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

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.