International Research Training Group 1874/1

Diabetic Microvascular Complications

Specific projects first funding period

Workpackage 1 – General Mechanisms

  • Detoxifying enzymes in diabetes target tissues – mitochondrial-dependent and independent effects of methylglyoxalOpen or Close

    Principal Investigator (Heidelberg): Peter P. Nawroth
    Co-PI (Heidelberg): Michael Mendler
    PIs (Groningen): Reinold B. Gans, Stephan Bakker
    Graduate: Nadine Volk
    Start of PhD project: January 01, 2013

    Project description:

    Late diabetic complications are not only explained by glucose, but also by glucose-dependent or –independent generation of toxic metabolites. Methylglyoxal (MG), formed from the spontaneous degradation of triose phosphates (GAD3P and DHAP) and from other non-enzymatic and enzymatic pathway, accumulates in diabetic tissues due to an increased glycemic flux and the diabetesassociated down-regulation of its detoxifying enzyme glyoxalase-1 (GLO-1). Accumulation of MG causes mitochondrial dysfunction and modification of mitochondrial proteins, increased reactive oxygen species (ROS) generation and NFkB-driven proinflammatory gene expression, finally resulting in vascular complications. While model organisms have demonstrated, that glucose toxicity can be prevented by both, improving GLO-1-dependent MG-detoxification and preventing mitochondrial dysfunction, the impact of mitochondrial dependent and -independent effects of MG on cellular dysfunction in late diabetic complications is yet unknown.

    • Riddle MC et al. Diabetes Care. 2010; 33: 983-90
    • Fleming T, Cuny J, Nawroth G, Djuric Z, Humpert PM, Zeier M, Bierhaus A, Nawroth PP, Diabetologia. 2012 Apr;55(4):1151-5
    • Jack MM et al. Diabetologia. 2011; 54:2174-82
    • Schlotterer A, Kukudov G, Bozorgmehr F, Hutter H, Du X, Oikonomou D, Ibrahim Y, Pfisterer F, Rabbani N, Thornalley P, Sayed A, Fleming T, Humpert P, Schwenger V, Zeier M, Hamann A, Stern D, Brownlee M, Bierhaus A, Nawroth P, Morcos M, Diabetes. 2009; 58: 2450-6
    • Morcos M, Du X, Pfisterer F, Hutter H, Sayed AA, Thornalley P, Ahmed N, Baynes J, Thorpe S, Kukudov G, Schlotterer A, Bozorgmehr F, El Baki RA, Stern D, Moehrlen F, Ibrahim Y, Oikonomou D, Hamann A, Becker C, Zeier M, Schwenger V, Miftari N, Humpert P, Hammes HP, Buechler M, Bierhaus A, Brownlee M, Nawroth PP, Aging CELL. 2008; 7: 260-269
    • Rabbani N, et al. 2008; 36:1045-50
    • Pal A et. al. Mol Immunol. 2009;46: 2039-44
    Methods that will be used:

    Study the consequences mitochondrial dysfunction on cellular activity either by pharmacology intervention or through the development to mitochondrial free cells. Study the effect of acute and chronic exposure to high glucose and methylglyoxal on mitochondrial dysfunction by assessment of methylglyoxal, ROS generation, activation of antioxidant defence system as well as induction of NFkB and NFkB dependent gene expression by biochemical analytics, Electrophoretic Mobility Shift Assays (EMSA), Chromatin immune precipitationassay (ChIP), Western Blots and Real time-PCR (RQ-PCR).

    Collaboration Partners:

    Prof. Hans-Peter Hammes, Dr. Jens Kroll, Prof. Markus Hecker

  • Dysfunction of compartmentalized cyclic AMP signaling in the diabetic vasculatureOpen or Close

    Principal Investigator (Mannheim): Thomas Wieland
    Co-PI (Mannheim): Yuxi Feng
    PI (Groningen): Martina Schmidt
    Graduate: Jaspal Garg
    Start of PhD project: May 15, 2013

    Project description:

    The versatile second messenger cAMP controls diverse physiological and pathological processes. Subcellular compartmentalization and spatiotemporal cAMP dynamics, maintained by protein kinase A (PKA), exchange factor directly activated by cAMP (Epac), AKAPs and phosphodiesterases (PDE), promote the distinct signal transduction events driven by cAMP. Dysregulation of cAMP compartmentalization is associated with several diseases including diabetes mellitus. The activation of Epac1 inhibited angiogenesis in vitro and in vivo. Subtle alterations in local activation of Epac likely contribute to pathological changes in the diabetic vasculature. The aim of the projest is to study the subcellular compartmentalization and spatiotemporal cAMP dynamics regulated by the Epac in different diabetes models.

    • Grandoch M, Roscioni SS, Schmidt M. The role of Epac proteins, novel cAMP mediators, in the regulation of immune, lung and neuronal function. Br J Pharmacol. 2010;159:265-284
    • Houslay MD. Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci.35:91-100
    • Doebele RC, Schulze-Hoepfner FT, Hong J, et al. A novel interplay between Epac/Rap1 and mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) regulates thrombospondin to control angiogenesis. Blood. 2009;114:4592-4600
    • Pfister F, Wang Y, Schreiter K, et al. Retinal overexpression of angiopoietin-2 mimics diabetic retinopathy and enhances vascular damages in hyperglycemia. Acta Diabetol. 2010;47:59-64
    • Wang Q, Pfister F, Dorn-Beineke A, vom Hagen F, Lin J, Feng Y, Hammes HP. Low-dose erythropoietin inhibits oxidative stress and early vascular changes in the experimental diabetic retina. Diabetologia. 2010;53:1227-1238
    Methods that will be used:

    Cell culture, gene transfer, Pull-down assay, Epac-based fluorescence resonance energy transfer (FRET), diabetic animal models, mouse model of retinopathy of prematurity, in vitro angiogenesis models, retinal morphometry, PCR, Western blot, immunofluorescence.

    Collaboration Partners:

    Martina Schmidt, Molecular Pharmacology, Groningen Research Institute of Pharmacy, University of Groningen.
    Hans-Peter Hammes, V. Medical Department, Medical Faculty Mannheim, University of Heidelberg
    Jens Kroll, Vascular Biology & Tumorangiogenesis, Medical Faculty Mannheim, University of Heidelberg
    Rudolf Schubert, Cardiovascular Physiology, Medical Faculty Mannheim, University of Heidelberg

  • Hyperglycemia-induced monocyte-macrophage differentiationOpen or Close

    Principal Investigator (Mannheim): Julia Kzhyshkowska
    Co-PI (Mannheim): Alexei Gratchev
    PI (Groningen): Marco Harmsen
    Graduate: Kondaiah Moganti
    Start of PhD project: February 01, 2013

    Project description:

    This project identifies molecular mechanism of hyperglycaemic memory in monocytes/macrophages and establishes the role of hyperglycemia-induced macrophages in microangiopathy common for all target tissues. Low-grade inflammation and pro-fibrotic processes are involved in diabetic microangiopathy. Type 2 macrophages (M2) regulate inflammation and release of pro-fibrotic factors. Monocytes from diabetic patients respond to hyperglycemia by amplification of TGFbeta signaling. We have shown that M2 express increased levels of TGFbetaRII on their surface and respond to TGFbeta by activation of low-grade inflammatory and pro-fibrotic activities including induction of the new transcription factor FoxQ1. In this project we aim to identify the mechanism of glucose-induced TGFbeta responsiveness of M2, to establish the role of hyperglycemic M2 in pro-microangiopathyc activities common to all target tissues, to identify the role of M2 in the development of hyperglycaemic memory, and to develop tools for targeting of TGFbeta/FoxQ1 pathway in the circulation. Using our model of human primary macrophages, we will establish how increased levels of glucose amplify TGFbetasignaling in M2. We will identify epigenentic changes in monocytes induced by glucose and TGFbeta that lead to hyperglycemic memory of resident M2. Bisulphite sequencing, ChIP, and miRNA analysis will be applied. Pro-microangiopatic genes activated by FoxQ1 will be identified using Affymetrix chip assays, Real-time PCR, and reporter systems. The mechanism of crass-talk between hyperglycaemic M2 and microvascular ECs will be established. The results of the project will be used in future to block hyperglycaemic programming of circulating monocytes by targeting TGFbeta/FoxQ1 pathway with blocking antibodies and si/shRNA.

    • Donath, M.Y., Shoelson, S.E. (2011). Nat Rev Immunol 11, 98-107.
    • Gordon, S., Martinez, F.O. (2010). Immunity 32, 593-604.
    • Wu, L., Derynck, R. (2009). Dev Cell 17, 35-48.
    • Gratchev, A., Kzhyshkowska, J., Kannookadan, S., Ochsenreiter, M., Popova, A., Yu, X., Mamidi, S., Stonehouse-Usselmann, E., Muller-Molinet, I., Gooi, L. and Goerdt, S. (2008). J Immunol 180, 6553-6565.
    • Martinez, F.O., Helming, L. and Gordon, S. (2009). Annu Rev Immunol 27, 451-483.
    • Mosig, S., Rennert, K., Krause, S., Kzhyshkowska, J., Neunubel, K., Heller, R. and Funke, H. (2009). FASEB J 23, 866-874.
    • Kzhyshkowska, J., Marciniak-Czochra, A. and Gratchev, A. (2008). Immunobiology 212, 813-825.
    • Gratchev, A., Kzhyshkowska, J., Kothe, K., Muller-Molinet, I., Kannookadan, S., Utikal, J. and Goerdt, S. (2006). Immunobiology 211, 473-486.
    • Kzhyshkowska, J., Mamidi, S., Gratchev, A., Kremmer, E., Schmuttermaier, C., Krusell, L., Haus, G., Utikal, J., Schledzewski, K., Scholtze, J. and Goerdt, S. (2006). Blood 107, 3221-3228.
    • Kzhyshkowska, J., Workman, G., Cardo-Vila, M., Arap, W., Pasqualini, R., Gratchev, A., Krusell, L., Goerdt, S. and Sage, E.H. (2006). J Immunol 176, 5825-5832.
    • Yang, X.F., Fang, P., Meng, S., Jan, M., Xiong, X., Yin, Y. and Wang, H. (2009). Front Biosci (Schol Ed) 1, 420-436.
    Methods that will be used:

    Bisulphite sequencing, ChIP, miRNA analysis, Real-time PCR, FACS, immunoblotting, ELISA, immunofluorescence/confocal microscopy, Affymetrix Gene Array analysis; Illumina arrays; proteomics techniques, cell-based functional systems, isolation and differentiation of human primary cells.

    Collaboration Partners:

    Martin C. Harmsen, PhD, University Medical Center Groningen, University of Groningen, the Netherlands
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg and Groningen

  • Kv channels in myogenic and perivascular fat-dependent autoregulation and small artery diabetic dysfunctionOpen or Close

    Principal Investigator (Mannheim): Rudolf Schubert
    Principal Investigator (Groningen): Hendrik Buikema
    Graduate: Bettina Müller
    Start of PhD project: May 01, 2013

    Project description:

    This project addresses the hypothesis that dysfunction of vascular smooth muscle voltage-gated potassium (Kv) channels, in particular Kv7 channels, contributes to dysregulation of myogenic and perivascular fat-dependent autoregulation in diabetes and related renal damage. The study will be performed cooperatively in order to use a larger range of complimentary methods from the molecular to the whole animal level to determine in diabetes (i) the functional role of Kv, in particular Kv7, channels in myogenic and perivascular fat-dependent autoregulation (ii) the expression of these channels and of members of caveolae-related channel protein complexes and (iii) the feasibility of the use of openers of these channels as a therapeutic option. Thus, this project provides unique opportunities for the doctoral student involved and will result in new insights into the mechanisms governing small vessel dysfunction in diabetes.

    • Schubert R, Lidington D, Bolz SS. The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction. Cardiovasc Res. 2008;77:8-18.
    • Xu Y, Henning RH, Sandovici M, van der Want JJ, van Gilst WH, Buikema H. Enhanced myogenic constriction of mesenteric artery in heart failure relates to decreased smooth muscle cell caveolae numbers and altered AT1- and epidermal growth factor-receptor function. Eur J Heart Fail. 2009;11:246-255.
    • Gollasch M. Vasodilator signals from perivascular adipose tissue. Br J Pharmacol. 2012;165:633-642.
    • Schleifenbaum J, Kohn C, Voblova N, Dubrovska G, Zavarirskaya O, Gloe T, Crean CS, Luft FC, Huang Y, Schubert R, Gollasch M. Systemic peripheral artery relaxation by KCNQ channel openers and hydrogen sulfide. J Hypertens. 2010;28:1875-1882.
    Methods that will be used:

    diabetic animal models, expression analysis (qPCR, Western blot), ion channel function (patch-clamp technique), in vitro vessel contractility (isometric and isobaric myography), in vivo vessel contractility (intravital microscopy), intracellular calcium determination (FURA-2 fluorimetry), renal function (histology, protein excretion), in vivo blood pressure measurement (telemetry)

    Collaboration Partners:

    Dr. Hendrik Buikema, Dept. Clinical Pharmacology; Faculty of Medicine, UMCG, University of Groningen; Ant. Deusinglaan 1, FB20, 9713 AV Groningen
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

Workpackage 2 – Diabetic Retinopathy

  • Hyperglycemic memory–mechanisms relevant to the diabetic retinaOpen or Close

    Principal Investigator (Mannheim): Hans-Peter Hammes
    Principal Investigator (Groningen): Grietje Molema
    Graduate: Patrick Friedrichs
    Start of PhD project: January 01, 2013

    Project description:

    Clinical and experimental evidence suggests that hyperglycemic memory is part of the complex pathogenesis of diabetic retinopathy. Perpetuation of oxidative stress, irreversible accumulation of advanced glycation end products (AGEs) and hyperglycemia-induced epigenetic changes are possible underlying mechanisms. However, given the unsolved question which pathogenetic mechanisms prevail at retinopathy onset, the gap needs to be closed between cellular memory experiments which reflects days, and animal experiments and human disease which represent years. The aim of the current project is to determine a. the clusters of retinal genes involved in hyperglycemic memory using state-of-the-art array technologies, b. the cellular compartments in which these genes are regulated using laser microdissection technologies and c. the underlying pathogenetic concept by comparing gene expressions patterns during euglycemic reentry versus metabolic signal blockade.

    • Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res. 2010; 107: 1058-70
    • Hammes HP, Du X, Edelstein D, et al. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat Med 2003; 9: 294-299
    • Yao D, Taguchi T, Matsumura T, et al. High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A. J Biol Chem 2007; 282: 31038-45
    • El-Osta A, Brasacchio D, Yao D et al. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. J Exp Med 2008; 205:2409-17
    • Wang Q, Gorbey S, Pfister F, et al. Long-term treatment with suberythropoietic Epo is vaso- and neuroprotective in experimental diabetic retinopathy. Cell Physiol Biochem 2011; 27:769-82
    • Zhong Q, Kowluru RA. Epigenetic changes in mitochondrial superoxide dismutase in the retina and the development of diabetic retinopathy. Diabetes. 2011; 60:1304-13
    Methods that will be used:

    Spontaneous and induced animal models of diabetes; Affymetrix Gene Array analysis; laser micro dissection microscopy; Illumina arrays; quantitative retinal morphometry, immunofluorescence and laser scanning microscopy, mass spectrometry, immunoblotting techniques.

    Collaboration Partners:

    Grietje Molema, PhD, University Medical Center Groningen, University of Groningen, the Netherlands
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

  • Impact of adipose-derived stem cells (ADSC) on diabetic and proliferative retinopathyOpen or Close

    Principal Investigator (Mannheim): Hans-Peter Hammes
    Principal Investigator (Groningen): Marco Harmsen
    Graduate: Vincenzo Terlizzi
    Start of PhD project: July 01, 2013

    Project description:

    Pericyte loss is the earliest morphological sign of diabetic retinopathy We hypothesize that diabetic retinopathy can be delayed or prevented through restoration of pericytes and pericyte function by adipose tissue-derived stem/stromal cells (ADSC). According to surface and internal marker validation, ADSCs share pericyte characteristics, and may have a therapeutic benefit n injured microvessels in the eye. The main aims of this project are to 1) dissect the molecular mechanisms of ADSC-driven pericyte restoration, 2) to investigate the impact of diabetes i.e. long-term hyperglycemia, on the molecular phenotype and function of ADSC and 3) whether ADSC support long-term normalization of the vasculature in the diabetic retina.

    • Hammes HP, Feng Y, Pfister F, Brownlee M. Diabetic retinopathy: Targeting vasoregression. Diabetes. 2011;60:9-16.
    • Corselli M, Chen CW, Crisan M, Lazzari L, Peault B. Perivascular ancestors of adult multipotent stem cells. Arterioscler Thromb Vasc Biol. 2010;30:1104-1109.
    • Zimmerlin L, Donnenberg VS, Pfeifer ME, Meyer EM, Peault B, Rubin JP, Donnenberg AD. Stromal vascular progenitors in adult human adipose tissue. Cytometry A. 2010;77:22-30.
    • Przybyt E, Pfister F, Hammes HP, Harmsen MC. Adipose tissue-derived stromal cell (ADSC) restore pericyte loss in mouse retinopathy of prematurity. (Manuscript in preparation).
    Methods that will be used:

    ADSC isolation and culture, gene and protein expression analyses, lentiviral transduction, in vitro angiogenesis models, immunofluorescence (confocal laser microscopy), mouse model of retinopathy of prematurity and diabetic animal models

    Collaboration Partners:

    Dr. Martin Harmsen, Dept. of Pathology & Laboratory Medicine, Cardiovascular Regenerative Medicine Research Group (CAVAREM), UMCG, University of Groningen;
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

  • MicroRNA-induced microangiopathy in diabetesOpen or Close

    Principal Investigator (Mannheim): Hans-Peter Hammes
    Principal Investigator (Groningen): Guido Krenning
    Graduate: Julian Friedrich
    Start of MD project: February 01, 2013

    Project description:

    Diabetic retinopathy is a chronic microvascular complication affecting virtually all patients with diabetes. Diabetic retinopathy is characterized by progressive alterations in the retinal microvasculature, i.e. endothelial dysfunction, vascular hyperpermeability, hyperglycemia-induced ROS production, pericyte dropout (Hammes et al. Diabetes 2011. MicroRNAs are small non-coding nucleotides that affect cell function by translational repression of their target genes. MicroRNA dysregulation is associated with diabetic retinopathy (Kovacs et al. Invest. Ophtalmol. Vis. Sci. In Press) and affects endothelial function and angiogenesis (Shantikumar et al. Cardiovasc.Res. 2012). We have recently identified several specific microRNAs that augment endothelial dysfunction by modulating crucial endothelial signaling transduction pathways and hypothesize that dysregulation of microRNAs occurs when diabetic complications develop.

    The main aim of this project is to clarify how novel diabetes-related microRNA (diamiR) ‘master switches’ can be manipulated to restore endothelial phenotype and function in diabetic retinopathy. Hereto we aim to (1) identify diamiRs which are associated with diabetic retinopathy and their gene targets, (2) to investigate the influence of diamiRs on the development of endothelial dysfunction and diabetic retinopathy, and (3) to therapeutically modulate diamiR expression to alleviate diabetic retinopathy in a mouse model.

    • Hammes HP, Feng Y, Pfister F, Brownlee M. Diabetic retinopathy: targeting vasoregression. Diabetes 2011;60:9-16
    • Kovacs B, Lumayag S, Cowan C, Xu S. microRNAs in Early Diabetic Retinopathy in Streptozotocin-Induced Diabetic Rats. Invest. Ophtalmol. Vis. Sci. In Press
    • Shantikumar S, Caporali A, Emanueli C. Role of microRNAs in diabetes and its cardiovascular complications.Cardiovasc.Res. 2012;93:583-93.
    Methods that will be used:

    Spontaneous and induced animal models of diabetes; laser micro dissection microscopy, Illumina arrays, quantitative retinal morphometry, immunofluorescence and laser scanning microscopy, mass spectrometry, immunoblotting techniques, microRNA in situ hybridization, permeability assays, ROS assays, transfection.

    Collaboration Partners:

    Guido Krenning, PhD, University Medical Center Groningen, University of Groningen, the Netherlands;
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

Workpackage 3 – Diabetic Nephropathy

  • The function of the Rac1 regulator ELMO1 in the vasculature and in renal development under high glucose in zebrafish and in human diabetic nephropathyOpen or Close

    Principal Investigator (Mannheim): Jens Kroll
    Principal Investigator (Groningen): Jan-Luuk Hillebrands
    Graduate: Krishna R. Sharma
    Start of PhD project: May 01, 2013

    Project description:

    Glucose-induced microangiopathy and nephropathy are diabetes-associated diseases. The Rac1 regulator ELMO1 is an important regulator of vascular development in zebrafish and polymorphisms in different regions of the human ELMO1 gene are associated with diabetes-associated nephropathy. The aim of this project is to analyze ELMO1’s function in the development of glucose-associated pathophysiological disorders in zebrafish and in diabetic patients. The project will include expression analyses in zebrafish and in diabetic patient samples; loss-of-function and gain-of-function experiments in zebrafish and biochemical studies in vitro.

    • Hassel D, Cheng P, White MP, Ivey KN, Kroll J, Augustin HG, Katus HA, Stainier DYR, Srivastava D. miR-10 regulates the angiogenic behavior of zebrafish and human endothelial cells by promoting VEGF signaling. Circ. Res. in press, 2012.
    • Urbich C, Kaluza D, Frömel T, Knau A, Bennewitz, K, Boon R, Bonauer A, Döbele C, Böckel JN, Hergenreider E, Zeiher AM, Kroll J, Fleming I, and Dimmeler S. MicroRNA-27a/b controls repulsion and angiogenesis by targeting semaphorin 6A in endothelial cells. Blood 119:1607-1616, 2012.
    • Stoll SJ, and Kroll J. HOXC9: A key regulator of endothelial cell quiescence and vascular morphogenesis. Trends Cardiovasc Med, 22: 7-11, 2012.
    • Jörgens k, Hillebrands JL, Hammes HP, and Kroll J. Zebrafish: A model for understanding diabetic complications. Exp Clin Endocrinol Diabetes, 120: 186-187, 2012.
    • Kaluza D, Kroll J, Gesierich S, Yao TP, Boon RA, Hergenreider E, Tjwa M, Rössig L, Seto E, Augustin HG, Zeiher AM, Dimmeler S, and Urbich C. Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin. EMBO J 30: 4142–4156, 2011.
    • Stoll SJ, Bartsch S, Augustin HG, and Kroll J. The transcription factor HOXC9 regulates endothelial cell quiescence and vascular morphogenesis in zebrafish via inhibition of interleukin 8. Circ Res 108: 1367-1377, 2011.
    • Jörgens K, Schäker K, and Kroll J. Der Modellorganismus Zebrafisch in der biomedizinischen Grundlagenforschung: Anwendungen und Perspektiven in der vaskulären Biologie und Medizin. Dtsch Med Wochenschr, 136: 1865-1868, 2011.
    • Epting D, Wendik B, Bennewitz K, Dietz CT, Driever W, and Kroll J. The Rac1 regulator ELMO1 controls vascular morphogenesis in zebrafish. Circ Res 107: 45-55, 2010.
    Methods that will be used:

    Expression studies and functional analyses in zebrafish; Imaging of zebrafish, Expression studies in human patient samples; Biochemical studies for Rac1 in cultured cells; Functional studies in cultured cells; Localization studies in cells. For further details see: and

    Collaboration Partners:

    Hans-Peter Hammes, Mannheim; Peter Nawroth, Heidelberg; Thomas Wieland, Mannheim; Hellmut Augustin, Mannheim/Heidelberg and Stefanie Dimmeler, Frankfurt.

  • Oxidative protein modification in diabetic nephropathy – friend or foe?Open or Close

    Principal Investigator (Heidelberg): Markus Hecker
    Co-PI (Heidelberg): Andreas Wagner
    PI (Groningen): Robert H. Henning
    Graduate: Tanja Wiedenmann
    Start of PhD project: January 01, 2013

  • Carnosinase in hyperglycemia: Influence on CN-1 secretion and relevance for damage to glomerular endothelial cellsOpen or Close

    Principal Investigator (Mannheim): Benito Yard
    Principal Investigator (Groningen): Jacob van den Born
    Graduate: Shiqi Zhang
    Start of MD project: January 01, 2013

    Project description:

    Serum carnosinase (CN-1) is a risk factor to develop diabetic nephropathy (DN). Both genetic factors and hyperglycemia influence CN-1 expression. This project underlies the hypothesis that hyperglycemia leads to epigenetic changes, i.e. methylation(s) of histones associated with the promoter of the CNDP1 gene, thereby influencing CN-1 transcription. Increased glomerular CN-1 expression subsequently counteracts the beneficial properties of carnosine through its degradation. The aims of the study are as follows: 1) to demonstrate that hyperglycemia provokes methylation of hisones associated with the promoter of the CNDP1 gene, 2) to demonstrate that this has functional consequences, 3) to understand why the rs2887 SNP in the 3’-UTR of CN-1 mRNA influences CN-1 expression, 4) to demonstrate the deleterious effect of CN-1 over-expression in endothelial cells on hyperglycemic damage. This project will make use of clinical epidemiological, molecular and cell biological studies.

    • Janssen B, Hohenadel D, Brinkkoetter P, Peters V, et al. Carnosine as a protective factor in diabetic nephropathy: association with a leucine repeat of the carnosinase gene CNDP1. Diabetes. 2005; 54 (8): 2320-2327
    • Riedl E, Koeppel H, Yard B et al. A CTG polymorphism in the CNDP1 gene determines the secretion of serum carnosinase in Cos-7 transfected cells. Diabetes. 2007;56(9):2410-2413.
    • McDonough CW, Hicks PJ, Lu L et al. The influence of carnosinase gene polymorphisms on diabetic nephropathy risk in African-Americans. Hum Genet. 2009;126(2):265-75.
    • Riedl E, Koeppel H, Yard B et al. N-glycosylation of carnosinase influences protein secretion and enzyme activity: implications for hyperglycemia. Diabetes. 2010;59(8):1984-90.
    • Brasacchio D, Okabe J, Tikellis C et al. Hyperglycemia induces a dynamic cooperativity of histone meythylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail.Diabetes 2009; 58 (5) :1229-1236
    Methods that will be used:

    In clinically well characterized contemporary cohorts of diabetic patients with and without DN (established in Mannheim and Groningen) and in aged matched healthy controls, mono-di- and trimethylated lysines at different positions of histone 3 (H3) will be assessed by ChIP and will be related to serum CN-1 concentrations by ELISA. In the Mannheim cohort all diabetic patients and controls have been genotyped for the (CTG)n allele and serum CN-1 concentrations at the time of blood collection are known. Genotyping of the rs2887 SNP will be performed. The 3’-UTR of CNDP1 will be screened in silico for putative miR target sequences. Subsequently the expression of identified miRs will be investigated in hepatocytes as well as the influence of hyperglycemia on their expression. Functional studies using a lucerferase reporter containing the 3’-UTR of CNDP1, real time qPCR and various cell culture models will be used.

    Collaboration Partners:

    Jaap van den Born, PhD, University Medical Center Groningen, University of Groningen, the Netherlands;
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

  • Diabetic nephropathy in the BTBR Ob/Ob mouse as a pre-clinical model for therapeutic interventionsOpen or Close

    Principal Investigator (Mannheim): Sibylle Hauske
    Principal Investigator (Groningen): Jacob van den Born
    Graduate: Thomas Albrecht
    Start of MD project: July 01, 2013

    Project description:

    The carnosine-carnosinase-system may play an important role in diabetes associated complications. Although the beneficial effect of carnosine on hyperglycemic damage has been well documented, in vivo evidence for positive effects of carnosine on renal pathology in diabetic nephropathy is still lacking. We hypothesize that diabetic nephropathy can be delayed or prevented by L-carnosine treatment. To test the hypothesis we will make use of the BTBR ob/ob model. This model offers an ideal opportunity to test the therapeutic efficacy of carnosine treatment because in this model glucose toxicity leads to progressive kidney damage which resembles the pathological changes of advanced human DN, i.e. podocyte loss, mesangial expansion, basement membrane thickening and interstitial fibrosis. The main aims of this project are 1) to test the efficacy of L-carnosine treatment on renal damage, 2) to assess whether carnosine prevents hyperglycaemic damage via reducing toxic metabolites and how this is accomplished and 3) to assess whether L-carnosine influences hyperglycaemic memory. Overall aim is identifying potential pathways by which carnosine counteracts glucose toxicity in renal tissue in a relevant preclinical model for DN.

    • Hudkins KL, Alpers CE et al. BTBR Ob/Ob mutant mice model progressive diabetic nephropathy. JASN. 2010; 21(9):1533-42
    • Janssen B, Hohenadel D, Brinkkoetter P, Peters V, et al. Carnosine as a protective factor in diabetic nephropathy: association with a leucine repeat of the carnosinase gene CNDP1. Diabetes. 2005; 54 (8): 2320-2327
    • Riedl E, Koeppel H, Yard B et al. A CTG polymorphism in the CNDP1 gen determines the secretion of serum carnosinase in Cos-7 transfected cells. Diabetes 2007; 56(9):2410-2413
    • Sauerhoefer S, Yuan G, Moeller MJ et al. L-carnosine, a substrate of carnosinase-1, influences glucose metabolism. Diabetes. 2007; 56(10):2425-32
    • Brasacchio D, Okabe J, Tikellis C et al. Hyperglycemia induces adynamic cooperativity of histone meythylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail. Diabetes. 2010; 59(8):1984-90
    • El-Osta A., Brasachio D, Brownlee M et al. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. JEM. 2008; 205(10): 2409-2417
    Methods that will be used:

    Diabetic animal models, renal histological analysis, immunofluorescence (confocal laser microscopy), affymetrix promoter arrays, expression analysis on PCR and protein base, immunohistochemistry, westernblotting.

    Collaboration Partners:

    Dr. Jacob van den Born, Laboratory of Experimental Nephrology, UMCG, University of Groningen;
    PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

Workpackage 4 – Diabetic Neuropathy

  • Altered cell-cell interactions in experimental diabetic neuropathy – Are they contributing to pain?Open or Close

    Principal Investigator (Heidelberg): Peter P. Nawroth
    Co-PI (Heidelberg): Thomas Fleming
    PI (Groningen): Reinhold B. Gans
    Graduate: Divija Deshpande
    Start of PhD project: March 01, 2013

    Project description:

    Activation of the transcription factor NFkB and subsequent proinflammatory gene expression contributes to pain and loss of pain perception in diabetic neuropathy. The receptor for Advanced Glycation Endproducts (RAGE) is central in regulating NFkB and diabetic RAGE-/--mice are partly protected from loss of pain perception. Since pain is not solely mediated by neurons, but can also be modulated by opioid releasing immune cells, recruitment of opioid containing cells by the Mac-1 counter receptors RAGE and ICAM-1 might act additively, synergistically or supplementary on the local control of inflammatory pain and further promote loss of pain perception as a consequence of sustained inflammatory responses.This project will seek to define whether recruitment of opioid releasing immunocytes modulates pain and promotes loss of pain perception in experimental diabetic neuropathy.

    • Machelska H et al. J Neurosci. 2002; 22: 5588-5596
    • Mousa SA et al. Brain Behav Immun. 2010; 24:1310-23
    • Frommhold D, Kamphues A, Hepper I, Pruenster M, Lukic IK, Socher I, Zablotskaya V, Buschmann K, Lange-Sperandio B, Schymeinsky J, Ryschich E, Poeschl J, Kupatt C, Nawroth PP, Moser M, Walzog B, Bierhaus A, Sperandio M, BLOOD. 2010 116: 841-849
    • Frommhold D, Kamphues A, Dannenberg S, Buschmann K, Zablotskaya V, Tschada R, Lange-Sperandio B, Nawroth PP, Poeschl J, Bierhaus A, Sperandio M., BMC Immunology. 2011; 12: 56-68
    • Chavakis T, Bierhaus A, Al-Fakhri N, Schneider D, Witte S, Linn T, Nagashima M, Morser J, Arnold B, Preissner KT, Nawroth PP, J. Exp. Med. 2003; 198; 1507-1515
    • Bierhaus A, Haslbeck KM, Humpert PM, Liliensiek B, Dehmer T, Morcos M, Sayed AA, Andrassy M, Schiekofer S, Schneider JG, Schulz JB, Heuss D, Neundörfer B, Dierl S, Huber J, Tritschler H, Schmidt AM, Schwaninger M, Haering HU, Schleicher E, Kasper M, Stern DM, Arnold B, Nawroth PP, J. Clin. Invest. 2004; 114: 1741- 1751
    Methods that will be used:

    Analyze of peripheral nerves and dorsal root ganglia (DRG) from healthy and diabetic C57Bl/6 wildtype, RAGE-/-, ICAM-1-/- and RAGE-/- x ICAM-1-/--mice.

    Study of the onset and progress of neuronal dysfunction by function assays (mechanical and thermal nociception using Hot-plate, Hargreaves, Tail-flick an Frey-filament assays), determination of motor and sensory nerve conduction velocity (NCV) and neuronal blood flow (NBF).

    Biochemical and molecular analysis of the expression and activation of RAGE, ICAM-1, NFkB-subunits, leukocyte-activation markers, ß-endorphins, opioid-receptors and genes involved in neuronal survival and destruction, as well as PGP9.5-positive neuronal cell bodies and Intraepidermal nerve fiber density (IENFD).

    Electrophoretic Mobility Shift Assays (EMSA) and Chromatin Immune Precipitation (ChIP)-assays for the binding activity of the NFkB-subunits p50, p65 and cRel and real time-PCR for NFkB -regulated gene products (ICAM-1, RAGE, MCP-1, IL1ß, IL-6 and MnSOD) from Sciatic nerves and DRG

    Collaboration Partners:

    Dr. Aimo Kant/Dr. Ralph Elvert at Sanofi , Prof. Hans-Peter Hammes, Dr. Jens Kroll

  • Diabetic thin fiber neuropathy - objective assessment and mechanismsOpen or Close

    Principal Investigator (Mannheim): Martin Schmelz
    Co-PI (Mannheim): Otilia Obreja
    PI (Groningen): Gerbrand J. Groen
    Graduate: Robin Jonas
    Start of PhD project: February 01, 2013

    Project description:

    Small fiber diabetic neuropathy is clinically important and advanced glycolysation end products (AGE) such as methylglyoxal have been suggested as its mediators. We hypothesize that methylglyoxal increases neuronal excitability in unmyelinated fibers. Excitability changes of single nociceptors upon acute methylglyoxal challenge will be assessed electrophysiologically in-vivo in the pig. This animal model provides axonal characteristics of unmyelinated nociceptor classes corresponding to human. In addition to suprathreshold responses to mechanical, heat and electrical stimuli also frequency dependent slowing of conduction velocity and conduction failure will be tested in C-afferent and sympathetic fibers. In a translational approach we will use corresponding stimulation protocols in diabetic patients with and without neuropathic pain in order to assess clinical neuronal hyperexcitability and its presumed relation to methylglyoxal levels in the patients.

    • Bierhaus A, Fleming T, … Brownlee M, Reeh PW, Nawroth PP. Methylglyoxal modification of Na(v)1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy. Nat Med 2012
    • Kalliomaki M, Kieseritzky JV, Schmidt R, Hagglof B, Karlsten R, Sjogren N, Albrecht P, Gee L, Rice F, Wiig M, Schmelz* M, Gordh T. Structural and functional differences between neuropathy with and without pain? Exp Neurol 2011;231:199-206.
    • Kleggetveit IP, Namer B, Schmidt R, Helas T, Ruckel M, Orstavik K, Schmelz* M, Jorum E. High spontaneous activity of C-nociceptors in painful polyneuropathy. Pain 2012;153:2040-2047.
    • Obreja* O, Ringkamp M, Namer B, Forsch E, Klusch A, Rukwied R, Petersen M, Schmelz* M. Patterns of activity-dependent conduction velocity changes differentiate classes of unmyelinated mechano-insensitive afferents including cold nociceptors, in pig and in human. Pain 2010;148:59-69.
    • Obreja* O, Ringkamp M, Turnquist B, Hirth M, Forsch E, Rukwied R, Petersen M, Schmelz* M. Nerve growth factor selectively decreases activity-dependent conduction slowing in mechano-insensitive C-nociceptors. Pain 2011;152:2138-2146.
    Methods that will be used:

    In vivo electrophysiology in animal models, human pain models, electrophysiology and psychophysics in patients

    Collaboration Partners:

    Prof. Dr. Gerbrand Groen, Pain Center, Dept of Anesthesiology UMCG, University of Groningen; Prof. Jorum, Dept. Neurology, University Oslo; Prof. Reeh, Dept. Physiology Erlangen; PIs of GRK 1874 DIAMICOM in Mannheim, Heidelberg, and Groningen

  • Identification of tissue specific changes within the nociceptive system in diabetic peripheral neuropathy – contribution of TRP channelsOpen or Close

    Principal Investigator (Mannheim): Rolf-Detlef Treede
    Co-PIs (Mannheim): Wolfgang Greffrath, Uta Binzen
    PI (Groningen): Jan-Luuk Hillebrands
    Graduate: Bastian Schlickenrieder
    Start of MD project: January 01, 2013

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