A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.     

Arterial-venous network formation during brain vascularization involves hemodynamic regulation of chemokine signaling

During angiogenic sprouting, newly forming blood vessels need to connect to the existing vasculature in order to establish a functional circulatory loop. Previous studies have implicated genetic pathways, such as VEGF and Notch signaling, in controlling angiogenesis. We show here that both pathways similarly act during vascularization of the zebrafish central nervous system. In addition, we find that chemokine signaling specifically controls arterial-venous network formation in the brain. Zebrafish mutants for the chemokine receptor cxcr4a or its ligand cxcl12b establish a decreased number of arterial-venous connections, leading to the formation of an unperfused and interconnected blood vessel network. We further find that expression of cxcr4a in newly forming brain capillaries is negatively regulated by blood flow. Accordingly, unperfused vessels continue to express cxcr4a, whereas connection of these vessels to the arterial circulation leads to rapid downregulation of cxcr4a expression and loss of angiogenic characteristics in endothelial cells, such as filopodia formation. Together, our findings indicate that hemodynamics, in addition to genetic pathways, influence vascular morphogenesis by regulating the expression of a proangiogenic factor that is necessary for the correct pathfinding of sprouting brain capillaries.     

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.     

Flow-Regulated Endothelial S1P Receptor-1 Signaling Sustains Vascular Development

During angiogenesis, nascent vascular sprouts fuse to form vascular networks, enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P(1)) stabilizes the primary vascular network. A gradient of S1P(1) expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P(1), adherens junctions are destabilized, barrier function is breached, and flow is perturbed, resulting in abnormal vascular hypersprouting. Interestingly, S1P(1) responds to S1P?as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in?vitro and in?vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P(1) to stabilize blood vessels in development and homeostasis.     

VEGF and vascular fusion: implications for normal and pathological vessels.

The avian embryo is well suited for the study of blood vessel morphogenesis. This is especially true of investigations that focus on the de novo formation of blood vessels from mesoderm, a process referred to as vasculogenesis. To examine the cellular and molecular mechanisms regulating vasculogenesis, we developed a bioassay that employs intact avian embryos. Among the many bioactive molecules we have examined, vascular epithelial growth factor (VEGF) stands out for its ability to affect vasculogenesis. Using the whole-embryo assay, we discovered that VEGF induces a vascular malformation we refer to as hyperfusion. Our studies showed that microinjection of recombinant VEGF165 converted the normally discrete network of embryonic blood vessels into enlarged endothelial sinuses. Depending on the amount of VEGF injected and the time of postinjection incubation, the misbehavior of the primordial endothelial cells can become so exaggerated that for all practical purposes the embryo contains a single enormous vascular sinus; all normal vessels are subsumed into a composite vascular structure. This morphology is reminiscent of the abnormal vascular sinuses characteristic of certain neovascular pathologies. (J Histochem Cytochem 47:1351-1355, 1999)     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

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.     

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF‐ß, and Ang‐1,2. Among these, the role of VEGF‐A signaling in vessel morphogenesis is best understood. The biological activity of VEGF‐A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF‐A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1‐tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1‐tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development. genesis 50:561–571, 2012. © 2012 Wiley Periodicals, Inc.     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.     

Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling

The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth.     

The role of CEA-related cell adhesion molecule-1 (CEACAM1) in vascular homeostasis

Carcinoembryonic antigen (CEA)-related cell adhesion molecules belong to the immunoglobulin superfamily, are expressed in a broad spectrum of tissues and cell types and exert context-dependent activating as well as inhibitory effects. Among these molecules, the CEA-related cell adhesion molecule-1 (CEACAM1) is a transmembrane molecule with an extracellular, a transmembrane and a cytoplasmic domain. The latter contains immunoreceptor tyrosine-based inhibitory motifs and functions as a signaling molecule. CEACAM1 can form homo- and heterodimers which is relevant for its signaling activities. CEACAM1 acts as co-receptor that modulates the activity of different receptor types including VEGFR-2, and B and T cell receptors. CEACAM1 is expressed in endothelial cells, in pericytes of developing and newly formed immature blood vessels and in angiogenically activated adult vessels, e.g., tumor blood vessels. However, it is either undetectable or only weakly expressed in quiescent blood vessels. Recent studies indicated that CEACAM1 is involved in the regulation of the endothelial barrier function. In CEACAM1 ^?/? mice, increased vascular permeability and development of small atherosclerotic lesions was observed in the aortae. CEACAM1 is also detectable in activated lymphatic endothelial cells and plays a role in tumor lymphangiogenesis. This review summarizes the vascular effects of CEACAM1 and focuses on its role in vascular morphogenesis and endothelial barrier regulation.     

VEGF signaling inside vascular endothelial cells and beyond

Vascular endothelial growth factor-A (VEGF-A) has long been recognized as the key regulator of vascular development and function in health and disease. VEGF is a secreted polypeptide that binds to transmembrane tyrosine kinase VEGF receptors on the plasma membrane, inducing their dimerization, activation and assembly of a membrane-proximal signaling complex. Recent studies have revealed that many key events of VEGFR signaling occur inside the endothelial cell and are regulated by endosomal receptor trafficking. Plasma membrane VEGFR interacting molecules, including vascular guidance receptors Neuropilins and Ephrins also regulate VEGFR endocytosis and trafficking. VEGF signaling is increasingly recognized for its roles outside of the vascular system, notably during neural development, and blood vessels regulate epithelial branching morphogenesis. We review here recent advances in our understanding of VEGF signaling and its biological roles.     

The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.     

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

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

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.     

Mechanoresponse of stem cells for vascular repair

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.     

Constitutive over-expression of VEGF results in reduced expression of Hand-1 during cardiac development in Xenopus

During heart development, various signaling cascades are tightly regulated in a stage- and region-dependent manner. Vascular endothelial growth factor (VEGF) is one of the important molecules required for both vascular development and cardiac morphogenesis. VEGF receptors are present in the embryonic heart, so we focused on heart formation in VEGF-over-expressing Xenopus embryos. Over-expression of VEGF(170) caused disorganized vessels, while the expression of an endothelial marker, Tie-2, was increased. The embryo's heart was distinctly larger than that of control, and showed abnormal morphology. Histological analysis of these embryos showed failure of heart looping. In situ hybridization with Hand-1, which controls intrinsic morphogenetic pathways, revealed that the expression level of Hand-1 was decreased in the heart region. These results suggest that increased VEGF(170) levels disturb Hand-1 expression in the region required for normal heart morphogenesis. VEGF expression level may be important in heart morphology during embryonic development.     

The VEGF family, the inside story

The role of paracrine signaling by vascular endothelial growth factor (VEGF) in the formation and maintenance of blood vessels has been studied extensively. In this issue, Lee et al. (2007) report unexpected results showing that endogenous VEGF produced by endothelial cells is also crucial for vascular homeostasis.     

Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement

Angiogenesis is a complex process, which is accomplished by reiteration of modules such as sprouting, elongation and bifurcation, that configures branching vascular networks. However, details of the individual and collective behaviors of vascular endothelial cells (ECs) during angiogenic morphogenesis remain largely unknown. Herein, we established a time-lapse imaging and computer-assisted analysis system that quantitatively characterizes behaviors in sprouting angiogenesis. Surprisingly, ECs moved backwards and forwards, overtaking each other even at the tip, showing an unknown mode of collective cell movement with dynamic 'cell-mixing'. Mosaic analysis, which enabled us to monitor the behavior of individual cells in a multicellular structure, confirmed the 'cell-mixing' phenomenon of ECs that occurs at the whole-cell level. Furthermore, an in vivo EC-tracking analysis revealed evidence of cell-mixing and overtaking at the tip in developing murine retinal vessels. In parametrical analysis, VEGF enhanced tip cell behavior and directed EC migration at the stalk during branch elongation. These movements were counter-regulated by EC-EC interplay via γ-secretase-dependent Dll4-Notch signaling, and might be promoted by EC-mural cell interplay. Finally, multiple regression analysis showed that these molecule-mediated tip cell behaviors and directed EC migration contributed to effective branch elongation. Taken together, our findings provide new insights into the individual and collective EC movements driving angiogenic morphogenesis. The methodology used for this analysis might serve to bridge the gap in our understanding between individual cell behavior and branching morphogenesis.