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Arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single-cell level, little is known about how collective cell migration contributes to the organization of the balanced distribution between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from early vasculogenesis throughout arteriovenous remodeling. We reveal that the genesis of arteries and veins relies on the coordination of 10 types of collective cell dynamics. Within these behavioral categories, we identify a heterogeneity of collective cell motion specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that cell-intrinsic Notch signaling and cell-extrinsic blood flow act as regulators in maintaining the heterogeneity of collective endothelial cell behavior, which, in turn, instructs the future territory of arteriovenous remodeling.

During vascular development, arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single cell level, little is known about how cell populations migrate in a finely tuned and coordinated manner, and distribute precisely between arteries and veins. Here, we use an endothelial-specific zebrafish reporter,arteriobow, to label small cohorts of arterial cells and trace their progeny from the initial events of vasculogenesis through the process of arterio-venous remodeling. We reveal that the genesis of both arteries and veins relies on the coordination of ten types of collective cell behaviors originating from discrete endothelial cell clusters. Within these behavioral categories, we identify a heterogeneity of collective cell dynamics specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that factors known to control vascular patterning such as cell-intrinsic Notch signaling and cell-extrinsic blood flow, potentially act as regulators by coordinating endothelial cohorts behavior, which in turn instructs the future territory of arterio-venous remodeling.

During vascular development, arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single cell level, little is known about how cell populations migrate in a finely tuned and coordinated manner, and distribute precisely between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from the initial events of vasculogenesis through the process of arterio-venous remodeling. We reveal that the genesis of both arteries and veins relies on the coordination of ten types of collective cell behaviors originating from discrete endothelial cell clusters. Within these behavioral categories, we identify a heterogeneity of collective cell dynamics specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that factors known to control vascular patterning such as cell-intrinsic Notch signaling and cell-extrinsic blood flow, potentially act as regulators by coordinating endothelial cohorts behavior, which in turn instructs the future territory of arterio-venous remodeling.

Key message Not only the first systematic characterization ofCAOgene in bamboo species, but also the first attempt to study the relationship betweenCAOgene expression and bamboo leaf color variation.

In angiosperms, NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the only light-dependent step in chlorophyll biosynthesis. There is a wide variety of gene organization and light- and development-dependent regulatory mechanisms for the POR genes. In this study, a POR homologue, designated PjPORB, was isolated from Pseudosasa japonica cv. Akebonosuji, which is a cultivar of high ornamental value in landscape due to its unique green-white striped leaf phenotype. The full-length PjPORB cDNA was 1567 bp long with a 1185 bp ORF that encoded 394 amino acids. Multiple amino acid sequence alignment showed that the putative PjPORB shared a high similarity to POR homologues from other plant species, and that the cofactor (NADPH)-binding motif and active site motif in particular were highly conserved among all the PORs. Further, PjPORB was overexpressed in Arabidopsis thaliana; and the Pchlide contents of the genetically modified plants were reduced to a larger extent than that of wild type plants, thereby indicating its important role in Pchlide photoreduction. In real-time quantitative RT-PCR analysis, PjPORB was expressed at higher levels in leaf samples than in culms and roots, and showed a first increasing-then decreasing expression pattern during the development of white, stripe, and green leaf samples, thus indicating its essential role in leaf development. Although the mRNA levels for PjPORB did not differ considerably among white, stripe, and green leaf samples within the two almost same earlier developmental stages, its expression levels in the late leaf developmental stage fell into two distinct classes: high expression levels in strip and green leaf samples, and low expression levels in white leaf. Collectively, these expression data suggested that PjPORB may be involved in the leaf color variation for P. japonica cv. Akebonosuji.

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84-99% enantiomeric excess and with 21-73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.

Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and ∼85% of kringle 5. In culture, cancer cell surface globular β-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface β-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized β-actin similarly, with a Kd of ∼140 nmol/L. The binding is inhibited by ε-aminocaproic acid (εACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from β-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 μmol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface β-actin, and the truncated kringle 5 in AS4.5 results in its release from β-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites. (Cancer Res 2006; 66(14): 7211-5)

Angiostatin4.5 (AS4.5) is a naturally occurring human angiostatin isoform, consisting of plasminogen kringles 1–4 plus 85% of kringle 5 (amino acids Lys78 to Arg529). Prior studies indicate that plasminogen is converted to AS4.5 in a two-step reaction. First, plasminogen is activated to plasmin. Then plasmin undergoes autoproteolysis within the inner loop of kringle 5, which can be induced by a free sulfhydryl donor or an alkaline pH. We now demonstrate that plasminogen can be converted to AS4.5 in a cell membrane-dependent reaction. Actin was shown previously to be a surface receptor for plasmin(ogen). We now show that β-actin is present on the extracellular membranes of cancer cells (PC-3, HT1080, and MDA-MB231), and β-actin can mediate plasmin binding to the cell surface and autoproteolysis to AS4.5. In the presence of β-actin, no small molecule-free sulfhydryl donor is needed for generation of AS4.5. Antibodies to actin reduced membrane-dependent generation of AS4.5 by 70%. In a cell-free system, addition of actin to in vitro-generated plasmin resulted in stoichiometric conversion to AS4.5. Annexin II and α-enolase have been reported to be plasminogen receptors, but we did not demonstrate a role for these proteins in conversion of plasminogen to AS4.5. Our data indicate that membrane-associated β-actin, documented previously as a plasminogen receptor, is a key cell membrane receptor capable of mediating conversion of plasmin to AS4.5. This conversion may serve an important role in regulating tumor angiogenesis, invasion, and metastasis, and surface β-actin may also serve as a prognostic marker to predict tumor behavior.

During placentation, the concentration of fibrinous deposits on the surfaces of maternal vasculature plays a role in villous development and has been strongly implicated in the pathophysiology of human fetal growth restriction (FGR). Fibrinous deposits are conspicuous sites of platelet aggregation where there is local activation of the hemostatic cascade. During activation of the hemostatic cascade, a number of pro- and antiangiogenic agents may be generated at the cell surface, and an imbalance in these factors may contribute to the placental pathology characteristic of FGR. We tested the hypothesis that angiostatin(4.5) (AS(4.5)), a cleavage fragment of plasminogen liberated at the cell surface, is capable of causing FGR in mice. Increased maternal levels of AS(4.5) in vivo result in reproducible placental pathology, including an altered vascular compartment (both in decidual and labyrinthine layers) and increased apoptosis throughout the placenta. In addition, there is significant skeletal growth delay and conspicuous edema in fetuses from mothers that received AS(4.5). Maternally generated AS(4.5), therefore, can access maternal placental vasculature and have a severe effect on placental architecture and inhibit fetal development in vivo. These findings strongly support the hypothesis that maternal AS(4.5) levels can influence placental development, possibly by directly influencing trophoblast turnover in the placenta, and contribute to fetal growth delay in mice.

Angiostatin4.5 (AS4.5), the naturally occurring human angiostatin isoform, is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and ~85% of kringle 5. An important question related to tumor angiogenesis is the mechanism by which tumor cells convert plasminogen to angiostatin locally and yet the angiostatin acts systemically. We recently reported that cancer cell surface globular β-actin mediates plasmin autoproteolysis to AS4.5. We now characterize the binding of plasminogen, plasmin and AS4.5 to globular β-actin. Plasminogen and plasmin bind comparably to immobilized globular β-actin, with a KD of approximately 140 nM. The binding is inhibited by ε-aminocaproic acid (EACA), a lysine analog, indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from β-actin in competitive binding studies, we show that the actin domain necessary for binding of plasminogen is within “GDEAQSKRGILTLKY”, (amino acids 55 to 69), which includes lys61 and lys68. Substitution of lys61 or Lys68 with arginine results in the loss of the peptide’s ability to block plasminogen binding, indicating that lys61 and Lys68 are necessary for plasminogen binding. Other actin peptides, including peptides with other lysines, did not inhibit the plasminogen-actin interaction. While plasminogen and plasmin bind to actin efficiently, AS4.5 did not bind, even at high concentrations (500 nM). Plasminogen, plasmin, and AS4.5 all contain kringles 1–4, however, kringle 5 is truncated in AS4.5, suggesting that the kringle 5 is the domain by which plasminogen and plasmin bind to actin. We thus tested pure recombinant kringle 5 for its ability to specifically bind actin. Isolated kringle 5 was shown to bind to actin supporting the hypothesis that intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface β-actin, and the truncated kringle 5 in AS4.5 results in its release from β-actin. We propose the following paradigm: Plasminogen binds cell surface globular β-actin, where uPA mediates activation to plasmin which remains bound to actin. Actin then mediates plasmin autoproteolysis, with an intra-kringle 5 cleavage, resulting in AS4.5 formation. With the truncation of kringle 5, the AS4.5 dissociates from the surface actin and enters the systemic circulation. These data may help explain why AS4.5 is formed locally, on cancer cell surfaces, and yet acts systemically.