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Responses to hypoxia via mTOR : role in endothelial cell proliferation and HIF-1[alpha] stabilization

Li, Weimin. Responses to hypoxia via mTOR : role in endothelial cell proliferation and HIF-1[alpha] stabilization. 2005, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7138

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Abstract

Ischemic cardiovascular disease and cancer are life-threatening disorders in human
mortality. Intensive studies on the etiology of and therapeutics for the diseases are
ongoing. One of the most intriguing and promising fields for studies in both disorders
is angiogenesis. Ischemic myocardium and cancer are closely associated with
hypoxia and require de novo blood vessels for tissue survival. However,
therapeutically, angiogenesis needs to be induced in the ischemic myocardium
whereas it ought to be suppressed in cancer. For both purposes, a thorough
understanding of the mechanisms of hypoxia-induced angiogenesis is indispensable.
Angiogenesis, in response to ischemia, requires factors that sense hypoxia and that
relay the signals to effectors. Mammalian target of rapamycin (mTOR), a key energysensor
for cell survival, has recently been shown to be involved in hypoxic signaling.
It remains unclear whether mTOR acts as part of the oxygen-sensing machinery and
how mTOR regulates the hypoxia-induced signaling transducers. On the other hand,
transcription factor hypoxia-inducible factor-1α (HIF-1α) is critical for hypoxic-driven
induction of angiogenic molecules. Again, it is unclear how mTOR affects HIF-1α
function. To unravel these questions, we have assessed mTOR activity as well as its
relationship to HIF-1α in rat aortic endothelial cells (RAECs) in response to hypoxia.
Previous studies in the lab had found that hypoxia potentiates angiogenesis of
explants from rat aorta in an mTOR dependent way. In this study, we have extended
this observation to proliferation of RAEC in vitro and a RAEC-spheroid sprouting
assay (angiogenesis in vitro). Rapamycin, an inhibitor of mTOR, inhibited
proliferation of RAEC and sprouting of endothelial cells in vitro under hypoxia.
Interestingly, upon hypoxic stimulation, mTOR is highly phosphorylated; both mTOR
and phospho-mTOR accumulate in cell nucleus, as does HIF-1α. However, S6k and
4E-BP1, two downstream targets of mTOR that are involved in translational control
are hypophosphorylated at the same time. In low O2 tension (1% O2), increased
nuclear HIF-1α levels are observed over time as well as with decreased O2
saturation. Similarly, the growth factor PDGF-BB induces HIF-1α nuclear
accumulation under normoxic conditions. Hypoxia and PDGF-BB synergistically
enhance HIF-1α nuclear levels. mTOR inhibition strongly reduces nuclear HIF-1α
levels under hypoxia or/and PDGF-BB stimulation while MEK1/2 blockage only
reduces PDGF-BB-induced nuclear HIF-1α accumulation in normoxia. Neither JNK
nor p38 inhibition alters nuclear HIF-1α protein levels. HIF-1α mRNA levels remain
stable under different oxygen saturations and upon mTOR or MEK1/2 inhibition.
Notably, rapamycin-decreased HIF-1α nuclear accumulation can be rescued by
proteasomal inhibition under hypoxia. Finally, mouse embryonic fibroblasts lacking
HIF-1α significantly decreased proliferation rates under hypoxia when compared to
wild type cells. However, mTOR over expression restores and further augments
hypoxia-triggered proliferation both in HIF-1α wild type and in HIF-1α deficient cells.
Taken together, hypoxia activates both HIF-1α-dependent and HIF-1α-independent
regulation of cell proliferation and angiogenesis. Hypoxia-induced mTOR activation
reduces S6K1 and 4E-BP1 phosphorylation. mTOR activity is required for protecting
HIF-1α from proteasomal degradation. Further investigations on mTOR and HIF-1α
during hypoxia in RAEC proliferation, spheroid sprouting assays, and angiogenesis in
vivo are required. Thus, targeting mTOR to enhance or reduce angiogenesis in
response to hypoxia may be clinically relevant.
Advisors:Hall, Michael N.
Committee Members:Christofori, Gerhard M. and Battegay, Edouard
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Hall)
UniBasel Contributors:Hall, Michael N. and Christofori, Gerhard M.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7138
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:116
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 15:06

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