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Hypoxia-induced signaling in angiogenesis : role of mTOR, HIF and Angiotensin II

Petrimpol, Marco Renato. Hypoxia-induced signaling in angiogenesis : role of mTOR, HIF and Angiotensin II. 2007, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7945

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Abstract

This thesis includes the work of four different projects I have been following during my
time as a PhD student; (1) the characterization of mTOR-associated signaling and
endothelial cell proliferation in response to hypoxia, and (2) identification of signaling
pathways responsible for HIF stabilization during hypoxia. A side project aimed at (3)
elucidating mechanisms of angiotensin II-induced angiogenesis. Furthermore, I have
contributed to a review about antihypertensive drugs and microvascular rarefaction.
Hypoxia is the main stimulus for angiogenesis, the formation of new microvessels from
pre-existing ones. To maintain adequate metabolism and supply of energy, eukaryotic
cells adapt when oxygen levels drop. β-Oxidation is switched off while enzymes for
glycolysis are induced. In most cells, cell cycle is arrested to reduce the number of
oxygen consuming cells.
When oxygen levels are low for a longer period, erythropoiesis and angiogenesis are
induced to increase tissue oxygenation. Specialized cells such as vascular endothelial
cells (EC) and smooth muscle cells (SMC) are activated and increase proliferation and
gene expression in response to hypoxia. EC proliferation and angiogenesis in response
to hypoxia is, amongst others, rapamycin-sensitive. Thus, we hypothesized that
mammalian target of rapamycin (mTOR) is involved in the response to hypoxia in
endothelial cells. mTOR is central in regulating cell growth and proliferation, and
integrates signals from nutrients, growth factors, energy status and stress such as
hypoxia. Recent studies have identified two structurally distinct mTOR multi protein
complexes (mTORC1 containing raptor and mTORC2 containing rictor) with individual
downstream targets.
Study 1: In the first project, we have investigated mTOR-associated signaling
components under hypoxia and their role in cell proliferation in rat aortic endothelial cells
(RAECs). By analyzing mTOR and the distinct downstream targets of mTORC1 (S6
kinase) and mTORC2 (PKB/AKT), we found that hypoxia activates mTOR signaling in a
timed program, leading to early activation and late inhibition of mTORC1 and a delayed
but sustained activation of mTORC2. Raptor and rictor knock down demonstrated that
rictor (mTORC2) is essential for hypoxia-induced endothelial proliferation, whereas
raptor knock down only partially inhibited increased proliferation.
When studying the pathways directing the hypoxic stimulus to mTOR, we found that
hypoxia-induced cell proliferation is independent of regulation by TSC (tuberous
sclerosis complex). TSC is upstream of mTORC1 and directs growth factor signals and
energy and nutrient status into this signaling pathway. Thus, hypoxia impinges on mTOR
TSC-independently; rapid mTOR phosphorylation under hypoxia rather suggests a direct
activation step. All together, our data suggest cooperating mechanisms between signals
from both mTOR complexes in the response to hypoxia in EC.
Study 2: To study potential downstream effectors of mTOR-dependent proliferation in
response to hypoxia we have focused on Hypoxia inducible factors (HIF). HIFs mainly
control transcription of genes for angiogenesis, erythropoiesis and glycolysis in response
to hypoxia. In normoxia HIF-α’s are constantly degraded. Degradation is prevented in
hypoxia, the HIF-α’s form heterodimers with HIF-β’s, translocate to the nucleus and
become transcriptionally active. HIF-1α stabilization in hypoxia was shown to be
rapamycin sensitive, and therefore to potentially require active mTOR signaling. How
mTORCs stabilize HIF-α’s is unclear.
In this study we have investigated the regulation and role of HIF1-α in hypoxia-induced
proliferation of aortic endothelial cells. Hypoxia and growth factor stimulation induced
stabilization and translocation of HIF-1α to the nucleus. By using siRNA constructs, we
found that HIF-1α knock down reduces RAEC proliferation in hypoxia. The pathways
potentially regulating HIF-1α have been investigated by using specific inhibitors of
signaling relay enzymes. We show that mTOR is required for HIF-1α accumulation
during hypoxia and growth factor stimulation, and is partially responsible for the
increased proliferation of RAECs in hypoxia. Inhibition of MEK1/2 signaling only affected
growth factor-induced HIF-1α stabilization under normoxia and endothelial proliferation
under normoxia and hypoxia to a similar extent, thus not specifically affecting the
hypoxic response. Knock down of raptor and rictor should answer the central question,
which of the two mTORCs is responsible for HIF-1α stabilization in hypoxia. These
experiments are ongoing.
Review: Hypertension and impaired angiogenesis are intrinsically linked. Angiogenesis
is impaired in most hypertensive patients, and microvascular rarefaction contributes to
hypertension-induced end organ damage. In the framework of a review we summarized
and discussed the effects of antihypertensive drugs on microvessel structure. Studies
done with diuretics, α- and β- adrenergic receptor blockers and calcium antagonists are
inconclusive. Most promising for an induction of angiogenesis or normalization of
microvessel structure are angiotensin II type1 receptor blockers (AT1 receptor blockers,
ARBs) and ACE (angiotensin converting ezyme) inhibitors.
Study 3: ARBs and ACE inhibitors both influence the renin-angiotensin-aldosterone
system (RAAS). RAAS controls blood pressure by regulating vasodilation and
vasoconstriction. The vasoactive peptide Angiotensin II (Ang II) is generated by cleaving
Ang I by ACE. Ang II causes vasoconstriction by activating the AT1 receptor. The AT2
receptor is the other potential binding domain for Ang II and can interact with the
bradykinin receptor B2 (BK-B2 receptor). Bradykinin binds the BK-B1 and BK-B2 -
receptors to up regulate nitric oxide, growth factors and was shown to induce
angiogenesis.
Using an angiogenesis assay in vitro and tissue from left ventricular myocardium of AT1
and AT2 –knock out and wild type mice, we investigated the mechanism underlying the
angiogenic effects of angiotensin II. AT1 and AT2 –receptors were expressed in normoxia
and hypoxia. Ang II induced angiogenesis dose-dependently but only in hypoxia.
Induction of angiogenesis by Ang II was dependent on the availability of the AT2 and B2
receptor, as blockade or knock out of AT2 inhibited angiogenesis in vitro. Also, Ang-IIinduced
angiogenesis was nitric oxide (NO) dependent. Inhibiting the formation of
bradykinin with a specific kininogenase inhibitor completely abrogated Ang II-induced
angiogenesis. Taken together, this study suggests an obligatory role of hypoxia in the
angiogenic effect of Ang II via the AT2 receptor through a mechanism that involves
bradykinin, its B2 receptor and NO as a downstream effector.
Angiogenesis occurs in physiological but also in pathological situations and may be
activated or inhibited in a therapeutic approach: Inhibiting hypoxia-driven tumor
angiogenesis may reduce cancer growth whereas stimulation of angiogenesis after
myocardial infarction may speed up tissue regeneration. Induction of microvessel growth
may also decrease peripheral resistance and thereby reduce hypertension.
Thus, mechanisms and pathways studied in this thesis are involved in the process of
angiogenesis and may contribute to the identification of potential targets to develop
drugs for modulating angiogenesis in patients.
Advisors:Hofbauer, Karl G.
Committee Members:Brink, Marijke and Aebi, Ueli
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Pharmacology/Neurobiology (Hofbauer)
UniBasel Contributors:Hofbauer, Karl G. and Brink, Marijke and Aebi, Ueli
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7945
Thesis status:Complete
Number of Pages:109
Language:English
Identification Number:
edoc DOI:
Last Modified:02 Aug 2021 15:05
Deposited On:13 Feb 2009 16:07

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