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In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which was partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre / Phd2 knockout mice compared with wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and patients with PAH all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds the potential for developing new treatments for endothelial dysfunction in PH. NEW & NOTEWORTHY HIF activation alone does not conclusively lead to human PH, suggesting that HIF-independent signaling may also contribute to hypoxia-induced PH. This study demonstrated that PHD2 silencing-induced pseudohypoxia in human lung endothelial cells suppresses apoptosis and activates STAT, effects that persist despite HIF2α inhibition or knockdown and are attributed to AKT and ERK activation, JNK inhibition, and AIP1 loss. These findings align with observations in lung endothelial cells and tissues from PAH rodent models and patients.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction. NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2 -silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2 -silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

Interferonopathies, interferon (IFN)-α/β therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1 -/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH., Competing Interests: Competing interest statement: The NIH Clinical Center Pulmonary Arterial Hypertension Program received support from Aadi Bioscience through a Cooperative Research and Development Agreement. The authors have no personal financial relationship with Aadi Bioscience or any other entity.

Inflammatory cell infiltrates are a prominent feature of aberrant vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that immune effector cells contribute to disease progression. Genome-wide blood expression profiling studies have attempted to better define this inflammatory component of PAH pathobiology but have been hampered by small sample sizes, methodological differences, and very little gene-level reproducibility. The current meta-analysis (seven studies; 156 PAH patients/110 healthy controls) was performed to assess the comparability of data across studies and to possibly derive a generalizable transcriptomic signature. Idiopathic (IPAH) compared with disease-associated PAH (APAH) displayed highly similar expression profiles with no differentially expressed genes, even after substantially relaxing selection stringency. In contrast, using a false discovery rate of ≤1% and I 2 < 40% (low-to-moderate heterogeneity across studies) both IPAH and APAH differed markedly from healthy controls with the combined PAH cohort yielding 1,269 differentially expressed, unique gene transcripts. Bioinformatic analyses, including gene-set enrichment, which uses all available data independent of gene selection thresholds, identified interferon, mammalian target of rapamycin/p70S6K, stress kinase, and Toll-like receptor signaling as enriched mechanisms within the PAH gene signature. Enriched biological functions and diseases included tumorigenesis, autoimmunity, antiviral response, and cell death consistent with prevailing theories of PAH pathogenesis. Although otherwise indistinguishable, APAH (predominantly PAH due to systemic sclerosis) had a somewhat stronger interferon profile than IPAH. Meta-analysis defined a robust and generalizable transcriptomic signature in the blood of PAH patients that can help inform the identification of biomarkers and therapeutic targets.

The major surface glycoprotein (Msg) is the most abundant surface protein among Pneumocystis species. Given that Msg is present on both the cyst and trophic forms of Pneumocystis and that dendritic cells play a critical role in initiating host immune responses, we undertook studies to examine activation of bone marrow-derived myeloid dendritic cells by Msg purified from Pneumocystis murina. Incubation of dendritic cells with Msg did not lead to increased expression of CD40, CD80, CD86, or major histocompatibility complex class II or to increased secretion of any of 10 cytokines. Microarray analysis identified very few differentially expressed genes. In contrast, lipopolysaccharide-activated dendritic cells had positive results of all of these assays. However, Msg did bind to mouse mannose macrophage receptor and human DC-SIGN, 2 C-type lectins expressed by dendritic cells that are important in recognition of pathogen-associated high-mannose glycoproteins. Deglycosylation of Msg demonstrated that this binding was dependent on glycosylation. These studies suggest that Pneumocystis has developed a mechanism to avoid activation of dendritic cells, potentially by the previously identified loss of genes that are responsible for the high level of protein mannosylation found in other fungi.

Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

β-glucans, which can activate innate immune responses, are a major component in the cell wall of the cyst form of Pneumocystis In the current study, we examined whether β-1,3-glucans are masked by surface proteins in Pneumocystis and what role β-glucans play in Pneumocystis-associated inflammation. For 3 species, including Pneumocystis jirovecii, which causes Pneumocystis pneumonia in humans, Pneumocystis carinii, and Pneumocystis murina, β-1,3-glucans were masked in most organisms, as demonstrated by increased exposure following trypsin treatment. Using quantitative polymerase chain reaction and microarray techniques, we demonstrated in a mouse model of Pneumocystis pneumonia that treatment with caspofungin, an inhibitor of β-1,3-glucan synthesis, for 21 days decreased expression of a broad panel of inflammatory markers, including interferon γ, tumor necrosis factor α, interleukin 1β, interleukin 6, and multiple chemokines/chemokine ligands. Thus, β-glucans in Pneumocystis cysts are largely masked, which likely decreases innate immune activation; this mechanism presumably was developed for interactions with immunocompetent hosts, in whom organism loads are substantially lower. In immunosuppressed hosts with a high organism burden, organism death and release of glucans appears to be an important contributor to deleterious host inflammatory responses., (Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.

Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Intense regional warming was observed in the western Antarctic Peninsula (WAP) over the last 50 years. Here, we investigate the impact of climate change on primary production (PP) in this highly productive region. This study is based on temporal data series of ozone thickness (1972-2010), sea ice concentration (1978-2010), sea-surface temperature (1990-2010), incident irradiance (1988-2010) and satellite-derived chlorophyll a concentration (Chl-a, 1997-2010) for the coastal WAP. In addition, we apply a photosynthesis/photoinhibition spectral model to satellite-derived data (1997-2010) to compute PP and examine the separate impacts of environmental forcings. Since 1978, sea ice retreat has been occurring earlier in the season (in March in 1978 and in late October during the 2000s) while the ozone hole is present in early spring (i.e. August to November) since the early 1990s, increasing the intensity of ultraviolet-B radiation (UVBR, 280-320 nm). The WAP waters have also warmed over 1990-2010. The modelled PP rates are in the lower range of previously reported PP rates in the WAP. The annual open water PP in the study area increased from 1997 to 2010 (from 0.73 to 1.03 Tg C yr(-1) ) concomitantly with the increase in the production season length. The coincidence between the earlier sea ice retreat and the presence of the ozone hole increased the exposure to incoming radiation (UVBR, UVAR and PAR) and, thus, increased photoinhibition during austral spring (September to November) in the study area (from 0.014 to 0.025 Tg C yr(-1) ). This increase in photoinhibition was minor compared to the overall increase in PP, however. Climate change hence had an overall positive impact on PP in the WAP waters., (© 2015 John Wiley & Sons Ltd.)

Background: Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues., Results: The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature., Conclusion: Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes.

Dendritic cells (DCs) comprise distinct populations with specialized immune-regulatory functions. However, the environmental factors that determine the differentiation of these subsets remain poorly defined. Here, we report that retinoic acid (RA), a vitamin A derivative, controls the homeostasis of pre-DC (precursor of DC)-derived splenic CD11b(+)CD8α(-)Esam(high) DCs and the developmentally related CD11b(+)CD103(+) subset within the gut. Whereas mice deprived of RA signaling significantly lost both of these populations, neither pre-DC-derived CD11b(-)CD8α(+) and CD11b(-)CD103(+) nor monocyte-derived CD11b(+)CD8α(-)Esam(low) or CD11b(+)CD103(-) DC populations were deficient. In fate-tracking experiments, transfer of pre-DCs into RA-supplemented hosts resulted in near complete conversion of these cells into the CD11b(+)CD8α(-) subset, whereas transfer into vitamin A-deficient (VAD) hosts caused diversion to the CD11b(-)CD8α(+) lineage. As vitamin A is an essential nutrient, we evaluated retinoid levels in mice and humans after radiation-induced mucosal injury and found this conditioning led to an acute VAD state. Consequently, radiation led to a selective loss of both RA-dependent DC subsets and impaired class II-restricted auto and antitumor immunity that could be rescued by supplemental RA. These findings establish a critical role for RA in regulating the homeostasis of pre-DC-derived DC subsets and have implications for the management of patients with immune deficiencies resulting from malnutrition and irradiation.

Plasma hemoglobin (Hb) released during intravascular hemolysis has been associated with numerous deleterious effects that may stem from increased nitric oxide (NO) scavenging, but has also been associated with reactive oxygen species generation and platelet activation. Therapies that convert plasma oxyHb to metHb, or metHb to iron-nitrosyl Hb, could be beneficial because these species do not scavenge NO. In this study, we investigated the effects of Angeli's salt (AS; sodium α-oxyhyponitrite, Na2N2O3), a nitroxyl (HNO) and nitrite (NO2(-)) donor, on plasma Hb oxidation and formation of iron-nitrosyl Hb from metHb and on the vasoactivity of plasma Hb. We hypothesized that AS could ameliorate hemolysis-associated pathology via its preferential reactivity with plasma Hb, as opposed to red-cell-encapsulated Hb, and through its intrinsic vasodilatory activity. To test this hypothesis, we infused (n=3 per group) (1) cell-free Hb and AS, (2) cell-free Hb+0.9% NaCl, (3) AS+3% albumin, and (4) 3% albumin+0.9% NaCl (colloid controls for Hb and AS, respectively) in a canine model. Co-infusion of AS and cell-free Hb led to preferential conversion of plasma Hb to metHb, but the extent of conversion was lower than anticipated based on the in vivo concentration of AS relative to plasma Hb. This lower metHb yield was probably due to reactions of nitroxyl-derived AS with plasma components such as thiol-containing compounds. From a physiological and therapeutic standpoint, the infusion of Hb alone led to significant increases in mean arterial pressure (p=0.03) and systemic vascular resistance index (p=0.01) compared to controls. Infusion of AS alone led to significant decreases in these parameters and co-infusion of AS along with Hb had an additive effect in reversing the effects of Hb alone on the systemic circulation. Interestingly, in the pulmonary system, the decrease in pressure when AS was added to Hb was significantly less than would have been expected compared to the effects of Hb and AS alone, suggesting that inactivation of scavenging with AS reduced the direct vasodilatory effects of AS on the vasculature. We also found that AS reduced platelet activation when administered to whole blood in vitro. These data suggest that AS-like compounds could serve as therapeutic agents to counteract the negative vasoconstrictive consequences of hemolysis that occur in hemolytic anemias, transfusion of stored blood, and other diseases. Increases in metHb in the red blood cell, the potential of AS for neurotoxicity, and hypotension would need to be carefully monitored in a clinical trial., (Published by Elsevier Inc.)

Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and β-catenin signaling axis, and accumulated β-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Supporting its therapeutic application in sepsis, p38 mitogen-activated protein kinase (MAPK) inhibition decreases cardiopulmonary injury and lethality with lipopolysaccharide challenge. However, only one preclinical study has reported the survival effects of a p38 inhibitor (SB203580, 100 mg/kg) during infection. We therefore tested SB203580 in mice (n = 763) challenged with intratracheal Escherichia coli and treated with antibiotics and fluids., Methods and Results: Compared with placebo, high dose SB203580 (100 mg/kg) pretreatment increased the hazards ratio of death (95% confidence interval) (3.6 [2.1, 6.1], p < 0.0001). Decreasing doses (10, 1, or 0.1 mg/kg) went from being harmful to having no significant effect (p < 0.0001 for the effect of decreasing dose). At 48 hours, but not 24 hours after E. coli, high and low dose SB203580 pretreatment decreased cardiac phosphorylated p38 MAPK levels and improved cardiac output either (p

Carbon monoxide (CO) is an endogenous messenger that suppresses inflammation, modulates apoptosis and promotes vascular remodeling. Here, microarrays were employed to globally characterize the CO (250 ppm) suppression of early (1 h) LPS-induced inflammation in human monocytic THP-1 cells. CO suppressed 79 of 101 immediate-early genes induced by LPS; 19% (15/79) were transcription factors and most others were cytokines, chemokines and immune response genes. The prototypic effects of CO on transcription and protein production occurred early but decreased rapidly. CO activated p38 MAPK, ERK1/2 and Akt and caused an early and transitory delay in LPS-induced JNK activation. However, selective inhibitors of these kinases failed to block CO suppression of LPS-induced IL-1beta, an inflammation marker. Of CO-suppressed genes, 81% (64/79) were found to have promoters with putative NF-kappaB binding sites. CO was subsequently shown to block LPS-induced phosphorylation and degradation of IkappaBalpha in human monocytes, thereby inhibiting NF-kappaB signal transduction. CO broadly suppresses the initial inflammatory response of human monocytes to LPS by reshaping proximal events in TLR4 signal transduction such as stress kinase responses and early NF-kappaB activation. These rapid, but transient effects of CO may have therapeutic applications in acute pulmonary and vascular injury.

UV irradiance has a broad range of effects on marine planktonic organisms. Direct and indirect effects on individual organisms have complex impacts on food-web structure and dynamics, with implications for carbon and nutrient cycling. Mesocosm experiments are well suited for the study of such complex interrelationships. Mesocosms offer the possibility to conduct well-controlled experiments with intact planktonic communities in physical, chemical and light conditions mimicking those of the natural environment. In allowing the manipulation of UV intensities and light spectral composition, the experimental mesocosm approach has proven to be especially useful in assessing the impacts at the community level. This review of mesocosm studies shows that, although a UV increase even well above natural intensities often has subtle effects on bulk biomass (carbon and chlorophyll), it can significantly impact the food-web structure because of different sensitivity to UV among planktonic organisms. Given the complexity of UV impacts, as evidenced by results of mesocosm studies, interactions between UV and changing environmental conditions (e.g. eutrophication and climate change) are likely to have significant effects on the function of marine ecosystems.

The impact of UV-B radiation (UVBR; 280-320 nm) on lower levels of a natural plankton assemblage (bacteria, phytoplankton and microzooplankton) from the St. Lawrence Estuary was studied during 9 days using several immersed outdoor mesocosms. Two exposure treatments were used in triplicate mesocosms: natural UVBR (N treatment, considered as the control treatment) and lamp-enhanced UVBR (H treatment, simulating 60% depletion of the ozone layer). A phytoplankton bloom developed after day 3, but no significant differences were found between treatments during the entire experiment for phytoplankton biomass (chlorophyll a and cell carbon) nor for phytoplankton cell abundances from flow cytometry and optical microscopy of three phytoplankton size classes (picoplankton, nanoplankton and microplankton). In contrast, bacterial abundances showed significantly higher values in the H treatment, attributed to a decrease in predation pressure due to a dramatic reduction in ciliate biomass (approximately 70-80%) in the H treatment relative to the N treatment. The most abundant ciliate species were Strombidinium sp., Prorodon ovum and Tintinnopsis sp.; all showed significantly lower abundances under the H treatment. P. ovum was the less-affected species (50% reduction in the H treatment compared with that of the N control), contrasting with approximately 90% for the other ones. Total specific phytoplanktonic and bacterial production were not affected by enhanced UVBR. However, both the ratio of primary to bacterial biomass and production decreased markedly under the H treatment. In contrast, the ratio of phytoplankton to bacterial plus ciliate carbon biomass showed an opposite trend than the previous results, with higher values in the H treatment at the end of the experiment. These results are explained by the changes in the ciliate biomass and suggest that UVBR can alter the structure of the lower levels of the planktonic community by selectively affecting key species. On the other hand, linearity between particulate organic carbon (POC) and estimated planktonic carbon was lost during the postbloom period in both treatments. On the basis of previous studies, our results can be attributed to the aggregation of carbon released by cells to the water column in the form of transparent exopolymer particles (TEPs) under nutrient limiting conditions. Unexpectedly, POC during such a period was higher in the H treatment than in controls. We hypothesize a decrease in the ingestion of TEPs by ciliates, in coincidence with increased DOC release by phytoplankton cells under enhanced UVBR. The consequences of such results for the carbon cycle in the ocean are discussed.

A microcosm approach was designed to study the combined effects of tributyltin (TBT) from antifouling paints and ultraviolet-B radiation (UVBR: 280-320 nm), on a natural planktonic assemblage (<150 microm) isolated from the St. Lawrence Estuary at the end of the springtime. Microcosms (9l, cylindrical Teflon bags, 75 cm heightx25 cm width) were immersed in the water column of mesocosms (1800 l, polyethylene bags, 2.3 m depth) and exposed to two different UVBR regimes: natural ambient UVBR (NUVBR), and enhanced level of UVBR (HUVBR). During consecutive 5 days, effects of TBT (120 ng l -1) and enhanced UVBR (giving a biologically weighted UVBR 2.15-fold higher than natural light condition) were monitored in the samples coming from following treatments: (i) NUVBR light condition without TBT (NUVBR), (ii) NUVBR light condition with TBT-added (NUVBR+TBT), (iii) HUVBR light condition without TBT (HUVBR) and (iv) HUVBR light condition with TBT-added (HUVBR+TBT). Each treatment was conducted in triplicate microcosms. Different parameters were then measured during 5 days, including TBT analysis, bacterial abundance and productivity, phytoplankton abundance, cellular characteristics and growth rates, as well as in vivo chlorophyll a (Chl a) fluorescence. Following TBT addition (NUVBR+TBT treatment), Chl a concentrations never exceeded 1 microg l-1 whereas final values as high as 54 microg l-1 were observed in TBT-free treatments (NUVBR and HUVBR). TBT addition resulted also in the lost of fluorescence signal of the maximum efficiency of the photosystem II in phytoplankton assemblage. TBT toxicity caused on phytoplankton <20 microm an increase of mean cell size and changes in shape reflected a drastic disturbance of the cell cycle leading to an inhibition of the apparent growth rate. These negative effects of TBT resulted in a final abundance of phytoplankton <20 microm of 591+/-35 cells ml-1 in NUVBR+TBT relative to NUVBR treatment (i.e., 31,846+/-312 cells ml-1). Moreover, when cells were submitted to TBT under enhanced UVBR (HUVBR+TBT treatment), final abundance of phytoplankton <20 microm was only 182+/-90 cells ml-1, with a significant interaction between TBT and UVBR during the last 2 days of the experiment. The same type of interaction was also observed for bacterial abundance in NUVBR+TBT and HUVBR+TBT with stimulation of 226 and of 403%, respectively due to TBT addition relative to NUVBR treatment. When considering bacterial productivity, TBT addition resulted in an inhibition of 32%, and this inhibition was significantly more pronounced under dual stresses (i.e., 77% in HUVBR+TBT). These results clearly demonstrate that the combination of TBT and UVBR stresses have synergistic effects affecting the first trophic levels of the marine food web.

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca(2+)-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts. The administration of the NF-kappaB inhibitor pyrrolidine-dithiocharbamate (PDTC) blocked the light-induced phase advance at circadian time 18 (vehicle+light pulse: 2.08+/-0.46 h, PDTC+light: 0.36+/-0.35 h). These results demonstrate the presence and activity of Rel/NF-kappaB family proteins in the hamster SCN and suggest that these proteins may be related to the entrainment and regulation of circadian rhythms.

Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the hypothalamic suprachiasmatic nuclei (SCN). We have tested the participation of heme oxygenase (HO) in the SCN, by assessing HO specific activity at different time points and photic conditions. HO activity was determined by the conversion of hemin to bilirubin. HO enzymatic activity in the SCN was significantly higher during the night than during the day; this difference persisted when animals were placed under constant darkness, suggesting an endogenous circadian control. HO inhibition by Zn-protoporphyrin did not affect light-induced phase shifts in vivo, suggesting that the enzyme is not necessary for light input to the clock.

Circadian rhythms are entrained by light-activated signal transduction pathways in the biological clock. Among these, circadian and photic control of mouse suprachiasmatic ERK MAP kinase activation has been reported. In this paper we extend these results to hamsters and to the two other major members of the MAPK family: JNK and p38. The three kinases are rhythmically phosphorylated under light-dark and constant conditions, with maximal values during the day or subjective day. Light pulses during the subjective night induce rapid activation of the three enzymes, suggesting that the three MAP kinases might be implicated in mammalian photic entrainment.

Mammalian circadian rhythms are generated by the hypothalamic suprachiasmatic nuclei and finely tuned to environmental periodicities by neurochemical responses to the light-dark cycle. Light reaches the clock through a direct retinohypothalamic tract, primarily through glutamatergic innervation, and its action is probably regulated by a variety of other neurotransmitters. A key second messenger in circadian photic entrainment is calcium, mobilized through membrane channels or intracellular reservoirs, which triggers the activation of several enzymes, including a calcium/calmodulin-dependent protein kinase and nitric oxide synthase. Other enzymes activated by light are mitogen-activated- and cGMP-dependent protein kinase; all of the above have been reported to be involved in the circadian responses to nocturnal light pulses. These mechanisms lead to expression of specific clock genes which eventually set the phase of the clock and of clock-controlled circadian rhythms.

Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the suprachiasmatic nuclei (SCN). Pharmacological inhibition of kinases such as cGMP-dependent kinase (PKG) or Ca2+/calmodulin-dependent kinase, but not cAMP-dependent kinase, blocks the circadian responses to light in vivo. Here we show a diurnal and circadian rhythm of cGMP levels and PKG activity in the hamster SCN, with maximal values during the day or subjective day. This rhythm depends on phosphodiesterase but not on guanylyl cyclase activity. Five-minute light pulses increased cGMP levels at the end of the subjective night [circadian time 18 (CT18)], but not at CT13.5. Western blot analysis indicated that the PKG II isoform is the one present in the SCN. Inhibition of PKG or guanylyl cyclase in vivo significantly attenuated light-induced phase shifts at CT18 (after 5-min light pulses) but did not affect c-Fos expression in the SCN. These results suggest that cGMP and PKG are related to SCN responses to light and undergo diurnal and circadian changes.

The levels of cyclic AMP and protein kinase A, as well as the activity of this enzyme, were measured in the hamster suprachiasmatic nuclei at different time points throughout the daily or circadian cycle. Significant diurnal variations for levels of AMPc and the catalytic subunit of protein kinase A and the activity of this enzyme were found. All of these parameters tended to increase throughout the nocturnal phase, reaching higher values at the end of the night and the beginning of the day and minimal values around the time of lights off. This rhythmicity appears to be under exogenous control, since constant darkness abolished fluctuations throughout the circadian cycle. In vitro incubation in the presence of melatonin during the day significantly decreased cyclic AMP levels and basal protein kinase A activity in the SCN, while neither neuropeptide Y nor light pulses affected these parameters. These results suggest a significant diurnal regulation of the cyclic AMP-dependent system in the hamster circadian clock.

Circadian rhythms are controlled by an endogenous clock, which in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN). A role for nitric oxide in circadian responses to light has been indicated. To test the role of nitric oxide synthase (NOS) in the SCN and in circadian responses to light, we examined NOS specific activity at different time points and photic conditions. NOS activity was determined by the conversion of 3H-arginine to 3H-citrulline. NOS enzymatic activity in the SCN was significantly higher during the dark phase than during the day, without any changes in the levels of the NOS protein. However, this difference disappeared when animals were placed under constant darkness, and NOS activity was similar at CT 8 and CT 18 (with CT 12 defined as the onset of the subjective night). When 5-min light pulses were administered at these time points (when light would induce no phase shift or a phase advance, respectively), NOS activity was significantly increased almost equally. A spectrophotometric assay was used to determine NO content in the SCN, showing relatively high constitutive levels enhanced by 100 microM glutamate. These results suggest that NOS activity is not controlled by the circadian clock, although it might mediate some of the effects of light on biological rhythms., (Copyright 1998 Elsevier Science B.V. All rights reserved.)