Projects

Vladan Čokić, Olivera Mitrović Ajtić, Dragoslava Đikić, Teodora Dragojević, Tijana Subotički, Milica Vukotić

Institute for Medical Research, National Institute for Medical Research, University of Belgrade

In order to discover the mechanism underlying frequent thrombosis in hematologic malignancies, it will be initiated the inflammation dependent activation and interplay of endothelial cells, platelets, and leukocytes with subsequent formation of microparticles and nitric oxide (NO) linked to thrombus generation by induction of coagulation and fibrinolysis. Cancer-associated thrombosis (CAT) is the second leading cause of death in cancer patients including both venous and arterial thromboembolism. While CAT can be treated with anticoagulants, benefits of therapy must be balanced with the increased bleeding risks. The coagulation related cells and biomarkers will be analyzed and selected by flow cytometry from the following hematologic malignancies: multiple myeloma, lymphoma, acute myeloid leukemia, and transplanted patients with graft-versus-host disease (GvHD). Besides clinical follow-up of the patients, extensive preclinical studies will be performed using in vitro cell cultures (hypoxia chamber, RT-qPCR, immunoassays), in vivo mouse models (NO analyzer), and in silico profiling. Activation of the coagulation factors will be observed through the prothrombotic properties of endothelium (microchip flow-chamber system), neutrophil extracellular traps (NETs, spectrophotometry) and anticancer treatments. The proposed study will show inflammatory stimulation of hypercoagulability, chemotaxis of immune response, and NET formation as well as modulation of fibrinolytic system and miRNA regulated platelet activation. In addition, the equilibrium between NO production and oxygen tension will largely influence the inflammation-dependent thrombus formation, with a prognostic significance of hypoxia related gene signature. Understanding the principal mechanisms may allow the development of new therapies to safely prevent CAT in the hematologic malignancies and GvHD, while determination of the specific biomarkers in each malignancy can be used as predictors of thrombosis.

PI Vladan Čokić, P2 Juan F. Santibanez, P3 Olivera Mitrović Ajtić, P4 Milica Vukotić, P5 Dragoslava Đikić, P6 Tijana Subotički, P7 Teodora Dragojević

Severe acute coronavirus virus syndrome 2 (SARS-CoV-2)-induced hyperinflammation is a major reason for severe clinical outcome of coronavirus disease 19 (COVID-19), principally in male patients. Cell entry of SARS-CoV-2 depends on binding to angiotensin-converting enzyme 2 (ACE2) and androgen-regulated transmembrane serine protease 2 (TMPRSS2). We propose a novel androgen-dependent mechanism of ACE2 activation by SARS-CoV-2 in endothelial and immune cells; as well as endothelial dysfunction and hyperinflammation stimulated immune response predispose to thrombus formation in COVID-19. SARS-CoV-2 will be isolated, in vitro expanded, and used to monitor cell signaling and cytokine storm by immune assays and viral load by RT-qPCR. Related androgen pathways will be pharmacologically targeted. Immune-endothelial cell co-cultures will be performed for thrombosis development. We expect androgen-dependent SARS-CoV-2 host cell infection and mediated inflammation in thrombotic complications. This translational research will reveal androgen pathways implication in SARS-CoV-2 infection and therapeutic strategies to speed up patient recovery.

Teodora Dragojević, Tijana Subotički, Miloš Diklić, Olivera Mitrović Ajtić, Vladan Čokić

The aim of this project is to investigate potential involvement of NOS enzymes in the molecular mechanism of action of the chemotherapeutic agent hydroxyurea, which is used for the treatment of myeloproliferative neoplasms, chronic myeloid leukemia, and solid tumors. Obtained results should answer the following scientific questions: 1) if HU activates NOS enzymes via direct interaction or indirectly, 2) if inhibition or reduced protein expression of NOS enzymes at least partially rescues HU-induced proliferation block and apoptosis and, 3) which of the NOS isoforms is involved in these processes? Primary mouse erythroid cells isolated from bone marrow and human erythroleukemic cell lines will be used in this study. The relevance of the results obtained in vitro will be confirmed by in vivo treatment of Nos2- or Nos3- deficient mice. Comprehensive knowledge of the molecular mechanism of HU might help improve its beneficial properties and decrease adverse effects.