Sven Zukunft

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays

Abstract Measuring NADPH oxidase (Nox)‐derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH‐stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1‐Nox2‐Nox4‐/‐) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH‐stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH‐stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH‐stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH‐dependent signal in assays utilizing lucigenin, L‐012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR‐/‐) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR‐/‐ abolished the signal in NADPH‐stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH‐dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays. Graphical abstract Figure. No Caption available. HighlightsNox activity of intact cells could not be recapitulated in membranes treated with NADPH.Proteomics of membranes show P450 reductase as source of NADPH‐dependent signal.Microsomes overexpressing Cytochrome P450 system produce a NADPH‐dependent signal.Knockout of P450 reductase (CRISPR/Cas9) abolished lucigenin signal in HEK cell membranes.Knockout of POR but not p22phox abolishes the basal and Angiotensin II‐stimulated NADPH‐dependent signal in SMC membranes.

Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte–endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

AMP-Activated Protein Kinase alpha 2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1 alpha and a Network of Proteins Affecting Metabolism and Apoptosis

Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPK&agr;1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPK&agr;2 subunit in vascular repair. Objective: To determine the role of the AMPK&agr;2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPK&agr;2−/− versus wild-type mice, a phenotype reproduced in mice lacking AMPK&agr;2 in myeloid cells (AMPK&agr;2&Dgr;MC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPK&agr;2&Dgr;MC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPK&agr;2&Dgr;MC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPK&agr;2&Dgr;MC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1&agr; induction was attenuated in AMPK&agr;2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPK&agr;2&Dgr;MC mice. Mechanistically, isocitrate dehydrogenase expression and the production of &agr;-ketoglutarate, which negatively regulate hypoxia-inducible factor-1&agr; stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPK&agr;2&Dgr;MC mice. Conclusions: AMPK&agr;2 regulates &agr;-ketoglutarate generation, hypoxia-inducible factor-1&agr; stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPK&agr;2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.

Tyrosine phosphorylation of eNOS regulates myocardial survival after an ischaemic insult: role of PYK2

Aims Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. Methods and results Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. Conclusion The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection.

Hydrogen sulfide preserves endothelial nitric oxide synthase function by inhibiting proline-rich kinase 2: Implications for cardiomyocyte survival and cardioprotection

Hydrogen sulfide (H2S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H2S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H2S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H2O2 or pharmacologic inhibition of H2S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na2S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H2S-reversible manner. The viability of cardiomyocytes exposed to Η2Ο2 was reduced and declined further after the inhibition of H2S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H2O2 on H9c2 cardiomyocyte survival. Moreover, H2S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H2S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na2S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na2S did not result in additive effects on infarct size. We conclude that H2S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H2S-NO interaction and identify PYK2 as a crucial target for the protective effects of H2S under conditions of oxidative stress.

Mitochondrial fragmentation in human macrophages attenuates palmitate-induced inflammatory responses

Macrophages in adipose tissue contribute to inflammation and the development of insulin resistance in obesity. Exposure of macrophages to saturated fatty acids alters cell metabolism and activates pro-inflammatory signaling. How fatty acids influence macrophage mitochondrial dynamics is unclear. We investigated the mechanism of palmitate-induced mitochondrial fragmentation and its impact on inflammatory responses in primary human macrophages. Fatty acids, such as palmitate, caused mitochondrial fragmentation in human macrophages. Increased mitochondrial fragmentation was also observed in peritoneal macrophages from hyperlipidemic apolipoprotein E knockout mice. Fatty acid-induced mitochondrial fragmentation was independent of the fatty acid chain saturation and required dynamin-related protein 1 (DRP1). Mechanistically, mitochondrial fragmentation was regulated by incorporation of palmitate into mitochondrial phospholipids and their precursors. Palmitate-induced endoplasmic reticulum stress and loss of mitochondrial membrane potential did not contribute to mitochondrial fragmentation. Macrophages treated with palmitate maintained intact mitochondrial respiration and ATP levels. Pharmacological or genetic inhibition of DRP1 enhanced palmitate-induced mitochondrial ROS production, c-Jun phosphorylation, and inflammatory cytokine expression. Our results indicate that mitochondrial fragmentation is a protective mechanism attenuating inflammatory responses induced by palmitate in human macrophages.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1β via CD36 and Nicotinic Acetylcholine Receptors

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

Effects of soluble CPE on glioma cell migration are associated with mTOR activation and enhanced glucose flux

Carboxypeptidase E (CPE) has recently been described as a multifunctional protein that regulates proliferation, migration and survival in several tumor entities. In glioblastoma (GBM), the most malignant primary brain tumor, secreted CPE (sCPE) was shown to modulate tumor cell migration. In our current study, we aimed at clarifying the underlying molecular mechanisms regulating anti-migratory as well as novel metabolic effects of sCPE in GBM. Here we show that sCPE activates mTORC1 signaling in glioma cells detectable by phosphorylation of its downstream target RPS6. Additionally, sCPE diminishes glioma cell migration associated with a negative regulation of Rac1 signaling via RPS6, since both inhibition of mTOR and stimulation of Rac1 results in a reversed effect of sCPE on migration. Knockdown of CPE leads to a decrease of active RPS6 associated with increased GBM cell motility. Apart from this, we show that sCPE enhances glucose flux into the tricarboxylic acid cycle at the expense of lactate production, thereby decreasing aerobic glycolysis, which might as well contribute to a less invasive behavior of tumor cells. Our data contributes to a better understanding of the complexity of GBM cell migration and sheds new light on how tumor cell invasion and metabolic plasticity are interconnected.Carboxypeptidase E (CPE) has recently been described as a multifunctional protein that regulates proliferation, migration and survival in several tumor entities. In glioblastoma (GBM), the most malignant primary brain tumor, secreted CPE (sCPE) was shown to modulate tumor cell migration. In our current study, we aimed at clarifying the underlying molecular mechanisms regulating anti-migratory as well as novel metabolic effects of sCPE in GBM. Here we show that sCPE activates mTORC1 signaling in glioma cells detectable by phosphorylation of its downstream target RPS6. Additionally, sCPE diminishes glioma cell migration associated with a negative regulation of Rac1 signaling via RPS6, since both inhibition of mTOR and stimulation of Rac1 results in a reversed effect of sCPE on migration. Knockdown of CPE leads to a decrease of active RPS6 associated with increased GBM cell motility. Apart from this, we show that sCPE enhances glucose flux into the tricarboxylic acid cycle at the expense of lactate production, thereby decreasing aerobic glycolysis, which might as well contribute to a less invasive behavior of tumor cells. Our data contributes to a better understanding of the complexity of GBM cell migration and sheds new light on how tumor cell invasion and metabolic plasticity are interconnected.

A selective and sensitive method for quantification of endogenous polysulfide production in biological samples

Hydrogen sulfide (H2S) is a gasotransmitter that regulates cellular homeostasis and impacts on multiple physiological and pathophysiological processes. However, it exerts many of its biological actions indirectly via the formation of H2S-derived sulfane sulfur species/polysulfides. Because of the high reactivity of sulfur species, the detection of H2S-derived polysulfides in biological systems is challenging and currently used methods are neither sensitive nor quantitative. Herein, we describe a LC-MS/MS-based method that makes use of Sulfane Sulfur Probe 4 to detect endogenously generated polysulfides in biological samples in a selective, sensitive and quantitative manner. The results indicate a large variability in the activity of the H2S-generating enzymes in different murine organs, but the method described was able to detect intracellular levels of polysulfides in the nanomolar range and identify cystathionine γ-lyase as the major intracellular source of sulfane sulfur species/polysulfides in murine endothelial cells and hearts. The protocol described can be applied to a variety of biological samples for the quantification of the H2S-derived polysulfides and has the potential to increase understanding on the control and consequences of this gaseous transmitter.

Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells

Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.During atherosclerosis, endothelial cells release purines in response to oxidized phospholipids. Here, Hitzel et al. show that oxidized phospholipids activate an MTHFD2-regulated gene network in endothelial cells which reprograms amino acid metabolism towards production of purines and thus compensates for their loss.