BIOANALYTICAL AND PROTEOMIC APPROACHES IN THE STUDY OF PATHOLOGIC ECS DYSFUNCTIONALITY, OXIDATIVE STRESS AND THE EFFECTS OF PFKFB3 MODULATORS.
Creators: name: NUKALA, SARATH BABU
Publisher: name: Universit� degli Studi di Milano
Year: 2019

description: Vascular dysfunction is one of the primary factors in the onset and progression of atherosclerosis and other vascular-related diseases such as acute myocardial infarction (AMI) and chronic thromboembolic pulmonary hypertension (CTEPH). Emerging evidence indicates that pathological blood vessel responses and endothelial dysfunction are associated with metabolic alterations in endothelial cells (ECs). The identification of the insights and causes resulting in dysfunctional ECs is crucial for the understanding of the disease and to the development of new therapeutic tools. Therefore, to study the characteristics of such EC dysfunction in AMI and CTEPH diseases, we aim to determine the protein profile and/or the altered redox status of patient-derived EC lines by using mass spectrometry-based label-free quantitative proteomic and/or, spectrophotometric and fluorometric approaches. Moreover, quantitative proteomic approach was used to explore the underlying mechanisms of 3PO [(3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), PFKFB3 inhibitor] at cellular level. 3PO is a compound able to inhibit the glycolytic flux partially and transiently and to reduce pathological angiogenesis in a variety of disease models. Bioinformatic and network analyses performed on pathologic HCAEC-AMI cells revealed the alteration of a) metabolism of RNA, b) platelet activation, signaling and aggregation, c) neutrophil degranulation, d) metabolism of amino acids and derivatives, e) cellular responses to stress and, f) response to elevated platelet cytosolic Ca2+ pathways. Similarly, network analysis in pathologic CTEPH-ECs, revealed the differentially regulation of pathways related to a) neutrophil and platelet degranulation, b) metabolism of lipids, amino acids and selenoamino acids, c) response to elevated cytosolic Ca2+, d) detoxification of reactive oxygen species. In addition, the main parameters, indicators of the redox status of both HCAEC-AMI and CTEPH-ECs were significantly increased: advanced oxidation protein products (AOPPs), protein carbonyls (PCO) content, and intracellular reactive oxygen species (ROS). Interestingly, the amount of GSH/GSSG (reduced glutathione/oxidized glutathione) and NADPH/NADP (reduced/oxidized form of nicotinamide adenine dinucleotide phosphate) ratios in dysfunctional ECs were reduced, a clear indication of oxidative stress involvement in both the pathological ECs. Finally, 3PO has multiple targets in the ECs, targeting mitochondrial inner membrane and it inhibits the important cellular pathways including the tricarboxylic acid cycle, the mitochondrial respiratory chain, and vasculogenesis, that may be useful for understanding the inhibitory effect of 3PO on EC proliferation and migration. Therefore, the present data suggest a potential application of this molecule as a starting point in designing novel molecules to prevent diseases where inflammatory reactions are involved, such as in atherosclerosis, cancer or neurodegenerative diseases.