BACKGROUND AND PURPOSE NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available ...l‐arginine, reducing NO and increasing superoxide (O2.−) production via NOS uncoupling. Elevated O2.− combines with NO to form peroxynitrite (ONOO‐), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide‐induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC‐activated RhoA/Rho kinase (ROCK) pathway.
EXPERIMENTAL APPROACH Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO‐ generator (SIN‐1) or H2O2. Pretreatment with inhibitors of Rho kinase (Y‐27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation.
KEY RESULTS Exposure to SIN‐1 (25 µM, 24 h) or H2O2 (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y‐27632, PKC inhibitor, Gö6976 or siRNA to p115‐Rho GEF. There was an early activation of p115‐Rho GEF (SIN‐1, 2 h; H2O2, 1 h) and Rho A (SIN‐1, 4 h; H2O2, 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN‐1 and H2O2 also reduced NOS activity, which was blocked by pretreatment with p115‐RhoGEF siRNA.
CONCLUSIONS AND IMPLICATIONS Our data indicate that the oxidative species ONOO‐ and H2O2 increase arginase activity/expression through PKC‐mediated activation of RhoA/Rho kinase pathway.
Abstract
Land vegetation is currently taking up large amounts of atmospheric CO
2
, possibly due to tree growth stimulation. Extant models predict that this growth stimulation will continue to cause ...a net carbon uptake this century. However, there are indications that increased growth rates may shorten trees′ lifespan and thus recent increases in forest carbon stocks may be transient due to lagged increases in mortality. Here we show that growth-lifespan trade-offs are indeed near universal, occurring across almost all species and climates. This trade-off is directly linked to faster growth reducing tree lifespan, and not due to covariance with climate or environment. Thus, current tree growth stimulation will, inevitably, result in a lagged increase in canopy tree mortality, as is indeed widely observed, and eventually neutralise carbon gains due to growth stimulation. Results from a strongly data-based forest simulator confirm these expectations. Extant Earth system model projections of global forest carbon sink persistence are likely too optimistic, increasing the need to curb greenhouse gas emissions.
Aims/hypothesis
A reduction in retinal blood flow occurs early in diabetes and is likely to be involved in the development of diabetic retinopathy. We hypothesise that activation of the arginase ...pathway could have a role in the vascular dysfunction of diabetic retinopathy.
Methods
Experiments were performed using a mouse and rat model of streptozotocin (STZ)-induced diabetes for in vivo and ex vivo analysis of retinal vascular function. For in vivo studies, mice were infused with the endothelial-dependent vasodilator acetylcholine (ACh) or the endothelial-independent vasodilator sodium nitroprusside (SNP), and vasodilation was assessed using a fundus microscope. Ex vivo assays included pressurised vessel myography, western blotting and arginase activity measurements.
Results
ACh-induced retinal vasodilation was markedly impaired in diabetic mice (40% of control values), whereas SNP-induced dilation was not altered. The diabetes-induced vascular dysfunction was markedly blunted in mice lacking one copy of the gene encoding arginase I and in mice treated with the arginase inhibitor 2(S)-amino-6-boronohexanoic acid. Ex vivo studies performed using pressure myography and central retinal arteries isolated from rats with STZ-induced diabetes showed a similar impairment of endothelial-dependent vasodilation that was partially blunted by pretreatment of the isolated vessels with another arginase inhibitor, (S)-2-boronoethyl-
l-
cysteine. The diabetes-induced vascular alterations were associated with significant increases in both arginase I protein levels and total arginase activity.
Conclusions/interpretation
These results indicate that, in the mouse and rat model, diabetes-induced increases in arginase I were involved in the diabetes-induced impairment of retinal blood flow by a mechanism involving vascular endothelial cell dysfunction.
Collectively, angiogenic ocular conditions represent the leading cause of irreversible vision loss in developed countries. In the US, for example, retinopathy of prematurity, diabetic retinopathy and ...age-related macular degeneration are the principal causes of blindness in the infant, working age and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor (VEGF), a 40kDa dimeric glycoprotein, promotes angiogenesis in each of these conditions, making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can influence cell proliferation, cell migration, proteolysis, cell survival and vessel permeability in a wide variety of biological contexts. This article will describe the roles played by VEGF in the pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the rationales for targeting other VEGF-related modulators of angiogenesis.
We report results from a reanalysis of data from the Cryogenic Dark Matter Search (CDMS II) experiment at the Soudan Underground Laboratory. Data taken between October 2006 and September 2008 using ...eight germanium detectors are reanalyzed with a lowered, 2 keV recoil-energy threshold, to give increased sensitivity to interactions from weakly interacting massive particles (WIMPs) with masses below ∼10 GeV/c(2). This analysis provides stronger constraints than previous CDMS II results for WIMP masses below 9 GeV/c(2) and excludes parameter space associated with possible low-mass WIMP signals from the DAMA/LIBRA and CoGeNT experiments.
Objectives. We tested the hypothesis that pravastatin (PRA) activates endothelial nitric oxide synthase (eNOS).
Background. Pravastatin has been found to have clinical benefits beyond those predicted ...by its actions in reducing plasma low density lipoprotein cholesterol (LDL). Both PRA and simvastatin (SIM) are equally effective in reducing LDL, but only PRA reduces platelet aggregation and is an effective vasodilator. Nitric oxide (NO) also inhibits platelet aggregation and vasodilates.
Methods. We determined PRA and SIM effects on vasorelaxation in aortic rings and NO production by cultured bovine aortic endothelial cells. Nitric oxide was measured by using a NO electrode and by an assay for conversion of hemoglobin to methemoglobin. Specificity of NOS activation was tested by using the NOS inhibitor nitro-l-arginine methyl ester (l-NAME, 1 mmol/liter) in the presence or absence of excess l-arginine (l-ARG, 1 mmol/liter).
Results. Endothelium-dependent vasorelaxation was maximal with acetylocholine (ACH, 100%), followed by PRA (62.8%) and then SIM (37.1%). Direct measurement of NO confirmed that vasorelaxation is due to NO release and showed that PRA and ACH had similar dose-dependent effects on NO production, while SIM was only 25% to 30% as effective. Methemoglobin assay confirmed these results and demonstrated their specificity for NOS activity. The l-NAME blunted the responses to 45% of initial values. Excess l-ARG reversed this effect and potentiated NO production to 133% of initial levels.
Conclusions. Both PRA and SIM activate eNOS, but SIM is much less effective. Clinical benefits with PRA not explained by LDL reductions may be the result of an independent action of PRA on eNOS activation.
Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here ...we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1β and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia.
The CDMS low ionization threshold experiment (CDMSlite) uses cryogenic germanium detectors operated at a relatively high bias voltage to amplify the phonon signal in the search for weakly interacting ...massive particles (WIMPs). Results are presented from the second CDMSlite run with an exposure of 70 kg day, which reached an energy threshold for electron recoils as low as 56 eV. A fiducialization cut reduces backgrounds below those previously reported by CDMSlite. New parameter space for the WIMP-nucleon spin-independent cross section is excluded for WIMP masses between 1.6 and 5.5 GeV/c^{2}.
Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a ...major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism.
Retinal vascular injury is a major cause of vision impairment in ischemic retinopathies. Insults such as hyperoxia, oxidative stress and inflammation contribute to this pathology. Previously, we ...showed that hyperoxia-induced retinal neurodegeneration is associated with increased polyamine oxidation. Here, we are studying the involvement of polyamine oxidases in hyperoxia-induced injury and death of retinal vascular endothelial cells. New-born C57BL6/J mice were exposed to hyperoxia (70% O2) from postnatal day (P) 7 to 12 and were treated with the polyamine oxidase inhibitor MDL 72527 or vehicle starting at P6. Mice were sacrificed after different durations of hyperoxia and their retinas were analyzed to determine the effects on vascular injury, microglial cell activation, and inflammatory cytokine profiling. The results of this analysis showed that MDL 72527 treatment significantly reduced hyperoxia-induced retinal vascular injury and enhanced vascular sprouting as compared with the vehicle controls. These protective effects were correlated with significant decreases in microglial activation as well as levels of inflammatory cytokines and chemokines. In order to model the effects of polyamine oxidation in causing microglial activation in vitro, studies were performed using rat brain microvascular endothelial cells treated with conditioned-medium from rat retinal microglia stimulated with hydrogen peroxide. Conditioned-medium from activated microglial cultures induced cell stress signals and cell death in microvascular endothelial cells. These studies demonstrate the involvement of polyamine oxidases in hyperoxia-induced retinal vascular injury and retinal inflammation in ischemic retinopathy, through mechanisms involving cross-talk between endothelial cells and resident retinal microglia.
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•Polyamine oxidase inhibitor, MDL 72527, reduces hyperoxia-induced vascular injury.•Treatment with MDL 72527 limits hyperoxia-induced microglial activation.•Activated microglia induce microvascular endothelial cell death in vitro.•Targeting polyamine oxidase offers a new therapy for CNS vascular injury.
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