattern is for the gastrointestinal vessels to arise as segmental arteries from the dorsal aorta, which is formed from the confluence of left and right aortae with similar diameters. The pattern is well conserved with only two major variations. First, in several clades of reptiles (testudines, crocodilians, lizards of the genera Varanus and Hydrosaurus) a substantial portion of the gastrointestinal arteries arises from the left aorta, leaving the diameter of the left aorta smaller than the right at their confluence. I hypothesize that this vascular arrangement facilitates growth by allowing more alkaline blood to flow to the somatic (body wall) and appendicular circulations, which may promote bone deposition and inhibit resorption, whereas hypercapnic, acidic blood flows to the digestive viscera, which may provide CO₂ as a substrate for the synthesis of gastric acid, bicarbonate, fatty acids, glutamine, purine rings, as well as glucose from lactate. Second, in some snakes and lizards with snake‐like body forms, such as Amphisbaenidae, the diameters of left and right aortae are asymmetrical at their confluence with the left aorta exceeding the right, but in members of the amphibian order Gymnophiona the right generally exceeds the left. This condition is associated with asymmetrical development of the lungs. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

A mutant receptor tyrosine phosphatase,CD148, causes defects in vascular development

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.     

Regulation and metamorphosis of the abdominal histoblasts ofDrosophila melanogaster

Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.     

Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern

We are using a monoclonal antibody, QH-1, as a label for angioblasts in quail embryos to study vascular development. Our previous experiments showed that major embryonic blood vessels, such as the dorsal aortae and posterior cardinal veins, develop from angioblasts of mesodermal origin that appear in the body of the embryo proper (Coffin and Poole: Development, 102:735-748, '88). We theorized that there are two separate processes for blood vessel development that occur in quail embryos. One mechanism termed "vasculogenesis" forms blood vessels in place by the aggregation of angioblasts into a cord. The other mechanism, termed "angiogenesis," is the formation of new vessels by sprouting of capillaries from existing vessels. Here we report the results of microsurgical transplantation experiments designed to determine the extent of cell migration taking place during blood vessel formation. Comparison of the chimeras to normal embryos suggests that the vascular pattern develops, in part, from the normally restricted points of entry of angioblasts into the head from the ventral and dorsal aortae. Transplantations of quail mesoderm (1-15 somite stage) into the head of 5-15 somite chick hosts resulted in extensive sprouting and in migration of single and small groups of angioblasts away from the graft sites. Transplantations into the trunk resulted in incorporation of the graft into the normal vascular pattern of the host. Lateral plate mesoderm was incorporated into the dorsal aortae and individual sprouts grew between somites and along the neural tube to contribute to the intersomitic and vertebral arteries, respectively.     

Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants

Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell‐specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms. genesis 48:146–150, 2010. © 2010 Wiley‐Liss, Inc.     

Syk and Zap-70 function redundantly to promote angioblast migration

Spleen tyrosine kinase (Syk) plays critical roles in B-cell and T-cell development, the maintenance of vascular integrity, and proper partitioning of the blood vascular and lymphatic vascular system. Here, we utilize the zebrafish as an in vivo system to demonstrate novel roles for Syk and the related kinase Zeta associated protein (Zap-70) in promoting angioblast migration. Partial knockdown of either gene results in early angiogenic delay of the intersegmental vessels, dorsal intersegmental vessel patterning defects, and partial loss of the thoracic duct. Higher dose knockdown of both genes results in little to no angiogenic sprouting of the intersegmental vessels, a phenotype which resembles knockdown of vegfa. Di-phosphorylated ERK, an effector of the vegfa pathway, is also downregulated in the aorta of syk:zap double morphants. Over-expression of syk under the control of a blood-specific or vascular-specific promoter rescues sprouting defects after loss of vegfa. Together these results suggest that syk and zap-70 function redundantly in an early progenitor to promote the migration of intersegmental vessel angioblasts and lymphangioblasts that contribute to the thoracic duct, either downstream of, or in parallel to vegfa.     

A critical role for calponin 2 in vascular development

Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.     

Cardiovascular ephrinB2 function is essential for embryonic angiogenesis

EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. We have generated mice in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. We find that such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells.     

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.     

Characterization of vascular mural cells during zebrafish development

Development and maturation of the nascent cardiovascular system requires the recruitment of mural cells (MCs) around the vascular tree in a process called vascular myogenesis. Understanding the origin and development of vascular MCs has been hampered by difficulties in observing these cells in vivo and performing defined genetic and experimental manipulations in available model organisms. Here, we investigate the origin of vascular MCs using molecular and genetic tools in zebrafish. We show that vascular MCs are present around the lateral dorsal aortae (LDA) and anterior mesenteric arteries (AMA) of developing animals, and that they also contribute to the outflow tract of the developing heart and ventral aorta (VA). Genetic data indicate that the vascular MCs of the LDA and AMA do not arise from blood or endothelial progenitors but from other derivatives of the lateral plate mesoderm. We further show that zebrafish vascular MCs share many of the morphological, molecular and functional characteristics of vascular smooth muscle cells and pericytes found in higher vertebrates. These data establish the zebrafish as a useful cellular and genetic model to study vascular myogenesis as well as tumor angiogenesis and other MC-associated diseases.