Angiogenesis and vascular remodelling in normal and cancerous tissues

Vascular development and homeostasis are underpinned by two fundamental features: the generation of new vessels to meet the metabolic demands of under-perfused regions and the elimination of vessels that do not sustain flow. In this paper we develop the first multiscale model of vascular tissue growth that combines blood flow, angiogenesis, vascular remodelling and the subcellular and tissue scale dynamics of multiple cell populations. Simulations show that vessel pruning, due to low wall shear stress, is highly sensitive to the pressure drop across a vascular network, the degree of pruning increasing as the pressure drop increases. In the model, low tissue oxygen levels alter the internal dynamics of normal cells, causing them to release vascular endothelial growth factor (VEGF), which stimulates angiogenic sprouting. Consequently, the level of blood oxygenation regulates the extent of angiogenesis, with higher oxygenation leading to fewer vessels. Simulations show that network remodelling (and de novo network formation) is best achieved via an appropriate balance between pruning and angiogenesis. An important factor is the strength of endothelial tip cell chemotaxis in response to VEGF. When a cluster of tumour cells is introduced into normal tissue, as the tumour grows hypoxic regions form, producing high levels of VEGF that stimulate angiogenesis and cause the vascular density to exceed that for normal tissue. If the original vessel network is sufficiently sparse then the tumour may remain localised near its parent vessel until new vessels bridge the gap to an adjacent vessel. This can lead to metastable periods, during which the tumour burden is approximately constant, followed by periods of rapid growth.     

Angiogenesis in the Developing Spinal Cord: Blood Vessel Exclusion from Neural Progenitor Region Is Mediated by VEGF and Its Antagonists

Blood vessels in the central nervous system supply a considerable amount of oxygen via intricate vascular networks. We studied how the initial vasculature of the spinal cord is formed in avian (chicken and quail) embryos. Vascular formation in the spinal cord starts by the ingression of intra-neural vascular plexus (INVP) from the peri-neural vascular plexus (PNVP) that envelops the neural tube. At the ventral region of the PNVP, the INVP grows dorsally in the neural tube, and we observed that these vessels followed the defined path at the interface between the medially positioned and undifferentiated neural progenitor zone and the laterally positioned differentiated zone. When the interface between these two zones was experimentally displaced, INVP faithfully followed a newly formed interface, suggesting that the growth path of the INVP is determined by surrounding neural cells. The progenitor zone expressed mRNA of vascular endothelial growth factor-A whereas its receptor VEGFR2 and FLT-1 (VEGFR1), a decoy for VEGF, were expressed in INVP. By manipulating the neural tube with either VEGF or the soluble form of FLT-1, we found that INVP grew in a VEGF-dependent manner, where VEGF signals appear to be fine-tuned by counteractions with anti-angiogenic activities including FLT-1 and possibly semaphorins. These results suggest that the stereotypic patterning of early INVP is achieved by interactions between these vessels and their surrounding neural cells, where VEGF and its antagonists play important roles.     

Angiopoietin 1 causes vessel enlargement, without angiogenic sprouting, during a critical developmental period

Early in development, endothelial cells proliferate, coalesce, and sprout to form a primitive plexus of undifferentiated microvessels. Subsequently, this plexus remodels into a hierarchical network of different-sized vessels. Although the processes of proliferation and sprouting are well studied and are dependent on the angiogenic growth factor VEGF, the factors involved in subsequent vessel remodeling are poorly understood. Here, we show that angiopoietin 1 can induce circumferential vessel enlargement, specifically on the venous side of the circulation. This action is due to the ability of angiopoietin 1 to promote endothelial cell proliferation in the absence of angiogenic sprouting; vessel growth without sprouting has not been ascribed to other vascular growth factors, nor has specificity for a particular segment of the vasculature. Moreover, angiopoietin 1 potently mediates widespread vessel enlargement only during a brief postnatal period, in particular, prior to the fourth postnatal week, corresponding to stages in which VEGF inhibition causes widespread vessel regression. These findings show that angiopoietin 1 has a potentially unique role among the vascular growth factors by acting to enlarge blood vessels without inducing sprouting, and also define a critical window of vascular plasticity in neonatal development. Finding the key molecular factors that regulate this plasticity may prove crucial to the further development of pro- and anti-angiogenic therapies.     

Angioarchitechtonics of the intervertebral disks in human fetuses and newborns]

The ingrowth of the blood vessel branches into the area of the intervertebral disk takes place at the end of the second month of the intrauterine life. The primary vascular elements have a shape of stretched vascular loops, which grow into the fibrous ring of the disk from the front, from behind and from the sides. Within the fibrous tissue the blood vessels are disposed in the slit-shaped spaces between the layers of the fibrous ring. Here the vascular network is formed by anastomosing. These networks have the smaller meshes the closer to the central zones of the intervertebral disk. No blood vessel branches have been found in the jelly-like nucleus. Intraorganic vessels of the intervertebral disk do not penetrate the vertebrum body. The connection between the disk vessels and the vessels of the vertebrum body takes place in the field of the perichondrium.     

Early effects of ionizing radiation on the microvascular networks in normal tissue

Microvascular networks, which control the delivery of oxygen and nutrients and the removal of metabolic waste, are the most sensitive part of the vascular system to ionizing radiation. Structural and functional changes in microvascular networks were studied in locally irradiated (single 10-Gy dose) hamster cremaster muscles observed 3, 7 and 30 days post-irradiation. Networks were selected in reference to a well-defined location in the tissue to reduce heterogeneity due to spatial variations. Intravital microscopy was used to measure structural and functional parameters in vivo. A factorial design was used to examine the effects of radiation status, time postirradiation, and network vessel type on the structure and function of microvascular networks. While the diameter of microvessels in control animals increased significantly with age, vessel diameter in irradiated vessels decreased significantly with age. Red blood cell velocity in irradiated networks at 3 and 30 days postirradiation was significantly lower than in control networks. There was a significant decrease in capillary surface area and a significant increase in vessel hematocrit in irradiated animals. Blood flow in irradiated vessels was significantly lower than in control vessels. Changes in functional parameters were evident at 3 days postirradiation while changes in structural parameters occurred later. All vessel types were not damaged equally by radiation at every time examined.     

Theoretical comparison of wall-derived and erythrocyte-derived mechanisms for metabolic flow regulation in heterogeneous microvascular networks

The objective of this study is to compare the effectiveness of metabolic signals derived from erythrocytes and derived from the vessel wall for regulating blood flow in heterogeneous microvascular networks. A theoretical model is used to simulate blood flow, mass transport, and vascular responses. The model accounts for myogenic, shear-dependent, and metabolic flow regulation. Metabolic signals are assumed to be propagated upstream along vessel walls via a conducted response. Arteriolar tone is assumed to depend on the conducted metabolic signal as well as local wall shear stress and wall tension, and arteriolar diameters are calculated based on vascular smooth muscle mechanics. The model shows that under certain conditions metabolic regulation based on wall-derived signals can be more effective in matching perfusion to local oxygen demand relative to regulation based on erythrocyte-derived signals, resulting in higher extraction and lower oxygen deficit. The lower effectiveness of the erythrocyte-derived signal is shown to result in part from the unequal partition of hematocrit at diverging bifurcations, such that low-flow vessels tend to receive a reduced hematocrit and thereby experience a reduced erythrocyte-derived metabolic signal. The model simulations predict that metabolic signals independent of erythrocytes may play an important role in local metabolic regulation of vascular tone and flow distribution in heterogeneous microvessel networks.     

Mouse corneal lymphangiogenesis model

This protocol describes a powerful in vivo method to quantitatively study the formation of new lymphatic vessels in the avascular cornea without interference of pre-existing lymphatics. Implantation of 100 ng of lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-A, VEGF-C or fibroblast growth factor-2, together with slow-release polymers, into a surgically created micropocket in the mouse cornea elicits a robust lymphangiogenic response. Newly formed lymphatic vessels are detected by immunohistochemical staining of the flattened corneal tissue with lymphatic endothelial-specific markers such as lymphatic vessel endothelial hyaluronan receptor-1; less-specific markers such as vascular endothelial growth factor receptor 3 may also be used. Lymphatic vessel growth in relation to hemangiogenesis can be readily detected starting at day 5 or 6 after pellet implantation and persists for ～14 d. This protocol offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function.     

When It Is Better to Regress: Dynamics of Vascular Pruning

Blood vascular networks in vertebrates are essential to tissue survival. Establishment of a fully functional vasculature is complex and requires a number of steps including vasculogenesis and angiogenesis that are followed by differentiation into specialized vascular tissues (i.e., arteries, veins, and lymphatics) and organ-specific differentiation. However, an equally essential step in this process is the pruning of excessive blood vessels. Recent studies have shown that pruning is critical for the effective perfusion of blood into tissues. Despite its significance, vessel pruning is the least understood process in vascular differentiation and development. Two recently published PLOS Biology papers provide important new information about cellular dynamics of vascular regression.Vascular biology is a rapidly emerging field of research. Given the critical role the vasculature frequently plays in a wide range of common and serious diseases such as arteriosclerosis, ischemic diseases, cancer, and chronic inflammatory diseases, a better understanding of the formation, maintenance, and remodeling of blood vessels is of major importance.A mature vascular network is a highly anisotropic, hierarchical, and dynamic structure that has evolved to provide optimal oxygen delivery to tissues under a variety of conditions. Whilst much has been learned about early steps in vascular development such as vasculogenesis and angiogenesis, we still know relatively little about how such anatomical and functional organization is achieved. Furthermore, the dynamic nature of mature vascular networks, with its potential for extensive remodeling and a continuing need for stability and maintenance, is even less understood. The issue of optimal vascular density in tissue is of particular importance as several recent studies demonstrated that excessive vascularity may, in fact, reduce effective perfusion [1–3]. Since all neovascularization processes initially result in the formation of excessive amounts of vasculature, be that capillaries, arterioles, or venules, pruning must occur to return the vascular density to its optimal value in order to achieve effective tissue perfusion.Yet despite its functional importance, little is known about how regression of the once formed vasculature actually happens. While several potential mechanisms have been proposed including apoptosis of endothelial cells, intussusception vascular pruning, and endothelial cell migration away from the regressing vessel, cellular and molecular understanding of how this might happen is conspicuously lacking. Two articles recently published in PLOS Biology describe migration of endothelial cells as the key mechanism of apoptosis-independent vascular pruning and place it in a specific biologic context. This important advance offers not only a new understanding of a poorly understood aspect of vascular biology but may also prove to be of considerable importance in the development of pro- and anti-angiogenic therapies.To put vessel regression in context, it helps to briefly outline the current understanding of vessel formation. During embryonic development, vasculature forms in several distinct steps that begin with vasculogenesis, a step that involves differentiation of stem cells into primitive endothelial cells that then form initial undifferentiated and nonhierarchically organized lumenized vascular structures termed the primary plexus [4]. The primary plexus is then remodeled, by the process termed angiogenesis, into a more mature vascular network [5]. This remodeling event involves both formation of new vessels accomplished either by branching angiogenesis, a process dependent on tip cell-driven formation of new branches [6], or intussusception, a poorly understood process of splitting an existing vessel into two [7]. This incompletely differentiated and still nonhierarchical vasculature then further remodels into a number of distinctly different types of vessels such as capillaries, arteries, and veins. This requires fate specification, differentiation, and incorporation of various mural cells into evolving vascular structures. Finally, additional specialization of the vascular network occur in an organ-specific manner.Once formed, vascular networks require active maintenance as withdrawal of key signals, such as of ongoing fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) stimulation, can lead to a rapid loss of vascular integrity and even changes in endothelial cell fate [8–12]. In addition, mature vessels retain the capacity for extensive remodeling and new growth as can be seen in a number of conditions from cancer to myocardial infarction and wound healing responses, among many others [5].A key issue common to both embryonic and adult vessel remodeling is how an existing lumenized vessel connected to the rest of the vasculature undergoes a change that results in its remodeling into something else. Such a change may involve either a new branch formation or regression of an existing branch, while the patency and integrity of the remaining circulation is maintained. Two types of cellular process leading to branching have been described—sprouting and intussusception. Formation of vascular branches by sprouting involves VEGF-A-induced expression of high levels of delta-like ligand 4 (Dll4) in a subset of endothelial cells at the leading edge of the vascular sprouts that are lying closest to the source of VEGF, thus converting them to a “tip cell” phenotype. Some of the key features of tip cells include the presence of cytoplasmic processes that extend into avascular (or hypoxic) tissue that form nascent branches. Dll4 expressed on tip cells binds Notch-1 receptor in neighboring endothelial cells, thereby activating their downstream Notch signaling. In turn, Notch signaling shuts down the formation of additional filopodia processes, converting these cells to a “stalk cell” phenotype and thereby avoiding excessive branching [13–15]. The bone morphogenetic protein signaling pathway provides further input in determining stalk cell fate [16]. Importantly, tip cells are only partially lumenized; only once they have converted to a stalk phenotype does the lumen extend to what was a tip cell and its sprouts.An alternative mechanism of branching involves intussusception, a process by which a tissue pillar from the surrounding tissue splits the existing endothelial tube into two along its long axis, creating two adjusting vessels. While this process has been described morphologically, virtually nothing is known about its molecular and cellular regulation. In development, angiogenesis by intussusception occurs in vessels previously formed by sprouting angiogenesis [17,18]. Importantly, however, both sprouting angiogenesis and intussusception allow growth and remodeling of vascular network without any integrity compromise, thereby avoiding bleeding and related complications.There are certain parallels between vessel formation and branching and vessel regression. While growth occurs either via sprouting (a process linked to endothelial cell-migration) or intussusception, regression involves either “reverse intussusception,” endothelial migration-dependent regression, or apoptosis. The latter is the primary means of regression of the hyaloid vasculature in the eye and of the vascular loss seen in oxygen-induced retinopathy (OIR). In the case of hyaloid vasculature, secretion of WNT7b by macrophages invading the hyaloid membrane induces apoptosis of hyaloid endothelial cells leading to the regression of the entire hyaloid vasculature [19]. This total apoptosis-induced loss of hyaloid blood vessels contrasts with a less extensive vascular regression seen in the setting of OIR. In this condition, exposure of the developing retinal vasculature to abnormally high oxygen levels leads to vascular damage characterized by capillary pruning [20]. The pruning is the consequence of apoptosis of endothelial cells due to the toxic effect of a combination of high oxygen and low VEGF level. Interestingly, larger vessels and mature capillaries are not sensitive to hyperoxia [21].Intussusception vascular pruning was also described in a low VEGF level context in the chick chorioallantoic membrane. Application of VEGF-releasing hydrogels to the membrane surface results in formation of an excessive vasculature. Removal or degradation of the hydrogel induces an abrupt VEGF withdrawal. In this context, formation of transluminal pillars, similar to the ones seen in intussusception angiogenesis, is observed in vessels undergoing pruning [22]. The same process is observed in the tumor vasculature in the setting of anti-angiogenic therapy [23]. Finally, apoptosis-independent vascular regression, driven by endothelial cell migration, has been described in the mouse retina, yolk vessels of the chick and mouse embryos, branchial arches, and the zebrafish brain [24–28].In all of these cases, only a subset of vessels is designated for pruning, and the selection of these vessels is highly regulated. Yet, factors involved in choosing a particular vascular branch for pruning remain ill-defined. One such factor is low blood flow [27,28]. Another is Notch signaling that has been shown to at least partially control vascular pruning in mouse retina and in intersegmental vessels (ISVs) in zebrafish [24]. Loss of Notch-regulated ankyrin repeat protein (Nrarp), target gene of Notch signaling, leads to an increase in vascular regression in these tissues due to a decrease in Wnt signaling-induced stalk cell proliferation. Similarly, in Dll4 ^+/- mice, developmental retinal vascular regression and OIR-induced vascular pruning are reduced [29], confirming the involvement of the Notch pathway in the control of vascular regression.The two factors may be linked, as low flow can affect endothelial shear stress and lead to a decrease in Notch activation. Such a link is suggested by studies on vascular regression in mice with endothelial expression of dominant negative NFκB pathway inhibitor that demonstrate excessive vascular growth but reduced tissue perfusion [2]. Molecular studies showed inhibition of flow- or cytokine-induced NFκB activation results in decreased Dll4 expression [2].Another important issue is the fate of endothelial cells from vessels undergoing pruning. In PLOS Biology, two groups recently described endothelial cell behavior during vascular pruning in three different models: the mouse retina, the ISVs in zebrafish, and the subintestinal vessel in zebrafish [30,31]. Using a high resolution time-lapse microscopy technique, Lenard and collaborators showed that vascular pruning during the subintestinal vessel formation occurs in two different ways. In type I pruning, the first step is the collapse of the lumen. Once that occurs, endothelial cells migrate and incorporate into the neighboring vessels. In type II pruning, the lumen is maintained. One endothelial cell in the center of the pruning vessel undergoes self-fusion, leading to a unicellular lumenized vessel. At the same time, other endothelial cells migrate away and incorporate into the neighboring vessels. The eventual lumen collapse is the last step after which the remaining single endothelial cell migrates and incorporates into one of the major vessels.Franco and collaborators described a pruning mechanism similar to the type I pruning described by Lenard et al., showing lumen disruption as an initial step in pruning of retinal vasculature in mice and ISVs in zebrafish [31]. By analyzing the first axial polarity map of endothelial cells in these models, they demonstrated that axial orientation predicts endothelial cell migration, and that migration-driven pruning occurs in vessels with low flow. Interestingly, migrating endothelial cells in regressing vessel display a tip cell phenotype with filopodia.The cellular dynamic of vessel pruning described here is the reverse of the cellular dynamic during anastomosis and angiogenesis [32]. Given the crucial role of factors as VEGF for the migration of endothelial cells during angiogenesis, can we go further and propose that other cytokines or cell–cell signaling may be involved in the migration of these endothelial cells? Indeed, low blood flow seems to be the cause of vessel pruning, but how can we explain the direction of endothelial cell migration, moreover with a tip cell morphology? Also, what determines the choice between type I and type II pruning? The collapse of lumen suggests a reorganization of the cytoskeleton, and a loss of polarity and electrostatic repulsion of endothelial cells. Molecular mechanisms leading from low shear stress to loss of endothelial cell polarity need further investigation. As defective vascular pruning could be involved in poor recovery after injury or ischemic accident, a better understanding of the molecular control of this phenomenon appears to have medical consequences. Another question that is still unanswered is the fate of mural cells that surrounded the pruned vessels. Small vessels are covered by pericytes, which have strong interaction with endothelial cells. How and when are these interactions disrupted? Are pericytes integrated into the neighboring vessel, or do they undergo apoptosis? Further studies are needed to understand the molecular and cellular mechanisms by which vasculature can adapt, even at the adult stage, to support the nutrient and oxygen needs of each cell.Overall, taking the results of these studies together with other recent developments in this field, the following picture is emerging (Fig 1). Under conditions of low blood flow in certain vascular tree branches, pruning will occur via endothelial cell migration out of these branches to the neighboring (presumably higher blood flow) vessels. This results in decreased total vascular cross-sectional area and increased average blood flow, thereby terminating further pruning. Importantly, this occurs without the loss of luminal integrity and without reduction in the total endothelial cell mass. At the same time, vessels that suddenly find themselves in a low VEGF environment will regress either by apoptosis of endothelial cells or by intussusception. In both cases, there is a reduction in the total vasculature without an increase in blood flow to this tissue. Thus, the local context determines the mechanism: migratory regression and remodeling in low shear stress versus apoptotic pruning in low VEGF milieu.Open in a separate windowFig 1Vessel regression under low flow versus low VEGF conditions.Vessel regression under low flow conditions proceeds by endothelial cell (EC) migration-driven regression, resulting in a decrease in total vessel areas but an increase in blood flow (left panel). Vessel regression under low VEGF conditions proceeds by EC apoptosis or intussusception regression, resulting in decreased vessel number and decreased flow to tissues subtended by the regressing vasculature (right panel). Image credit: Nicolas Ricard & Michael Simons.This distinction is likely to be of a significant practical importance, in particular in the context of therapies designed to facilitate vessel normalization in tumors after VEGF-targeting treatments and therapies designed to promote vascularization of mildly ischemic tissues as occurs, for example, in the setting of chronic stable angina and other similar conditions. In the former case, a precipitous drop in VEGF levels is likely to induce vascular regression by induction of endothelial apoptosis, and further promotion of apoptosis may facilitate this process. In contrast, in the latter case, low flow in newly formed collateral arteries may induce their regression by stimulating outmigration of endothelial cells, thereby limiting their beneficial functional impact. Therapies designed to inhibit this mechanism, therefore, may promote growth of the new functional vasculature.     

Structural response of microcirculatory networks to changes in demand: information transfer by shear stress

Matching blood flow to metabolic demand in terminal vascular beds involves coordinated changes in diameters of vessels along flow pathways, requiring upstream and downstream transfer of information on local conditions. Here, the role of information transfer mechanisms in structural adaptation of microvascular networks after a small change in capillary oxygen demand was studied using a theoretical model. The model includes diameter adaptation and information transfer via vascular reactions to wall shear stress, transmural pressure, and oxygen levels. Information transfer is additionally effected by conduction along vessel walls and by convection of metabolites. The model permits selective blocking of information transfer mechanisms. Six networks, based on in vivo data, were considered. With information transfer, increases in network conductance and capillary oxygen supply were amplified by factors of 4.9 +/- 0.2 and 9.4 +/- 1.1 (means +/- SE), relative to increases when information transfer was blocked. Information transfer by flow coupling alone, in which increased shear stress triggers vascular enlargement, gave amplifications of 4.0 +/- 0.3 and 4.9 +/- 0.5. Other information transfer mechanisms acting alone gave amplifications below 1.6. Thus shear-stress-mediated flow coupling is the main mechanism for the structural adjustment of feeding and draining vessel diameters to small changes in capillary oxygen demand.     

Pro-angiogenic signaling by the endothelial presence of CEACAM1

Here, we demonstrate the expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in angiogenic sprouts but not in large mother blood vessels within tumor tissue. Correspondingly, only human microvascular endothelial cells involved in in vitro tube formation exhibit CEACAM1. CEACAM1-overexpressing versus CEACAM1-silenced human microvascular endothelial cells were used in migration and tube formation assays. CEACAM1-overexpressing microvascular endothelial cells showed prolonged survival and increased tube formation when they were stimulated with vascular endothelial growth factor (VEGF), whereas CEACAM1 silencing via small interfering RNA blocks these effects. Gene array and LightCycler analyses show an up-regulation of angiogenic factors such as VEGF, VEGF receptor 2, angiopoietin-1, angiopoietin-2, tie-2, angiogenin, and interleukin-8 but a down-regulation of collagen XVIII/endostatin and Tie-1 in CEACAM1-overexpressing microvascular endothelial cells. Western blot analyses confirm these results for VEGF and endostatin at the protein level. These results suggest that constitutive expression of CEACAM1 in microvascular endothelial cells switches them to an angiogenic phenotype, whereas CEACAM1 silencing apparently abrogates the VEGF-induced morphogenetic effects during capillary formation. Thus, strategies targeting the endothelial up-regulation of CEACAM1 might be promising for antiangiogenic tumor therapy.     

Structural adaptation of microvascular networks: functional roles of adaptive responses

Terminal vascular beds continually adapt to changing demands. A theoretical model is used to simulate structural diameter changes in response to hemodynamic and metabolic stimuli in microvascular networks. Increased wall shear stress and decreased intravascular pressure are assumed to stimulate diameter increase. Intravascular partial pressure of oxygen (PO(2)) is estimated for each segment. Decreasing PO(2) is assumed to generate a metabolic stimulus for diameter increase, which acts locally, upstream via conduction along vessel walls, and downstream via metabolite convection. By adjusting the sensitivities to these stimuli, good agreement is achieved between predicted network characteristics and experimental data from microvascular networks in rat mesentery. Reduced pressure sensitivity leads to increased capillary pressure with reduced viscous energy dissipation and little change in tissue oxygenation. Dissipation decreases strongly with decreased metabolic response. Below a threshold level of metabolic response flow shifts to shorter pathways through the network, and oxygen supply efficiency decreases sharply. In summary, the distribution of vessel diameters generated by the simulated adaptive process allows the network to meet the functional demands of tissue while avoiding excessive viscous energy dissipation.     

Spatial and temporal control of angiogenesis and arterialization using focal applications of VEGF164 and Ang-1

Microvascular networks undergo patterning changes that determine and reflect functional adaptations during tissue remodeling. Alterations in network architectures are a result of complex and integrated signaling events. To understand how two growth factor signals interact to stimulate angiogenesis and arterialization, we engineered spatially directed microvascular pattern changes in vivo by using combinations of focally delivered exogenous growth factors. We implanted microdelivery beads containing recombinant vascular endothelial growth factor-164 (VEGF(164)) and recombinant angiopoietin-1* (Ang-1*) into the dorsal subcutaneous tissue of fully anesthetized male Fischer 344 rats implanted with backpack window chambers, and we quantified vascular patterning changes by using intravital microscopy, a combination of architectural metrics, and immunohistochemistry. Focal delivery of VEGF(164) caused spatially directed increases in both the total number and the density of vessels with diameters <25 microm 7 days after microbead implantation. Increases were maintained out to 14 days but were reduced to control values by day 21. The addition of Ang-1* on day 7 maintained these increases out to day 21, induced vessel order ratios comparable to control levels, and was accompanied by increases in the length density of smooth muscle alpha-actin-positive vessels. We achieved spatial control of patterning changes in vivo by using multisignal stimulation via focal delivery of exogenous growth factor combinations and conclude that Ang-1* administered subsequent to VEGF(164) stimulation induces vascular growth while maintaining a network pattern consistent with native patterns that persist in the presence of vehicle control stimulation.     

Astrocyte-Derived Vascular Endothelial Growth Factor Stabilizes Vessels in the Developing Retinal Vasculature

Vascular endothelial growth factor (VEGF) plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.     

Simulation of angiogenesis in three dimensions: Application to cerebral cortex

The vasculature is a dynamic structure, growing and regressing in response to embryonic development, growth, changing physiological demands, wound healing, tumor growth and other stimuli. At the microvascular level, network geometry is not predetermined, but emerges as a result of biological responses of each vessel to the stimuli that it receives. These responses may be summarized as angiogenesis, remodeling and pruning. Previous theoretical simulations have shown how two-dimensional vascular patterns generated by these processes in the mesentery are consistent with experimental observations. During early development of the brain, a mesh-like network of vessels is formed on the surface of the cerebral cortex. This network then forms branches into the cortex, forming a three-dimensional network throughout its thickness. Here, a theoretical model is presented for this process, based on known or hypothesized vascular response mechanisms together with experimentally obtained information on the structure and hemodynamics of the mouse cerebral cortex. According to this model, essential components of the system include sensing of oxygen levels in the midrange of partial pressures and conducted responses in vessel walls that propagate information about metabolic needs of the tissue to upstream segments of the network. The model provides insights into the effects of deficits in vascular response mechanisms, and can be used to generate physiologically realistic microvascular network structures.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis.     

Vascular remodeling by intussusceptive angiogenesis

Intussusception (growth within itself) is an alternative to the sprouting mode of angiogenesis. The protrusion of opposing microvascular walls into the capillary lumen creates a contact zone between endothelial cells. The endothelial bilayer is perforated, intercellular contacts are reorganized, and a transluminal pillar with an interstitial core is formed, which is soon invaded by myofibroblasts and pericytes leading to its rapid enlargement by the deposition of collagen fibrils. Intussusception has been implicated in three processes of vascular growth and remodeling. (1) Intussusceptive microvascular growth permits rapid expansion of the capillary plexus, furnishing a large endothelial surface for metabolic exchange. (2) Intussusceptive arborization causes changes in the size, position, and form of preferentially perfused capillary segments, creating a hierarchical tree. (3) Intussusceptive branching remodeling (IBR) leads to modification of the branching geometry of supplying vessels, optimizing pre- and postcapillary flow properties. IBR can also lead to the removal of branches by pruning in response to changes in metabolic needs. None of the three modes requires the immediate proliferation of endothelial cells but rather the rearrangement and plastic remodeling of existing ones. Intussusception appears to be triggered immediately after the formation of the primitive capillary plexus by vasculogenesis or sprouting. The advantage of this mechanism of growth over sprouting is that blood vessels are generated more rapidly in an energetically and metabolically more economic manner, as extensive cell proliferation, basement membrane degradation, and invasion of the surrounding tissue are not required; the capillaries thereby formed are less leaky. This process occurs without disrupting organ function. Improvements in our understanding of the process should enable the development of novel pro- and anti-angiogenic therapeutic treatments.Electronic Supplementary Material Supplementary material is available in the online version of this article at This research was supported by the Swiss National Science Foundation (grant no. 3100-055895.98/2) and the Bernese Cancer League     

Hypoxia-induced insulin-like growth factor II contributes to retinal vascularization in ocular development

In ocular development, retinal physiological hypoxia in response to the retinal metabolic activity controls retinal vascular development, which is regulated by variable angiogenic factors. Herein, we demonstrated that hypoxia-induced IGF-II could contribute to retinal vascularization in ocular development. In the developing retina, IGF-II expression appears to be predominant on retinal vessels, which was chronologically increased and peaked during active retinal angiogenesis similar to VEGF expression. Under hypoxic condition, IGF-II as well as VEGF was significantly up-regulated in retinal vascular endothelial cells. In addition, IGF-II treatment could also increase VEGF expression in retinal vascular endothelial cells. The VEGF expression induced by IGF-II was mediated by ERK-1/2 activation. Moreover, IGF-II strongly promoted angiogenic processes of migration and tube formation of retinal microvascular endothelial cells. In conclusion, our results provided that hypoxia-induced IGF-II may regulate retinal vascular development not only directly by IGF-II-mediated angiogenic activity, but also indirectly by IGF-II-induced VEGF expression. Therefore, the potential contribution of IGF-II to pathological retinal angiogenesis should be furthermore explored for the development of novel treatments to vaso-proliferative retinopathies.     

VEGF regulates cell behavior during vasculogenesis

Prominent among molecules that control neovascular processes is vascular endothelial growth factor (VEGF). The VEGF ligands comprise a family of well-studied mitogens/permeability factors that bind cell surface receptor tyrosine kinases. Targets include VEGF receptor-1/Flt1 and VEGF receptor-2/Flk1. Mice lacking genes for VEGF ligand or VEGF receptor-2 die early in gestation, making it difficult to determine the precise nature of underlying endothelial cellular behavior(s). To examine the effect(s) of VEGF signaling on cell behavior in detail, we conducted loss-of-function studies using avian embryos. Injection of soluble VEGFR-1 results in malformed vascular networks and the absence of large vessels. In the most severe cases embryos exhibited vascular atresia. Closely associated with the altered phenotype was a clear endothelial cell response-a marked decrease in cell protrusive activity. Further, we demonstrate that VEGF gain of function strikingly increased cell protrusive activity. Together, our data show that VEGF/VEGF receptor signaling regulates endothelial cell protrusive activity, a key determinant of blood vessel morphogenesis. We propose that VEGF functions as an instructive molecule during de novo blood vessel morphogenesis.     

Numerical evaluation reveals the effect of branching morphology on vessel transport properties during angiogenesis

Blood flow governs transport of oxygen and nutrients into tissues. Hypoxic tissues secrete VEGFs to promote angiogenesis during development and in tissue homeostasis. In contrast, tumors enhance pathologic angiogenesis during growth and metastasis, suggesting suppression of tumor angiogenesis could limit tumor growth. In line with these observations, various factors have been identified to control vessel formation in the last decades. However, their impacts on the vascular transport properties of oxygen remain elusive. Here, we take a computational approach to examine the effects of vascular branching on blood flow in the growing vasculature. First of all, we reconstruct a 3D vascular model from the 2D confocal images of the growing vasculature at postnatal day 5 (P5) mouse retina, then simulate blood flow in the vasculatures, which are obtained from the gene targeting mouse models causing hypo- or hyper-branching vascular formation. Interestingly, hyper-branching morphology attenuates effective blood flow at the angiogenic front, likely promoting tissue hypoxia. In contrast, vascular hypo-branching enhances blood supply at the angiogenic front of the growing vasculature. Oxygen supply by newly formed blood vessels improves local hypoxia and decreases VEGF expression at the angiogenic front during angiogenesis. Consistent with the simulation results indicating improved blood flow in the hypo-branching vasculature, VEGF expression around the angiogenic front is reduced in those mouse retinas. Conversely, VEGF expression is enhanced in the angiogenic front of hyper-branching vasculature. Our results indicate the importance of detailed flow analysis in evaluating the vascular transport properties of branching morphology of the blood vessels.