Publications by authors named "Dörthe M Katschinski"

60 Publications

Precisely Tuned Inhibition of HIF Prolyl Hydroxylases Is Key for Cardioprotection After Ischemia.

Circ Res 2021 Apr 25;128(8):1208-1210. Epub 2021 Feb 25.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany (A.J., A.Z., K.B.-C., A.M.V., D.M.K.).

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http://dx.doi.org/10.1161/CIRCRESAHA.120.318216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048377PMC
April 2021

Hippocampal neurons respond to brain activity with functional hypoxia.

Mol Psychiatry 2021 Feb 9. Epub 2021 Feb 9.

Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

Physical activity and cognitive challenge are established non-invasive methods to induce comprehensive brain activation and thereby improve global brain function including mood and emotional well-being in healthy subjects and in patients. However, the mechanisms underlying this experimental and clinical observation and broadly exploited therapeutic tool are still widely obscure. Here we show in the behaving brain that physiological (endogenous) hypoxia is likely a respective lead mechanism, regulating hippocampal plasticity via adaptive gene expression. A refined transgenic approach in mice, utilizing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to CreERT2 recombinase, allows us to demonstrate hypoxic cells in the performing brain under normoxia and motor-cognitive challenge, and spatially map them by light-sheet microscopy, all in comparison to inspiratory hypoxia as strong positive control. We report that a complex motor-cognitive challenge causes hypoxia across essentially all brain areas, with hypoxic neurons particularly abundant in the hippocampus. These data suggest an intriguing model of neuroplasticity, in which a specific task-associated neuronal activity triggers mild hypoxia as a local neuron-specific as well as a brain-wide response, comprising indirectly activated neurons and non-neuronal cells.
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http://dx.doi.org/10.1038/s41380-020-00988-wDOI Listing
February 2021

Inhibition of Prolyl-Hydroxylase Domain Enzymes Protects From Reoxygenation Injury in Engineered Human Myocardium.

Circulation 2020 Oct 26;142(17):1694-1696. Epub 2020 Oct 26.

Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (E.L., C.N., L.C.Z., W-H.Z., M.T.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044471DOI Listing
October 2020

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via -linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

Circ Res 2020 05 5;126(10):e80-e96. Epub 2020 Mar 5.

From the Department of Pharmacology, University of California, Davis School of Medicine (S.L., Z.L., J.B., D.M.B.).

Rationale: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) -linked attachment of N-acetylglucosamine (-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality.

Objective: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII.

Methods And Results: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca release events (Ca sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca leak in cardiomyocytes. To test the involvement of -GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation.

Conclusions: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires -GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210078PMC
May 2020

Now a Nobel gas: oxygen.

Pflugers Arch 2019 12 22;471(11-12):1343-1358. Epub 2019 Nov 22.

Institute of Physiology and National Center of Competence in Research "Kidney.CH", University of Zürich, CH-8057, Zürich, Switzerland.

The recent bestowal of the Nobel Prize 2019 in Physiology or Medicine to Gregg L. Semenza, Sir Peter J. Ratcliffe, and William G. Kaelin Jr. celebrates a series of remarkable discoveries that span from the physiological research question on how oxygen deficiency (hypoxia) induces the red blood cell forming hormone erythropoietin (Epo) to the first clinical application of a novel family of Epo-inducing drugs to treat patients suffering from renal anemia. This review looks back at the most important findings made by the three Nobel laureates, highlights current research trends, and sheds an eye on future perspectives of hypoxia research, including emerging and potential clinical applications.
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http://dx.doi.org/10.1007/s00424-019-02334-8DOI Listing
December 2019

Mononuclear phagocytes orchestrate prolyl hydroxylase inhibition-mediated renoprotection in chronic tubulointerstitial nephritis.

Kidney Int 2019 08 5;96(2):378-396. Epub 2019 Mar 5.

Department of Internal Medicine 4-Nephrology and Hypertension, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and University Hospital Erlangen, Erlangen, Germany. Electronic address:

Prolyl hydroxylase domain enzyme inhibitors (PHDIs) stabilize hypoxia-inducible factors (HIFs), and are protective in models of acute ischemic and inflammatory kidney disease. Whether PHDIs also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDI treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury, tubular dilation and renal crystal deposition were significantly attenuated in PHDI-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine-induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates of F4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine-enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDIs that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.
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http://dx.doi.org/10.1016/j.kint.2019.02.016DOI Listing
August 2019

Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium.

Front Physiol 2019 4;10:272. Epub 2019 Apr 4.

Institute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, Germany.

Redox signaling affects all aspects of cardiac function and homeostasis. With the development of genetically encoded fluorescent redox sensors, novel tools for the optogenetic investigation of redox signaling have emerged. Here, we sought to develop a human heart muscle model for in-tissue imaging of redox alterations. For this, we made use of (1) the genetically-encoded Grx1-roGFP2 sensor, which reports changes in cellular glutathione redox status (GSH/GSSG), (2) human embryonic stem cells (HES2), and (3) the engineered heart muscle (EHM) technology. We first generated HES2 lines expressing Grx1-roGFP2 in cytosol or mitochondria compartments by TALEN-guided genomic integration. Grx1-roGFP2 sensor localization and function was verified by fluorescence imaging. Grx1-roGFP2 HES2 were then subjected to directed differentiation to obtain high purity cardiomyocyte populations. Despite being able to report glutathione redox potential from cytosol and mitochondria, we observed dysfunctional sarcomerogenesis in Grx1-roGFP2 expressing cardiomyocytes. Conversely, lentiviral transduction of Grx1-roGFP2 in already differentiated HES2-cardiomyocytes and human foreskin fibroblast was possible, without compromising cell function as determined in EHM from defined Grx1-roGFP2-expressing cardiomyocyte and fibroblast populations. Finally, cell-type specific GSH/GSSG imaging was demonstrated in EHM. Collectively, our observations suggests a crucial role for redox signaling in cardiomyocyte differentiation and provide a solution as to how this apparent limitation can be overcome to enable cell-type specific GSH/GSSG imaging in a human heart muscle context.
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http://dx.doi.org/10.3389/fphys.2019.00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460052PMC
April 2019

O affects mitochondrial functionality ex vivo.

Redox Biol 2019 04 23;22:101152. Epub 2019 Feb 23.

Institute for Cardiovascular Physiology, University Medical Center Göttingen, Georg-August-University, Humbdoltallee 23, 37077 Göttingen, Germany. Electronic address:

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (E) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct E of the mitochondrial matrix (-278.9 ± 0.4 mV) in isolated cardiomyocytes is observed. When analyzing the E in isolated mitochondria from the transgenic hearts, however, the E in the mitochondrial matrix is significantly oxidized (-247.7 ± 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% O) conditions that mimics mitochondrial pO levels in cellulo. The reduced E is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.
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http://dx.doi.org/10.1016/j.redox.2019.101152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396017PMC
April 2019

Hypoxia suppresses myofibroblast differentiation by changing RhoA activity.

J Cell Sci 2019 02 18;132(5). Epub 2019 Feb 18.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August University Göttingen, 37073 Göttingen, Germany

Fibroblasts show a high range of phenotypic plasticity, including transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor β can induce the myofibroblastic program, whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemia-induced tissue remodeling and the fibrotic response.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.223230DOI Listing
February 2019

Red fluorescent redox-sensitive biosensor Grx1-roCherry.

Redox Biol 2019 02 7;21:101071. Epub 2018 Dec 7.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia; Pirogov Russian National Research Medical University, Moscow 117997, Russia. Electronic address:

Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of - 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local HO production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging.
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http://dx.doi.org/10.1016/j.redox.2018.101071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302151PMC
February 2019

Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia.

Cell Rep 2018 10;25(3):561-570.e6

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, 37073 Göttingen, Germany.

Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1α signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1α regulation but rather HIF-1α gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-κB activation and concomitantly HIF-1α gene expression. Tafazzin-deficient mice hearts display reduced HIF-1α levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1α signaling due to a lack of NF-κB activation through reduced mitochondrial ROS production, decreasing HIF-1α transcription.
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http://dx.doi.org/10.1016/j.celrep.2018.09.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205837PMC
October 2018

Transgenic Organisms Meet Redox Bioimaging: One Step Closer to Physiology.

Antioxid Redox Signal 2018 08 16;29(6):603-612. Epub 2018 Feb 16.

2 Institute of Cardiovascular Physiology, University Medical Center Göttingen, Georg-August University of Göttingen , Göttingen, Germany .

Significance: Redox signaling is a common mechanism in the cellular response toward a variety of stimuli. For analyzing redox-dependent specific alterations in a cell, genetically encoded biosensors were highly instrumental in the past. To advance the knowledge about the importance of this signaling mechanism in vivo, models that are as close as possible to physiology are needed. Recent Advances: The development of transgenic (tg) redox biosensor animal models has enhanced the knowledge of redox signaling under patho(physio)logical conditions. So far, commonly used small animal models, that is, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio, and genetically modified mice were employed for redox biosensor transgenesis. However, especially the available mouse models are still limited.

Critical Issues: The analysis of redox biosensor responses in vivo at the tissue level, especially for internal organs, is hampered by the detection limit of the available redox biosensors and microscopy techniques. Recent technical developments such as redox histology and the analysis of cell-type-specific biosensor responses need to be further refined and followed up in a systematic manner.

Future Directions: The usage of tg animal models in the field of redox signaling has helped to answer open questions. Application of the already established models and consequent development of more defined tg models will enable this research area to define the role of redox signaling in (patho)physiology in further depth. Antioxid. Redox Signal. 29, 603-612.
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http://dx.doi.org/10.1089/ars.2017.7469DOI Listing
August 2018

Loss of PHD3 in myeloid cells dampens the inflammatory response and fibrosis after hind-limb ischemia.

Cell Death Dis 2017 08 10;8(8):e2976. Epub 2017 Aug 10.

Institute of Cardiovascular Physiology, University Medical Center, Göttingen, Germany.

Macrophages are essential for the inflammatory response after an ischemic insult and thereby influence tissue recovery. For the oxygen sensing prolyl-4-hydroxylase domain enzyme (PHD) 2 a clear impact on the macrophage-mediated arteriogenic response after hind-limb ischemia has been demonstrated previously, which involves fine tuning a M2-like macrophage population. To analyze the role of PHD3 in macrophages, we performed hind-limb ischemia (ligation and excision of the femoral artery) in myeloid-specific PHD3 knockout mice (PHD3) and analyzed the inflammatory cell invasion, reperfusion recovery and fibrosis in the ischemic muscle post-surgery. In contrast to PHD2, reperfusion recovery and angiogenesis was unaltered in PHD3 compared to WT mice. Macrophages from PHD3 mice showed, however, a dampened inflammatory reaction in the affected skeletal muscle tissues compared to WT controls. This was associated with a decrease in fibrosis and an anti-inflammatory phenotype of the PHD3 macrophages, as well as decreased expression of Cyp2s1 and increased PGE2-secretion, which could be mimicked by PHD3 bone marrow-derived macrophages in serum starvation.
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http://dx.doi.org/10.1038/cddis.2017.375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596563PMC
August 2017

μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.

PLoS One 2017 8;12(2):e0170597. Epub 2017 Feb 8.

Institute for Diagnostic and Interventional Radiology, University Medical Center, Goettingen, Germany.

The small size of the adult and developing mouse heart poses a great challenge for imaging in preclinical research. The aim of the study was to establish a phosphotungstic acid (PTA) ex-vivo staining approach that efficiently enhances the x-ray attenuation of soft-tissue to allow high resolution 3D visualization of mouse hearts by synchrotron radiation based μCT (SRμCT) and classical μCT. We demonstrate that SRμCT of PTA stained mouse hearts ex-vivo allows imaging of the cardiac atrium, ventricles, myocardium especially its fibre structure and vessel walls in great detail and furthermore enables the depiction of growth and anatomical changes during distinct developmental stages of hearts in mouse embryos. Our x-ray based virtual histology approach is not limited to SRμCT as it does not require monochromatic and/or coherent x-ray sources and even more importantly can be combined with conventional histological procedures. Furthermore, it permits volumetric measurements as we show for the assessment of the plaque volumes in the aortic valve region of mice from an ApoE-/- mouse model. Subsequent, Masson-Goldner trichrome staining of paraffin sections of PTA stained samples revealed intact collagen and muscle fibres and positive staining of CD31 on endothelial cells by immunohistochemistry illustrates that our approach does not prevent immunochemistry analysis. The feasibility to scan hearts already embedded in paraffin ensured a 100% correlation between virtual cut sections of the CT data sets and histological heart sections of the same sample and may allow in future guiding the cutting process to specific regions of interest. In summary, since our CT based virtual histology approach is a powerful tool for the 3D depiction of morphological alterations in hearts and embryos in high resolution and can be combined with classical histological analysis it may be used in preclinical research to unravel structural alterations of various heart diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298245PMC
August 2017

The Three "Musketairs" - Lasker Prize 2016 goes to the protagonists of hypoxia research.

Hypoxia (Auckl) 2016 25;4:161-162. Epub 2016 Nov 25.

Institute of Cardiovascular Physiology, Georg-August University Göttingen, Göttingen, Germany.

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http://dx.doi.org/10.2147/HP.S126290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135071PMC
November 2016

PHD2 Is a Regulator for Glycolytic Reprogramming in Macrophages.

Mol Cell Biol 2017 01 19;37(1). Epub 2016 Dec 19.

Institute for Cardiovascular Physiology, Georg August University Göttingen, Göttingen, Germany

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior.
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http://dx.doi.org/10.1128/MCB.00236-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192080PMC
January 2017

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice.

Circ Res 2016 Oct 23;119(9):1004-1016. Epub 2016 Aug 23.

From the Institute of Cardiovascular Physiology, Georg August University Göttingen, Germany (L.S., A.K., A.Z., A.G., A.J., A.B., M.S.N., D.M.K.); Institute of Pharmacology, Technical University Dresden, Germany (S.M.-R., A.E.-A.); Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany (C.V.); Cellular Biochemistry, Department of Biology, University of Kaiserslautern, Germany (B.M.); Department of Cellular Biochemistry, University Medical Center Göttingen, Germany (S.D.); Cardiovascular Division, King's College London, British Heart Foundation Centre, United Kingdom (A.M.S.); and German Center for Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (V.O.N.); and Institute of Experimental Cardiovascular Research, Hamburg, Germany (V.O.N.).

Rationale: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors.

Objective: Establishment of mouse models, which allow the quantification of the glutathione redox potential (E) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2).

Methods And Results: We generated transgenic mice with cardiac myocyte-restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward HO, diamide, and dithiothreitol was titrated and used to determine the E in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct E were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction.

Conclusions: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of E in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.309551DOI Listing
October 2016

Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen.

Cell Rep 2015 Dec 25;13(10):2048-55. Epub 2015 Nov 25.

Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany; Institute of Clinical Microbiology and Hygiene, University Hospital of Regensburg and University of Regensburg, 93053 Regensburg, Germany. Electronic address:

Both hypoxic and inflammatory conditions activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here, we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of cosubstrate oxygen versus deprivation of co-factor iron).
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http://dx.doi.org/10.1016/j.celrep.2015.11.005DOI Listing
December 2015

RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes.

PLoS One 2015 8;10(10):e0137519. Epub 2015 Oct 8.

Institute of Pharmacology, University Medical Center Göttingen, Georg-August-University, Germany; DZHK (German Center for Cardiovascular Research), partner site Göttingen, Germany.

Introduction: RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models.

Results: Downregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-β and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues.

Conclusion: RhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137519PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598021PMC
June 2016

Circulating Endothelial Cells Expressing the Angiogenic Transcription Factor Krüppel-Like Factor 4 are Decreased in Patients with Coronary Artery Disease.

Microcirculation 2015 Nov;22(8):700-10

Department of Cardiology and Pulmonary Medicine, University Medical Center Göttingen, Göttingen, Germany.

Objective: The zinc finger transcription factor KLF4 is known to control diverse EC functions.

Methods: The functional role of KLF4 for angiogenesis and its association with CAD was examined in HUVECs and human CECs.

Results: In two different angiogenesis assays, siRNA-mediated KLF4 downregulation impaired HUVEC sprouting and network formation. Conversely, KLF4 overexpression increased HUVEC sprouting and network formation. Similar findings were observed after incubation of HUVECs with CdM from KLF4 cDNA-transfected cells, suggesting a role of paracrine factors for mediating angiogenic KLF4 effects. In this regard, VEGF expression was increased in KLF4-overexpressing HUVECs, whereas its expression was reduced in HUVECs transfected with KLF4 siRNA. To examine the relevance of our in vitro findings for human endothelial dysfunction, we analyzed the expression of KLF4 in CECs of patients with stable CAD. Flow cytometry analyses revealed decreased numbers of KLF4-positive CECs in peripheral blood from CAD patients compared to healthy controls.

Conclusions: Our findings suggest that KLF4 may represent a potential biomarker for EC dysfunction. In the future, (therapeutic) modulation of KLF4 may be useful in regulating EC function during vascular disease processes.
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http://dx.doi.org/10.1111/micc.12226DOI Listing
November 2015

Cardiomyocyte-Specific Transgenic Expression of Prolyl-4-Hydroxylase Domain 3 Impairs the Myocardial Response to Ischemia.

Cell Physiol Biochem 2015 27;36(3):843-51. Epub 2015 May 27.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany.

Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far.

Methods And Results: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice.

Conclusion: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.
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http://dx.doi.org/10.1159/000430260DOI Listing
March 2016

Pre- and post-conditional inhibition of prolyl-4-hydroxylase domain enzymes protects the heart from an ischemic insult.

Pflugers Arch 2015 Oct 13;467(10):2141-9. Epub 2015 Jan 13.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.

Several genetically modified mouse models implicated that prolyl-4-hydroxylase domain (PHD) enzymes are critical mediators for protecting tissues from an ischemic insult including myocardial infarction by affecting the stability and activation of hypoxia-inducible factor (HIF)-1 and HIF-2. Thus, the current efforts to develop small-molecule PHD inhibitors open a new therapeutic option for myocardial tissue protection during ischemia. Therefore, we aimed to investigate the applicability and efficacy of pharmacological HIFα stabilization by a small-molecule PHD inhibitor in the heart. We tested for protective effects in the acute phase of myocardial infarction after pre- or post-conditional application of the inhibitor. Application of the specific PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) resulted in HIF-1α and HIF-2α accumulation in heart muscle cells in vitro and in vivo. The rapid and robust responsiveness of cardiac tissue towards ICA was further confirmed by induction of the known HIF target genes heme oxygenase-1 and PHD3. Pre- and post-conditional treatment of mice undergoing myocardial infarction resulted in a significantly smaller infarct size. Tissue protection from ischemia after pre- or post-conditional ICA treatment demonstrates that there is a therapeutic time window for the application of the PHD inhibitor (PHI) post-myocardial infarction, which might be exploited for acute medical interventions.
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http://dx.doi.org/10.1007/s00424-014-1667-zDOI Listing
October 2015

Lights on for HIF-1α: genetically enhanced mouse cardiomyocytes for heart tissue imaging.

Cell Physiol Biochem 2014 30;34(2):455-62. Epub 2014 Jul 30.

Institute of Cardiovascular Physiology, Georg-August-University Göttingen, Göttingen, Germany.

Background/aims: The hypoxia inducible factor-1 (HIF-1) is a suitable marker for tissue oxygenation. We intended to develop cardiomyocytes (CMs) expressing the oxygen-dependent degradation domain of HIF-1α fused to the firefly luciferase (ODD-Luc) followed by proof-of-concept for its applicability in the assessment of heart muscle oxygenation.

Methods And Results: We first generated embryonic stem cell (ESC) lines (ODD-Luc ESCs) from a Tg ROSA26 ODD-Luc/+ mouse. Subsequent CMs selection was facilitated by stable integration of an antibiotic resistance expressed under the control of the αMHC promoter. ODD-Luc ESCs showed a strong Luc-signal within 1 h of hypoxia (1% oxygen), which coincided with endogenous HIF-1α. Engineered heart muscle (EHM) constructed with ODD-Luc CMs confirmed the utility of the model to sense hypoxia, and monitor reoxygenation also in a multicellular heart muscle model. Pharmacologically induced inotropy/chronotropy under isoprenaline resulted in enhanced Luc-signal suggesting enhanced oxygen consumption, leading to notable myocardial hypoxia.

Conclusions: ODD-Luc-CMs can be used to monitor dynamic changes of cardiomyocyte oxygenation in living heart muscle samples. We provide proof-of-concept for pharmacologically induced myocardial interventions and envision applications of the developed model in drug screens and fundamental studies of ischemia/reperfusion injury.
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http://dx.doi.org/10.1159/000363014DOI Listing
April 2015

Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions.

J Leukoc Biol 2014 Sep 13;96(3):365-75. Epub 2014 Mar 13.

Institute of Cardiovascular Physiology, University Medical Center, Georg August University Göttingen, Germany; and

On a molecular level, cells sense changes in oxygen availability through the PHDs, which regulate the protein stability of the α-subunit of the transcription factor HIF. Especially, PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore, myeloid-specific PHD3(-/-) mice were created and analyzed. PHD3(-/-) BMDM showed no altered HIF-1α or HIF-2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2 polarization was unchanged likewise. Compared with macrophages from WT littermates, PHD3(-/-) BMDM exhibited a significant reduction in TUNEL-positive cells after serum withdrawal or treatment with stauro and SNAP. Under the same conditions, PHD3(-/-) BMDM also showed less Annexin V staining, which is representative for membrane disruption, and indicated a reduced early apoptosis. In an unbiased transcriptome screen, we found that Angptl2 expression was reduced in PHD3(-/-) BMDM under stress conditions. Addition of rAngptl2 rescued the antiapoptotic phenotype, demonstrating that it is involved in the PHD3-mediated response toward apoptotic stimuli in macrophages.
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http://dx.doi.org/10.1189/jlb.2HI1013-533RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395934PMC
September 2014

Editorial: an introduction and welcome to .

Hypoxia (Auckl) 2013 31;1:29-30. Epub 2013 Oct 31.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, Georg-August University Göttingen, Göttingen, Germany.

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http://dx.doi.org/10.2147/HP.S52866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045049PMC
October 2013

Hypoxia modulates fibroblastic architecture, adhesion and migration: a role for HIF-1α in cofilin regulation and cytoplasmic actin distribution.

PLoS One 2013 18;8(7):e69128. Epub 2013 Jul 18.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University Goettingen, Goettingen, Germany.

Cells can adapt to hypoxia by various mechanisms. Yet, hypoxia-induced effects on the cytoskeleton-based cell architecture and functions are largely unknown. Here we present a comprehensive analysis of the architecture and function of L929 fibroblasts under hypoxic conditions (1% O2). Cells cultivated in hypoxia showed striking morphological differences as compared to cells cultivated under normoxic conditions (20% O2). These changes include an enlargement of cell area and volume, increased numbers of focal contacts and loss of cell polarization. Furthermore the β- and γ-actin distribution is greatly altered. These hypoxic adjustments are associated with enhanced cell spreading and a decline of cell motility in wound closure and single cell motility assays. As the hypoxia-inducible factor-1α (HIF-1α) is stabilised in hypoxia and plays a pivotal role in the transcriptional response to changes in oxygen availability we used an shRNA-approach to examine the role of HIF-1α in cytoskeleton-related architecture and functions. We show that the observed increase in cell area, actin filament rearrangement, decrease of single cell migration in hypoxia and the maintenance of p-cofilin levels is dependent on HIF-1α stabilisation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069128PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715466PMC
February 2014

Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice.

Mol Cell Biol 2013 Sep 24;33(17):3426-38. Epub 2013 Jun 24.

Emmy Noether Research Group, University of Technology, Dresden, Germany.

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.
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http://dx.doi.org/10.1128/MCB.00609-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753847PMC
September 2013

Endothelial cell HIF-1α and HIF-2α differentially regulate metastatic success.

Cancer Cell 2012 Jan;21(1):52-65

Department of Physiology, Development and Neuroscience, University of Cambridge, UK.

The hypoxia inducible transcription factors (HIFs) control many mediators of vascular response, including both angiogenic factors and small molecules such as nitric oxide (NO). In studying how endothelial HIF response itself affects metastasis, we found that loss of HIF-1α in endothelial cells reduces NO synthesis, retards tumor cell migration through endothelial layers, and restricts tumor cell metastasis, and that loss of HIF-2α has in each case the opposite effect. This results from differential regulation of NO homeostasis that in turn regulates vascular endothelial growth factor expression in an NO-dependent feedback loop. These opposing roles for the two HIF factors indicate that both they and endothelial cells regulate metastasis as malignancy progresses.
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http://dx.doi.org/10.1016/j.ccr.2011.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334270PMC
January 2012

Unfavourable consequences of chronic cardiac HIF-1α stabilization.

Cardiovasc Res 2012 Apr 18;94(1):77-86. Epub 2012 Jan 18.

Department of Cardiovascular Physiology, Universitätsmedizin, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.

Aims: The hypoxia-inducible factor-1 (HIF-1) is the master modulator of hypoxic gene expression. The effects of chronically stabilized cardiac HIF-1α and its role in the diseased heart are not precisely known. The aims of this study were as follows: (i) to elucidate consequences of HIF-1α stabilization in the heart; (ii) to analyse long-term effects of HIF-1α stabilization with ageing and the ability of the HIF-1α overexpressing hearts to respond to increased mechanical load; and (iii) to analyse HIF-1α protein levels in failing heart samples.

Methods And Results: In a cardiac-specific HIF-1α transgenic mouse model, constitutive expression of HIF-1α leads to changes in capillary area and shifts the cardiac metabolism towards glycolysis with a net increase in glucose uptake. Furthermore, Ca(2+) handling is altered, with increased Ca(2)(+) transients and faster intracellular [Ca(2+)] decline. These changes are associated with decreased expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a but elevated phosphorylation of phospholamban. HIF-1α transgenic mice subjected to transverse aortic constriction exhibited profound cardiac decompensation. Moreover, cardiomyopathy was also seen in ageing transgenic mice. In parallel, we found an increased stabilization of HIF-1α in heart samples of patients with end-stage heart failure.

Conclusion: Changes induced with transgenic cardiac HIF-1α possibly mediate beneficial effects in the short term; however, with increased mechanical load and ageing they become detrimental for cardiac function. Together with the finding of increased HIF-1α protein levels in samples from human patients with cardiomyopathy, these data indicate that chronic HIF-1α stabilization drives autonomous pathways that add to disease progression.
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http://dx.doi.org/10.1093/cvr/cvs014DOI Listing
April 2012

Targeted luminescent near-infrared polymer-nanoprobes for in vivo imaging of tumor hypoxia.

Anal Chem 2011 Dec 2;83(23):9039-46. Epub 2011 Nov 2.

Department of Hematology and Oncology, University Medical Center Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.

Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.
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http://dx.doi.org/10.1021/ac201870bDOI Listing
December 2011