Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.

Neuropeptide Y (NPY), a 36-aa peptide, is widely distributed in the brain and peripheral tissues. Whereas physiological roles of NPY as a hormone͞neurotransmitter have been well studied, little is known about its other peripheral functions. Here, we report that NPY acts as a potent angiogenic factor in vivo using the mouse corneal micropocket and the chick chorioallantoic membrane (CAM) assays. Unlike vascular endothelial growth factor (VEGF), microvessels induced by NPY had distinct vascular tree-like structures showing vasodilation. This angiogenic pattern was similar to that induced by fibroblast growth factor-2, and the angiogenic response was dose-dependent. (2). Although the neuropeptides were isolated and characterized several decades ago and the NPY receptor cDNAs have been cloned for 5-10 yr, surprisingly little is known about the molecular mechanisms that regulate NPY receptor activity and the biological significance of NPY in the periphery.It has been found that NPY regulates the vascular tone by inducing contractions of blood vessels (3). NPY also stimulates growth of vascular smooth muscle cell and hypertrophy of ventricular cardiomyocytes (4, 5). The trophic effect of NPY on blood vessels does not seem to be limited to vascular smooth muscle cells.

No abstract

We have previously shown that angiomotin (Amot) plays an important role in growth factor-induced migration of endothelial cells in vitro. Genetic knock-down of Amot in zebrafish also results in inhibition of migration of intersegmental vessels in vivo. Amot is expressed as two different isoforms, p80-Amot and p130-Amot. Here we have analyzed the expression of the two Amot isoforms during retinal angiogenesis in vivo and demonstrate that p80-Amot is expressed during the migratory phase. In contrast, p130-Amot is expressed during the period of blood vessel stabilization and maturation. We also show that the N-terminal domain of p130-Amot serves as a targeting domain responsible for localization of p130-Amot to actin and tight junctions. We further show that the relative expression levels of p80-Amot and p130-Amot regulate a switch between a migratory and a non-migratory cell phenotype where the migratory function of p80-Amot is dominant over the stabilization and maturation function of p130-Amot. Our data indicates that homo-oligomerization of p80-Amot and hetero-oligomerization of both isoforms are critical for this regulation.

In the originally published version of this article, author Bà rbara Laviñ a was mistakenly listed as ''Bà rbara Laviñ a Siemsen.'' This error has now been corrected in the article online. We apologize for the error and any inconvenience that may have resulted.

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.

Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione-disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD + -dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.proteomics | cardiovascular system

BackgroundEtoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.Methodology/Principal FindingsTo examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.Conclusions/SignificanceThese results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.