Ralf H. Adams

Molecular regulation of angiogenesis and lymphangiogenesis

Blood vessels and lymphatic vessels form extensive networks that are essential for the transport of fluids, gases, macromolecules and cells within the large and complex bodies of vertebrates. Both of these vascular structures are lined with endothelial cells that integrate functionally into different organs, acquire tissue-specific specialization and retain plasticity; thereby, they permit growth during tissue repair or in disease settings. The angiogenic growth of blood vessels and lymphatic vessels coordinates several biological processes such as cell proliferation, guided migration, differentiation and cell–cell communication.

The Notch Ligands Dll4 and Jagged1 Have Opposing Effects on Angiogenesis

The Notch pathway is a highly conserved signaling system that controls a diversity of growth, differentiation, and patterning processes. In growing blood vessels, sprouting of endothelial tip cells is inhibited by Notch signaling, which is activated by binding of the Notch receptor to its ligand Delta-like 4 (Dll4). Here, we show that the Notch ligand Jagged1 is a potent proangiogenic regulator in mice that antagonizes Dll4-Notch signaling in cells expressing Fringe family glycosyltransferases. Upon glycosylation of Notch, Dll4-Notch signaling is enhanced, whereas Jagged1 has weak signaling capacity and competes with Dll4. Our findings establish that the equilibrium between two Notch ligands with distinct spatial expression patterns and opposing functional roles regulates angiogenesis, a mechanism that might also apply to other Notch-controlled biological processes.

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Essential Role of p38α MAP Kinase in Placental but Not Embryonic Cardiovascular Development

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

Murine semaphorin D/collapsin is a member of a diverse gene family and creates domains inhibitory for axonal extension

Members of the collapsin/semaphorin gene family have been proposed to act as growth cone guidance signals in vertebrates and invertebrates. To identify candidate molecules involved in axonal pathfinding during mouse embryogenesis, we isolated cDNAs encoding five new members of the semaphorin family (Sem A-Sem E). The murine semaphorin genes are differentially expressed in mesoderm and neuroectoderm before and during the time when axons select their pathways in the embryo. In explant cultures, recombinant Sem D/collapsin converts a matrix permissive for axonal growth into one that is inhibitory for neurites of peripheral ganglia. Our data demonstrate that semaphorins are a diverse family of molecules that may provide local signals to specify territories nonaccessible for growing axons.

Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone

The mammalian skeletal system harbours a hierarchical system of mesenchymal stem cells, osteoprogenitors and osteoblasts sustaining lifelong bone formation. Osteogenesis is indispensable for the homeostatic renewal of bone as well as regenerative fracture healing, but these processes frequently decline in ageing organisms, leading to loss of bone mass and increased fracture incidence. Evidence indicates that the growth of blood vessels in bone and osteogenesis are coupled, but relatively little is known about the underlying cellular and molecular mechanisms. Here we identify a new capillary subtype in the murine skeletal system with distinct morphological, molecular and functional properties. These vessels are found in specific locations, mediate growth of the bone vasculature, generate distinct metabolic and molecular microenvironments, maintain perivascular osteoprogenitors and couple angiogenesis to osteogenesis. The abundance of these vessels and associated osteoprogenitors was strongly reduced in bone from aged animals, and pharmacological reversal of this decline allowed the restoration of bone mass.

Ephrin-B2 Controls Cell Motility and Adhesion during Blood-Vessel-Wall Assembly

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αv integrin subunit because various αv-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride–induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αv integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αv-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Dynamics of endothelial cell behavior in sprouting angiogenesis

The vertebrate body contains an extensive blood vessel network that forms, with a few exceptions, by endothelial sprouting from the existing vasculature. This process, termed angiogenesis, involves complex and highly dynamic interactions between endothelial cells and their environment. Pro-angiogenic signals, such as VEGF, promote endothelial motility, filopodia extension and proliferation, and, together with Notch signaling, controls whether specific endothelial cells become lead tip cells or trailing stalk cells. Sprouts then convert into endothelial tubules and form connections with other vessels, which requires the local suppression of motility and the formation of new cell-cell junctions. We here review the dynamics of angiogenesis in the context of key molecules and pathways controlling tip cell selection, sprouting and the formation of new vessels.

The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration.

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, we replaced the endogenous gene by cDNAs encoding either carboxyterminally truncated (ephrinB2(DeltaC)) or, as a control, full-length ligand (ephrinB2(WT)). While homozygous ephrinB2(WT/WT) animals were viable and fertile, loss of the ephrinB2 cytoplasmic domain resulted in midgestation lethality similar to ephrinB2 null mutants (ephrinB2(KO)). The truncated ligand was sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2(DeltaC/DeltaC) embryos showed defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2(KO/KO) animals. Our results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo.