Role of NADPH oxidase Nox4 in endothelial cell senescence and vascular ageing

NADPH oxidases are enzymes, which produce superoxide, which is used for various purposes, such as the host defence in the case of Nox2. In addition to prototypical Nox2, which is active mostly in macrophages, new members of the Nox gene family have been identified. These genes referred to as Nox1-Nox5 play important roles in cellular signalling processes. In the case of Nox4 it has been shown that this enzyme is expressed in many cell types including vascular endothelial cells; in contrast to other enzymes of this class, Nox4 is constitutively active. Many downstream targets of Nox4 and reactive oxygen species derived therefrom have been proposed, and we recently demonstrated that Nox4 induces nuclear DNA damage in human endothelial cells. Based on our unpublished findings that Nox4 induces mitochondrial dysfunction, a major goal of the present project is to study molecular mechanisms by which Nox4 induces mitochondrial dysfunction and subsequent nuclear DNA damage. Nox4 is well known for its capability to regulate the expression of a large variety of cellular genes; however, molecular mechanisms underlying this process are incompletely understood. We propose here to test the hypothesis that regulation of gene expression by Nox4 involves functional interactions with nuclear transcription factor ZSCAN 18 and the AMPK/FOXO3a axis. We will also address the question if Nox4 is required for stress-induced senescence in human endothelial cells in vitro, and address changes in the expression and function of Nox4 in human vascular aging and age-associated vascular dysfunction.

Our work addressed the role of NADPH oxidase Nox4 in the regulation of endothelial cell senescence. We found that Nox4, known as an important player in cellular signal transduction, can induce DNA damage and subsequent cellular senescence if overactive. We also found a functional interaction between Nox4 and the mitochondrial respiratory chain. Using high resolution respiromatry we found that complex I of the respiratory chain is specifically inhibited by Nox4. These results established a new mechanism by which mitochondria are damaged by members of the Nox family. In subsequent experiments we isolated nine small molecules derived from edible plants which have a significant inhibitory activity on Nox4. The best inhibitors isolated in this way are characterized by inhibitory coefficient (IC50) in the micromolar range. Thereby these molecules are comparable in their activity with the first-in-class pharmacological inhibitors of Nox 4, some of which are presently being tested in clinical trials. Finally, we addressed the role of NADPH oxidases in prostate carcinoma. We found that in PC3 prostate carcinoma cells NADPH oxidase Nox5 is selectively expressed. In functional studies we could demonstrate that Nox 5 contributes to proliferation and survival of PC3 prostate carcinoma cells, suggesting Nox5 as a potential new target for prostate cancer therapy.