Search

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (μCT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1 μm and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA., (© 2021 American Association for Anatomy.)

The therian-specific gene paternally expressed 10 (Peg10) plays an essential role in placenta formation: Peg10 knockout mice exhibit early embryonic lethality as a result of severe placental defects. The PEG10 protein exhibits homology with long terminal repeat (LTR) retrotransposon GAG and POL proteins; therefore, we generated mice harboring a mutation in the highly conserved viral aspartic protease motif in the POL-like region of PEG10 because this motif is essential for the life cycle of LTR retrotransposons/retroviruses. Intriguingly, frequent perinatal lethality, not early embryonic lethality, was observed with fetal and placental growth retardation starting mid-gestation. In the mutant placentas, severe defects were observed in the fetal vasculature, where PEG10 is expressed in the three trophoblast cell layers that surround fetal capillary endothelial cells. Thus, Peg10 has essential roles, not only in early placenta formation, but also in placental vasculature maintenance from mid- to late-gestation. This implies that along the feto-maternal placenta interface an interaction occurs between two retrovirus-derived genes, Peg10 and retrotransposon Gag like 1 (Rtl1, also called Peg11), that is essential for the maintenance of fetal capillary endothelial cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Objective: The objective of the current study was to evaluate the embryo-toxicity of omega-3 fatty acids., Methods: Firstly, the embryo-toxicity of docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), as well as their interaction with Bcl-2 family members, were predicted using an in silico assay. In the next step, the embryonic pathological lesions and amniotic fluid biochemical changes following omega-3 treatment were investigated using a chick embryo model. Finally, the drug's vascular apoptotic effect on the chick's yolk sac membrane (YSM) was assessed., Results: In silico simulations revealed the embryo-toxicity, tissue-toxicity (respiratory and cardiovascular), and vascular-toxicity (apoptotic activity) of DHA and EPA. There was also an accurate interaction between DHA and EPA with Bax (Binding affinity: -7.6 and -10.6 kcal/mol) and Bcl-2 (Binding affinity: -8.0 and -12.2 kcal/mol), respectively. Moreover, DHA and EPA administrations were related to various adverse consequences, including weight loss and lesions in the respiratory and cardiovascular systems. Histopathological findings consisted of pulmonary edema, airway dilatation, increased interstitial tissue, and hyperemia in the lungs, heart, liver, kidney, and brain. Morphometric evaluation of the YSM vasculature revealed that the vascular apoptotic effect of omega-3was associated with a significant reduction in mean capillary area. In immunohistochemistry assay, increased expression of BAX and low expression of Bcl-2 affirmed apoptosis in YSM vessels., Conclusion: According to the results of this study, one could confirm that the possible embryo-toxicity of omega-3 was approved by data presented in this research. The obtained results also support the suspicion that alteration of the apoptotic-related proteins in vessels is an essential pathway in embryo-toxicity of omega-3., (Copyright © 2021 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

During embryonic development, the heart undergoes complex morphogenesis from a liner tube into the four chambers consisting of ventricles, atria and valves. At the same time, the cardiomyocytes compact into a dense, aligned, and highly vascularized myocardium. The extracellular matrix (ECM) is known to play an important role in this process but understanding of the expression and organization remains incomplete. Here, we performed 3D confocal imaging of ECM in the left ventricle and whole heart of embryonic chick from stages Hamburger-Hamilton 28-35 (days 5-9) as an accessible model of heart formation. First, we observed the formation of a fibronectin-rich, capillary-like networks in the myocardium between day 5 and day 9 of development. Then, we focused on day 5 prior to vascularization to determine the relative expression of fibronectin, laminin, and collagen type IV. Cardiomyocytes were found to uniaxially align prior to vascularization and, while the epicardium contained all ECM components, laminin was reduced, and collagen type IV was largely absent. Quantification of fibronectin revealed highly aligned fibers with a mean diameter of ~500 nm and interfiber spacing of ~3 µm. These structural parameters (volume, spacing, fiber diameter, length, and orientation) provide a quantitative framework to describe the organization of the embryonic ECM., Competing Interests: The authors declare no conflict of interest.

Glomerular capillary formation is one of the fundamental mysteries in renal developmental biology. However, there are still debates on this issue, and its detailed formation process has not been clarified. To resolve this problem, we performed antibody staining with ultra-thick section on embryonic and postnatal mouse kidneys. We obtained the expression patterns of several genes that play an important role in the development of glomerular capillaries. We found that blood vessel of the fetal kidneys expanded through proliferation and sprouting. During the comma-stage and S-shaped stage, 3-4 capillaries began to bud and migrate into the glomerular cleft, forming a capillary bed in the Bowman's capsule. Then, the capillary bed expanded into mature glomerular capillary by intussusceptive angiogenesis. The afferent and efferent arterioles were formed through pruning. The distribution of VEGFA in the nephron epithelial cells but not only in podocytes, induced multiple capillaries sprouted into the glomerular cleft. And CXCR4 played an important role in the differentiation and expansion of capillary bed into glomerular capillary. Immunofluorescence performed with ultra-thick section allowed us to investigate the development of complex structure tissues systematically and comprehensively., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To assess the cerebral and vascular development in fetuses with hypoplastic left heart syndrome (HLHS)., Methods: Pregnant women carrying fetuses diagnosed with HLHS who decided to interrupt their pregnancies were included in our study. Aortic size and blood flow were assessed based from fetal echocardiography. Immunohistochemical staining was performed in brain sections obtained from pathology in fetuses with HLHS and control fetuses without heart disease., Results: Twenty-seven midgestation fetal HLHS were included (gestational age, 23.3 ± 3.4 weeks). Head circumference z scores were lower in HLHS fetuses. Middle cerebral artery pulsatility index, a measure of cerebrovascular resistance, was inversely correlated with the ascending aortic z score (P < 0.05). Fetuses with HLHS had lower capillary density in the germinal matrix and their capillaries were larger compared with control fetuses with (P < 0.05). The expression of neuronal differentiation marker, FGFR1, and oligodendrocyte precursor, O4, were lower in HLHS brains compared with controls (P < 0.05)., Conclusion: Our study identified abnormalities of vascular flow and structural brain abnormalities in fetal HLHS associated with impaired neuronal and oligodendrocyte differentiation, as well as cerebral growth impairment, early in gestation. These findings may be related in part to early vascular abnormalities., (© 2018 The Authors Prenatal Diagnosis Published by John Wiley & Sons Ltd.)

The alveolar microvascular network is significant for the lung development of vertebrates, which is consisted of tree-like main veins and interconnected capillaries between the main veins. However, it is still unclear how the meshwork pattern is formed by the multigenerational branches. Based on the reaction-diffusion model, we find that the meshwork pattern is transformed from the branching pattern in a spherical shell domain. Furthermore, we find that it is the branch tip fusion that facilitates the meshwork pattern formation, which is guided by the activator peak fusion, and it is the insufficient space for branch tip growth that promotes the branch tip fusion. In addition, we notice that the consumption rate of substrate regulates the meshwork pattern formation and an asymmetric shell domain composed of prolate and oblate hemi-ellipsoidal shell facilitates the dense meshwork pattern formation. Our model provides a qualitative understanding of how the vascular system remodels from the branching pattern to the meshwork pattern and how to make a dense meshwork pattern. Both researches associated with branching and network morphogenesis can benefit from our work., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Formation and homeostasis of the vascular system requires several coordinated cellular functions, but their precise interplay during development and their relative importance for vascular pathologies remain poorly understood. Here, we investigated the endothelial functions regulated by Cdc42 and their in vivo relevance during angiogenic sprouting and vascular morphogenesis in the postnatal mouse retina. We found that Cdc42 is required for endothelial tip cell selection, directed cell migration and filopodia formation, but dispensable for cell proliferation or apoptosis. Although the loss of Cdc42 seems generally compatible with apical-basal polarization and lumen formation in retinal blood vessels, it leads to defective endothelial axial polarization and to the formation of severe vascular malformations in capillaries and veins. Tracking of Cdc42-depleted endothelial cells in mosaic retinas suggests that these capillary-venous malformations arise as a consequence of defective cell migration, when endothelial cells that proliferate at normal rates are unable to re-distribute within the vascular network., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

l-Arginine is required for regulating synapse formation/patterning and angiogenesis in the developing brain. We hypothesized that this requirement would be met by increased transporter-mediated supply across the blood-brain barrier (BBB). Thus, the purpose of this work was to test the idea that elevation of blood-to-brain l-arginine transport across the BBB in the postnatal period coincides with up-regulation of cationic acid transporter 1 (CAT1) expression in developing brain capillaries. We found that the apparent brain-to-plasma concentration ratio (Kp, app) of l-arginine after intravenous administration during the first and second postnatal weeks was 2-fold greater than that at the adult stage. Kp, app of l-serine was also increased at the first postnatal week. In contrast, Kp, app of d-mannitol, a passively BBB-permeable molecule, did not change, indicating that increased transport of l-arginine and l-serine is not due to BBB immaturity. Double immunohistochemical staining of CAT1 and a marker protein, glucose transporter 1, revealed that CAT1 was localized on both luminal and abluminal membranes of brain capillary endothelial cells during the developmental and adult stages. A dramatic increase in CAT1 expression in the brain was seen at postnatal day 7 (P7) and day 14 (P14) and the expression subsequently decreased as the brain matured. In accordance with this, intense immunostaining of CAT1 was observed in brain capillaries at P7 and P14. These findings strongly support our hypothesis and suggest that the supply of blood-born l-arginine to the brain via CAT1 at the BBB plays a key role in meeting the elevated demand for l-arginine in postnatal brain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lung morphogenesis is a highly orchestrated process beginning with the appearance of lung buds on approximately embryonic day 9.5 in the mouse. Endodermally derived epithelial cells of the primitive lung buds undergo branching morphogenesis to generate the tree-like network of epithelial-lined tubules. The pulmonary vasculature develops in close proximity to epithelial progenitor cells in a process that is regulated by interactions between the developing epithelium and underlying mesenchyme. Studies in transgenic and knockout mouse models demonstrate that normal lung morphogenesis requires coordinated interactions between cells lining the tubules, which end in peripheral saccules, juxtaposed to an extensive network of capillaries. Multiple growth factors, microRNAs, transcription factors, and their associated signaling cascades regulate cellular proliferation, migration, survival, and differentiation during formation of the peripheral lung. Dysregulation of signaling events caused by gene mutations, teratogens, or premature birth causes severe congenital and acquired lung diseases in which normal alveolar architecture and the pulmonary capillary network are disrupted. Herein, we review scientific progress regarding signaling and transcriptional mechanisms regulating the development of pulmonary vasculature during lung morphogenesis.

Background/aims: Intussusceptive angiogenesis (IA) is a dynamic process which contributes to vascular expansion and remodeling. Intraluminal pillars have long been the distinctive structural indicator of IA. However, the mechanism of their formation has not been fully elucidated., Methods: Using light and electron microscopy, we studied intussusceptive vascular growth in the developing porcine metanephric kidney., Results: We observed intraluminal pillars formed by endothelial cells in the vasculature of developing glomeruli. Their diameter was < 2.5 µm, consistent with the diameter of nascent pillars. TEM revealed that the majority of these pillars consisted only of endothelium. However, a central core of extracellular matrix (ECM) covered by endothelium, reminiscent of a more mature intussusceptive pillar, was also found in the lumen of a glomerular capillary. Perivascular cells or pericytes were not involved in the pillar structure during these stages of formation., Conclusion: This study shows ECM presence in a mature intussusceptive pillar without any perivascular cell involvement in the structure. This leads to the hypothesis that ECM deposition precedes the participation of these cells in the formation of intraluminal pillars during IA in porcine metanephric glomerular capillaries., (© 2018 S. Karger AG, Basel.)

Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images. Here, we develop and test advanced methods that allow mapping the flow fields in branched vessel networks at the resolution of 10 to 20  μm. The methods, based on the application of spatiotemporal image correlation spectroscopy and its extension to cross-correlation analysis, are applied here to the case of early stage embryos of zebrafish., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Objective: The chromatin remodeling enzyme BRG1 (brahma-related gene 1) transcriptionally regulates target genes important for early blood vessel development and primitive hematopoiesis. However, because Brg1 deletion in vascular progenitor cells results in lethal anemia by embryonic day 10.5 (E10.5), roles for BRG1 in embryonic vascular development after midgestation are unknown. In this study, we sought to determine whether endothelial cell BRG1 regulates genes important for vascular development or maintenance later in embryonic development., Approach and Results: Using mice with temporally inducible deletion of endothelial BRG1 ( Brg1 fl/fl ;Cdh5(PAC)-Cre ERT2 ), we found that Brg1 excision between E9.5 and 11.5 results in capillary dilation and lethal hemorrhage by E14.5. This phenotype strongly resembles that seen when the SRF (serum response factor) transcription factor is deleted from embryonic endothelial cells. Although expression of Srf and several of its known endothelial cell target genes are downregulated in BRG1-depleted endothelial cells, we did not detect binding of BRG1 at these gene promoters, indicating that they are not direct BRG1 target genes. Instead, we found that BRG1 binds to the promoters of the SRF cofactors Mrtfa and Mrtfb (myocardin-related transcription factors A and B) in endothelial cells, and these genes are downregulated in Brg1 -deficient endothelial cells., Conclusions: BRG1 promotes transcription of endothelial Mrtfa and Mrtfb , which elevates expression of SRF and SRF target genes that establish embryonic capillary integrity. These data highlight a new and temporally specific role for BRG1 in embryonic vasculature and provide novel information about epigenetic regulation of Mrtf expression and SRF signaling in developing blood vessels., (© 2017 American Heart Association, Inc.)

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31 + F4/80 + hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31 + F4/80 + cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31 + F4/80 + cells transdifferentiated into NG2/PDGFRβ/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.

Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.

Blood vessels have a unified mission to circulate blood throughout the body; however, they have additional diverse and specialized roles in various organs. For example, in the liver, discontinuous sinusoids, which are fenestrated capillaries with intercellular gaps and a fragmented basement membrane, facilitate delivery of macromolecules to highly metabolic hepatocytes. During embryonic development, discontinuous sinusoids also allow circulating hematopoietic progenitor and stem cells to populate the liver and promote blood cell differentiation. In this issue of the JCI, Géraud et al. describe an essential role for the transcription factor GATA4 in promoting the development of discontinuous sinusoids. In the absence of liver sinusoidal GATA4, mouse embryos developed hepatic capillaries with upregulated endothelial cell junction proteins and a continuous basement membrane. These features prevented hematopoietic progenitor cells from transmigrating into the developing liver, and Gata4-mutant embryos died from subsequent liver hypoplasia and anemia. This study highlights the surprising and extensive transcriptional control GATA4 exercises over specialized liver vascular development and function.

The renal vasculature is integral to the physiologic function of the kidneys in regulating hemodynamics of the body and maintaining organ health. The close inter-relationship of capillaries and the renal epithelium is key to renal physiology, but how renal tubules regulate capillary development remains unclear. Our previous work showed that Wnt7b is expressed in the ureteric trunk epithelium and activates canonical Wnt signaling in the surrounding medullary interstitium, where the capillaries reside. In this study, we showed by immunofluorescence that the target interstitial cells of Wnt7b/canonical Wnt signaling are mural cells of periureteric bud capillaries in the nascent renal medulla of embryonic mice. Genetic ablation of Wnt7b enhanced the proliferation of Wnt7b target mural cells, an effect that associated with decreased expression of PDGFRβ and p57kip2, a cyclin-dependent kinase inhibitor, in these cells. Furthermore, Wnt7b regulated lumen formation of the capillary endothelium in the renal medulla. In the absence of Wnt7b signaling, the periureteric bud medullary capillaries displayed narrower lumens lined with less flattened endothelial cells and a significantly increased presence of luminal endothelial cell-cell junctions, a transient configuration in the forming blood vessels in the controls. Moreover, the absence of Wnt7b led to greatly diminished levels of vascular endothelial (VE)-cadherin at the cell surface in these blood vessels. VE-cadherin is essential for blood vessel lumen formation; thus, Wnt7b may regulate lumen formation through modulation of VE-cadherin localization. Overall, these results indicate a novel role of Wnt7b signaling and the ureteric bud epithelium in renal medullary capillary development., (Copyright © 2016 by the American Society of Nephrology.)

The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time in-depth cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium., Competing Interests: The authors declare no competing or financial interests., (Copyright © 2016 Elsevier Inc. All rights reserved.)

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

CITED2 is a transcriptional co-factor with important roles in many organs of the developing mammalian embryo. Complete deletion of this gene causes severe malformation of the placenta, and results in significantly reduced embryonic growth and death from E14.5. The placenta is a complex organ originating from cells derived from three lineages: the maternal decidua, the trophectoderm, and the extra-embryonic mesoderm. Cited2 is expressed in many of these cell types, but its exact role in the formation of the placenta is unknown. Here we use a conditional deletion approach to remove Cited2 from overlapping subsets of trophectoderm and extra-embryonic mesoderm. We find that Cited2 in sinusoidal trophoblast giant cells and syncytiotrophoblasts is likely to have a non-cell autonomous role in patterning of the pericytes associated with the embryonic capillaries. This function is likely to be mediated by PDGF signaling. Furthermore, we also identify that loss of Cited2 in syncytiotrophoblasts results in the subcellular mislocalization of one of the major lactate transporters in the placenta, SLC16A3 (MCT4). We hypothesize that the embryonic growth retardation observed in Cited2 null embryos is due in part to a disorganized embryonic capillary network, and in part due to abnormalities of the nutrient transport functions of the feto-maternal interface., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vascularization is essential for organ and tissue development. Teeth develop through interactions between epithelium and mesenchyme. The developing capillaries in the enamel organ, the dental epithelial structure, occur simultaneously by mechanisms of vasculogenesis and angiogenesis at the onset of dentinogenesis. The vascular neoformation in the dental mesenchyme has been reported to start from the cap stage. However, the mechanisms of vascularization in the dental mesenchyme remain unknown. In the hope of understanding the mechanisms of the formation of dental mesenchymal vasculature, mouse lower molar germs from embryonic day (E) 13.5 to E16.5 were processed for immunostaining of CD31 and CD34, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and transmission electron microscopy (TEM). In addition, the role of apoptosis for the vascularization in dental mesenchyme was examined by in vitro culture of E14.0 lower molars in the presence of the apoptosis inhibitor (z-VAD-fmk) and a subsequent subrenal culture. Our results showed that CD31- and CD34-positive cells progressively entered the central part of the dental papilla from the peridental mesenchyme. For TEM, angioblasts, young capillaries with thick endothelium and endothelial cells containing vacuoles were observed in peripheral dental mesenchyme, suggesting vasculogenesis was taking place. The presence of lateral sprouting, cytoplasmic filopodia and transluminal bridges in the dental papilla suggested angiogenesis was also occurring. Inhibition of apoptosis delayed the angiogenic vascularization of the dental papilla. Therefore, these data demonstrated that molar mesenchyme is progressively vascularized by mechanisms of both vasculogenesis and angiogenesis and apoptosis partially contributes to the vascularization of the dental papilla.

The importance of Transforming Growth Factor β (TGFβ) signalling during early development has been well established. In particular, Nodal ligands have been shown to play essential roles for the specification and the patterning of the mesendoderm, axes formation and organogenesis. Activin ligands, like Nodal, signal by inducing the phosphorylation of the intracellular signal transducers Smad2 and Smad3. However, the roles of Activins during embryonic development are much less understood. Here, we report that during Xenopus tropicalis development two waves of Smad2 phoshorylation can be observed, first during gastrulation and then a second one after neurulation. Using a knock-down approach, we show that the second wave of Smad2 phosphorylation depends on activinβa (actβa) and activinβb (actβb) expression. Knocking down the expression of actβa, or treating the embryos with a chemical inhibitor inhibiting TGFβ receptor I (TGFβRI) activity after neurulation result in a decrease of the expression of endothelial cell markers and a lack of blood flow in Xenopus tadpoles. Taken together these data suggest that Activin ligands play an important role during vascular development in Xenopus tropicalis embryos.

During vertebrate development, the lung inaugurates as an endodermal bud from the primitive foregut. Dichotomous subdivision of the bud results in arborizing airways that form the prospective gas exchanging chambers, where a thin blood-gas barrier (BGB) is established. In the mammalian lung, this proceeds through conversion of type II cells to type I cells, thinning, and elongation of the cells as well as extrusion of the lamellar bodies. Subsequent diminution of interstitial tissue and apposition of capillaries to the alveolar epithelium establish a thin BGB. In the noncompliant avian lung, attenuation proceeds through cell-cutting processes that result in remarkable thinning of the epithelial layer. A host of morphoregulatory molecules, including transcription factors such as Nkx2.1, GATA, HNF-3, and WNT5a; signaling molecules including FGF, BMP-4, Shh, and TFG- β and extracellular proteins and their receptors have been implicated. During normal physiological function, the BGB may be remodeled in response to alterations in transmural pressures in both blood capillaries and airspaces. Such changes are mitigated through rapid expression of the relevant genes for extracellular matrix proteins and growth factors. While an appreciable amount of information regarding molecular control has been documented in the mammalian lung, very little is available on the avian lung.

How the fetoplacental arterial tree grows and expands during late gestational development is largely unknown. In this study, we quantified changes in arterial branching in the fetal exchange region of the mouse placenta during late gestation, when capillarization increases rapidly. We studied two commonly used mouse strains, CD1 and C57Bl/6 (B6), at embryonic days (E)13.5, 15.5, and 17.5. B6 mice differ from CD1 mice by exhibiting a blunted fetal weight gain in late gestation. We found that B6 capillarization and interhemal membrane thinning were reduced and placental hypoxia-inducible factor-1α and VEGF-A expression were higher than CD1 near term. Automated vascular segmentation of microcomputed tomography data sets revealed that the number of arterial vessels ≥50 μm remained constant during late gestation in both strains, despite large increases in downstream capillary volume quantified by stereology (+65% in B6 mice and +200% in CD1 mice). Arterial diameters expanded in both strains from E13.5 to E15.5; however, diameters continued to expand to E17.5 in B6 mice only. The diameter scaling coefficient at branch sites was near optimal (-3.0) and remained constant in CD1 mice, whereas it decreased, becoming abnormal, in B6 mice at term (-3.5 ± 0.2). Based on arterial tree geometry, resistance remained constant throughout late gestation (∼0.45 mmHg·s·μl(-1)) in CD1 mice, whereas it decreased by 50% in late gestation in B6 mice. Quantification of the fetoplacental vasculature revealed significant strain-dependent differences in arterial and capillary expansion in late gestation. In both strains, enlargement of the fetoplacental arterial tree occurred primarily by increased arterial diameters with no change in segment numbers in late gestation.

Background: The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development., Methodology/principal Findings: We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping., Conclusions/significance: We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue-engineered matrices. The aim of our study was to investigate the impact of a well-characterized murine embryonal EPC line (T17b-EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time-points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β-galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell-free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

During embryonic development, blood flow is needed not only to nourish the developing embryo but is also important for shaping the vascular network such that it becomes hemodynamically efficient. The first blood vessels form a network called the capillary plexus. After the onset of blood flow, the capillary plexus remodel into a more hierarchical tree-shaped network. Mechanical forces created by blood flow are required for remodelling to occur and these forces are believed to induce a maturation of the blood vessels that stabilizes the growing vascular network. The role of mechanical force has been extensively studied in the mature cardiovascular system. Though the events induced by blood flow during development are thought to be similar to what occurs in the adult, there are several important differences between the embryo and the adult. We therefore discuss what is known about the role of mechanical forces in vascular remodelling from the adult cardiovascular system and highlight how embryonic development differs from the adult. We consider the role of blood flow in altering branching morphology, arterial-venous identity and the formation of the blood vessel wall during early vascular development., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The documented data regarding the three-dimensional structure of the air capillaries (ACs), the ultimate sites of gas exchange in the avian lung is contradictory. Further, the mode of gas exchange, described as cross-current has not been clearly elucidated. We studied the temporal and spatial arrangement of the terminal air conduits of the chicken lung and their relationship with the blood capillaries (BCs) in embryos as well as the definitive architecture in adults. Several visualization techniques that included corrosion casting, light microscopy as well as scanning and transmission electron microscopy were used. Two to six infundibulae extend from each atrium and give rise to numerous ACs that spread centrifugally. Majority of the ACs are tubular structures that give off branches, which anastomose with their neighboring cognates. Some ACs have globular shapes and a few are blind-ending tapering tubes. During inauguration, the luminal aspects of the ACs are characterized by numerous microvillus-like microplicae, which are formed during the complex processes of cell attenuation and canalization of the ACs. The parabronchial exchange BCs, initially inaugurated as disorganized meshworks, are reoriented via pillar formation to lie predominantly orthogonal to the long axes of the ACs. The remodeling of the retiform meshworks by intussusceptive angiogenesis essentially accomplishes a cross-current system at the gas exchange interface in the adults, where BCs form ring-like patterns around the ACs, thus establishing a cross-current system. Our findings clarify the mode of gas exchange in the parabronchial mantle and illuminate the basis for the functional efficiency of the avian lung., (Copyright © 2010 Wiley-Liss, Inc.)

Vascular architecture of the human pancreas was investigated by corrosion casting combined with scanning electron microscopy in fetuses aged 20 and 25 gestational weeks. The general pattern of the microvascular system was similar to that of the postnatal pancreas, with an evident insulo-acinar portal system and with three types of capillary networks: capillaries of exocrine lobules, islet capillaries and periductal capillaries around large ducts located in the interlobular septa. All these capillary networks were supplied by arteriolar branches of the interlobular arteries. As compared with the postnatal pancreas, capillaries of exocrine lobules formed denser meshworks, had a more sinusoidal character and revealed morphological features indicative of angiogenesis (blind capillary sprouts). The number of efferent (portal) capillaries per islet was lower and the predominant pattern of islet vasculature was top to bottom rather than inner to outer, as observed in adults. These results show that in the second trimester the human pancreatic vascular architecture is almost completely developed and requires only minor remodeling to be fully functional in the postnatal period.

The native (pre-existing) collateral circulation minimizes tissue injury if obstructive vascular disease develops. Evidence suggests that large differences in collateral extent exist among healthy individuals, presumably from as-yet unknown genetic and/or environmental factors. Little is known regarding when or how native collaterals form-information needed to identify these factors. We examined collateral development between the middle and anterior cerebral artery trees in BALB/c and C57BL/6 mouse embryos-strains with marked differences in adult collateral density and diameter (85% fewer, 50% smaller in BALB/c). The circulation was dilated, fixed and stained. By E15.5, a "primary collateral plexus" was beginning to form in both strains. By E18.5, plexus vessel number peaked but was 60% less and diameter smaller in BALB/c (P<0.001). Earlier time points were examined to determine if these differences correlated with differences in patterning of the general circulation. At approximately E9.0, the primary capillary plexus was similar between strains, but by E12.5 branching was less and diameter larger in BALB/c (P<0.05). Between E12.5-E18.5-during pial artery tree development-small differences in tree size, branch number and distance between branches did not correlate with the large difference in collaterogenesis. Pruning of nascent collaterals between P1-P21 was comparable in both strains, yielding the adult density, but diameter and tortuosity increased less in BALB/c. Pericyte recruitment to nascent collaterals was comparable, despite lower VEGF-A and PDGF-B expression in BALB/c mice. These findings demonstrate that collaterals form late during vascular development and undergo postnatal maturation and that differences in genetic background have dramatic effects on these processes.

Systemic sclerosis (SSc, scleroderma) is a chronic, multisystem connective tissue disorder affecting the skin and various internal organs. Although the disease is characterized by a triad of widespread microangiopathy, fibrosis and autoimmunity, increasing evidence indicates that vascular damage is a primary event in the pathogenesis of SSc. The progressive vascular injury includes persistent endothelial cell activation/damage and apoptosis, intimal thickening, delamination, vessel narrowing and obliteration. These profound vascular changes lead to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and severe clinical manifestations, such as digital ulceration or amputation, pulmonary arterial hypertension and scleroderma renal crisis. The resulting tissue hypoxia induces complex cellular and molecular mechanisms in the attempt to recover endothelial cell function and tissue perfusion. Nevertheless, in SSc patients there is no evidence of significant angiogenesis and the disease evolves towards chronic tissue ischemia, with progressive and irreversible structural changes in multiple vascular beds culminating in the loss of capillaries. A severe imbalance between pro-angiogenic and angiostatic factors may also lead to impaired angiogenic response during SSc. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers and functional defects of bone marrow-derived endothelial progenitor cells may contribute to the vascular pathogenesis of SSc. The purpose of this article is to review the contribution of recent studies to the understanding of the complex mechanisms of impaired vascular repair in SSc. Indeed, understanding the pathophysiology of SSc-associated vascular disease may be the key in dissecting the disease pathogenesis and developing novel therapies. Either angiogenic or vasculogenic mechanisms may potentially become in the future the target of therapeutic strategies to promote capillary regeneration in SSc.

Background: Although bronchopulmonary dysplasia is closely associated with an arrest of alveolar development and pulmonary capillary dysplasia, it is unknown whether these two features are causally related. To investigate the relationship between pulmonary capillaries and alveolar formation, we partially embolized the pulmonary capillary bed., Methods: Partial pulmonary embolization (PPE) was induced in chronically catheterized fetal sheep by injection of microspheres into the left pulmonary artery for 1 day (1d PPE; 115d gestational age; GA) or 5 days (5d PPE; 110-115d GA). Control fetuses received vehicle injections. Lung morphology, secondary septal crests, elastin, collagen, myofibroblast, PECAM1 and HIF1 alpha abundance and localization were determined histologically. VEGF-A, Flk-1, PDGF-A and PDGF-R alpha mRNA levels were measured using real-time PCR., Results: At 130d GA (term approximately 147d), in embolized regions of the lung the percentage of lung occupied by tissue was increased from 29 +/- 1% in controls to 35 +/- 1% in 1d PPE and 44 +/- 1% in 5d PPE fetuses (p < 0.001). Secondary septal crest density was reduced from 8 +/- 0% in controls to 5 +/- 0% in 1d PPE and 4 +/- 0% in 5d PPE fetuses (p < 0.05), indicating impaired alveolar formation. The deposition of differentiated myofibroblasts (23 +/- 1% vs 28 +/- 1%; p < 0.001) and elastin fibres (3 +/- 0% vs 4 +/- 0%; p < 0.05) were also impaired in embolized lung regions of PPE fetuses compared to controls. PPE did not alter the deposition of collagen or PECAM1. At 116d GA in 5d PPE fetuses, markers of hypoxia indicated that a small and transient hypoxic event had occurred (hypoxia in 6.7 +/- 1.4% of the tissue within embolized regions of 5d PPE fetuses at 116d compared to 0.8 +/- 0.2% of tissue in control regions). There was no change in the proportion of tissue labelled with HIF1 alpha. There was no change in mRNA levels of the angiogenic factors VEGF and Flk-1, although a small increase in PDGF-R alpha expression at 116d GA, from 1.00 +/- 0.12 in control fetuses to 1.61 +/- 0.18 in 5d PPE fetuses may account for impaired differentiation of alveolar myofibroblasts and alveolar development., Conclusions: PPE impairs alveolarization without adverse systemic effects and is a novel model for investigating the role of pulmonary capillaries and alveolar myofibroblasts in alveolar formation.

Purpose: To isolate and characterize primary fetus retinal microvascular endothelial cells (RMECs) in order to facilitate the study of their properties in vitro., Methods: Human RMECs were isolated from abortive fetuses whose gestational ages were between 20 and 28 weeks by mechanical morcel and trypsin-collagenase digestion, plated on fibronectin-coated wells and purified gradually in high-selective condition. The RMECs were characterized for expression and localization of endothelial cell markers by indirect immunocytochemical staining of von Willebrand factor (vWF) and CD34, the distribution of cell markers was observed by confocal immunofluorescence microscope. The ability of these cells to form capillary-like networks was assessed subsequently. Cobalt dichloride (CoCl2) was used to simulate hypoxia in the culture condition and Western blot was used to detect the alternation of VEGF protein level under normoxia and hypoxia., Results: Isolation of fetus RMECs has been very difficult and primary culture of these special premature RMECs has not been previously reported due to many reasons. Here we established a simple and convenient method for the primary culture of fetus human RMECs. Our results show that delicate RMECs could be obtained readily and grow very fast, and the purity of RMECs was up to 95% demonstrated by specific staining for vWF in the cytoplasm. Our research also indicates that nearly 100% of cells could express CD34. The cells were successfully passaged and maintained in culture for 5-7 passages without a significant loss in expression of endothelia cell markers. The fetus RMECs formed capillary-like networks on the Matrigel well. Expression of VEGF protein increased significantly under hypoxia., Conclusions: Pure fetus RMECs could be obtained rapidly and easily by our method.Fetus RMECs have some specific properties. Hypoxia could induce expression of VEGF. Cell properties related with age of the donors should be considered while studying in vitro, especially in some age-specific diseases such as retinopathy of prematurity and diabetic retinopathy., (Copyright 2010 S. Karger AG, Basel.)

Purpose: The purpose of this study was to examine the structural and functional maturation of the choriocapillaris (CC) and to determine when fenestrations form, the capillaries are invested with pericytes, and the endothelial cells (ECs) became functional., Methods: Immunohistochemistry was performed on cryopreserved sections of embryonic/fetal human eyes from 7 to 22 weeks' gestation (WG), using antibodies against PAL-E, PV-1 (fenestrations), carbonic anhydrase IV (CA IV), eNOS, and alpha-smooth muscle actin (alphaSMA) and NG2 (two pericyte markers) and the EC marker (CD31). Alkaline phosphatase (APase) enzymatic activity was demonstrated by enzyme histochemistry. Transmission electron microscopy (TEM) was performed on eyes at 11, 14, 16, and 22 WG. Adult human eyes were used as the positive control., Results: All EC markers were present in the CC by 7 WG. PAL-E, CA IV, and eNOS immunoreactivities and APase activity were present in the CC by 7 to 9 WG. TEM analysis demonstrated how structurally immature this vasculature was, even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. The few fenestrations that were observed were often present within the luminal space in the filopodia. Contiguous fenestrations and significant PV-1 were not observed until 21 to 22 WG. alphaSMA was prominent at 22 WG, and the maturation of pericytes was confirmed by TEM., Conclusions: It appears that ECs and their precursors express enzymes present in adult CC well before they are structurally mature. Although ECs make tight junctions early in development, contiguous fenestrations and mature pericytes occur much later in development.

The excitatory neurons of the mammalian cerebral cortex arise from asymmetric divisions of radial glial cells in the ventricular zone and symmetric division of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ) of the embryonic cortex. Little is known about the microenvironment in which IPCs divide or whether a stem cell niche exists in the SVZ of the embryonic cortex. Recent evidence suggests that vasculature may provide a niche for adult stem cells but its role in development is less clear. We have investigated the vasculature in the embryonic cortex during neurogenesis and find that IPCs are spatially and temporally associated with blood vessels during cortical development. Intermediate progenitors mimic the pattern of capillaries suggesting patterns of angiogenesis and neurogenesis are coordinated during development. More importantly, we find that IPCs divide near blood vessel branch points suggesting that cerebral vasculature establishes a stem cell niche for intermediate progenitors in the SVZ. These data provide novel evidence for the presence of a neurogenic niche for intermediate progenitors in the embryonic SVZ and suggest blood vessels are important for proper patterning of neurogenesis.

Hypoxic heart disease is a predominant cause of disability and death worldwide. Since adult mammalian hearts are incapable of regeneration after hypoxia, attempts to modify this deficiency are critical. As demonstrated in zebrafish, recall of the embryonic developmental program may be the key to success. Because thymosin beta4 (TB4) is beneficial for myocardial cell survival and essential for coronary development in embryos, we hypothesized that it reactivates the embryonic developmental program and initiates epicardial progenitor mobilization in adult mammals. We found that TB4 stimulates capillary-like tube formation of adult coronary endothelial cells and increases embryonic endothelial cell migration and proliferation in vitro. The increase of blood vessel/epicardial substance (Bves) expressing cells accompanied by elevated VEGF, Flk-1, TGF-beta, Fgfr-2, Fgfr-4, Fgf-17 and beta-Catenin expression and increase of Tbx-18 and Wt-1 positive myocardial progenitors suggested organ-wide recall of the embryonic program in the adult epicardium. TB4 also positively regulated the expression and phosphorylation of myristoylated alanine-rich C-kinase substrate (Marcks), a direct substrate and indicator of protein kinase C (PKC) activity in vitro and in vivo. PKC inhibition significantly reduced TB4 initiated epicardial thickening, capillary growth and the number of myocardial progenitors. Our results demonstrate that TB4 is the first known molecule capable of organ-wide activation of the embryonic coronary developmental program in the adult mammalian heart after systemic administration and that PKC plays a significant role in the process.

A restructuring of the capillary bed-from the embryonic structure with a three-dimensional network of wide and long protocapillaries to the mature structure with high density of thin and short capillaries along the fibers-has been demonstrated in the chick skeletal muscle on embryonic days 10-19 by morphometric analysis. In this case, the volumetric flow rate and capillary luminal area per cm3 of the muscle remained unaltered, while the blood volume in it significantly dropped. The response of muscle circulation to nitroprusside (increase) and noradrenaline (decrease) appeared in 19-day-old embryos, but this response could develop only under conditions of initially low or high bloodstream, respectively. We propose that the arterial trunk lumen area to the total capillary lumen area remains constant as the intraorganic circulation is formed, which provides for the required linear blood velocity in capillaries.

Controlled regulation of Rho GTPase activity is an essential component mediating growth factor-stimulated migration. We have previously shown that angiomotin (Amot), a membrane-associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have used peptide pull-down and yeast 2-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase exchange factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. Using FRET analysis, we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knockdown of Syx in zebrafish results in inhibition of migration of intersegmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that controls local GTPase activity.

Glomerulus is an important filtrating apparatus in the body. Three types of cells--endothelial, mesangial and visceral epithelial cells can be identified in the capillary tuft. Glomeruli develop during nephrogenesis which starts in the 8th week and ends between the 32nd and 36th week of gestation. The nephron develops through stages described as the vesicle, the comma-shaped, S-shaped with the developing glomerulus and the mature glomerulus. Glomerular differentiation involves the expansion of the original capillary component into the plexus that consists of 6-8 loops and the migration of podocytes that are arranged around these glomerular capillaries. Glomerular capillary differentiation represents a set of developmental changes of the glomerular endothelial and epithelial cells. The active differentiation of glomerular capillaries starts in the hemisphere of an inferior arm of S-shaped bodies. Endothelial precursors unite into precapillaries devoid of lumen. Further differentiation includes the flattening of endothelial cells on the basement membrane, the loss of superfluous cells, the development of lumen and the formation of fenestrae. The glomerular basement membrane is differentiated by the fusion of epithelial and endothelial basement membrane. The differentiation of visceral epithelial cells includes the development of cytoplasmic processes and the flattening of cell bodies. Primary cytoplasmic processes are formed from the podocyte bodies and develop secondary and tertiary processes or foot processes. Foot processes from one podocyte interdigitate with foot processes from other podocytes. In the developing glomeruli, there is a difference in the level of differentiation of visceral epithelial cells. Cells with differentiated foot processes and cells with no cytoplasmic processes are observed within the same glomerulus.

Oxygen availability is one of the necessary prerequisites for normal embryonic development. In our previous study we found that quail embryos incubated under hypoxic conditions (16% O(2)) die at embryonic day (ED) 9 with signs of heart failure. By ED4 and ED6 we found thinner ventricular wall and increased capillary density. We thus hypothesized that the cause of death would lie in severe myocardial and coronary maldevelopment. ED6 and 7 hypoxic hearts had thinner ventricular wall, especially left. There was a simultaneous increase in capillary density, most pronounced in the interventricular septum. This site corresponds to an area of tissue hypoxia and ensuing increased angiogenesis, and also formation of ventricular conduction system. Hypoxia had a positive effect on normal sequence of maturation of the conduction system evaluated by optical mapping at ED7. In sections from ED9 hypoxic hearts we found, in addition to thinner ventricular walls, irregularities in development of coronary tree (missing coronary ostia, absence of one coronary artery, and irregular arterial wall). This deficiency was due to decreased myocyte proliferation rather than to increased apoptosis. By Indian ink injection through the left ventricle we found in normoxic hearts regular coronary branching pattern, while in the hypoxic ones there was often only an irregular plexus. Embryonic hypoxia thus leads to increased capillarity and trabeculation to minimize diffusion distance. In the subsequent period there is a failure in organization of vascular plexus into normal vasculature, resulting in thin compact myocardium that likely leads to heart failure and embryonic death.

We investigated whether the use of antenatal corticosteroids could improve the maturation of choroid plexus capillaries in fetal mice. The study was carried out in two groups of preterm mice. The study group consisted of pregnant mice that received dexamethasone at a dose of 4 mg/kg intraperitoneally. This group was further subdivided into four subgroups according to the timing of steroid administration as follows: day 13, day 14, day 15, and day 16 of pregnancy, and each subgroup included 12 premature mice. All animals received a second injection 24 hours after the first injection. The control group was given normal saline. The pregnant mice were operated on to obtain premature mice. The choroid plexus capillaries were assessed for integrity and thickness of their basement membranes by electronmicroscopy. We found that, in the study group, the maturity of the basement membrane of the choroid plexus capillaries was more precise, in that capillaries were better in control animals at term for the following reasons: (a) the basement membrane becomes more intact, (b) thickness of the basement membrane increased, and (c) the protein particles become tighter. Antenatal dexamethasone improved the maturation of the choroid plexus capillaries in fetal mice but also decreased the incidence of periventricular-intraventricular hemorrhage (PIVH). Using two doses of steroids had no influence on birthweight or brainweight of the mice. In conclusion, these findings provide an experimental basis for the use of antenatal steroids for decreasing the incidence of PIVH in premature infants.

The microvascular architecture of developing lateral ventricle choroid plexus was investigated by corrosion casting and scanning electron microscopy in human fetuses aged 20 gestational weeks. The areas with different microvascular patterns corresponded to the particular parts of the mature plexus: anterior part, glomus, posterior part, the villous fringe and the free margin. In the posterior part, densely packed parallel arterioles and venules were surrounded by sheath-like capillary networks. Other areas contained compact capillary plexuses of the primary villi: the most prominent, protruding basket- and leaf-shaped plexuses were observed in the villous fringe, whilst less numerous and smaller plexuses occurred in the anterior part and glomus. The capillaries of the plexuses had a large diameter and sinusoidal dilations, and showed the presence of occasional short, blind sprouts indicative of angiogenesis. Short anastomoses between arterioles supplying the plexuses and venules draining them were only rarely observed. In the upper area of the choroid plexus, the superior choroidal vein was surrounded by a capillary network forming small, glomerular or rosette-shapes plexuses. The free margin of the choroid plexus was characterized by flat, multiple, arcade-like capillary loops. The general vascular architecture of the human choroid plexus at 20 gestational weeks seems to be similar to that of postnatal/mature plexus, still lacking, however, the complex vascular plexuses of the secondary villi.

Nidogens have been proposed to play a key role in basement membrane (BM) formation. However, recent findings using genetic approaches and organotypic coculture models demonstrated distinct tissue requirements thus changing the classical view of BM assembly. Toward this end, we have analyzed the dermo-epidermal junction and the microvasculature in skin of nidogen-deficient mice for their BM composition and structural assembly. Histology of nidogen double-null embryos at embryonic day (E)18.5 revealed overall normal skin morphology with a regularly differentiated epidermis. However, in the dermis, numerous erythrocytes had extravasated out of the microvasculature. Residual composition and ultrastructure of the dermo-epidermal BM are not altered in the absence of nidogens, demonstrating that the deposition of laminin, collagen IV, and perlecan occurs and allows cutaneous BM formation. In contrast, in capillaries, BM formation is severely impaired in the absence of nidogens, showing an irregular, patchy distribution and a dramatically reduced deposition of collagen IV, perlecan, and particularly laminin-411. Ultrastructure revealed thin fragile walls in the small blood vessels next to the epidermis, completely lacking a distinct endothelial BM. In summary, our results indicate that in skin the laminin composition of the various BMs determines whether nidogens are required for their assembly and stabilization.

Using an nlacZ reporter gene inserted at the Msx1 and Msx2 loci, we could analyze the expression of these homeogenes in the adult mouse. We observed that Msx genes are prominently expressed in a subset of blood vessels. The Msx2nlacZ allele is mainly expressed in a restricted population of mural cells in peripheral arteries and veins. Msx1nlacZ is expressed to a lesser extent by vascular smooth muscle cells of peripheral arteries, but is highly expressed in arterioles and capillaries, making Msx1 a novel marker for a subpopulation of pericytes. Expression is set up early in developing vessels and maintained throughout life. In addition, expression of both genes is observed in a few endothelial cells of the aorta at fetal stages, and only Msx2 continues to be expressed in this layer at the adult stage. These results suggest major functions for Msx genes in vascular mural cell formation and remodeling., (Copyright (c) 2008 Wiley-Liss, Inc.)

Angiogenesis-related treatments have a broad spectrum of potential applications ranging from cancer to macular degeneration, to wound healing. Thus, the identification of pharmacological agents that modulate new blood vessel formation has attracted much attention. In the present study, we investigated the effects of the poly(ADP-ribose) polymerase (PARP) inhibitor PJ-34 [N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide] on angiogenesis. Treatment of chicken chorioallantoic membranes (CAM) with PJ-34 reduced vascular length in these tissues; paradoxically, lower doses of PJ-34 (0.03 or 0.3 nmol/cm2) were more effective in inhibiting neovascularisation than higher doses (3 or 30 nmol/cm2). In vitro, incubation of endothelial cells (EC) with PJ-34 (300 nM to 10 microM) inhibited their proliferation in a concentration-dependent manner with maximal inhibition of 22.3% being observed at 10 microM. Capillary morphogenesis of EC grown on Matrigel was also negatively affected by PJ-34. In addition, PJ-34 abolished the migratory response to the prototype angiogenic factor vascular endothelial growth factor (VEGF) and reduced VEGF-stimulated activation of members of the mitogen activated protein kinase family (ERK1/2, p38), as well as Akt. PJ-34 also inhibited VEGF-induced NO release and cGMP accumulation. In conclusion, we provide evidence that PARP inhibition blocks angiogenesis-related EC properties by interfering with multiple signalling pathways leading to the inhibition of new blood vessel formation.

Vascular morphogenesis encompasses a temporally regulated set of morphological changes that endothelial cells undergo to generate a network of interconnected tubules. Such a complex process inevitably involves multiple cell signaling pathways that must be tightly coordinated in time and space. The formation of a new capillary involves endothelial cell activation, migration, alignment, proliferation, tube formation, branching, anastomosis, and maturation of intercellular junctions and the surrounding basement membrane. Each of these stages is either known or suspected to fall under the influence of the vascular endothelial growth factor, notch, and transforming growth factor-beta/bone morphogenetic protein signaling pathways. Vascular endothelial growth factor is essential for initiation of angiogenic sprouting, and also regulates migration of capillary tip cells, proliferation of trunk cells, and gene expression in both. Notch has been implicated in the regulation of cell fate decisions in the vasculature, especially the choice between arterial and venular endothelial cells, and between tip and trunk cell phenotype. Transforming growth factor-beta regulates cell migration and proliferation, as well as matrix synthesis. In this review, we emphasize how crosstalk between these pathways is essential for proper patterning of the vasculature and offer a transcriptional oscillator model to explain how these pathways might interact to generate new tip cells during retinal angiogenesis.

The juxtaposition of a dense capillary network to lung epithelial cells is essential for air-blood gas exchange. Defective lung vascular development can result in bronchopulmonary dysplasia and alveolar capillary dysplasia. Although vascular endothelial growth factor A (Vegfa) is required for formation of the lung capillary network, little is known regarding the factors that regulate the density and location of the distal capillary plexus and the expression pattern of Vegfa. Here, we show that fibroblast growth factor 9 (FGF9) and sonic hedgehog (SHH) signaling to lung mesenchyme, but not to endothelial cells, are each necessary and together sufficient for distal capillary development. Furthermore, both gain- and loss-of-function of FGF9 regulates Vegfa expression in lung mesenchyme, and VEGF signaling is required for FGF9-mediated blood vessel formation. FGF9, however, can only partially rescue the reduction in capillary density found in the absence of SHH signaling, and SHH is unable to rescue the vascular phenotype found in Fgf9(-/-) lungs. Thus, both signaling systems regulate distinct aspects of vascular development in distal lung mesenchyme. These data suggest a molecular mechanism through which FGF9 and SHH signaling coordinately control the growth and patterning of the lung capillary plexus, and regulate the temporal and spatial expression of Vegfa.

There is increasing evidence that epithelial-vascular interactions are essential for tissue patterning. Here we identified components of the molecular cross talk between respiratory epithelial cells and pulmonary capillaries necessary for the formation of the gas exchange surface of the lung. Selective inactivation of the Vegf-A gene in respiratory epithelium results in an almost complete absence of pulmonary capillaries, demonstrating the dependence of pulmonary capillary development on epithelium-derived Vegf-A. Deficient capillary formation in Vegf-A deficient lungs is associated with a defect in primary septae formation, a morphogenetic process critical for distal lung morphogenesis, coupled with suppression of epithelial cell proliferation and decreased hepatocyte growth factor (Hgf) expression. Lung endothelial cells express Hgf, and selective deletion of the Hgf receptor gene in respiratory epithelium phenocopies the malformation of septae, confirming the requirement for epithelial Hgf signaling in normal septae formation and suggesting that Hgf serves as an endothelium-derived factor that signals to the epithelium. Our findings support a mechanism for primary septae formation dependent on reciprocal interactions between respiratory epithelium and the underlying vasculature, establishing the dependence of pulmonary capillary development on epithelium-derived Vegf-A, and identify Hgf as a putative endothelium-derived factor that mediates the reciprocal signaling from the vasculature to the respiratory epithelium.

Crim1, a transmembrane cysteine-rich repeat-containing protein that is related to chordin, plays a role in the tethering of growth factors at the cell surface. Crim1 is expressed in the developing kidney; in parietal cells, podocytes, and mesangial cells of the glomerulus; and in pericytes that surround the arterial vasculature. A gene-trap mouse line with an insertion in the Crim1 gene (Crim1(KST264/KST264)) displayed perinatal lethality with defects in multiple organ systems. This study further analyzed the defects that are present within the kidneys of these mice. Crim1(KST264/KST264) mice displayed abnormal glomerular development, illustrated by enlarged capillary loops, podocyte effacement, and mesangiolysis. When outbred, homozygotes that reached birth displayed podocyte and glomerular endothelial cell defects and marked albuminuria. The podocytic co-expression of Crim1 with vascular endothelial growth factor-A (VEGF-A) suggested a role for Crim1 in the regulation of VEGF-A action. Crim1 and VEGF-A were shown to interact directly, providing evidence that cysteine-rich repeat-containing proteins can bind to non-TGF-beta superfamily ligands. Crim1(KST264/KST264) mice display a mislocalization of VEGF-A within the developing glomerulus, as assessed by immunogold electron microscopy and increased activation of VEGF receptor 2 (Flk1) in the glomerular endothelial cells, suggesting that Crim1 regulates the delivery of VEGF-A by the podocytes to the endothelial cells. This is the first in vivo demonstration of regulation of VEGF-A delivery and supports the hypothesis that Crim1 functions to regulate the release of growth factors from the cell of synthesis.

The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells. LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis. Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation. Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells.