Domokos Gero

By Domokos Gerő

Hypoxia-reoxygenation injury is a commonly used in vitro model of ischemia, which is useful to study the recovery processes following the hypoxic period. Hypoxia can be rapidly induced in vitro by replacing the culture atmosphere with hypoxic or anoxic gas mixture. Cellular injury mostly occurs as a result of energetic failure in this model: the lack of oxygen blocks the mitochondrial respiration and anaerobic metabolism becomes the major source of high-energy molecules in the cells. In the absence of glucose, glycolysis and pentose phosphate pathway fail to suffice the cellular energy prerequisite and longer periods of oxygen-glucose deprivation (OGD) can completely deplete the cellular NAD+ and ATP pools. The lack of NAD+ results in severe metabolic suppression and predisposes the cells to other injury types. This includes oxidant-induced damage, since oxidative stress activates poly(ADP-ribose) polymerase (PARP) that further depletes the cellular NAD+ pool and leads to excessive cell death. The impaired mitochondrial respiration also leads to an increase in the mitochondrial membrane potential and augments the mitochondrial superoxide generation leading to oxidative stress. The above processes ultimately lead to necrotic cell death, but in certain cell types, mitochondrial damage can also trigger apoptosis.

Part of the book: Hypoxia and Human Diseases

By Domokos Gero

Glucose-induced endothelial dysfunction plays a fundamental role in the development of diabetic vascular complications and glycemic control (the foundation of diabetes care) provides limited protection against the cardiovascular complications. Therefore, identification of novel drug targets and treatment approaches for diabetes complications represent a key direction of current pharmaceutical research. The “unifying theory” of hyperglycemia-induced endothelial cell injury organizes the events of cellular dysfunction in a linear cascade and identifies mitochondrial superoxide generation as the triggering event of the injury. Exposure to high glucose concentration for long periods or repeated glycemic swings may induce changes in metabolic substrate availability and lead to mitochondrial hyperpolarization. Changes in the mitochondrial membrane potential induce superoxide production by the electron transport chain and result in oxidative stress. Mitochondrial superoxide is also responsible for the induction of other sources of reactive oxygen species (ROS) within the cells, including advanced glycation end products (AGEs) and the NADPH oxidase. Mitochondria also show morphological changes and impaired assembly of the respiratory complexes occurs, which results in cellular energy failure, cell senescence and vascular dysfunction. Current intervention strategies aim to inhibit the mitochondrial ROS production and novel therapeutic approaches are expected to provide valuable tools in diabetes therapy in the upcoming years.

Part of the book: Endothelial Dysfunction

By Domokos Gerő

Prevention of cellular injury and consequent cell death is expected to provide therapeutic benefit in various diseases, but with the complexity of cell damaging pathways involved, identification and validation of novel potential drug targets is not a trivial task. New drug targets are expected to take part in complex responses with wide-ranging effects on gene expression and cellular function and drug candidates rather modify these effects than act as simple agonists or antagonists to ultimately protect the cells from an injury. Phenotypic screening may help identify cytoprotective compounds in diseases, in which the lack of drug targets makes target-based approaches unfeasible. This chapter gives an overview of the strategy of cell-based assay development, primary screening, hit selection and confirmation. Considerations about the choice of small molecule compound libraries utilized in cell-based models are discussed as well as the use of clinical drugs for drug repurposing or repositioning. The choice of cell types and issues associated with cell culture techniques are overviewed and the most common assays and readouts are briefly described. Finally, the potential pitfalls of data analysis and hit selection are discussed.

Part of the book: Drug Discovery