P5: NO-cGMP mediated effects on platelet lipid metabolism and their role in governing thromboinflammation in stroke
Aims
To characterize the association between cGMP levels in platelets and the platelet lipidome driving thromboinflammation during stroke in murine stroke models and cerebral stroke patients. Further, we would like to validate the potential therapeutic impact of NO donors and cGMP analogs in regulating the platelet lipidome and, thereby, thrombotic disposition and thromboischaemic events.
Questions and Methods
cGMP and Platelet Lipid Metabolism
- We will employ both targeted and untargeted lipidomic analysis of platelets from megakaryocyte-platelet lineage specific genetically deficient mice fed a high cholesterol diet (HCD). The platelet lipidomic profile at baseline will be compared with that of plasma cholesterol/ceramides/tryglyceride levels at the end of the HCD administration period and by co-investigating the respective control siblings.
- We aim to assess the impact of endogenous eNOS and cGMP signaling in platelets on cerebral blood flow (CBF), extent of cerebral injury and recovery following ischaemic stroke under hyperlipidaemic conditions utilizing mice with perturbed cGMP signaling in platelets.
- We will characterize platelet thrombotic functions in cell-specific mutants that lack different components of the cGMP pathway at strategic time points before and during HCD feeding. In parallel, haemostatic parameters will be measured. Together, these data will provide us with insight how endogenous NO and cGMP signals influence thrombotic and haemostatic platelet functions under hyperlipidaemic conditions.
- Finally, the project will translate experimental findings into clinical validation of our hypothesis that NO and cGMP can regulate the platelet lipidome to influence thrombo-ischaemic disposition in stroke patients. We will analyze clinical samples from stroke patients (progressive disease severity) at the University Clinic of Tübingen for genetic polymorphisms in NOS and NO-GC isoforms, NO generation (NOS activity, NO metabolites by biochemical assays and flow cytometry), stable NO metabolites (nitrite and nitrate), and cGMP levels (ELISA) in plasma.
Boston Internship
Atochin Lab
In the Atochin lab in Boston, the doctoral researchers will be taught the
- middle cerebral artery occlusion (MCAO) stroke model,
- state of the art technologies to measure cerebral blood flow (CBF), and
- functional analysis of platelets in the murine stroke models.
Supported by the long standing expertise of the Atochin lab, CBF will be evaluated under basal conditions and following stroke in mice with perturbed cGMP signaling in platelets as well as in eNOS-/- bone marrow chimeras fed different diets.
Boston Co-mentor
Doctoral Students
Marcel Kremser
Marcel Kremser earned his B.Sc. in Molecular Biology at the University in Bielefeld with a focus on cell biology. After earning his M.Sc. in Medical Biology in Essen, Marcel began his doctoral studies in the laboratory of Meinrad Gawaz. Here, his research focusses on the role of cGMP signaling in platelets and further deepens our understanding of cardiovascular diseases.
Valerie Dicenta
Valerie Dicenta studied Biotechnology at the University of applied Science in Esslingen. In her bachelor’s thesis she established a capillary electrophoresis mass spectrometry method for the characterization of the primary structure of therapeutic monoclonal antibodies at the Sanofi-Aventis Deutschland GmbH in Frankfurt. She completed her Master’s degree in Industrial Biotechnology at the University of Ulm and Biberach. In her thesis she focused on the inhibition of virulence associated proteases from prominent bacterial pathogens as a possible alternative to antibiotics. Valerie joined the group of Prof. Meinrad Gawaz working on the role of platelets and their signaling to understand cardiovascular diseases and to find possible ways for treatments.
Daniel Pinto Quintero (associated PhD student)
Daniel Pinto Quintero obtained his Bachelor's degree in Human biology from Phillips University in Marburg where he developed a special interest in tumor biology. During his Bachelor thesis, he investigated the aberrant expression pattern of GAPDH on salivary gland tumors.Following his interest in biomedical research, he then continued his Master studies in Human biology at the University of Marburg. For his Master thesis, Daniel was working in a radiation oncology laboratory investigating a putative detrimental role of serum amyloid A limiting the success of radiotherapy in head and neck cancer. Daniel currently focuses on the investigation of the relationship between shear stress and cGMP signaling in platelets. In the future, this might lead to improved treatment options for patients with blood clotting disorders (thrombosis).
Key Publications
Vogel S, Bodenstein R, Chen Q, Feil S, Feil R, Rheinlaender J, Schäffer TE, Bohn E, Frick JS, Borst O, Munzer P, Walker B, Markel J, Csanyi G, Pagano PJ, Loughran P, Jessup ME, Watkins SC, Bullock GC, Sperry JL, Zuckerbraun BS, Billiar TR, Lotze MT, Gawaz M, Neal MD. Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest. 2015;125:4638-54
Chatterjee M, Rath D, Schlotterbeck J, Rheinlaender J, Walker-Allgaier B, Alnaggar N, Zdanyte M, Müller I, Borst O, Geisler T, Schäffer TE, Lämmerhofer M, Gawaz M. Regulation of oxidized platelet lipidome: implications for coronary artery disease. Eur Heart J. 2017;38:1993-2005