The effect of selected substances on the modulation of energy metabolism in human endothelium

Endotelijske celice v krvnih žilah so prve celice v organizmu, ki pridejo v stik z zdravilom po intravenski aplikaciji. Celice se navadno nahajajo v mirujočem stanju, v katerem zadostijo energijskim potrebam preko glikolitične poti. Vendar se v primeru stresa oziroma patološkega stanja hitro prilagodijo z diferenciacijo v proliferirajoče celice. To privede do večjih potreb po energiji, kar spodbudi večji obseg oksidativne fosforilacije. Pri tem pride do nastanka oksidativnega stresa, ki predstavlja enega ključnih dejavnikov tveganja za razvoj endotelijske disfunkcije. Uravnavanje energijske presnove ima zato velik potencial v terapiji preprečevanja srčno-žilnih bolezni. V magistrski nalogi smo preverili vpliv izbranih učinkovin na energijsko presnovo človeške endotelijske celične linije Ea.hy926 v fizioloških (pH 7,4) in acidoznih pogojih (pH 7,2). Analizator izvenceličnega pretoka Agilent Seahorse XFe24 nam je omogočil spremljanje hitrosti izvencelične acidifikacije (glikolize) in hitrosti porabe kisika (mitohondrijskega dihanja) v realnem času. Preizkusili smo 13 učinkovin iz štirih različnih skupin – neopioidnih analgetikov (paracetamol, ibuprofen, acetilsalicilno kislino), antipsihotikov (haloperidol, flufenazin, risperidon), antidepresivov (citalopram, amitriptilin, fluoksetin) in učinkovin z delovanjem na žilno steno (adrenalin, N-ω-nitro arginin, acetilholin, sildenafil). Po pregledu literature smo določili koncentracijo posamezne učinkovine v zgornjem terapevtskem oziroma spodnjem toksičnem območju. Vzporedno z določevanjem energijskega fenotipa endotelija smo z metodo redukcije resazurina pri enakih eksperimentalnih pogojih preverili tudi viabilnost celic. Na podlagi naših poskusov smo ocenili vpliv izbranih učinkovin na energijsko presnovo endotelijskih celic v izbranih koncentracijah. Inkubacija s paracetamolom je vplivala na oksidativno fosforilacijo celic. Ibuprofen je imel škodljive učinke na mitohondrije. Predvidevamo da je deloval kot odklopnik elektrokemijskega potenciala na notranji membrani mitohondrijev. Poskusi z učinkovinami z delovanjem na žilno steno so pokazali povezavo med razpoložljivostjo dušikovega oksida in glikolizo. Manj kot je bilo dušikovega oksida, bolj je bila aktivna glikoliza. Ostale učinkovine niso imele značilnega vpliva na energijsko presnovo endotelijskih celic, kar pomeni, da niso delovale mitotoksično.Endothelial cells in the blood vessels are the first cells that come in direct contact with a drug after intravenous application. These cells are usually in a quiescent state in which they fulfil energy demands through glycolysis. They are highly adaptable and can quickly differentiate into proliferative cells in the presence of stress or pathologic changes. This requires higher energy demands that push the cells to increase energy production through oxidative phosphorylation. Mitochondrial respiration leads to oxidative stress, which is known to be the principal risk factor for the development of endothelial dysfunction. Therefore, the modulation of energy metabolism has a great potential in preventing cardiovascular disease. In this master thesis, we looked for the effect of selected substances on the energy metabolism in human endothelial cell line Ea.hy926 under physiological (pH 7,4) and acidic (pH 7,2) conditions. The extracellular flux analyser Agilent Seahorse XFe24 enabled us to observe the extracellular acidification rate (glycolysis) and the oxygen consumption rate (mitochondrial respiration) in real-time. Throughout the experiments, we made a screening of 13 substances from four different groups: non-opioid analgesics (paracetamol, ibuprofen, acetylsalicylic acid), antipsychotics (haloperidol, fluphenazine, risperidone), antidepressants (citalopram, amitriptyline, fluoxetine) and substances with effect on the vessel wall (adrenaline, N- ω-nitro arginine, acetylcholine, sildenafil). Following the literature review, we selected concentrations of the substances in the range from high-therapeutic-dose to low-toxic-dose. In parallel, we tested the cell viability under the same experimental conditions with a resazurin reduction assay. In conclusion, we were able to detect effects on energy metabolism. Incubation with paracetamol influenced oxidative phosphorylation in cells. Ibuprofen demonstrated a damaging effect on mitochondria, presumably due to uncoupling electrochemical potential on the inner mitochondrial membrane. Also, our experiments showed the correlation between nitric oxygen levels and glycolysis. The lower the levels of nitric oxygen, the more active glycolysis. Other studied drug substances did not have any significant effect on the energy metabolism of endothelial cells. Thus, these drug substances did not exhibit any mitochondrial toxicity