Pharmacotherapeutic mechanisms, spectroscopic signatures, and nanomechanics of dysfunctional endothelium in liver steatosis and heart failure; towards organ-specyfic mechanisms
Funding: National Research Centre (funding agency), Program SYMFONIA
Prof. Stefan Chłopicki (JCET, UJ)
Prof. Malgorzata Baranska (JCET, UJ)
Prof. Marek Szymoński (Faculty of Physics, Astronomy and Applied Computer Science, UJ)
Impairment of endothelial function is a primary cause or a result of many human diseases. Although the mechanisms causing the primary endothelial dysfunction are known (i.e. those leading to the development and progression of atherosclerotic plaque), still there are many diseases in which the secondary endothelial dysfunction occurs, but underlying mechanisms remains unknown. Particularly a dysfunction of liver sinusoidal endothelial cells (LSEC) mediated by alterations in hepatocyte-derived paracrine signals is essential for the development of liver steatosis, while dysfunction of cardiac microvascular endothelial cell (CMEC) mediated by changes in cardiomyocyte–derived paracrine signals is an important determinant of the development of heart failure. We hypothesize, that secondary endothelial dysfunction that develops in liver steatosis, is mediated by a decrease in the activity of hepatocyte-derived VEGF signaling that may be associated with increased sFLT activity, and results in impaired LCES-derived NO production. In turn, we claim that in the heart failure alterations in cardiomyocyte-derived PDGF signaling and excessive activity of myocardial angiotensin II-aldosterone pathway results in the impairment of NO production in CMEC. The aim of the project is to understand the signals that governs intracellular communication between hepatocytes and LSECs and between cardiomyocytes and CMECs, since these microvascular bed-specific mechanisms determine the phenotype of the endothelium in an organ-specific way and may constitute the pathophysiological basis for the hepato-selective and cardio-selective therapy of endothelial dysfunction.
In the presented project we aim to examine the progression of changes of various aspects of endothelial phenotype (structure, function, elasticity, glycocalix, biochemical profile, vasoprotective, pro-thrombotic and pro-inflammatory mechanisms) in LSECs in the course of the development of liver steatosis in mice (induced with high-fat diet), and in CMECs in the course of the development of heart failure in Tgg*44 mice in both cases in the relation to the comprehensive description of the development of these pathologies. In particular, we aim to investigate the role of intracellular signaling homeostasis depending on a) VEGFNO in the liver, and b) PDGF/Aldosterone –NO in the myocardium in the development of the secondary endothelial dysfunction in the above mentioned diseases.
Prostacyclin, nitric oxide and carbon monoxide-based pharmacotherapy of endothelial dysfunction and platelet activation-a novel strategy to inhibit cancer metastasis
Funding: Polish Strategical Framework Program STRATEGMED
Jagiellonian University in Kraków – JCET (project leader)
Medical University of Łódź;
Medical University of Gdańsk;
Medical University of Białystok;
Institute of Immunology and Experimental Therapy of Polish Academy of Sciences in Wrocław
Latvian Institute of Organic Synthesis
Our own preliminary studies indicate that endothelium-targeted pharmacotherapy bears potential to inhibit cancer metastasis. Aims of this project are:
- to characterize the endothelium related pharmacotherapeutic mechanisms that throught its vasoprotective, anti–platelet action may provide the novel strategy for prevention of cancer metastasis;
- to develop a novel rationally-designed, endothelium-targeted compounds effective in preventing of endothelial dysfunction, displaying anti–platelet, anti-thrombotic activity and thus inhibiting cancer metastasis, and finally
- to develop the novel panel of biomarkers of endothelial dysfunction, platelet activation and cancer metastasis, and its application to assess the efficacy of endothelium/platelet targeted pharmacotherapy in prevention of cancer metastasis in mice as well as its validation in humans to assess its usefulness as markers of metastasis.
This project will develop endothelium-targeted innovative pharmacotherapy for cancer metastasis. We will provide extensive preclinical dossier for novel molecules with endothelium-based anti-metastatic properties and prove their mechanisms of action, efficacy, profile of pharmacokinetics and safety.
The consortium of 6 partners (5 from Poland and one foreign), represents dynamic and interdisciplinary group with complementary research expertise.
In search of biochemical, mechanical and functional fingerprints of oxidative stress in RBCs
Funding Agency: National Science Centre, Poland, Program: OPUS
Project leader: Katarzyna Marzec, Ph.D.
The reactive oxygen species (ROS), reactive nitrogen species (RNS) as well as other free radical species may have damaging impact on our bodies on the cellular level, however their production is also an integral process necessary for many metabolic reactions. Blood play a special role in oxidative stress mechanism, as it is not only a connective tissue which supply oxygen and nutrients to tissues but also removes many wastes including oxidative species. Therefore all blood cells are exposed during oxidative stress to oxidative environments rich in ROS, NOS and free radical species. The foundation of the project is to investigate RBCs alternations. This is untypical approach, as previous studies were mostly focused on other blood fractions, mainly white blood cells. We believe, that biological understanding of biochemical changes due to oxidative stress in situ in isolated RBCs as well as RBCs in whole blood could expand or knowledge about oxidative stress action. RBCs exhibit oxidative damage through characteristic changes in the biochemical content of the hemoporphirins and cell membrane, the size and shape of cell morphology as well as mechanical properties such as for example stiffness and functional parameters. All those changes have impact or are connected with changes in other blood fractions and general blood properties like increased
blood viscosity and impaired flow.
In this project we focus on comprehensive assessment of oxidative stress in red blood cells (RBCs) with the use of innovative technology. The use of combination of Raman spectroscopy (RS), Fourier transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM) supported with referenced techniques (UV-Vis, biochemical parameters, stainings, blood analysis and ektocytometry) will provide the knowledge about biochemical, mechanical and functional fingerprints of level of oxidative stress in RBCs. The recognition of such fingerprints due to oxidative stress in situ in RBCs can be a biomarker of general oxidative stress status. Project will allow to create the innovative methodology for sensitive and fast diagnosis of the oxidative stress level in RBCs which will be tested to be apply to in vitro/ex vivo description of oxidative stress level in RBCs obtained from mice models of different diseases of affluence. Such diseases as atherosclerosis, or diabetes share etiologic pathways wherein oxidative stress plays the contributing role.
To the main aims of the project we may include:
- In situ characterization of oxidative stress chemically induced in isolated RBCs from healthy mice with the focus on haemoglobin structure; alternations of the lipids and proteins inside membranes of RBCs; mechanical properties of RBCs such as stiffness, topography, adhesion; functional properties such as deformability and aggregation; biochemical content of the macrovesicles produced by RBCs in oxidative stress conditions.
- Definition and description of the possible biochemical, mechanical and functional fingerprints of oxidative stress in RBCs will be carry out depending on type of chemically induce oxidative damage/pathology, type of RBCs and comparison between results obtained for isolated RBCs versus whole blood.
- Selection and testing of chosen fingerprints obtained with the use of IR or Raman or AFM for in vitro/ex vivo detection of the oxidative stress level in RBCs obtained from the different mice models of human disease of affluence which share etiologic pathways wherein oxidative stress plays the contributing role.
We believe that the planed in our studies use of Raman, IR and AFM techniques allows not only for detection of oxidative stress level of RBCs but maybe also a potential new set of techniques for detection of some RBCs alternations in the first place. Raman technique uses the advantages of immersion confocal spectroscopy as well as provides information about the biochemical changes of the sample on the molecular level. IR has additional advantage of not only detection but also easy quantification of many biochemical changes. It was already shown that both, IR and RS have huge diagnostic potential also in RBCs studies. If specific molecular change of RBCs has specific marker bands the diagnosis could take even up to several second what gives additional possibility of the fast in vitro/ex vivo determination of the oxidative stress in RBCs. AFM can provide a mechanical fingerprint of a single-cell pathological changes also for RBCs what makes this a promising technique in cell alternation diagnosis. Moreover, those techniques allow for an analysis of the molecular and mechanical changes in a single RBC in small quantity of blood without previous staining or fixation. Obtained data (the set of Raman/IR/AFM results) which keep the molecular information, can be easily stored for future analysis.
It is expected that the new methodology used in this project will be developed for characterization of the oxidative stress level in RBCs. Results of the project will give physical output such as formation of unique research group which uses new applications of imaging biospectroscopy and AFM to new concept of RBCs investigation. Moreover, results will be published in international journals with a high Impact Factor, present on international and Polish conferences. The project contribution to a higher level goal is creation of the methodology of RS/IR/AFM diagnosis of the oxidation damage in RBCs which can be apply in studies on animal models of different human diseases of affluence.
Diagnostics of the biochemical alterations in packed human red blood cells (PRBCs) with the use of vibrational spectroscopy
Funding Agency: National Centre for Research and Development “Lider VIII”
Project leader: Katarzyna Marzec, Ph.D.
A large number of patients worldwide are in need of periodic, therapeutic transfusions of packed red blood cells (PRBCs). This has led to an increased demand for an efficient technology providing information on the PRBCs alterations, which may affect their quality. The goal of this project is the development of the spectroscopic methodology for the Raman (RS) and Infrared (IR) diagnosis of the pre-transfusion quality of RBCs. In this project we are planning to study PRBCs fractions with the focus on biochemical changes of RBCs due to donor characteristics, age and characteristics of PRBCs, level of oxidative stress and damage in RBCs (steps I and II). RS and IR spectroscopies will provide fast determination of the RBCs condition before transfusions. To understand the biochemistry behind all changes observed by IR and RS, the RBCs samples will be studied also by other classical techniques. We can include here absorption spectrophotometry, blood gas analysis, flow cytometry, atomic force microcopy, ektocytometry, staining’s and the measurement of the chosen biochemical parameters. The focus on comprehensive assessment of PRBCs will provide the knowledge about biochemical fingerprints of RBCs condition. The last step of project (step III), which goal is an evaluation of the obtained SPECDIAG-RED methodology, will be focused on the studies of previously chosen spectroscopic biomarkers of biochemical state of PRBCs for a specific group of patient (patients with acute myeloid or/and acute lymphoblastic leukemia). The project will not affect the medical procedures performed routinely in hospital as the IR/RS measurement will be carry out on the small sample of PRBCs (1 mL) or by the direct measurement through the PRBCs wall. The PRBCs state will be assessed with the use of SPEC-DIAG-RED methodology and combine with the feedback from the clinics about patients condition before and after transfusion what will allow for the final evaluation of results.
Studies on antiplatelet and antithrombotic activity of CO-relasing molecules (CO-RM)
Funding: National Research Centre (funding agency), Program HARMONIA
Partner: Dr Roberto Motterlini (University Paris-Est, INSERM U955, Faculty of Medicine)
Purpose of research / hypothesis Carbon monoxide (CO) has significant vasodilator, antiplatelet and antithrombotic action, but the mechanism underlying the pharmacological activity of CO still remains unexplored. Until now it was impossible to separate the antiplatelet and antithrombotic effects of CO with vasodilator and toxic effects of CO. Our previous studies conducted in collaboration with Dr. Motterlini team showed that the release kinetics of the CO donor has a significant impact on the anti-platelet and anti-thrombotic effects of CO. These results constitute the background for the current collaborative project.
Scientific objectives of the project is to investigate the kinetics of release of CO from the CO releasing molecules in terms of its regulation by the oxidative stress, to characterize the effects of novel CO-RMs on platelets and endothelial cells in normoxia and hypoxia with particular emphasis on the mitochondria mechanisms and the role of guanylate cyclase in the pharmacological activity of CO. Furthermore, our aim is to examine the antithrombotic properties of CO-RMs, in the classical model of arterial thrombosis in the rat and in the state-of the art thrombosis model with confocal imaging in mice.
These studies should help not only to better understand the antiplatelet effect of carbon monoxide, but also may allow to point out a possible molecular target of CO in platelets.
Vascular endothelium in life-style diseases: from basic research to the offer of an innovative endothelium-targeted therapeutic
Funding: European Regional Development Fund, Operational Program – Innovative Economy, Action 1.1.2
Jagiellonian University in Kraków – JCET (project leader) 7 research teams from the following faculties: Faculty of Medicine; Faculty of Pharmacy; Faculty of Chemistry; Faculty of Biochemistry, Biophysics and Biotechnology; Faculty of Physics, Astronomy and Applied Computer Science;
Lodz University of Technology;
Nuclear Physics Institute of Polish Academy of Sciences in Kraków;
University of Agriculture in Kraków;
University School of Physical Education in Kraków;
Medical University of Łódź;
Nencki Institute of Experimental Biology of Polish Academy of Sciences in Warsaw;
Medical University of Gdańsk;
Medical University of Białystok;
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of Polish Academy of Sciences in Wrocław
Project period: 01.03.2009—30.06.2015
Project Budget: 63,5 mln PLN
The aim of the project is to carry out interdisciplinary research and development work on the vascular endothelium and its role in the development of lifestyle diseases, and, in particularly, development of original interdisciplinary research on the role of endothelium in the development of lifestyle diseases, with a particular interest in the new area of this knowledge; Development of interdisciplinary studies on synthesis,mechanisms of action and therapeutic and pharmacokinetic properties of the original chemical compounds (natural and synthetic) affecting the endothelium, which may be the basis for new therapies of lifestyle diseases; Tightening the collaboration between research units and the industry in the field of endothelial studies. The strategic aim of the project is the development of novel research strategies in the field of assessment endothelial dysfunction, as well as development of original, interdisciplinary studies on endothelium-targeted therapeutics including:
- a development of a unique specialized technology of a comprehension assessment of endothelial dysfunction and panel of assays for detection of endothelial activity of chemical compounds;
- an offer of innovative endothelium-targeted therapeutic.
Project fits into the main aim of the Operative Program – Innovative Economy, which is undertaking the actions assuring the increase in the competitiveness of Polish science and in a further perspective also the increase of the innovation of the industrial companies.
Jagiellonian Centre for Experimental Therapeutics – JCET
Funding: European Regional Development Fund, Operational Program – Innovative Economy, Action 2.2
Jagiellonian University in Kraków – JCET (project leader)
Lodz University of Technology
Henryk Niewodniczański Nuclear Physics Institute of Polish Academy of Sciences in Kraków
Project period: 02.01.2008—30.09.2012
Project Budget: 48 mln PLN
The main aim of the project is to create a modern research infrastructure by creating laboratories of the Jagiellonian Centre for Experimental Therapeutics (JCET) at the Jagiellonian University and equipping and modernization of the rooms of the Laboratory of Chemical Synthesis of the Lodz University of Technology as well as purchase of MRI for in vivo animal imaging and modernization of the rooms necessary for the equipment installation in the In vivo Imaging Laboratory of the Nuclear Physics Institute of Polish Academy of Sciences in Kraków.
Jagiellonian University has bought, within the project, research equipment worth approximately 22 million PLN; moreover JCET has allocated approximately 5.5 mln PLN for JCET laboratory adaptation. Lodz University of Technology acquired research equipment worth 3 mln PLN and already adapted the starting the Laboratory of Chemical Synthesis in the Institute of Radiation Technology of the Faculty o Chemistry. Nuclear Physics Institute of Polish Academy of Sciences in Kraków has bought the MRI equipment for almost 10 mln PLN and adapted rooms for In vivo Imaging Laboratory in the Department of Magnetic Resonance Imaging, necessary for setting-up the modern MRI equipment.
The purchase of research-scientific equipment made within the project executed by the consortium of units under the supervision of the Jagiellonian Centre for Experimental Therapeutics (JCET) was funded by structural fund of the European Union within European Regional Development Fund from the Operative Program – Innovative Economy.We invite research groups from national and international research units and companies interested in JCET offer to collaborate and take the opportunities to use available equipment and unique expertise of JCET research teams.