Vasculo-proliferative disorders such as atherosclerosis, postangioplasty restenosis, and pulmonary hypertension are complex processes that are especially related to vascular smooth muscle cells (VSMCs)45, 78. In the arterial media, VSMC are normally quiescent, however, for the development and progression of the above mentioned diseases it is prerequisite that quiescent VSMCs start to proliferate, migrate and undergo apoptosis. Different extracellular stimuli are responsible for regulating VSMC homeostasis, including growth factors, cytokines and mechanic stress. Through activating the intracellular phosphatidylinositol 3-kinase (PI3K)/Aktpathway these factors critically regulate the transcriptional activity of the forkhead box O (FoxO) transcription factors via phosphorylation. FoxOs have crucial roles in different biological processes such as proliferation, differentiation, metabolism, aging, cell survival and stress resistance. Intervening VSMC function by affecting FoxO1a function may represent an attractive approach for future therapeutic strategies in the prevention of vasculo-proliferative diseases.Part 1: Upon Akt-mediated phosphorylation under mitogenic conditions, FoxOs depart from the nucleus. However, stabilization and localization of the ranscription factors in the nucleus is a prerequisite for executing their regulatory function. Psammaplysene A, a natural product from the marine sponge Psammaplysilla sp., was revealed to promote retention of FoxO1a in the nucleus of VSMCs by directly regulating FoxO1a localization. The marine compound was demonstrated to affect cell viability and to inhibit VSMC proliferation in vitro and in vivo by inhibiting S-phase due to attenuating cyclin D1 expression. A Psammaplysene A-analogue named F10 similarly affected VSMC behavior. By applying Psammaplysene A and F10 simultaneously, single doses of each compound could be reduced. Dysfunction of pulmonary VSMCs (PASMCs) contributes to the development of pulmonary hypertension. In Part 2 of this thesis I identified FoxO1a to simultaneously modulate proliferation, migration and cell death of PASMCs. Following transduction with constitutive active FoxO1a, proliferation and migration were significantly attenuated, whereas the number of apoptotic cells increased. Caveolin-1 was suggested to mainly mediate this pro-apoptotic response, since only protein expression of caveolin-1 - but not that of any other FoxO1a target involved in apoptosis - was elevated. The effect of FoxO1a on pathologically modified PASMCs was comparable to that on normal cells. This demonstrates FoxO transcription factors to be important in the disease state as well.In Part 3, I showed that SIRT1, a class III histone deacetylase known for controlling longevity in organisms ranging from bacteria to complex eukaryotes, was able to regulate vascular homeostasis and remodeling processes in vitro and in vivo. It did so by deacetylating FoxO factors, thereby inducing their transcriptional activity. Under native conditions, SIRT1 physiologically interacts with FoxO1a. Pharmacological inhibition of SIRT1, as well as knockdown of SIRT1 reduced FoxO1a´s DNA-binding and transactivation capacity leading to attenuated FoxO1a target gene expression. In contrast, the SIRT1 activator resveratrol enhanced FoxO1a´s transcriptional activity. Upon stress conditions, the observed SIRT1/FoxO1a interaction was increased and led to expression of target genes involved in regulating the cells oxidative stress response (e.g. GADD45), thus, shifting FoxO1a´s transcriptional activity towards cell survival. Application of resveratrol protected from apoptotic cell death, whereas inhibition of SIRT1 activity by pharmacologic drugs or siRNA enhanced the apoptotic response. Moreover, embryonic fibroblasts derived from SIRT1 knockout mice were more resistant to oxidative stress-induced apoptosis as compared to wildtype cells.
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