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BDNF and nitric oxide signaling both contribute to plasticity at glutamatergic synapses. However, the role of combined signaling of both pathways at the same synapse is largely unknown. Using NO imaging with diaminofluoresceine in cultured hippocampal neurons we analyzed the time course of neurotrophin-induced NO signals. Application of exogenous BDNF, NT-4, and NT-3 (but not NGF) induced NO signals in the soma and in proximal dendrites of hippocampal neurons that were sensitive to NO synthase activity, TrkB signaling, and intracellular calcium elevation. The effect of NO signaling on neurotrophin secretion was analyzed in BDNF-GFP, and NT-3-GFP transfected hippocampal neurons. Exogenous application of the NO donor sodium-nitroprusside markedly inhibited neurotrophin secretion. However, endogenously generated NO in response to depolarization and neurotrophin stimulation, both did not result in a negative feedback on neurotrophin secretion. These results suggest that a negative feedback of NO signaling on synaptic secretion of neurotrophins operates only at high intracellular levels of nitric oxide that are under physiological conditions not reached by depolarization or BDNF signaling.

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RA(del) mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RA(del) mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs.

Background: Tight-junction (TJ) protein degradation is a decisive step in hypoxic blood-brain barrier (BBB) breakdown in stroke. In this study we elucidated the impact of acute cerebral ischemia on TJ protein arrangement and the role of the apoptotic effector protease caspase-3 in this context., Methodology/principal Findings: We used an in vitro model of the neurovascular unit and the guinea pig whole brain preparation to analyze with immunohistochemical methods the BBB properties and neurovascular integrity. In both methodological approaches we observed rapid TJ protein disruptions after 30 min of oxygen and glucose deprivation or middle cerebral artery occlusion, which were accompanied by strong caspase-3 activation in brain endothelial cells (BEC). Surprisingly only few DNA-fragmentations were detected with TUNEL stainings in BEC. Z-DEVD-fmk, an irreversible caspase-3 inhibitor, partly blocked TJ disruptions and was protective on trans-endothelial electrical resistance., Conclusions/significance: Our data provide evidence that caspase-3 is rapidly activated during acute cerebral ischemia predominantly without triggering DNA-fragmentation in BEC. Further we detected fast TJ protein disruptions which could be partly blocked by caspase-3 inhibition with Z-DEVD-fmk. We suggest that the basis for clinically relevant BBB breakdown in form of TJ disruptions is initiated within minutes during ischemia and that caspase-3 contributes to this process.

We present the preparation and isolation of different chemically modified BSA species with varying numbers of primary amino groups at the surface. Highly cationic albumin proteins with increased numbers of amino groups were achieved and complex formation with plasmid DNA was carefully investigated. We compare the transfection results, polyelectrolyte complexes morphologies with their impact on complex stabilities, cytotoxicities and DNA accessibility. This knowledge-driven approach led to the identification of the efficient non-viral DNA delivery agent cBSA-147, which showed high transfection efficacies and stability., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

C-reactive protein (CRP) has been demonstrated to induce blood-brain barrier disruption (BBB) involving NAD(P)H-oxidase dependent oxidative stress. It is unclear why CRP affects the BBB and not other vascular beds following stroke. Therefore we examined CRP receptor and NAD(P)H-oxidase expression levels in bovine brain- (BEC) and aortic endothelial cells. Dichlorodihydrofluorescein measurements revealed significantly higher CRP-induced reactive oxygen species (ROS) levels in BEC. Protein expression of the CRP-receptors CD16, CD32 and of the NAD(P)H-oxidase subunit p22phox were also significantly higher in BEC. In conclusion BEC show a higher vulnerability to CRP due to increased levels of CRP receptors and the NAD(P)H-oxidase., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: The dietary flavonoid apigenin (Api) has been demonstrated to exert multiple beneficial effects upon the vascular endothelium. The aim of this study was to examine whether Ca(2+)-activated K(+) channels (K(Ca)) are involved in endothelial nitric oxide (NO) production and antiangiogenic effects., Methods: Endothelial NO generation was monitored using a cyclic guanosine monophosphate radioimmunoassay. K(Ca) activity and changes of the intracellular Ca(2+) concentration [Ca(2+)](i) were analyzed using the fluorescent dyes bis-barbituric acid oxonol, potassium-binding benzofuran isophthalate, and fluo-3. The endothelial angiogenic parameters measured were cell proliferation, [(3)H]-thymidine incorporation, and cell migration (scratch assay). Akt phosphorylation was examined using immunohistochemistry., Results: Api caused a concentration-dependent increase in cyclic guanosine monophosphate levels, with a maximum effect at a concentration of 1 mum. Api-induced hyperpolarization was blocked by the small and large conductance K(Ca) inhibitors apamin and iberiotoxin, respectively. Furthermore, apamin and iberiotoxin blocked the late, long-lasting plateau phase of the Api-induced biphasic increase of [Ca(2+)](i). Inhibition of Ca(2+) signaling and the K(Ca) blockade both blocked NO production. Prevention of all three (NO, Ca(2+), and K(Ca) signaling) reversed the antiangiogenic effects of Api under both basal and basic fibroblast growth factor-induced culture conditions. Basic fibroblast growth factor-induced Akt phosphorylation was also reduced by Api., Conclusions: Based on our experimental results we propose the following signaling cascade for the effects of Api on endothelial cell signaling. Api activates small and large conductance K(Ca), leading to a hyperpolarization that is followed by a Ca(2+) influx. The increase of [Ca(2+)](i) is responsible for an increased NO production that mediates the antiangiogenic effects of Api via Akt dephosphorylation.

The proliferation of endothelial cells plays a crucial role in the development of intraplaque angiogenesis (IPA). IPA is a major source of intraplaque hemorrhage and therefore contributes to the destabilization of atherosclerotic plaques. Therefore, the aim of the present study was to examine, whether sildenafil inhibits endothelial cell growth. The proliferation of human endothelial cells derived from umbilical cord veins (HUVEC) was examined on DNA level by measurements of ((3)H)-thymidine incorporation. Cell viability was analyzed using trypan blue staining. The proliferation of cultured human endothelial cells was significantly decreased by 1 μmol/l (-48.4%) and 10 μmol/l (-89.6%) sildenafil (n=10, p<0.05). This was not a cytotoxic effect, because cell viability was only reduced at sildenafil concentrations of 50 μmol/l or greater. In addition sildenafil significantly reduced endothelial proliferation induced by bFGF (n=10, p<0.05). The presented results demonstrate an antiangiogenic effect of sildenafil that might be useful in the prevention of atherosclerotic plaque vascularization.

Objective: The autonomous proliferative response of endothelial cells to hypoxia has been shown to be dependent on activation of NAD(P)H oxidase, on the cytosolic Ca2+ load, and, consequently, on nuclear translocation of extracellular signal-regulated kinase (ERK)1/2 during transient hypoxia. The aim of the present study was to investigate whether poly(ADP-ribose) polymerase (PARP) is a downstream signal of NAD(P)H oxidase, mediating cytosolic Ca2+ load and hence nuclear translocation of ERK1/2 and endothelial cell proliferation., Methods: Porcine aortic endothelial cells were incubated under hypoxic conditions for 40 min. Cytosolic [Ca2+] and reactive oxygen species (ROS) formation were measured in fura-2- and DCF-loaded cells, respectively. PARP activation was detected by immunocytochemistry, and endothelial cell proliferation was determined 24 h after 60 min of transient hypoxia., Results: Inhibition of NAD(P)H oxidase with antisense oligonucleotide against the p22(phox) subunit, MEK/ERK signalling with UO 126 (30 microM), or PARP with PJ 34 (10 microM) leads to a marked reduction in hypoxia-induced cytosolic Ca2+ load and activation of PARP. Hypoxia-induced translocation of ERK1/2 and endothelial cell proliferation were also prevented when NAD(P)H oxidase or PARP were inhibited; however, hypoxic ROS formation was not affected in the presence of PARP inhibitor., Conclusion: PARP represents a downstream effector of NADP(H) oxidase and acts as a necessary intermediate step for the hypoxic proliferative response of endothelial cells.

Intracellular cGMP is an important second messenger in endothelial cells. Because Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to regulate endothelial cell functions, the aim of the present study was to examine whether sildenafil modulates BK(Ca) activity in cultured human endothelial cells. Changes of the endothelial cell membrane potential were analyzed using the fluorescence dye DiBAC. The patch-clamp technique was used to study BK(Ca) in human endothelial cells of umbilical cord veins (HUVEC). Intracellular Ca(2+) levels were analyzed using Fura-2 fluorescence imaging. Sildenafil caused a dose-dependent (0.05-5 micromol/l) hyperpolarization of the endothelial cells with a maximum at a concentration of 1 micromol/l. A significant increase of BK(Ca) activity was induced by sildenafil (1 micromol/l) perfusion. BK(Ca) open state-probability (NPo) was also increased by the cGMP-analogue 8-bromo-cGMP (0.5 mmol/l), whereas inhibition of the cGMP-dependent kinase (PKG) had no effect on NPo. PKG-inhibition abolished 8-bromo-cGMP induced BK(Ca) activation, and reduced sildenafil induced NPo. Furthermore, sildenafil caused a significant increase of intracellular calcium that was blocked by the BK(Ca) inhibitor iberiotoxin (100 nmol/l). In conclusion sildenafil activates BK(Ca) by a mechanism, which involves cGMP. The activation of the BK(Ca) is responsible for the sildenafil-induced increase of intracellular Ca(2+).

Background: The hepatocyte growth factor (HGF) has been shown to promote endothelial cell proliferation. In this study, the signaling cascade responsible for the HGF-induced proliferation was examined., Methods: The proliferation of human umbilical cord vein endothelial cells (HUCVEC) was determined using cell counts. Changes of the membrane potential were analyzed using the fluorescence dye DiBAC. Intracellular cGMP-levels were measured by means of [3H]-cGMP-radioimmunoassay. Phosphorylation of the p42/p44 MAP-kinase (MAPK) and the endothelial nitric oxide synthase (eNOS) was analyzed by immunocytochemistry., Results: A dose-dependent (1-30 ng mL(-1)) increase of HUCVEC proliferation with a maximum at a concentration of 15 ng mL(-1) was induced by HGF. This effect was significantly reduced by the addition of the K+ channel blocker iberiotoxin (100 nmol L(-1)), eNOS inhibitor L-NMMA (300 micromol L(-1)), or the MEK inhibitor PD 98059 (20 micromol L(-1)). A HGF-induced hyperpolarization that was blocked by iberiotoxin was observed. In addition, HGF-induced activation of the eNOS was blocked by the K+ channel inhibitor. An increase of +101% MAPK phosphorylation was induced by HGF, which was blocked, if the cells were treated with L-NMMA (n = 20; P < 0.05), whereas HGF-induced phosphorylation of the eNOS was not affected by MEK inhibition., Conclusions: Hepatocyte growth factor modulates endothelial K+ channels causing an activation of the eNOS; the increase of nitric oxide is necessary for the phosphorylation of the MAPK inducing the proliferation of HUCVEC.

Objective: Inward rectifier K+ currents (K(ir)) determine the resting membrane potential and thereby modulate essential Ca2+-dependent pathways, like cell growth and synthesis of vasoactive agents in endothelial cells. Basic fibroblast growth factor (bFGF) acts as a vasodilatator and angiogenic factor. Therefore, we investigated the effect of bFGF on K(ir) and assessed the role in proliferation and nitric oxide (NO) formation of endothelial cells., Methods and Results: Using the patch-clamp technique, we found characteristic K(ir) in human umbilical cord vein endothelial cells (HUVEC), which were dose-dependently blocked by barium (10 to 100 micromol/L). Perfusion with bFGF (50 ng/mL) caused a significant increase of K(ir), which was blocked by 100 micromol/L barium (n=18, P<0.01). The bFGF-induced HUVEC proliferation was significantly inhibited when using 50 to 100 micromol/L barium (n=6; P<0.01). NO production was examined using a cGMP radioimmunoassay. bFGF caused a significant increase of cGMP levels (n=10; P<0.05), which were blocked by barium., Conclusions: Modulation of K(ir) plays an important role in bFGF-mediated endothelial cell growth and NO formation.

Objective: Oxidized low-density lipoprotein (oxLDL) plays an important role in causing endothelial dysfunction and initiating atherosclerosis. Some of the endothelial functions have been shown to be modulated by changes in cellular electrophysiological properties. Therefore, we analysed the effect of oxLDL on endothelial Ca(2+)-activated K(+) channels (BK(Ca)) and its contribution to oxLDL-mediated changes of proliferation and syntheses of nitric oxide (NO)., Methods: The patch-clamp technique was used to study the behavior of BK(Ca) in human endothelial cells of umbilical cord veins (HUVEC). Changes of intracellular Ca(2+) were measured by means of Fura-2 imaging. Cell counts and [3H]-thymidine incorporation were used to analyse endothelial proliferation. Synthesis of NO was measured by means of [3H]-cGMP radioimmunoassay., Results: oxLDL (10 microg/ml) caused a significant increase of BK(Ca) activity, whereas preincubation of HUVEC with an antibody against the lectin-like-oxLDL-receptor-1 (LOX-1) abolished BK(Ca) activation. Fura-2 measurements revealed a biphasic increase of intracellular Ca(2+) after application of the atherogenic lipid. Endothelial proliferation was significantly increased by oxLDL. The highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/l IBX) blocked this proliferative response. Acetylcholine-induced NO synthesis was significantly decreased by IBX. Interestingly, oxLDL significantly decreased acetylcholine-induced NO synthesis if the production of superoxide was not blocked by antisense oligonucleotides against the NAD(P)H-oxidase., Conclusions: Our data demonstrate that oxLDL activates BK(Ca), which plays an important role in oxLDL-mediated endothelial proliferation. Acetylcholine-induced NO synthesis is modulated by BK(Ca), whereas the reduction of acetylcholine-induced NO-synthesis by oxLDL is related to an increase in superoxide production.