Tissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) . In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues.
► Expression of hlx1 is associated with angiogenic cell behavior in vivo ► hlx1 selectively marks sprouting endothelial cells during zebrafish development ► Hlx1 is required for intersegmental vessel angiogenesis in zebrafish embryos ► Hlx1 cell-autonomously maintains endothelial stalk cell potential
The healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. During angiogenesis tip cells lead the developing sprout in response to extracellular signals, amongst which vascular endothelial growth factor (VEGF) is critical. In order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled, which is primarily achieved by the Dll4-Notch1 signaling pathway. This study presents a novel multiscale model of osteogenesis and sprouting angiogenesis, incorporating lateral inhibition of endothelial cells (further denoted MOSAIC model) through Dll4-Notch1 signaling, and applies it to fracture healing. The MOSAIC model correctly predicted the bone regeneration process and recapitulated many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen for cell fates, an increased tip cell density due to the loss of Dll4 and an excessive number of tip cells in high VEGF environments. When VEGF concentration was even further increased, the MOSAIC model predicted the absence of a vascular network and fracture healing, thereby leading to a non-union, which is a direct consequence of the mutual inhibition of neighboring cells through Dll4-Notch1 signaling. This result was not retrieved for a more phenomenological model that only considers extracellular signals for tip cell migration, which illustrates the importance of implementing the actual signaling pathway rather than phenomenological rules. Finally, the MOSAIC model demonstrated the importance of a proper criterion for tip cell selection and the need for experimental data to further explore this. In conclusion, this study demonstrates that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation in a more mechanistic way and across different time and spatial scales.
The healing of a fracture largely depends on the development of a new blood vessel network (angiogenesis), which can be investigated and simulated with mathematical models. The current mathematical models of angiogenesis during fracture healing do not, however, implement all relevant biological scales (e.g. a tissue, cellular and intracellular level) rigorously in a multiscale framework. This study established a novel multiscale platform of angiogenesis during fracture healing (called MOSAIC) which allowed us to investigate the interactions of several influential factors across the different biological scales. We focused on the biological process of tip cell selection, during which a specific cell of a blood vessel, the “tip cell”, is selected to migrate away from the original vessel and lead the new branch. After showing that the MOSAIC model is able to correctly predict the bone regeneration process as well as many experimentally observed aspects of tip cell selection, we have used the model to investigate the influence of stimulating signals on the development of the vasculature and the progression of healing. These results raised an important biological question concerning the criterion for tip cell selection. This study demonstrates the potential of multiscale modeling to contribute to the understanding of biological processes like angiogenesis.
The ability to form and maintain a functional system of contiguous hollow tubes is a critical feature of vascular endothelial cells (ECs). Lumen formation, or tubulogenesis, occurs in blood vessels during both vasculogenesis and angiogenesis in the embryo. Formation of vascular lumens takes place prior to the establishment of blood flow and to vascular remodeling which results in a characteristic hierarchical vessel organization. While epithelial lumen formation has received intense attention in past decades, more recent work has only just begun to elucidate the mechanisms controlling the initiation and morphogenesis of endothelial lumens. Studies using in vitro and in vivo models, including zebrafish and mammals, are beginning to paint an emerging picture of how blood vessels establish their characteristic morphology and become patent. In this chapter, we review and discuss the molecular and cellular mechanisms driving the formation of vascular tubes, primarily in vivo, and we compare and contrast proposed models for blood vessel lumen formation.
blood vessel; lumen; endothelial cell; adhesion; cell polarity; vacuole; vasculogenesis; angiogenesis; cord hollowing; sialomucin; Rasip1; NMHCIIA; Arhgap29
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2+/−; Vegfr3+/− compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.
Angiogenesis, or the formation of new blood vessels from preexisting ones, is essential to establish the vascular circuit during embryonic development. During angiogenic sprouting, endothelial cells exhibit a diverse array of cellular behaviors. Endothelial tip cells must migrate extensively and proliferate in response to proangiogenic cues while trailing cells need to maintain their position and connection to the patent vasculature, despite exposure to the same proangiogenic molecules. Several new studies have now shed light on the underlying mechanisms that are responsible for coordinating this process. In particular, this work has identified a conserved role for the Notch signalling pathway in limiting the cellular angiogenic response, in part by reducing the level of vascular endothelial growth factor receptors in endothelial cells. In this overview, we discuss the emerging concepts elucidated by these studies and propose a model in which Notch acts reiteratively throughout the angiogenic process, likely by acting as a switch to determine a cell's response to Vegf.
blood vessel; angiogenesis; Notch; VEGF; zebrafish
Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.
Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully formed blood vessels) organize into a vessel network (vasculogenesis), or by sprouting or splitting of existing blood vessels (angiogenesis). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier–Graner–Hogeweg model (also called Cellular Potts Model) simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller–Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin–mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface–normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell–cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.
A better understanding of the mechanisms by which endothelial cells (the cells lining the inner walls of blood vessels) organize into blood vessels is crucial if we need to enhance or suppress blood vessel growth under pathological conditions, including diabetes, wound healing, and tumor growth. During embryonic development, endothelial cells initially self-organize into a network of solid cords via blood vessel growth. The vascular network expands by splitting of existing blood vessels and by sprouting. Using computer simulations, we have captured a small set of biologically plausible cell behaviors that can reproduce the initial self-organization of endothelial cells, the sprouting of existing vessels, and the immediately subsequent remodeling of the resulting networks. In this model, endothelial cells both secrete diffusible chemoattractants and move up gradients of those chemicals by extending and retracting small pseudopods. By itself, this behavior causes simulated cells to accumulate to aggregate into large, round clusters. We propose that endothelial cells stop extending pseudopods along a given section of cell membrane as soon as the membrane touches the membrane of another endothelial cell (contact inhibition). Adding such contact-inhibition to our simulations allows vascular cords to form sprouts under a wide range of conditions.
Notch signaling is an evolutionarily-conserved, intercellular signaling mechanism that plays myriad roles during vascular development and physiology in vertebrates. These roles include the regulation of arteriovenous specification and differentiation in both endothelial cells and vascular smooth muscle cells, regulation of blood vessel sprouting and branching during normal and pathological angiogenesis, and the physiological responses of vascular smooth muscle cells. Defects in Notch signaling also cause inherited vascular diseases, such as the degenerative vascular disorder Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). This review summarizes recent studies that highlight the multiple roles the Notch signaling pathway plays during vascular development and physiology.
Vascularization of the vertebrate brain takes place during embryonic development from a preformed perineural vascular plexus. As a consequence of the intimate contact with neuroectodermal cells the vessels, which are entering the brain exclusively via sprouting angiogenesis, acquire and maintain unique barrier properties known as the blood-brain barrier (BBB). The endothelial BBB depends upon the close association of endothelial cells with pericytes, astrocytes, neurons and microglia, which are summarized in the term neuro-vascular unit. Although it is known since decades that the CNS tissue provides the cues for BBB induction and differentiation in endothelial cells, the molecular mechanism remained obscure.
Only recently, the canonical Wnt/β-catenin pathway and the Wnt7a/7b growth factors have been implicated in brain angiogenesis on the one hand and in BBB induction on the other. This breakthrough in understanding the differentiation of the brain vasculature prompted us to review these findings embedded in the emerging concepts of Wnt signaling in the vasculature. In particular, interactions with other pathways that are crucial for vascular development such as VEGF, Notch, angiopoietins and Sonic hedgehog are discussed. Finally, we considered the potential role of the Wnt pathway in vascular brain pathologies in which BBB function is hampered, as for example in glioma, stroke and Alzheimer's disease.
Extracellular matrix (ECM) is essential for all stages of angiogenesis. In the adult, angiogenesis begins with endothelial cell (EC) activation, degradation of vascular basement membrane, and vascular sprouting within interstitial matrix. During this sprouting phase, ECM binding to integrins provides critical signaling support for EC proliferation, survival, and migration. ECM also signals the EC cytoskeleton to initiate blood vessel morphogenesis. Dynamic remodeling of ECM, particularly by membrane-type matrix metalloproteases (MT-MMPs), coordinates formation of vascular tubes with lumens and provides guidance tunnels for pericytes that assist ECs in the assembly of vascular basement membrane. ECM also provides a binding scaffold for a variety of cytokines that exert essential signaling functions during angiogenesis. In the embryo, ECM is equally critical for angiogenesis and vessel stabilization, although there are likely important distinctions from the adult because of differences in composition and abundance of specific ECM components.
The extracellular matrix provides basic support for endothelial cell proliferation, survival, and migration. In conjunction with cytokines (e.g., VEGF), it also regulates blood vessel morphogenesis and maturation.
The Notch signaling pathway is a critical component of vascular formation and morphogenesis in both development and disease. Compelling evidence indicates that Notch signaling is required for the induction of arterial-cell fate during development and for the selection of endothelial tip and stalk cells during sprouting angiogenesis. In mammals, two of the four Notch receptors (Notch1 and Notch4) and three of the five Notch ligands (Jagged1, Dll1, and Dll4) are predominantly expressed in vascular endothelial cells and are important for many aspects of vascular biology. During arterial cell-fate selection and angiogenesis, the roles of Notch1 and Notch4 are thought to be similar, and the function of Dll4 is well-characterized. However, the molecular mechanisms that determine the functional similarities and differences of Notch ligands in vascular endothelial cells remain largely unknown; consequently, additional research is needed to elucidate the ligand-specific functions and mechanisms associated with Notch activation in the vascular endothelium. Results from recent studies indicate that Dll1 and Dll4 have distinct roles in the specification and maintenance of arterial cell identity, while Dll4 and Jagged1 have opposing functions in tip- and stalk-cell selection during sprouting angiogenesis. This review will focus on the newly discovered, distinct functions of several Notch ligands in the regulation of blood vessel formation and will provide perspectives for future research in the field.
In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis.
Methods and Results
We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression.
Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression.
Angiogenesis is the process of developing vascular sprouts from existing blood vessels. Luminal endothelial cells convert into “tip” cells that contribute to the development of a multicellular stalk, which then undergoes lumen formation. In this review, we consider a variety of cellular and molecular pathways that mediate these transitions. We focus first on Notch signaling in cell fate determination as a mechanism to define tip and stalk cells. We next discuss the current models of lumen formation and describe new players in this process, such as chloride intracellular channel proteins. Finally, we consider the possible medical therapeutic benefits of understanding these processes and acknowledge potential obstacles in drug development.
During angiogenesis, VEGF/Notch interplay leads to the formation of vascular sprouts in existing blood vessels. Lumen formation involves vesicular trafficking that is mediated by ion channels (e.g., CLICs).
The discovery that Notch, a key regulator of cell fate determination, is functional in the vasculature has greatly improved our understanding of differentiation and specialization of vessels. Notch signaling has been proven to be critical for arterial specification, sprouting angiogenesis, and vessel maturation. In newly forming vascular sprouts, Notch promotes the distinction between the leading “tip” endothelial cell and the growing “stalk” cell, the endothelial cells that eventually form a new capillary. Notch signaling has also been implicated in vessel stability by regulating vascular mural cell function. More recently, macrophages carrying an activated Notch have been implicated in shaping the course of new sprout formation. Tumor vessels abide by similar principles and use Notch signaling in similar ways. An exciting discovery, made by several researchers, shows that blocking Notch function in tumor vasculature provides a means by which to suppress tumor growth. The authors discuss the developmental and physiological role of Notch in the vasculature and apply this knowledge to an overview of how Notch targeting in the tumor environment can affect tumor angiogenesis and growth.
Notch; VEGF; Dll4; sprouting angiogenesis
A key feature of angiogenesis is directional control of endothelial cell (EC) morphogenesis and movement . During angiogenic sprouting, endothelial “tip cells” directionally branch from existing vessels in response to biochemical cues such as VEGF or hypoxia, and migrate and invade the surrounding extracellular matrix (ECM) in a process that requires ECM remodeling by matrix metalloproteases (MMPs) [2–4]. Tip EC branching is mediated by directional protrusion of subcellular pseudopodial branches [5, 6]. Here we sought to understand how EC pseudopodial branching is locally regulated to directionally guide angiogenesis. We develop an in vitro 3D EC model system where migrating ECs display branched pseudopodia morphodynamics similar to those in living zebrafish. Using this system, we find that ECM stiffness and ROCK-mediated myosin II activity inhibit EC pseudopodial branch initiation. Myosin II is dynamically localized to the EC cortex, and is partially released under conditions that promote branching. Local depletion of cortical myosin II precedes branch initiation, and initiation can be induced by local inhibition of myosin II activity. Thus, local downregulation of myosin II cortical contraction allows pseudopodium initiation to mediate EC branching and hence guide directional migration and angiogenesis.
The heterotypic interactions of endothelial cells and mural cells (smooth muscle cells or pericytes) are crucial for assembly, maturation and subsequent function of blood vessels. Yet, the molecular mechanisms underlying their association have not been fully defined.
Our previous in vitro studies indicated that Notch3, which is expressed in mural cells, mediates these cell-cell interactions. To assess the significance of Notch3 on blood vessel formation in vivo, we investigated its role in retinal angiogenesis.
Methods and Results
We show that Notch3-deficient mice exhibit reduced retinal vascularization, with diminished sprouting and vascular branching. Moreover, Notch3 deletion impairs mural cell investment, resulting in progressive loss of vessel coverage. In an oxygen-induced retinopathy (OIR) model, we demonstrate that Notch3 is induced in hypoxia and interestingly, pathological neovascularization is decreased in retinas of Notch3 null mice. Analysis of OIR mediators revealed that Angiopoietin-2 expression is significantly reduced in the absence of Notch3. Further, in vitro experiments showed that Notch3 is sufficient for Angiopoietin-2 induction, and this expression is additionally enhanced in the presence of HIF1α.
These results provide compelling evidence that Notch3 is important for the investment of mural cells and is a critical regulator of developmental and pathological blood vessel formation.
Notch3; retina; angiogenesis; smooth muscle cell; pericytes; blood vessel
Blood vessels arose during evolution carrying oxygen and nutrients to distant organs via complex networks of blood vessels penetrating organs and tissues. Mammalian cells require oxygen and nutrients for survival, of which oxygen has a diffusion limit of 100 to 200 μm between cell and blood vessel. For growth beyond this margin, cells must recruit new blood vessels, first by vasculogenesis, where embryonic vessels form from endothelial precursors, then angiogenesis which is the sprouting of interstitial tissue columns into the lumen of preexisting blood vessels. Angiogenesis occurs in many inflammatory diseases and in many malignant disease states, including over 90% of solid tumours. Malignant melanoma (MM) is the most lethal skin cancer, highly angiogenic, highly metastatic, and refractory to all treatments. Raised serum levels of vascular endothelial growth factor (VEGF) strongly correlate MM disease progression and poor prognosis. Melanoma cells secrete several proangiogenic cytokines including VEGF-A, fibroblast growth factor (FGF-2), platelet growth factor (PGF-1), interleukin-8 (IL-8), and transforming growth factor (TGF-1) that modulate the angiogenic switch, changing expression levels during tumour transition from radial to invasive vertical and then metastatic growth. We highlight modern and historical lines of research and development that are driving this exciting area of research currently.
Angiogenesis is a critical process to form new blood vessels from pre-existing vessels under physiological and pathological conditions and involves cellular and morphological changes such as endothelial cell proliferation, migration, and vascular tube formation. Despite evidence that angiogenic factors, including vascular endothelial growth factor (VEGF) and Notch, control various aspects of angiogenesis, the molecular mechanisms underlying gene regulation in blood vessels and surrounding tissues are not fully understood. Importantly, recent studies demonstrate that Forkhead transcription factor Foxc2 directly regulates expression of various genes involved in angiogenesis, CXCR4, integrin β3, Delta-like 4 (Dll4) and angiopoietin (Ang)-2, thereby controlling angiogenic processes. Thus, Foxc2 is now recognized as a novel regulator of vascular formation and remodeling. This review summarizes current knowledge about the function of Foxc2 in angiogenesis and discusses prospects for future research in Foxc2-mediated pathological angiogenesis in cardiovascular disease.
The critical contribution of the Notch signaling pathway to vascular morphogenesis has been underscored by loss-of-function studies in mouse and zebrafish. Nonetheless, a comprehensive understanding as to how this signaling system influences the formation of blood vessels at the cellular and molecular level is far from reached. Here, we provide a detailed analysis of the distribution of active Notch1 in relation to its DSL (Delta, Serrate, Lag2) ligands, Jagged1, Delta-like1, and Delta-like4, during progressive stages of vascular morphogenesis and maturation. Important differences in the cellular distribution of Notch ligands were found. Jagged1 (Jag1) was detected in “stalk cells” of the leading vasculature and at arterial branch points, a site where Delta-like4 (Dll4) was clearly absent. Dll4 was the only ligand expressed in “tip cells” at the end of the growing vascular sprouts. It was also present in stalk cells, capillaries, arterial endothelium, and in mural cells of mature arteries in a homogenous manner. Delta-like1 (Dll1) was observed in both arteries and veins of the developing network, but was also excluded from mature arterial branch points. These findings support alternative and distinct roles for Notch ligands during the angiogenic process.
arteries; blood vessels; capillaries; delta-like1; delta-like4; endothelial; jagged1; vascular remodeling; vasculature; veins
Development of tissues in vitro with dimensions larger than 150 to 200 μm requires the presence of a functional vascular network. Therefore, we have studied capillary morphogenesis under controlled biological and biophysical conditions with the aim of promoting vascular structures in tissue constructs. We and others have previously demonstrated that physiological values of interstitial fluid flow normal to an endothelial monolayer in combination with vascular endothelial growth factor play a critical role during capillary morphogenesis by promoting cell sprouting. In the present work, we studied the effect that a range of interstitial flow velocities (0–50 μm/min) has in promoting the amount, length, and branching of developing sprouts during capillary morphogenesis. The number of capillary-like structures developed from human umbilical vein endothelial cell monolayers across the interstitial flow values tested was not significantly affected. Instead, the length and branching degree of the sprouts presented a significant maximum at flow velocities of 10 to 20 μm/min. More-over, at these same flow values, the phosphorylation level of Src also showed its peak. We discovered that capillary morphogenesis is restricted to patches of Src-activated cells (phosphorylated Src (pSrc)) at the monolayer, suggesting that the transduction pathway in charge of sensing the mechanical stimulus induced by flow is promoting predetermined mechanically sensitive areas (pSrc) to undergo capillary morphogenesis.
Angiogenesis, which is morphogenesis undertaken by endothelial cells (ECs) during new blood vessel formation, has been traditionally studied on natural extracellular matrix proteins. In this work, we aimed to regulate and guide angiogenesis on synthetic, bioactive poly(ethylene glycol)-diacrylate (PEGDA) hydrogels. PEGDA hydrogel is intrinsically cell nonadhesive and highly resistant to protein adsorption, allowing a high degree of control over presentation of ligands for cell adhesion and signaling. Since these materials are photopolymerizable, a variety of photolithographic technologies may be applied to spatially control presentation of bioactive ligands. To manipulate EC adhesion, migration, and tubulogenesis, the surface of PEGDA hydrogels was micropatterned with a cell adhesive ligand, Arg-Gly-Asp-Ser (RGDS), in desired concentrations and geometries. ECs cultured on these RGDS patterns reorganized their cell bodies into cord-like structures on 50-μm-wide stripes, but not on wider stripes, suggesting that EC morphogenesis can be regulated by geometrical cues. The cords formed by ECs were reminiscent of capillaries with cells participating in the self-assembly and reorganization into multicellular structures. Further, endothelial cord formation was stimulated on intermediate concentration of RGDS at 20 μg/cm2, whereas it was inhibited at higher concentrations. This work has shown that angiogenic responses can be tightly regulated and guided by micropatterning of bioactive ligands and also demonstrated great potentials of micropatterned PEGDA hydrogels for various applications in tissue engineering, where vascularization prior to implantation is critical.
Blood vessels form de novo through the tightly regulated programs of vasculogenesis and angiogenesis. Both processes are distinct but one of the steps they share is the formation of a central lumen, when groups of cells organized as vascular cords undergo complex changes to achieve a tube-like morphology. Recently, a protein termed epidermal growth factor-like domain 7 (EGFL7) was described as a novel endothelial cell-derived factor involved in the regulation of the spatial arrangement of cells during vascular tube assembly. With its impact on tubulogenesis and vessel shape EGFL7 joined the large family of molecules governing blood vessel formation. Only recently, the molecular mechanisms underlying EGFL7's effects have been started to be elucidated and shaping of the extracellular matrix (ECM) as well as Notch signaling might very well play a role in mediating its biological effects. Further, findings in knock-out animal models suggest miR-126, a miRNA located within the egfl7 gene, has a major role in vessel development by promoting VEGF signaling, angiogenesis and vascular integrity. This review summarizes our current knowledge on EGFL7 and miR-126 and we will discuss the implications of both bioactive molecules for the formation of blood vessels.
Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.
VEGF; endothelial cell; filopodia; astrocyte; migration; proliferation
The vascular system developed early in evolution. It is required in large multicellular organisms for the transport of nutrients, oxygen, and waste products to and from tissues. The vascular system is composed of hollow tubes, which have a high level of complexity in vertebrates. Vasculogenesis describes the de novo formation of blood vessels, e.g., aorta formation in vertebrate embryogenesis. In contrast, angiogenesis is the formation of blood vessels from preexisting ones, e.g., sprouting of intersomitic blood vessels from the aorta. Importantly, the lumen of all blood vessels in vertebrates is lined and formed by endothelial cells. In both vasculogenesis and angiogenesis, lumen formation takes place in a cord of endothelial cells. It involves a complex molecular mechanism composed of endothelial cell repulsion at the cell–cell contacts within the endothelial cell cords, junctional rearrangement, and endothelial cell shape change. As the vascular system also participates in the course of many diseases, such as cancer, stroke, and myocardial infarction, it is important to understand and make use of the molecular mechanisms of blood vessel formation to better understand and manipulate the pathomechanisms involved.
Cord hollowing is the major mechanism of vascular lumen formation. This involves endothelial cell repulsion at cell–cell contacts within the cords, junctional rearrangements, and endothelial cell shape changes.
Background/aim: During angiogenesis—that is, the outgrowth of new from pre-existing blood vessels, new capillaries undergo a period of “fine tuning” when vascular endothelial cells become apoptotic if sufficient supply of angiogenic factors is lacking. Morphologically, this period correlates with the absence of pericyte coverage of new vessels. Mature, pericyte covered vessels, in contrast, do not depend on elevated levels of angiogenic factors for survival. This study analyses whether, and if so when, pathological vessels in human corneal neovascularisation (CN) acquire pericyte coverage. This can be of importance for future angioregressive therapeutic strategies.
Methods: Vascularised human corneas obtained by keratoplasty were evaluated by electron microscopy for pericyte coverage of new vessels. These data were correlated with the duration of CN (mean 73 (SD 95) (range 0.5–360) months; n = 15). CN was secondary to keratitis, transplant rejection, aniridia, or trauma.
Results: Overall, 196 blood vessels were analysed ultrastructurally (72 (37%) capillaries, 122 (62%) venules, and two (1%) arterioles). Electron microscopically, 170 (87%) vessels were covered by pericytes and two (1%) in addition by smooth muscle cells. Pericyte recruitment increased with time, evolving between clinically noted onset of CN and keratoplasty. Already 2 weeks after onset of CN, more than 80% of new vessels were covered by pericytes.
Conclusion: Pathological new vessels in human corneal angiogenesis are rapidly covered by pericytes. Therapeutic strategies aimed at regression of immature, not yet pericyte covered vessels by antagonising angiogenic factors should thus be most effective if applied very early in the course of corneal neovascularisation.