Zinc oxide nanoparticles alleviate cadmium toxicity and promote tolerance by modulating programmed cell death in alfalfa (Medicago sativa L.).

Cadmium (Cd) can induce programmed cell death (PCD) and zinc oxide nanoparticles (ZnO NPs) effectively alleviate Cd stress. However, the mechanisms of ZnO NPs-mediated Cd detoxification in alfalfa (Medicago sativa L.) are limited. The pot experiment was conducted with Cd soil (19.2 mg kg-1) and foliar ZnO NPs (100 mg L-1) on alfalfa. The results showed that Cd reduced shoot height and biomass, and accumulated reactive oxygen species (ROS), resulting in oxidative stress and further PCD (plasmolysis, cytosolic and nuclear condensation, subcellular organelle swelling, and cell death). ZnO NPs positively regulated the antioxidant system, cell membrane stability, ultrastructure, osmotic homeostasis, and reduced PCD, indicating a multi-level coordination for the increased Cd tolerance. ZnO NPs up-regulated the activity and expression of antioxidant enzymes and regulated PCD-related genes to scavenge ROS and mitigate PCD caused by Cd. The genes related to ZnO NPs-mediated Cd detoxification were significantly enriched in cell death and porphyrin and chlorophyll metabolism. Overall, it elucidates the molecular basis of ZnO NPs-mediated Cd-tolerance by promoting redox and osmotic homeostasis, maintaining cellular ultrastructure, reducing Cd content, and attenuating Cd-induced PCD. it provides a promising application of ZnO NPs to mitigate Cd phytotoxicity and the related cellular and biochemical mechanisms. ENVIRONMENTAL IMPLICATION: Cd, one of the most toxic heavy metals, has caused serious environmental pollution. ZnO NPs can effectively alleviate Cd stress on plants and the environment. This study revealed that foliar-applied ZnO NPs alleviate Cd toxicity by mitigating the oxidative damage and regulating Cd-induced PCD via morphological, physiological, and transcriptomic levels. The findings elucidated the molecular basis of ZnO NPs-mediated Cd tolerance by promoting osmotic and redox homeostasis, reducing Cd content and lipid peroxidation, attenuating Cd-induced PCD features, and altering PCD-related genes in alfalfa. The study laid a theoretical foundation for the safe production of alfalfa under Cd pollution.

NME3 binds to phosphatidic acid and mediates PLD6-induced mitochondrial tethering.

Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

Individual and mutual effects of elevated carbon dioxide and temperature on salt and cadmium uptake and translocation by rice seedlings.

Plant kingdoms are facing increasingly harsh environmental challenges marked by the coexposure of salinity and pollution in the pedosphere and elevated CO2 and temperature in the atmosphere due to the rapid acceleration of industrialization and global climate change. In this study, we deployed a hydroponics-based experiment to explore the individual and mutual effects of different temperatures (low temperature, T1: 23°C; high temperature, T2: 27°C) and CO2 concentrations (ambient CO2: 360 ppm; medium CO2: 450 ppm; high CO2: 700 ppm) on the uptake and translocation of sodium chloride (NaCl, 0.0, 0.2, 0.6, and 1.1 g Na/L) and cadmium nitrate (Cd(NO3)2·4H2O, 0.0, 0.2, 1.8, and 5.4 mg Cd/L) by rice seedlings. The results indicated that Cd and Na exposure significantly (P< 0.05) inhibited plant growth, but T2 and medium/high CO2 alleviated the effects of Cd and Na on plant growth. Neither significant synergistic nor antagonistic effects of Cd and Na were observed, particularly not at T1 or high CO2. At increasing temperatures, relative growth rates increased despite higher concentrations of Cd and Na in both rice roots and shoots. Similarly, higher CO2 stimulated the growth rate but resulted in significantly lower concentrations of Na, while the Cd concentration was highest at medium CO2. Coexposure experiments suggested that the concentration of Cd in roots slightly declined with additional Na and more at T2. Overall, our preliminary study suggested that global climate change may alter the distribution of mineral and toxic elements in rice plants as well as the tolerance of the plants.

Transcriptional analysis of heavy metal P1B-ATPases (HMAs) elucidates competitive interaction in metal transport between cadmium and mineral elements in rice plants.

Cadmium (Cd) pollution has become a major threat to crop production and quality globally. The heavy metal P1B-ATPases (HMAs) play a crucial role in metal transport in plants. In the present study, we investigated the interaction in metal transport by HMAs between Cd and mineral elements in rice plants. Rice seedlings were treated with cadmium nitrate either in the nutrient solution ("Cd+M") or in the ultrapure water ("Cd-M"). Result showed that phytotoxicity of Cd to rice seedlings was evident from both Cd treatments, judged by relative growth rate (RGR), where more severe repression (p < 0.05) of RGR was observed in the "Cd-M" treatments than the "Cd+M" treatments. More Cd (p < 0.05) was accumulated in rice tissues from the "Cd-M" treatments than the "Cd+M" treatments, while there is a significant difference (p < 0.05) in distribution and translocation of mineral elements in rice tissues between the "Cd+M" and the "Cd-M" treatments. RT-qPCR analysis displayed that the expression patterns of HMAs related genes were quite different between "Cd+M" and "Cd-M" treatments, suggesting their different regulatory effects during the transport of Cd and mineral elements within rice plants. The competition in metal transport by HMAs mainly occurs between Cd and micro-elements of Zn and Cu in rice tissues during Cd exposure. Overall, this study provides new evidence to clarify the different translocation mechanisms of HMAs in metal transport between Cd and mineral elements in rice seedlings during Cd exposure.

Chemical Modification, Characterization, and Activity Changes of Land Plant Polysaccharides: A Review.

Plant polysaccharides are widely found in nature and have a variety of biological activities, including immunomodulatory, antioxidative, and antitumoral. Due to their low toxicity and easy absorption, they are widely used in the health food and pharmaceutical industries. However, low activity hinders the wide application. Chemical modification is an important method to improve plant polysaccharides' physical and chemical properties. Through chemical modification, the antioxidant and immunomodulatory abilities of polysaccharides were significantly improved. Some polysaccharides with poor water solubility also significantly improved their water solubility after modification. Chemical modification of plant polysaccharides has become an important research direction. Research on the modification of plant polysaccharides is currently increasing, but a review of the various modification studies is absent. This paper reviews the research progress of chemical modification (sulfation, phosphorylation, acetylation, selenization, and carboxymethylation modification) of land plant polysaccharides (excluding marine plant polysaccharides and fungi plant polysaccharides) during the period of January 2012-June 2022, including the preparation, characterization, and biological activity of modified polysaccharides. This study will provide a basis for the deep application of land plant polysaccharides in food, nutraceuticals, and pharmaceuticals.

Implications of the fate of hydrogen sulfide derived from assimilation of thiocyanate in rice plants.

Thiocyanate (SCN-) is a sulfur-containing pollutant, which is frequently detected in irrigation water and has negative effects on plant growth and crop yields. Uptake and assimilation of exogenous SCN- in rice plants was evident, in which two metabolic pathways, carbonyl sulfide (COS) and cyanate (CNO), are activated. Hydrogen sulfide (H2S) is an important concomitant derived from detoxification of exogenous SCN- in rice plants, which may cause coupling action on the endogenous source of H2S from sulfur metabolism. Since H2S has dual regulatory effects, the fate of H2S derived from assimilation of SCN- in plants is critical for clarifying the inclusiveness of H2S in various physiological activities. In fact, application of exogenous H2S not only positively changed the root phenotype traits of SCN--treated seedlings, but also effectively mitigated the toxic effects of SCN- in rice seedlings by stimulating the process of the PSII repair cycle. In this study, it is tempting to analyze and clarify the flux of the concomitant production of H2S from assimilation of exogenous SCN- into the innate pool, which may function in signaling regulation and other physiological processes in rice plants. This study would update our understanding of the fate of H2S derived from assimilation of SCN- in plants and provide new insights into the affirmative actions of H2S in direct proximity to SCN- exposure.

Tracing the pollution and human risks of potentially toxic elements in agricultural area nearby the cyanide baths from an active private gold mine in Hainan Province, China.

Mining activities are well-known sources of potentially toxic elements (PTEs) pollution, which often jeopardize the biosphere, pedosphere, and hydrosphere. However, the soil and groundwater pollution caused by active private mining activities has long been neglected. This study investigated the occurrence of PTEs and cyanide (CN) in agricultural soils, mine tailings, and groundwater nearby the cyanide baths from a private gold mine in Hainan Province, southern China. Results indicated that concentrations of Pb, As, Cd, Hg, and CN in different soil depths and mine tailings were up to ten thousand mg/kg, and relatively higher content of As and Pb was detected in groundwater. The chemical forms of Cd, Pb, As, and Hg varied greatly in different soil depths; over 80% of Cd distributed in the water-soluble fraction, suggesting its higher mobility in soils, while approximately 60-90% of Pb, As, and Hg distributed in other chemical fractions, indicating relatively lower mobility in soils. The pollution indices also revealed the serious pollution and deterioration of site quality in this area. Human risk assessments also reflected a high non-carcinogenic/carcinogenic health risk in this area. The framework of integrated management strategies for private metal mines was proposed to mitigate PTEs pollution and reduce health risks.

Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

Prevalent phthalates in air-soil-vegetable systems of plastic greenhouses in a subtropical city and health risk assessments.

Wide use of plastic greenhouses for vegetable production increases human exposure to phthalate (PAEs) through vegetable intake. However, little information is available about distribution of PAEs in air-soil-vegetable systems of plastic greenhouses and PAE estrogenic effects. This study was designed to investigate PAE distributions and corresponding health risk in plastic greenhouses in Guangzhou, a subtropical city in South China. PAEs were prevalent in plastic greenhouses, with sum concentrations of 16 PAE compounds (∑16PAEs) up to 5.76 mg/kg in soils, 5.27 mg/kg in vegetables and 4393 ng/m3 in air. Di (2-ethylhexyl) phthalate, di-isobutyl phthalate, and dibutyl phthalate were predominant compounds. Average concentrations and bioconcentration factor of ∑16PAEs and the predominant PAE compounds in vegetables of greenhouses were higher than those of open fields. Plastic greenhouses exhibited significantly higher air PAE levels than those of open fields due to higher indoor temperature, which enhanced PAE accumulation by vegetables. Both carcinogenic and non-carcinogenic risks of PAEs via dietary and non-dietary exposures for farmers decreased with an order of vegetable > air > soil. Consumption of vegetables from greenhouses resulted in significantly higher estrogenic effects compared to those from open field cultivation. This study emphasizes highly potential health risks of PAEs in air-soil-vegetable systems of plastic greenhouses.

Occurrence and human health risks of phthalates in indoor air of laboratories.

Phthalate acid esters (PAEs) are of serious concern as a human health risk due to their ubiquitous presence in indoor air. In the present study, fifteen PAEs in the indoor air samples from physical, chemical, and biological laboratories in Guangzhou, southern China were analysed using gas chromatography mass spectrometry. Extremely high levels of PAEs of up to 6.39 × 104 ng/m3 were detected in some laboratories. Diisobutyl phthalate (DiBP), di(methoxyethyl) phthalate (DMEP), and di-n-butyl phthalate (DBP) were the dominant PAEs with median levels of 0.48 × 103, 0.44 × 103, and 0.39 × 103 ng/m3, respectively, followed by di-(2-propylheptyl) phthalate (DPHP) and di(2-ethylhexyl) phthlate (DEHP) (median levels: 0.16 × 103 and 0.13 × 103 ng/m3, respectively). DMEP and DPHP were found for the first time in indoor air. Principal component analysis indicated that profiles of PAEs varied greatly among laboratory types, suggesting notable variations in sources. The results of independent samples t-tests showed that levels of PAEs were significantly influenced by various environmental conditions. Both the non-carcinogenic and carcinogenic health risks from human exposure to PAEs based on the daily exposure dose in laboratory air were acceptable. Further research should be conducted to investigate the long-term health effects of exposure to PAEs in laboratories.

Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats.

The aim of this study is to investigate the vascular outcome after intravitreal mesenchymal stem cell (MSC) administration in rats without or with damage to the neurovascular unit [transgenic (TGR) rats]. Male Sprague-Dawley (SD) and TGR rats received an intravitreal injection of 2 × 104 rat bone marrow-derived MSCs (BMSCs) or human adipose-derived stem cells (ASCs) at postnatal d 30. After 4 wk, vasculature, neuronal function, and gene expression in the retinas were evaluated using retinal morphometry, electroretinography, immunofluorescence, Western blot, and quantitative PCR. Intravitreal administration of rat BMSCs and human ASCs in both SD and TGR eyes induced cataract, loss of pericytes, and increased formation of acellular capillaries. BMSCs remained in the vitreous cavity and did not migrate into the retinas. Intravitreal administration of BMSCs impacted retinal neuronal function in neither SD nor TGR rats. Retinal glial activation, elevation of IL-1ß, C3, arginase 1, and heat shock protein 90 were detected in BMSC-injected SD rats. Intravitreal administration of MSCs induces cataract, retinal vasoregression, activation of retinal glial cells, and inflammatory response in rat eyes.-Huang, H., Kolibabka, M., Eshwaran, R., Chatterjee, A., Schlotterer, A., Willer, H., Bieback, K., Hammes, H.-P., Feng, Y. Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats.

O-GlcNAcylation of FoxO1 mediates nucleoside diphosphate kinase B deficiency induced endothelial damage.

Nucleoside diphosphate kinase B (NDPK-B) acts as a protective factor in the retinal vasculature. NDPK-B deficiency leads to retinal vasoregression mimicking diabetic retinopathy (DR). Angiopoetin 2 (Ang-2), an initiator of retinal vasoregression in DR, is upregulated in NDPK-B deficient retinas and in NDPK-B depleted endothelial cells (ECs) in vitro. We therefore investigated the importance of Ang-2 in NDPK-B deficient retinas and characterized the mechanisms of Ang-2 upregulation upon NDPK-B depletion in cultured ECs. The crucial role of retinal Ang-2 in the initiation of vasoregression was verified by crossing NDPK-B deficient with Ang-2 haplodeficient mice. On the molecular level, FoxO1, a transcription factor regulating Ang-2, was upregulated in NDPK-B depleted ECs. Knockdown of FoxO1 abolished the elevation of Ang-2 induced by NDPK-B depletion. Furthermore O-GlcNAcylated FoxO1 was found preferentially in the nucleus. An increased O-GlcNAcylation of FoxO1 was revealed upon NDPK-B depletion. In accordance, the inhibition of protein O-GlcNAcylation normalized NDPK-B depletion induced Ang-2 upregulation. In summary, we demonstrated that the upregulation of Ang-2 upon NDPK-B deficiency is driven by O-GlcNAcylation of FoxO1. Our data provide evidence for a central role of protein O-GlcNAcylation in NDPK-B associated vascular damage and point to the hexosamine pathway as an important target in retinal vasoregression.

Cystoid edema, neovascularization and inflammatory processes in the murine Norrin-deficient retina.

Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

Catecholamines facilitate VEGF-dependent angiogenesis via ß2-adrenoceptor-induced Epac1 and PKA activation.

Chronic stress has been associated with the progression of cancer and antagonists for ß-adrenoceptors (ßAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial ß2AR in angiogenesis can be envisioned. We therefore investigated the role of ß2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by ß2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2'-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, ß2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC.Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a ß2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion.

Nucleoside diphosphate kinase B regulates angiogenic responses in the endothelium via caveolae formation and c-Src-mediated caveolin-1 phosphorylation.

Nucleoside diphosphate kinase B (NDPK-B) is an enzyme required for nucleoside triphosphate homeostasis, which has been shown to interact with caveolin-1 (Cav-1). In endothelial cells (ECs), NDPK-B contributes to the regulation of angiogenesis and adherens junction (AJ) integrity. We therefore investigated whether an interaction of NDPK-B with Cav-1 in ECs is required for this regulation and the involvement of VEGF signaling herein. We report that simultaneous depletion of NDPK-B/Cav-1 in HUVECs synergistically impaired sprouting angiogenesis. NDPK-B depletion alone impaired caveolae formation, VEGF-induced phosphorylation of c-Src/Cav-1 but not of ERK1/2/AKT/eNOS. In vivo, Cav-1-/- mice showed impaired retinal vascularization at postnatal-day five, whereas NDPK-B-/- mice did not. Primary mouse brain ECs (MBMECs) from NDPK-B-/- mice showed no change in caveolae content and transendothelial-electrical resistance upon VEGF stimulation. Interestingly, NDPK-B-/- MBMECs displayed an accumulation of intracellular vesicles and increased Cav-1 levels. Dextran tracer analysis showed increased vascular permeability in the brain of NDPK-B-/- mice compared to wild type. In conclusion, our data indicate that NDPK-B is required for the correct localization of Cav-1 at the plasma membrane and the formation of caveolae. The genetic ablation of NDPK-B could partially be compensated by an increased Cav-1 content, which restored caveolae formation and some endothelial functions.

Transcription of Inflammatory Cytokine TNFα is Upregulated in Retinal Angiogenesis under Hyperoxia.

BACKGROUND/AIMS: Hypoxia induces angiogenesis while hyperoxia promotes vasoregression in the retina. We investigated herein the effect of prolonged hyperoxia on retinal angiogenesis and the underlying mechanism in an oxygen-induced retinopathy (OIR) model. METHODS: Vascular morphology was quantified in whole-mount retina from the mice subjected to the conventional OIR model (c-OIR) or the OIR model with prolonged hyperoxia (p-OIR). Expressions of genes related to angiogenesis were determined by real-time PCR. RESULTS: p-OIR retinas showed few intraretinal neovascular tufts at the border of avascular zones, lacking preretinal neovascularization, whereas c-OIR retinas had numerous preretinal neovascularizations. p-OIR retinas demonstrated outgrowth of capillaries in the deep layers despite persistent hyperoxia and possess a larger avascular zone compared with the c-OIR retinas. The capillaries in the p-OIR retinas were well-formed in contrast to those in the c-OIR retinas. p-OIR retinas expressed significantly higher TNFα (∼4 fold) than c-OIR retinas. The expression of vascular endothelial growth factor, Erythropoietin, Angiopoietin 1 and 2 remained unchanged. CONCLUSION: Our data demonstrate that TNFα transcription is increased in hyperoxia-promoted retinal angiogenesis, implicating it, in association with low VEGF levels, as a possible proponent in retinal angiogenesis under hyperoxia.

Nucleoside diphosphate kinase B deficiency causes a diabetes-like vascular pathology via up-regulation of endothelial angiopoietin-2 in the retina.

AIMS: Nucleoside diphosphate kinase B (NDPKB) is capable of maintaining the cellular nucleotide triphosphate pools. It might therefore supply UTP for the formation of UDP-GlcNAc from glucose. As NDPKB contributes to vascular dysfunction, we speculate that NDPKB might play a role in microangiopathies, such as diabetic retinopathy (DR). Therefore, we investigated the impact of NDPKB on retinal vascular damage using NDPKB(-/-) mice during development of DR and its possible mechanisms. METHODS: Pericyte loss and acellular capillary (AC) formation were assessed in streptozotocin-induced diabetic NDPKB(-/-) and wild-type (WT) mice. Expression of angiopoietin-2 (Ang2) and protein N-acetylglucosamine modification (GlcNAcylation) were assessed by western blot and/or immunofluorescence in the diabetic retinas as well as in endothelial cells depleted of NDPKB by siRNA and stimulated with high glucose. RESULTS: Similar to diabetic WT retinas, non-diabetic NDPKB(-/-) retinas showed a significant decrease in pericyte coverage in comparison with non-diabetic WT retinas. Hyperglycemia further aggravates pericyte loss in diabetic NDPKB(-/-) retinas. AC formation was detected in the diabetic NDPKB(-/-) retinas. Similar to hyperglycemia, NDPKB deficiency induced Ang2 expression and protein GlcNAcylation that were not further altered in the diabetic retinas. In cultured endothelial cells, stimulation with high glucose and NDPKB depletion comparably increased Ang2 expression and protein GlcNAcylation. CONCLUSIONS: Our data identify NDPKB as a protective factor in the retina, which controls Ang2 expression and the hexosamine pathway. NDPKB-deficient mice are a suitable model for studying mechanisms underlying diabetic retinal vascular damage.

Nucleoside diphosphate kinase B-activated intermediate conductance potassium channels are critical for neointima formation in mouse carotid arteries.

OBJECTIVE: Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca(2+)-activated K(+) (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (ISK4) in proliferating VSMC and neointima formation. APPROACH AND RESULTS: Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased ISK4, whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in ISK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. CONCLUSIONS: NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

Nucleoside diphosphate kinase B regulates angiogenesis through modulation of vascular endothelial growth factor receptor type 2 and endothelial adherens junction proteins.

OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.