Main Research Projects

Jagiellonian Centre for Experimental Therapeutics – JCET

Funding: European Regional Development Fund, Operational Program – Innovative Economy, Action 2.2
Partners:

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.

The role of nicotinamide N-methyltransferase and mitochondrial activity during endothelial dysfunction

Funding: National Research Centre (funding agency), Program OPUS
Project leader: Prof. Krzysztof Olaf Zabłocki (Nencki Institute of Experimental Biology Polish Academy of Sciences)
Copartner: Prof. Stefan Chłopicki (JCET, UJ)

mong many possible factors maintaining the healthy endothelial phenotype, the activity of nicotinamide N-methyltransferase (NNMT) still needs further examination. NNMT catalyses nicotinamide (NA) methylation, releasing 1-methylnicotinamide (MNA), which anti-inflammatory, anti-thrombotic and vasoprotective action has been reported in many animal models. Although beneficial effect of MNA treatment has been demonstrated in several studies, it is still little known about the role of endogenous MNA in dysfunctional endothelium, where NA methylation by NNMT seems to be linked with cellular bioenergetics. In our preliminary studies we demonstrated the upregulation of NNMT in endothelial cells stimulated with TNFα. According to the hypothesis, activation of endothelial NNMT stabilizes NAD- dependent sirtuins, histone deacetylases involved in genome expression and stability. The main aim of this project is to examine the role of NNMT and endogenous MNA during endothelial insulin resistance and endothelial dysfunction, triggered by different stressors: oxidative, metabolic, hypoxic and genotoxic. This project could provide necessary data to verify the hypothesis about vasoprotective, MNA-dependent effect of NNMT on mitochondrial sirtuin 3 and nuclear sirtuin 1 regulation, which is probably linked to biogenesis of mitochondria. The second aim of the project is to examine the cross-talk between NNMT activity, mitochondrial metabolism and endoplasmic reticulum, as well as potential MNA protective effect on above-mentioned organelles.

In search for endothelial mechanisms of TPO-induced neuroprotection

Funding: European Commission, Research Executive Agency, HORIZON2020, TONEURENDO
Project leader: Mateusz Adamski, PhD

Ageing is recognized as one of the greatest social and economical challenges of the 21century for European societies. With,,Vascular Cognitive Impairment (VCI) being one of the leading causes of age-related cognitive impairment and one of the major causes of disability in the elderly. Although, the concept of VCI was introduced in 1993, current treatment is limited to management of vascular risks and symptomatic  harmacotherapy targeting vascular dementia.

The overall objective of this project is to fill the significant gap in early detection, prevention and treatment of VCI. This will be achieved by explaining microvascular mechanism of protective effects of  hrombopoietin (TPO) in a novel unique mixed-risk animal model of VCI- specific to hypertension plus carotid-artery hypoperfusion (HH-VCI).

Given increased number of progenitor endothelial cells after TPO treatment, it is hypothesizes that protective effect of TPO is mediated by endothelium. Furthermore, protective effect of TPO is expected to be caused by activation of neoangiogenesis, anti-inflammatory and vasoprotective mechanisms driven by TPO action on endothelial. This hypothesis will be tested in two stages first, in-vitro and second, in-vivo. In-vitro models, will be used to investigate endothelial response to TPO in terms of its modulation of inflammatory response angiogenic potential and vasoprotective mechanisms. In-vivo models of TPOR KO mice and HH-VCI mice will be used to validate and confirm mechanisms identified in in vitro stage of experiments.

Induced chirality in “guest-host” supramolecular assemblies – developement of new methods of resonance Raman Optical Activity

Funding Agency: National Science Centre, Poland, Program: OPUS 13
Project leader: Agnieszka Kaczor, PhD, D.Sc. (JCET, UJ)

The project is devoted to studies of chirality induction in supramolecular ”host-guest” aggregates using chiroptical methods, in particular Raman Optical Activity (ROA) and Vibrational Circular Dichroism (VCD). The aim of the project is to synthesize and characterize supramolecular ”host-guest” systems of different structure and applications, but designed in such a way that they exhibit resonance ROA and enable induction of chiral information in these systems. Analysis of “guest-host” interactions and conformational effects in generated aggregates will be undertaken in order to propose optimal ”guest” and ”host” molecules, designed to create universal and efficient systems for inducing chirality, and thus the possibility of indirect detection of achiral molecules. Development of methods of resonantly enhanced Raman Optical Activity is a side, nonetheless relevant effect of the study. Analysis of mechanisms of chirality induction can become crucial in designing chiral sensors and understanding of chiral recognition processes. Thus, the submitted project brings significant cognitive potential and can provide a conceptual basis for application of supramolecular entities in controlled transfer of chiral information.

To catch lifestyle diseases red-handed – in vivo Raman spectroscopy

Funding Agency: National Science Centre, Poland, Program: OPUS 9
Project leader: Agnieszka Kaczor, PhD, D.Sc. (JCET, UJ)
Participants: Marta Pacia, Krzysztof Czamara, Ewelina Bik, Zuzanna Majka

Raman spectroscopy is successfully used to study biochemical changes in tissues and cells in in vitro and ex vivo models. These models, although undoubtedly informative, are based on several assumptions, for instance negligibility of the lack of the “tissue environment” (in case of in vitro cellular models), negligible influence of fixation procedures, transferability of conclusions based on studying animals on humans or various other limitations of the model systems that are often highly homogenous, gene knock-out or simplified, one-factor activated models. All these limitations can be eliminated with the application of the of fiber optic probes and in vivo approach, in which the tissue of interest is studied in the living organism. According to my best knowledge, in vivo Raman spectroscopy is not developed in Poland, although it is the method tested since a decade or two in studies of various pathologies. In the light of growing risk of various diseases of affluence, application of in vivo Raman spectroscopy to study these pathologies is an important factor that may help to contribute to their prevention. Therefore, the objective of this project is the application of the Raman fiber optic probe system to investigate and characterize biochemical changes in the organisms in vivo (in real physiological environment) upon development and progression of chosen lifestyle diseases.

Development of EPR oximetry methodology for evaluation of endothelial action of drugs

Funding: National Research Centre (funding agency), Program SONATA 3
Project leader: Patrycja Kaczara, PhD

The project is planned for original studies of perivascular adipose tissue The main goal of the project was to develop a methodology for measurements of endothelial cells respiration. It was necessary for better understanding of mechanisms, which underlie oxygen consumption and production of intracellular energy versus generation of reactive oxygen species, which can participate in variety of signaling processes, beneficial or detrimental for the cell. Endothelial cells are postulated to cover majority of energy demands not by oxidative phosphorylation in electron transport chain, but by aerobic glycolysis. These bioenergetics processes may be helpful in evaluation of endothelial cell activity in physiological and pathological conditions under treatment with newly developed pharmacological compounds.

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:

1. 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.
2. 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.
3. 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.

See more: Red Blood Cells Spectroscopy Research Team

Structural studies and chemical imaging of vitamins A, E and their metabolites in healthy and pathologically altered animal tissue

Funding: National Research Centre (funding agency), Program SONATA 4
Project leader: Katarzyna M. Marzec, PhD

The project was focused on finding the spectroscopic differences (fingerprints, connected with different molecular structures) between the title vitamins A, E and their metabolites. The structural studies were carried out on the basis of various spectroscopic techniques (infrared (IR) and Raman (RS) spectroscopy) as well as theoretical vibrational analysis based on DFT calculations. This allowed first recognizing and imaging the compounds in situ in the biological samples and second, location and study of the distribution of the title compounds in situ inside tissues of healthy and altered mice obtained from different organs such as liver, lungs, aorta, kidneys, brain and sight organs. The results were published in six manuscripts and presented in the form of nine poster presentations.

Design of SERS nanosensors for ex vivo detection of inflammatory state in blood vessels

Funding Agency: National Science Centre, Poland, Program: OPUS 11
Project leader: Kamilla Malek, PhD, D.Sc. (JCET, UJ)

Optical chemical- and bio-sensing is one of the most promising directions of diagnostic sensors development nowadays, offering advantages of high sensitivity, combined with non-invasive nature of analysis, portable devices suitable for in-point care analysis. The principle aim of the current project is to develop novel sensitive detection technologies combining a highly selective immunoassay approach with unique spectral signatures of Surface Enhanced Raman Spectroscopy (SERS) for detection of markers of inflammatory state. SERS is a highly sensitive Raman detection technique based on gold nanostructured substrates fabricated in chemical reaction (nanoparticles like nanostars, nanoshells) and in processes like anodization and photoreduction giving periodic in shape nano-sized structures. SERS has far-reaching applications in fields as diverse as biomedical diagnostics and biochemical studies and shows great potential for multiplex analysis, making it particularly suitable for the simultaneous diagnosis of multiple disease markers.

The general objectives of the project is to design two types of biosensing assays to detect ex vivo inflammatory vascular disease in tissue cross-section and blood plasma. One of them will provide multiplex staining combined with imaging of few antigens in arteries in atherosclerotic plaque while the second SERS biosensor will be a sandwich-type bioassay for quantitative detection of pro-inflammatory cytokines in blood plasma. Effectiveness and function of designed sensors will be validated in murine models expressing vascular inflammation (lipopolysaccharide-induced endotexia – LPS model and atherosclerosis – model ApoE/LDLR-/-) which perfectly reproduce human diseases. Their choice is dictated by the fact that elevated levels of several inflammatory mediators among apparently healthy men and women have proven to have predictive value for future vascular events. Actually, inflammation is a beginning stage of almost all diseases, including heart attack, stroke, cardiovascular complications of diabetes, cance, etc. The planned research are multidisciplinary linking of nanotechnology, extremely sensitive spectroscopic technique and medical technology supported by three groups of specialists in the field of the project.

The motivation of this research is a continuing need to develop advantages of SERS biosensing and solve pitfalls limiting translation of this diagnostic technology into clinics. The main beneficiaries of the project results will be nanotechnologists, spectroscopists and clinical scientists who will receive a potential novel tool and deeper insight into designing new ones for  nvestigation of biological systems of clinical significance. It seems, therefore, that the project is innovative because of the technology used and the object of study. Due to the fact that this kind of research is pioneering in Poland it should be highlighted that the project will have an impact on our country’s growing importance in world-wide biomedical research.

Low Dose Research towards Multidisciplinary Integration (DoReMi)

Funding: European Commission, Research Executive Agency, 7PR
Project leader: Prof. Stefan Chłopicki (JCET, UJ)

Coronary artery disease and atherothrombosis are preceded by and associated with the development of endothelial dysfunction that promotes vascular inflammation and thrombosis. It has been demonstrated that
exposures at low doses of low-LET radiation during various stages of atherosclerosis protect against the progression of the disease in genetically-modified mice. Recently, it has been demonstrated that exposures to low-level low-LET ionizing radiation attenuate the development and progression of various diabetic complications such as nephropathy, vascular and cardiac inflammation, inhibited proliferation of neurons, and impaired wound healing. Since, in contrast to high-dose exposures, irradiations at low doses of Xor γ-rays have been shown to stimulate anti-oxidant functions in various cells and tissues, it can be hypothesized that such beneficial effects of the low-level exposures result from the radiation-induced suppression of the oxidative damage to the endothelial lining of micro- and macrovasculature. Thus far, no direct evidence has been provided on the effects of low-level exposures to ionizing radiation on the development or reversal of the impaired function of endothelium in diabetes.
We presume that endothelial effects of low doses of low-LET radiation will afford protection against atherothrombotic complications of diabetes. The scientific objectives of the proposal include evaluation of the effects of low doses of X-rays on the structure and function of vascular endothelium in mice before and during the development of hyperglycaemia – a cause of the vascular complications in diabetes.

Towards experimental pharmacology of endothelium in vivo based on multiparameter method of assessment of the vascular endothelial function in mice with the use of MR imaging techniques

Funding: National Research Centre (funding agency), Program PRELUDIUM 12
Project leader: Anna Bar, PhD

Endothelial dysfunction accompanies development of many disease, not only diseases of the cardiovascular system and has pathophysiological, prognostic and therapeutic significance. Therefore, development of fast and reliable methods of assessment of endothelial phenotype in vivo, in mice models, constituting a basic model in preclinical studies, is essential for the better understanding research development on of the role of endothelial cells in the progression of disease and on the efficacy of novel research on the pharmacotherapeutic mechanisms in the treatment of endothelial dysfunction in vivo.

The aim of this study is: firstly, to develop and validate a sensitive method for endothelial imaging with using magnetic-resonance techniques (MR) in vivo, allowing for an effective assessment of impaired, endothelial-dependent vasodilatation response and permeability of endothelium, which are the main features of endothelial dysfunction; and secondly, to use the developed methodology to assess the effects of various endothelial pharmacotherapies in murine model of atherosclerosis (ApoE/LDLR-/- mice).

The novel methodology developed in this project, will allow for a comprehensive assessment of the main features of endothelial dysfunction in murine models in vivo, including evaluation of: a/ endothelial-dependent response to Ach in various vessels available for MR imaging, b/ FMD in mice, contributing to the development of preclinical studies of pharmacotherapeutic mechanism of endothelium, based on the technique used in humans, opening the way for translational research, c/ changes in endothelial permeability using a unique formulation of gadolinium contained in a liposome, which is a significant addition to assessment of the vasodilatation response of endothelium. Performed assessment of endothelial dysfunction progression in ApoE/LDLR-/- mice, and the effect of ACE-I and PPI and vitamin K2 on endothelial function in vivo, in comparison to the EPR measurements, which are the most sensitive method for disorders of NO production detection in a blood vessel ex vivo, altogether will open up a new perspectives for research on pharmacotherapy of endothelium and on profiling of drugs effects on endothelium in murine models in vivo.

To sum up, on the one hand the objective of this project is to develop and validate new and a unique methodology for measuring the phenotype of endothelial cells in vivo using MR imaging and on the other hand to characterize mechanisms of the vitamin K2 effects on the endothelium function in murine models of atherosclerosis in vivo.

Description of platelets role in metastasis and evaluation of anti-platelet treatment efficacy in mice model of metastatic breast cancer

Funding Agency: National Science Centre, Poland, Program: PRELUDIUM
Project leader: Elżbieta Buczek, M.Sc.,  Ph.D. student

The main purpose of the project is to investigate the role of platelets in the development of metastases in the lungs in experimental model of murine 4T1 mammary adenocarcinoma. This project allows for a comprehensive assessment of changes in platelet activation and reactivity in mice at different stages of metastases development using three complementary methods:

• a flow cytometry to examine platelet activation markers
• evaluation of thromboxane B2 generation in whole blood ex vivo
• assessment of the platelet adhesion and aggregation.

It is also relevant to prove that 4T1 breast cancer is a proper model to test antimetastatic potential of new substances. It is planned to assess the efficacy of antiplatelet therapy with different mechanisms of action for inhibition of the development of primary tumor and metastasis in this model.

Aptamer-based biosensor for simultaneous detection of thrombin and von Willebrand factor in plasma

Funding: National Research Centre (funding agency), Program PRELUDIUM 11
Project leader: Katarzyna Derszniak, PhD Student

The aim of the project is to develop an aptamer-based biosensor, with aptamers as molecules for capturing ligands, for the simultaneous detection of von Willebrand factor and thrombin in biological samples, including plasma. It turns out, that the detection of disorders of the coagulation system may help to predict the development of both atherosclerosis and cancer diseases. Thrombin, as a key component of the coagulation system, favors the development of atherosclerotic plaque and has influence on rising risk of thrombo-embolic incidents which are complications of atherosclerosis. It can also stimulate the proliferation of vascular endothelial cells and tumor cells. What is more, thrombin also promotes intravascular clotting and tumor-cell – platelet interactions. Moreover, it can cause that the endothelial cells are more sensitive to the action of Vascular Endothelial Growth Factor (VEGF). It leads with increasing of endothelial cells mitosis and cell migration. The occurrence of Venous Thromboembolism (VTE) among cancer patients is further evidence of the correlations between dysfunction of coagulation system and cancer diseases. One of the identified risk factors for VTE among cancer patients is hypercoagulable state, which results from increased thrombin generation. The von Willebrand factor is an another important component of the coagulation system which is released by activated endothelial cells, promoting the development of thrombosis and tumor metastasis. Therefore, developing a method to study the condition of the coagulation system is important to assess the state of cancer development and cardiovascular pathologies.

Aptamers in the study of thrombotic mechanisms dependent on platelets and erythrocytes functions by microfluidic devices

Funding Agency: National Science Centre, Poland, Program: ETIUDA 8
Project leader: Katarzyna Derszniak, PhD Student

The mechanism of erythrocytes and platelets adhesion to the vessel wall will be studied by the application of aptamers and the microfluidic vascularized 3D platform formed by selfassembling cells and developed by the team of Professor Christopher C. W. Hughes (University of California, Irvine, USA).

Resonance Raman spectroscopy studies of the in vitro and ex vivo haemoglobin adducts with gasotransmitters in biological systems

Funding: National Research Centre (funding agency), Program PRELUDIUM 12
Project leader: Jakub Dybaś, PhD

The aim of the project is to study an influence of endothelial gaseous mediators, nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) on porphyrins in various human- and murine-derived biological samples. Especially, the interaction between the iron ion in hemoglobin of red blood cells and NO, CO and H2S will be reevaluated, as well as with detection of the formed Hb-X adducts, which could have a potential importance in these gasotransmitters bioavailability. The principal assumption of the project is to carry out experiments ex vivo (whole human blood and isolated human red blood cells, murine isolated Kupffer cells) and in vitro (murine macrophage cell line) on biological samples instead on standard compounds. Due to the used methods (resonance Raman spectroscopy and spectrophotometry UV-Vis), it will be possible to deeply inquire into the changes both on molecular level and on quite bigger, macroscopic scale of studied systems. All the measurements will be done in pH and temperature controlled environment to the best reproduction of physiological conditions.

Obtained results should shed additional light towards better understanding the role, which formed adducts plays in the context of NO, CO and H2S bioavailability.

Molecular spectroscopy studies on chosen hemoprotein adducts and their changes in biological systems

Funding: National Research Centre (funding agency), Program ETIUDA 5
Project leader: Jakub Dybaś, PhD

As a part of this project, Jakub spent 6 months in Saint Louis University (SLU), where his main aims were to enhance knowledge about resonance Raman (rR) application to heme studies and to spectroscopically characterize cytochrome c (Cc) with the special attention on rR spectral response to changes in oxidation and spin states marker band positions as well as detection and differentiation of the modes arising from the peripheral substituents with the use of various excitation wavelengths.During this internship, Jakub performed also rR measurements of myoglobin, hemoglobin andcytochromes c adducts with isotopically labeled O2, CO, NO and CN– ligands to deeply inquire their molecular structure and biophysical properties. He was trained in use of highly specialized Raman instrumentation containing a high resolution 1250 M spectrometer (Horiba Scientific) equipped with a Princeton Instruments liquid nitrogen cooled array detector (PYLON CCD, Princeton Instruments), a Coherent Innova 300C Kr+ ion laser and a KimmonHe:Cd laser, as well as all necessary optics, including holographic Notch filters covering many laser lines in the range of 350 to 440 nm.

Bioanalitycal and pharmacological studies on the compensatory mechanism dependent on MNA/COX-2/PGI2 pathway in NO-deficient mice

Funding Agency: National Science Centre, Poland, Program: PRELUDIUM 9
Project leader: Agnieszka Kij, M.Sc. 

The goal of this project is to assess the compensatory role of MNA/COX-2/PGI₂ pathway in the development of endothelial dysfunction associated with the lack of NO bioavailability as well as the evaluation of pharmacological effects of upregulation of this pathway on platelets activity in NO-deficient mice.

We know that, exogenous MNA (1-methylnicotinamide) exerts anti-thrombotic and anti-inflammatory effect through activation of COX-2/PGI₂ pathway. The concentration of MNA in inflammatory states such as atherosclerosis or hepatitis is elevated what indicates that this molecule can be responsible for the counteract of endothelial dysfunction associated with those pathologies. We hypothesize, that endogenous MNA is a regulator of COX-2/PGI₂ pathway activity, thus activation of the compensatory effect in the lack of the endothelium-derived NO can be attributed to MNA/COX-2/PGI₂ mechanism. This project aims to verify this hypothesis using NO-deficient mice with hypertension induced by L-NAME which is an inhibitor of all isoforms of NO synthases. To confirm our thesis, we will evaluate the influence of impaired NO bioactivity on the action of MNA/COX-2/PGI₂ system by the quantification of MNA and its metabolites, evaluation of eicosanoids profile in urine and plasma specimens and also by platelets activity analysis. All measurements will be performed in different stages of hypertension before as well as after administration of MNA donor (MNACl) or MNA and NO source (MNANO₂, MNANO₃) to NO-deficient mice. The changes in platelets activity analysed by ex vivo dynamic TXB₂ generation assay in whole blood combined with alternation in MNA and PGI₂ synthesis in NO-deficient mice during progression of hypertension and after pharmacotherapy using MNA or MNA and NO donors will allow to assess the importance of MNA/COX-2/PGI₂ pathway in the regulation of platelets activity in different stages of endothelium-derived NO bioavailability impairment. The project is also concentrated on the development of bioanalytical method for quantification of selected eicosanoids in mice urine and plasma applying LC/MS/MS technique as well as method validation. Moreover, based on the results we will attempt to evaluate the PK/PD model encompassing the integration of pharmacokinetics, biochemical and pharmacodynamics parameters which describe the biological effect of studied substances and antiplatelet activity of two considered pathways – NO and COX-2/PGI₂.

Pharmacokinetic profiling to understand differences in biological activity of two novel liver-selective NO-releasing compounds V-PYRRO/NO and V-PROLI/NO in the treatment of experimental model of NAFLD in mice

Funding: National Research Centre (funding agency), Program PRELUDIUM 6
Project leader: Kamil Kuś, PhD

The main goal of the proposed project is to analyze in comprehensive way pharmacokinetic profile of two NO-releasing hepatoselective prodrugs: V-PYRRO/NO and V-PROLI/NO, that exert differential pharmacological effects in the murine model of NAFLD (Non-Alcoholic Fatty Liver Disease). In our preliminary experiments V-PYRRO/NO, but not V-PROLI/NO proved to be effective in inhibiting liver steatosis, improving insulin sensitivity and modifying hepatotoxic lipid profile in the liver. Based on the results of this studies, we presume that pharmacokinetic differences between these two agents are responsible for the differences in their pharmacological efficacy. Therefore in-depth examination of PK profile of studied compounds will help to understand the differences in their pharmacological activities. The specific objectives of the project are as follows: – (1) to optimize the methodology, including the development and validation of LC/MS/MS bioanalitical methods. – (2) to characterize pharmacokinetic profile of V-PYRRO/NO and V-PROLI/NO in vivo – (3) to study liver elimination process of V-PYRRO/NO and V-PROLI/NO ex vivo – (4) to study metabolism of V-PYRRO/NO and V-PROLI/NO using mouse liver microsomes and primary mouse liver hepatocytes in vitro – (5) to explain differences in V-PYRRO/NO and V-PROLI/NO pharmacological efficacy, based on pharmacokinetics studies planned in the current project.

Glycocalyx damage – the first stage of pathomechanism of endothelial dysfunction; measurements using capillary electrophoresis

Funding Agency: National Science Centre, Poland, Program: PRELUDIUM 16
Project leader: Karolina Matyjaszczyk-Gwarda, M.Sc.,  Ph.D.

An endothelial dysfunction is a crucial element of the pathophysiology of many different diseases, including atherosclerosis, diabetes and neurodegenerative diseases. The proper function of the vascular endothelium is closely associated with the correct function and structure of the glycocalyx (GLX) – a carbohydrate-rich ‘jelly-like’ layer covering the endothelium. There is more and more evidence that damage of GLX precedes the development of endothelial dysfunction, therefore the knowledge about changes in the quantitative and qualitative structure of GLX related to different pathological states may be extremely helpful in the understanding of the role of the particular glycosaminoglycans (GAG) in physiological processes. So far, published studies represent only a fraction of knowledge about quantitative and qualitative changes in GLX that occur during the development of endothelial dysfunction. The available analytical methods for GLX measurements have limitations: they can be used only for ex vivo material (immunohistochemistry, atomic force microscopy), they are based on the quantitative analysis of only one of the GLX components (enzyme immunoenzymatic methods, e.g. ELISA), or they are not sensitive enough.

To our knowledge, no studies have been carried out to explain how individual classes of glycosaminoglycans (GAG) – polysaccharides, which are one of the GLX building blocks, behave during subsequent stages of GLX damage. We suppose that the study of differences in the levels of GAG in plasma (heparan sulphate/ heparin, dermatan sulfate/chondroitin sulfate, keratan sulfate, hyaluronan) can reflect in more comprehensive way the complexity of the GLX damage process and consequently become a more sensitive biomarker of GLX degradation compared to currently used (ELISA, proteoglycan-based tests, or only one of GAG). The use of capillary electrophoresis (CE) in the proposed project is a unique approach that allows the simultaneous determination of several different GAG. By applying quantitative method, developed based on the special possibilities offered by CE, we can expect results that will answer the question of how changes in the entire GAG profile, look, during the subsequent stages of GLX damage leading to endothelial dysfunction. The developed methodology will be used to study GLX in an in vitro and in vivo model, in the acute GLX injury induced by LPS (endotoxaemia model).

The results obtained from in vitro experiments will be used to create a scheme of how GLX behaves under the influence of more or less specific enzymes and what changes in GAG levels are most characteristic of individual enzymes. This formula will serve as a basis for investigating whether specific stages can be expected in in vivo models correlated with changes representative of a particular GAG. The proposed project may become the first step towards the diagnosis of endothelial dysfunction at a very early stage based on the diagnosis of GLX injury. The use of CE, which is a high-throughput and relatively cheap technique, can introduce a new quality among the available methods and become a “gold standard” in the diagnosis of early vascular endothelial damage.

Raman spectroscopy in studies on cellular uptake of sinusoidal endothelial cells in vitros

Funding Agency: National Science Centre, Poland, Program: PRELUDIUM 14
Project leader: Ewelina Matuszyk (Szafraniec), M.Sc., Ph.D. student

The project is focused on application of Raman spectroscopy to study the process of cellular uptake (mainly by endocytosis) that takes place in Liver Sinusoidal Endothelial Cells (LSECs). The aim of the project is to develop an analytical method allowing to follow the uptake of molecules and to determine the kinetics of this process, taking an advantage of high chemical specificity and resolving power of Raman imaging technique. The outcomes will be referred to results achieved with the use of fluorescence microcopy, which will serve as a reference technique. The crucial stage of the project will be the validation of the developed method, which involves the inhibition of the cellular uptake by the use of appropriate inhibitors. This stage is intended to confirm the participation of known mechanisms mediating the uptake process. LSEC cells are characterized by high endocytic capacity allowing for blood purification from potentially dangerous molecules, while being a significant ‘player’ in maintaining liver homeostasis and immunity. Dysfunction of LSECs is a key event in multiple liver disorders, having a straight correlation with the loss of endocytic capacity.

The planned research will provide a new insight into the molecules uptake by LSECs. In addition, the use of Raman spectroscopy is an innovative approach in this field. This enhances the application potential of spectroscopic methods that offer research capabilities not available to conventional techniques used in subcellular investigations.

Raman spectroscopy in the study on non-alcoholic fatty liver disease using isolated liver cells and in vitro cell models

Funding Agency: National Science Centre, Poland, Program: ETIUDA
Project leader: Ewelina Matuszyk (Szafraniec), M.Sc., Ph.D. student

The 21st century is a period of increased incidence of related civilization diseaseswith an unhealthy, sedentary lifestyle. Many of these disorders are associated with endothelial dysfunction lining all blood vessels. This also applies to liver dysfunction such as non-alcoholic fatty liver disease (NAFLD). Despite the growing incidence, the exact mechanism of the emergence and development of this disease is still unknown. The ongoing work focuses on understanding the processes associated with the so-called secondary liver sinusoidal endothelial cell dysfunction (LSECs), which appears to be crucial in the development of NAFLD and contribute to the further progression of this disease towards hepatitis and cirrhosis. The work uses a unique approach involving the study of isolated liver cells derived from a mouse model of non-alcoholic fatty liver resulting from the use of a high-fat diet. The key aspect of the work is to find spectroscopic markers that allow to identify the appearance of dysfunction and track its development during disease progression. For this purpose, research was carried out at an early stage(2 weeks high-fat diet) and late (15-20 weeks) development of fatty liver. In addition, the purpose of the work is to explore the processes of lipid uptake and metabolism in LSECs and hepatocytes. Raman spectroscopy has become a valuable tool in research at the cellular and subcellular level, enabling detection and localization of biochemical changes that occur during the development of dysfunction, as well as to study various cellular processes. The use of this technique creates new research opportunities and at the same time, strengthens the application potential of vibrational spectroscopy methods.

Label-free imaging of subcellular structures of endothelium using Confocal Raman Spectroscopy supported by FT-IR Spectroscopy, AFM and Fluorescence Microscopy

Funding: National Research Centre (funding agency), Program PRELUDIUM 5
Project leader: Katarzyna B. Majzner, PhD

The project involves the development of novel methods with the use of confocal Raman microspectroscopy, infrared absorption spectroscopy, atomic force microscopy (AFM) and fluorescence microscopy to simultaneously evaluate changes in the level of intracellular morphological, biochemical and physicochemical, with particular emphasis on research formation and the role of lipid bodies endothelium. Lipid droplets (LDs), sometimes referred to in the literature lipid bodies are organelles generally known in the neutrophils, eosinophils, or tumor cells. The presence of LDs in the endothelium represents a new aspect of the research, especially that the role of LDs in cells is unexplored. Thus naturally comes the overall objective of the project, i.e. the development of a comprehensive research methodology to study the effect of supplementation with arachidonic acid on the presence of LDs in endothelial cells. Using the developed research methodology for study of LDs in the endothelium is the basis for a better understanding of the biological role of the LDs, including study the formation these structures and their functions.

The hypothesis of the potential protective role of lipid bodies, as a reservoir of arachidonic acid available for the production of prostacyclin (PGI2) will be verified using the methodology developed in the project.

Leading research method planned in the project is Raman spectroscopy. This is dictated by several factors:

• The opportunity to study the biochemical composition of cell at subcellular level in a non-destructive and sensitive way (possibility of a live cells studies),
• the possibility of rapid and unambiguous identification of LDs in a cell based on the unique spectroscopic “signature” of these organelles,
• the ease of obtaining information about the distribution of the main biochemical components with high spatial resolution (up to ca. 300 nm),
• using a confocal Raman imaging allow for the 3D reconstruction and tracking the distribution, shape and size of LDs.

In addition, examination of cells using Raman spectroscopy will be supported by AFM, what allows the simultaneous examination of the biochemical composition and topography of the analyzed sample. Applying a combination of Raman spectroscopy and AFM to study these same areas of the cells is possible to obtain complete information about the biochemical composition of endothelial cells in vitro and the distribution of the individual components and investigate the physical and mechanical properties of the sample. Knowledge about the changes taking place in the cell as a result of pharmacological stimulation will be supplemented by the results obtained from the use of infrared absorption spectroscopy and fluorescence microscopy (immunocytochemical staining).

Described project is particularly important because of the proposed research methodology taking into account the combination of vibrational spectroscopy, atomic force microscopy and fluorescent staining. This approach to research will allow for a significant increase in both knowledge of the biochemical changes associated with pharmacological stimulation of endothelial cells, the role of lipids in endothelial bodies, as well as presenting new research methodology that can be successfully applied to other systems. It should be noted that this project is focused on the theme, brand new in the context of research on the endothelium, because knowledge about lipid bodies observed in the endothelium is negligible. Although the results have no direct potential application, they will contribute to new information and reassessment of existing scientific data.

The results of the project will be incorporated to the PhD thesis of the Applicant. They will be also presented at the conference forum and published in pre-reviewed papers in the international chemical or chemistry-related journals. Experience of the Raman Imaging Group Faculty of Chemistry UJ in terms of Raman spectroscopy (measurements and advanced spectral analysis), as well JCET research groups in in vitro pharmacological and biological studies, and the availability of the necessary equipment for the project allow for the statement that the risk of this project is low.

Small scale, great significance: Raman spectroscopy, 3D imaging, atomic force microscopy and immunohistochemical staining in study of diabetes

Funding: National Research Centre (funding agency), Program PRELUDIUM 4
Project leader: Marta Z. Pacia (Pilarczyk), Ph.D.

The main aim of the project is to find spectroscopic changes in the vascular wall in the healthy and diabetic (db/db model) mice. The proposed study is aimed to determine the biochemical composition of vascular wall samples, to investigate the effect of diabetes on the biochemical composition and structure of blood vessel walls, find differences between healthy and diabetic tissue and explain them on the biochemical ground. In order to achieve the objectives of the project, an innovative and unique approach is offered, i.e. Raman scattering spectroscopy technique with an emphasis on Raman imaging, 3D imaging correlated well with other techniques: atomic force microscopy (AFM) and immunohistochemical staining (IHC). This project involves increasing the knowledge and awareness of changes taking place in tissue (vascular wall of mice) in study of diabetes using combination of Raman/AFM spectroscopy.

The subject of the research will be vascular wall samples of healthy and diabetic mice (model db/db). Two different types of preparations: the aortic cross-section of example thickness of 5 mm and aorta en face will be applied. Measurements of en face samples allows for studying of the inner side of aorta after opening (aorta is cut along and opened). The basic technique used in project is Raman imaging allowing for studying of the biochemical composition of the vascular wall and distribution of the individual components of the selected areas of tissue. Especially, for en face samples it seems to be a valuable idea to perform a series of Raman images for the same area of a tissue and visualize changes in the biochemical composition in the depth profile of tissue to obtain 3D Raman image. The project proposes a new approach using a combination of Raman spectroscopy, AFM and immunohistochemical staining to study of exactly the same areas in the sample. Multimodal approach of Raman imaging, combined with atomic force microscopy (AFM), immunohistochemical staining (IHC) and probably with scanning near-field optical microscopy (SNOM) provides complex information about chemical, physical and mechanical properties of the vascular wall with emphasis on the changes between healthy and diabetic mice.

Diabetes as a civilization disease is a major health problem for many people around the world. Currently, a lot of work focuses on developing methods to prevent and treat diabetes. This project is focused on understanding of the exact biochemical changes in vascular wall of diabetic subjects (db/db mouse model) compared with healthy animals. The results obtained in the project will expand the knowledge on the impact of diabetes on biochemical composition of vascular wall. Although the results are not directly applicable, they will contribute find new information about diabetes and reassess of existing scientific data. Application of Raman imaging combined with AFM and aided by SNOM and IHC staining to analyze the vascular wall is an innovative approach that allows obtaining multidimensional information about the studied system. The reached conclusions will help to increase knowledge about the mechanisms of vascular wall inflammation in diabetes, that is of the prime importance from the point of view of diabetes treatment and prevention.The results will be published in scientific journals and presented at conferences in Poland and abroad, which allows for their popularization.

The role of reactive oxygen species (ROS) generated during a maximal exercise by endothelial NOX2 i NOX4 in the regulation of post-exercise oxidant stress, postexercise haemostasis and exercise capacity

Funding Agency: National Science Centre, Poland, Program: PRELUDIUM 13
Project leader: Kamil Przyborowski Ph.D. 

Oxidative stress, resulting from overproduction of reactive oxygen species (ROS), may lead to damage of cellular structures, proteins, lipids and DNA and has been involved in a wide range of pathologies such as neurodegeneration, cardiovascular diseases and cancer. On the other hand, ROS play an important role as signaling molecules in regulation of physiological processes, including cell proliferation and differentiation, inflammation, innate immune response, vascular tone and endothelium phenotype.

Physical exercise results in excessive generation of ROS and could trigger oxidative stress resulting in impaired skeletal muscle contractility, muscular fatigue and endothelial dysfunction due to NO inactivation. On the other hand, there is a growing number of evidence that ROS generated during exercise could improve insulin sensitivity, enhance physical fitness and extend the lifespan. Currently, there is no data clearly defining the beneficial and detrimental effects of ROS generated during a single bout of acute exercise that could depend on the different enzymatic source of ROS.

The aim of this project is to investigate the role of ROS generated during a single bout of maximal exercise by endothelial NOX2 and NOX4 enzymes (Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase) in post-exercise oxidant stress and adaptation/maladaptation of endothelial cells, and in regulation of exercise capacity and post-exercise haemostatic/thrombotic parameters using unique animal models of endothelial-specific NOX knock-out mice.

The identification of beneficial or detrimental role of ROS generated in response to exercise depending on their enzymatic sources in endothelium may contribute to development of innovative pharmacological endothelium-targeted strategy which could improve exercise tolerance and protect against dangerous consequences of acute intense exercise in patients with endothelial dysfunction.

Vibrational spectroscopy in analysis of biochemical status of blood that mirrors vascular wall pathology in animals with systemic and pulmonary hypertension

Funding: National Research Centre (funding agency), Program PRELUDIUM 5
Project leader: Emilia Staniszewska, PhD

The main aim of the project is to determine the overall biochemical profile of blood components in animal models of systemic hypertension and pulmonary hypertension by using Fourier Transform Infrared (FTIR) and Raman spectroscopy. The study will be carried out for various stages of pathological changes in both models of hypertension. Planned research and defined methodology will allow developing a new strategy for predicting the pathology of blood vessel wall and related risks on the basis of fingerprint induced by hypertension in biochemical profiles of blood recorded in vibrational spectra.

The object of the studies will be platelet poor- and rich- plasma, serum, erythrocytes and leukocytes isolated from healthy animals (mice BL/6J C57 and rats type Wistar) and from animals with induced systemic hypertension (mouse model after 8-week administration of L-Name) and pulmonary hypertension (rat model after single administration of monocrotaline). A tool of investigations will be two techniques of vibrational spectroscopy: Infrared and Raman spectroscopy. Searching of spectral markers will be supported by chemometric methods (cluster analysis (CA), principal components analysis (PCA), etc.), and next spectral analysis will be compared with structural , biochemical, and functional markers of pathology of blood vessel wall.

The role of PGI2 and NO, produced by liver sinusoidal endothelial cells (LSEC), in the regulation of hapatic glycogenolysis and gluconeogenesis at an early stage of NAFLD development – studies using a unique isolated perfused liver system

Funding: National Research Centre (funding agency), Program MINIATURA
Project leader: Izabela Czyżyńska-Cichoń, PhD

The aim of the project is to assess the role of prostacyclin (PGI2) and nitric oxide (NO) produced by liver sinusoidal endothelial cells (LSEC) in paracrine regulation of hepatic glycogenolysis and gluconeogenesis at an early stage of the development of non-alcoholic fatty liver disease (NAFLD). This research may answer the questions: if LSEC mediators can regulate glucose metabolism in hepatocytes and whether LSEC-dependent glucose metabolism regulation is impaired during NAFLD? These studies will use the unique ex vivo method of isolated liver perfusion, which enables biochemical and functional profiling of the organ avoiding the influence of other tissues.

Analysis of the molecular mechanisms of metastasis in murine model of breast cancer via the comprehensive study on protein expression changes in lungs

Funding: National Research Centre (funding agency), Program MINIATURA 1
Project leader: Anna Kurpińska, PhD

Breast cancer is one of the most common cancers. It is characterized by a robust ability to metastasize, specifically to the lungs and consequently results in high mortality. Despite significant progress in diagnosis and treatment still not fully understood mechanisms of tumorigenesis and metastasis form a barrier for the selection of an effective and selective drugs.

The aim of this project was to analyse the mechanisms underlying dysfunction of the lungs in the course of cancer progression, via the search for biomarkers characteristic for early metastatic processes in the lungs and description of the metabolic shift in late phase of the cancer progression.

Application of the proteomic techniques enabled broadening the knowledge in the field of cancer metastasis, characterization of the molecular changes and to analyze the communication and adaptation mechanisms of the metastases development. An important result of the presented studies is the answer to the question on how lung tissue is remodeled in the early stages of cancer progression, when the metastatic cells are not observed or only few cancer cells are present in the lungs. The obtained results clearly indicate very early stimulation from cells forming the primary tumor and activation of mechanisms that prepare the tissue for the invasion of cancer cells.

Non-invasive MRI assessment of microvascular nitric oxide synthase dysfunctionand permeability in the lung endothelium in a mouse model of a chronic heartfailure

Funding: National Research Centre (funding agency), Program MINIATURA
Project leader: Grzegorz Kwiatkowski, PhD

Endothelial dysfunction is a key characteristic of several cardiovascular diseases including diabetics, atherosclerosis and hypertension. In particular, global endothelial status holds a prognostic value in the development of heart failure. Several reports shown endothelial dysfunction developed over the course heart failure including impairment ofnitricoxide synthesis. Moreover, at the advanced stage of heart failure, the endothelial impairment was also observed in the lungs. There is, however, very little known on a relationship between lung microvascular endothelial status and heart failure progression. In particular, it is not known whether the lung endothelium exhibits adaptive or maladaptive mechanisms towards heart failure progression. The lung endothelium covers up to 30% of the total endothelial surface and as such is potentially an important treatment target.

Recent developments in the Magnetic Resonance Imaging offer novel, dedicated methods to study microcirculation but have not been yet applied to the lungs. We plan to employed state-of-the-art MRI protocols to characterize changes in the lung endothelium phenotype during the progression of heart failure in a mouse model of this disease.

Changes in paracrine signaling of liver sinusoidal endothelial cells stimulated with fatty acids and lipid droplets formation in hepatocytes – studies with use of a platform for three-dimensional co-culture under micro-flow condition, OrganoPlate

Funding: National Research Centre (funding agency), Program MINIATURA 1
Project leader: Edyta Kuś (Maślak), PhD

The goal of the project was to investigate the effect of fatty acids on the paracrine signaling of liver sinusoidal endothelial cells (LSEC) and its contribution to the formation of lipid drops in hepatocytes. Research were conducted using a unique platform for three-dimensional co-cultures in micro-flow, OrganoPlate mimicking liver physiological environment, i.e. static conditions in the extracellular matrix for hepatocytes and controlled microflow for LSEC with maintained continuous communication between cells. The project aims to better understand the role of LSEC in the development of fatty liver disease.

Effect of visfatin on endothelial bioenergetics and phenotype

Funding: National Research Centre (funding agency), Program MINIATURA
Project leader: Łukasz Mateuszuk, PhD

Nicotinamide phosphoribosyltransferase (NAMPT) and 5’ectonucleotidase (CD73) are involved in nicotinamide (NA) metabolic pathway, leading to the synthesis of nicotinamide adenine dinucleotide (NAD), an important cofactor of redox reactions and substrate for sirtuins and poly-ADP-ribose polymerases (PARPs). Intracellular NAMPT is responsible for nicotinamide mononucleotide (NMN) synthesis from NA, which is a direct precursor of NAD. Extracellular form of NAMPT is known as visfatin (Vis), an adipokine with pro-inflammatory properties, which high plasma concentration has been previously detected in obesity, type 2 diabetes and metabolic syndrome. Vis increases IL1, IL6 and TNFα production, decreases endothelial nitric oxide synthesis and most probably is also released by dysfunctional endothelium, which may lead to vascular inflammation usually preceding the symptoms of cardiovascular diseases. Both NAMPT and Vis are encoded by the same gene, but Vis protein molecule is slightly bigger, which suggests post-translational modification of polypeptide chain. Although adipokine activity of Vis has been documented during previous studies, the extracellular enzymatic properties of this protein still requires additional studies. By ribosyl-phosphorylation of extracellular NA, Vis may act as an alternative source of NMN. The main aim of this project was to assess endothelial expression of Vis and CD73 in basal conditions and during endothelial disfunction, as well as examining the phosphoribosiltransferase potential of visfatin in a presence of NA.

Application of a new immunohistochemical technique for imaging of DMPO spin trap to image oxidative stress in cardiac muscle in mice

Funding: National Research Centre (funding agency), Program MINIATURA
Project leader: Bartosz Proniewski, PhD

Reactive oxygen species (ROS) serve as important physiological transmitters, however in inflammation and other pathologies, they can be overproduced and overpower the antioxidant capacity, leading to the so called oxidative stress. Clinical effects of oxidative stress have been reported in heart failure and in various mouse models of this disease, including earlier studies using the Tgαq*44 mouse model, where increased superoxide production was seen within the heart. Methods typically used to study oxidative stress are either unspecific (fluorescent probes) or require sample homogenization. What is more, in the literature often just the increased production of a reactive oxygen species (e.g. superoxide) is considered indicative of oxidative stress, whereas in fact it’s definition encompasses an increased ROS production that is unbalanced by the inefficient antioxidant defense.

Here, DMPO immuno-spin trapping (IST) has been used to image oxidative stress in the heart of mice, based on to the high reactivity of DMPO spin trap against protein/lipid radicals and the utilization of specific anti-DMPO antibodies, allowing the visualization of oxidative modifications within the hearts of transgenic Tgαq*44 mice, which develop heart failure over time due to a cardiomyocyte-specific overexpression of the Gαq protein. It was demonstrated that these detrimental changes appear much later, then increased superoxide production is detected, but parallel to the first functional symptoms of heart failure, as seen using in vivo imaging (USG/MRI). This is due to effective antioxidative mechanism that are activated in the studied mouse model. The results suggest, that the IST method in fact shows the “end effect” of oxidative stress, in contrast to measurements based solely on free radical detection, without monitoring of enzymatic activity of the antioxidative pathways in parallel.

Fluorescent imaging of DMPO-nitrone adducts in slices of the isolated myocardium allowed for determination that the ventricles are most affected by oxidative modifications, especially the left ventricle. Moreover, using a double staining procedure with lectin antibody to visualize the coronary endothelium, it was shown that it is not only the cardiomyocyte being affected. In fact, both large coronary vessels and small capillaries are under oxidative stress. These oxidative modifications seen in the endothelium suggest that there has to be a mechanism by which oxidative stress is propagated from the cardiomyocytes, where it originates based on the Gq protein modification, to the endothelium, and it’s identification and pharmacological modulation may lead to the design of novel, effective therapeutic route in heart failure.

Multiparametric analysis of pulmonary endothelial dysfunction in the murine model along the progression of breast cancer

Funding: National Research Centre (funding agency), Program MINIATURA 1
Project leader: Marta Smęda, PhD

Healthy endothelium constitutes an “impermeable barrier” for cancer cells. The aim of the project is to perform multiparametric characterization of endothelial dysfunction in the metastatic lungs throughout the progression of breast cancer that will comprise measurement of pulmonary NO production and eNOS activation, assessment of mesenchymal transformation of pulmonary endothelium (EndMT) as well as pulmonary endothelium permeability and activation. The study will be conducted in the orthotopic model allowing for spontaneous metastasis as well as in the model of experimental metastasis after intravenous inoculation of cancer cells. The study will contribute significantly to the field of experimental research regarding mechanisms of metastasis.

The PGI2 / SIRT1 pathway is a chance to revolutionize pharmacotherapy of cardiovascular disease

Funding: National Research Centre (funding agency), Program MINIATURA 2
Project leader: Magdalena Sternak, PhD

SIRT 1 is attributed to participation in the activation of repair mechanisms in endothelial dysfunction. Despite the extensive literature on sirtuins and in the context of the non-specificity of SIRT1 activators (Pacholec et al., 2010), their mechanism of action, and in particular the contribution of the PGI2/COX2 pathway to the SIRT1 regulation mechanism, remains unclear. There are speculations that the use of SIRT1-targeted drugs by acting on endogenous endothelial mediators can be a promising therapy for the pathogenesis of human diseases, which is one of the major challenges of medicine. The aim of the planned research in the project is to investigate the role of prostacyclin PGI2 in defense mechanisms associated with SIRT1 activity in improving the vascular endothelium function in the mouse transgenic model (endothelial CDK5R1 (EC-p25) with the overexpression of the EC-p25 protein. The EC-p25 mouse model that was used to implement the project is an innovative transgenic model, which may contribute to the explanation of important mechanisms of endothelial dysfunction, and may also contribute to finding effective therapy in the treatment of diseases based on interference in the pathway associated with SIRT1 and thus lead to the development of clinical trials.

Budget: 49 500 PLN, Time: 12 months,

Toward pharmacology of cancer cells adhesion and transendothelial migration using unique 3D microfluidic platforms

Funding: National Research Centre (funding agency), Program MINIATURA 1
Project leader: Marta Stojak, PhD

The main goal of the project was to learn and implement the unique approach of 3D microfluidic platforms (VMT, vascularized microtumors) during the internship in the laboratory of Professor Christopher C. W. Hughes (Department of Molecular Biology and Biochemistry, University of California, Irvine, USA) and the use of this methodology to study the adhesion and transendothelial migration of cancer cells. The main problem in understanding cancer cells interactions with the endothelium is lack of good in vitro models. Classical models put on simplicity and convenience, at the same time ignoring complex interplay between tumor cells and microenvironment. In the Hughes Lab, an innovative 3D microphysiological system was developed which ensures physiological flow and transport of nutrients and metabolites. In addition, the VMT platform is transparent, which allows non-invasive imaging of tissues or cells. All together VMT devices have great potential and advantage over other commercially available microfluidic devices as well as on the classic system I have used in previous studies.

The effect of the activators of Nrf2 transcription factor on endothelial permeability- ex vivo studies on B6.129X1-Nfe2l2tm1Ywk / J knockout mice

Funding: National Research Centre (funding agency), Program MINIATURA
Project leader: Ewa Szczęsny-Małysiak, PhD

The aim of the project is to investigate the role of the activation of Nrf2 transcription factor in changes in aortic endothelial cell permeability (which is a measure of endothelial dysfunction). Experiments will be performed ex vivo on blood vessels taken from knockout mice B6.129X1-Nfe2l2tm1Ywk / J Nrf2 characterized by impaired expression of Nrf2 factor and wild-type C57BL / 6J mice. Beforehand, the blood vessels will be treated with drugs – activators of factor Nrf2: methyl bardoxolone, dimethyl fumarate and L-sulforaphane. Showing differences in the action of individual substances will allow to determine whether the observed effects depend on the activation of Nrf2 or whether it will be necessary to determine another molecular mechanism of the occurring phenomena.