The release of nitric oxide from S-nitrosothiols promotes angiogenesis

Free nitric oxide (NO) reacts with sulphydryl residues to form S-nitrosothiols, which act as NO reservoirs. We sought to determine whether thiol-preserving agents and antioxidants, such as dithiothreitol (DTT) and vitamin C, induce NO release from S-nitrosylated proteins in endothelial cell cultures to promote angiogenesis. NO release was measured directly in cell supernatants using a Sievers NO Analyser, and in vitro angiogenesis was assessed by quantifying capillary-like tube network formation of porcine aortic endothelial cells (PAEC) on growth factor-reduced Matrigel. Incubation of PAEC with DTT or vitamin C significantly increased NO release in a concentration-dependent manner. However, the nitric oxide synthase (NOS) inhibitors, L-NNA and L-NIO, had no effect on DTT- or vitamin C-induced NO release, and there was no concomitant increase in the phosphorylation of endothelial NOS at serine-1177 following DTT or vitamin C treatment. DTT and vitamin C increased capillary-like tube network formation by nine- and two-fold, respectively, and the addition of copper ions doubled the effect of vitamin C. Surprisingly, DTT maintained endothelial tube networks for up to one month under serum-free conditions, and selective inhibitors of guanylyl cyclase (ODQ) and PKG (KT-5823) blocked this, demonstrating the requirement of cyclic GMP and PKG in this process. Both DTT and vitamin C are capable of releasing sufficient NO from S-nitrosothiols to induce capillary morphogenesis. This study provides the first evidence that increased denitrosylation leads to increased bioavailability of NO, independent of NOS activity, to promote sustained angiogenesis.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1

Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release.

Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase Cgamma1. Consistent with these findings, the VEGFR-1-specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2-activated phospholipase Cgamma1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II-induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II-mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system.

Angiopoietin-1 induces migration of monocytes in a tie-2 and integrin-independent manner

Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whether Ang-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in the progression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that is independent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1 promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulated transendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly to monocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primary monocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1 binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogated Ang-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. In summary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase and heparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role in inflammatory angiogenesis and may promote atherosclerosis.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Angiopoietin-2 confers Atheroprotection in apoE-/- mice by inhibiting LDL oxidation via nitric oxide

Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE-/- mice fed a Western diet significantly reduced atherosclerotic lesion size 8 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection.

Relationship between freight accessibility and logistics employment in US counties

This paper analyzes the relationship between freight accessibility and logistics employment in the US. It develops an accessibility measure relevant for logistics companies based on a gravity model. This allows for an analysis of the accessibility of US counties focusing on four different modes of transportation: road, rail, air, and maritime. Using a Partial Least Squares model, these four different freight accessibility measures are combined into two constructs, continental and intercontinental freight accessibility, and related to logistics employment. Results show that highly accessible counties attract more logistics employment than other counties. The analyses show that it is very important to control for the effect of the county population on both freight accessibility and logistics employment. While county population explains the most variation in the logistics employment per county, there is a significant relationship between freight accessibility and logistics employment, when controlling for this effect.

Recruitment and Retention Insights for the Hotel Industry

Recruiting talented managerial employees for the hotel industry is a constant challenge. Identifying and training supervisors and junior level managers for senior positions seems to be one solution. The authors explore the career influences confronting these individuals and make recommendations for recruitment and retention.

A high-resolution sedimentology and age determination of core PS2185 from the central Arctic Ocean

A high-resolution multiparameter stratigraphy allows the identification of late Quaternary glacial and interglacial cycles in a central Arctic Ocean sediment core. Distinct sandy layers in the upper part of the otherwise fine-grained sediment core from the Lomonosov Ridge (lat 87.5°N) correlate to four major glacials since ca. 0.7 Ma. The composition of these ice-rafted terrigenous sediments points to a glaciated northern Siberia as the main source. In contrast, lithic carbonates derived from North America are also present in older sediments and indicate a northern North American glaciation since at least 2.8 Ma. We conclude that large-scale northern Siberian glaciation began much later than other Northern Hemisphere ice sheets.

Beryllium, neodymium and metal composition of ODP Leg 194 sites and survey sites crusts

Be and Nd isotope compositions and metal concentrations (Mn, Fe, Co, Ni, and Cu) of surface and subsurface ferromanganese hardground crusts from Ocean Drilling Program Leg 194 Marion Plateau Sites 1194 and 1196 provide new insights into the crusts' genesis, growth rates, and ages. Metal compositions indicate that the hardgrounds, which have grown on erosional surfaces in water depths of <400 m because of strong bottom currents, are not pure hydrogenetic precipitates. Nevertheless, the ratios between cosmogenic 10Be and stable 9Be in hardgrounds from the present-day seafloor at Site 1196 between 1 x 10**-7 and 1.5 x 10**-7 are within the range of values expected for Pacific seawater, which shows that the hardgrounds recorded the isotope composition of ambient seawater. This is also confirmed by their Nd isotope composition (epsilon Nd between -3 and 0). The 10Be/9Be ratios in the up to 30-mm-thick and partly laminated hardgrounds do not show a decrease with depth, which suggests high growth rates on the present-day seafloor. The subsurface crust at Site 1194 (117 m below the seafloor) grew during a sedimentation hiatus, when bottom currents in the late Miocene prevented sediment accumulation on the carbonate platform during a sea level lowstand. The age of 8.65 ± 0.50 Ma for this crust obtained from 10Be-based dating agrees well with the combined seismostratigraphic and biostratigraphic evidence, which suggests an age for the hiatus between 7.7 and 11.8 Ma.

(Table 1) Elemental mean values of the upper Paleocene and lower Eocene in IODP Hole 302-M0004A

We reconstruct the latest Paleocene and early Eocene (~57-50 Ma) environmental trends in the Arctic Ocean and focus on the Paleocene-Eocene thermal maximum (PETM) (~55 Ma), using strata recovered from the Lomonosov Ridge by the Integrated Ocean Drilling Program Expedition 302. The Lomonosov Ridge was still partially subaerial during the latest Paleocene and earliest Eocene and gradually subsided during the early Eocene. Organic dinoflagellate cyst (dinocyst) assemblages point to brackish and productive surface waters throughout the latest Paleocene and early Eocene. Dinocyst assemblages are cosmopolitan during this time interval, suggesting warm conditions, which is corroborated by TEX86'-reconstructed temperatures of 15°-18°C. Inorganic geochemistry generally reflects reducing conditions within the sediment and euxinic conditions during the upper lower Eocene. Spectral analysis reveals that the cyclicity, recorded in X-ray fluorescence scanning Fe data from close to Eocene thermal maximum 2 (~53 Ma, presence confirmed by dinocyst stratigraphy), is related to precession. Within the lower part of the PETM, proxy records indicate enhanced weathering, runoff, anoxia, and productivity along with sea level rise. On the basis of total organic carbon content and variations in sediment accumulation rates, excess organic carbon burial in the Arctic Ocean appears to have contributed significantly to the sequestration of injected carbon during the PETM.

(Table 1) The 10Be and 9Be isotope record of IODP EXP202 composite Site

The upper 200 m of the sediments recovered during IODP Leg 302, the Arctic Coring Expedition (ACEX), to the Lomonosov Ridge in the central Arctic Ocean consist almost exclusively of detrital material. The scarcity of biostratigraphic markers severely complicates the establishment of a reliable chronostratigraphic framework for these sediments, which contain the first continuous record of the Neogene environmental and climatic evolution of the Arctic region. Here we present profiles of cosmogenic 10Be together with the seawater-derived fraction of stable 9Be obtained from the ACEX cores. The down-core decrease of 10Be/9Be provides an average sedimentation rate of 14.5 ± 1 m/Ma for the uppermost 151 m of the ACEX record and allows the establishment of a chronostratigraphy for the past 12.3 Ma. The age corrected 10Be concentrations and 10Be/9Be ratios suggest the existence of an essentially continuous sea ice cover over the past 12.3 Ma.