MOLECULAR ASPECTS OF ENDOTHELIAL CELL FUNCTION AND HYPOXIA-DEPENDENT TUMOR ANGIOGENESIS

Hypoxia, or deficient oxygen levels, is an inherent feature of rapidly expanding tumor tissue. In order to survive in the hostile, hypoxic environment and to restore the supply of oxygen and nutrients tumor cells activate a variety of adaptive mechanisms. Among them, hypoxia-induced angiogenesis, i.e. the formation of tumor blood vessels from pre-existing vasculature, has proved to be of critical importance, and has led to the development of anti-angiogenic therapy in the treatment of cancer patients. Anti-angiogenic drugs targeting the vascular endothelial growth factor (VEGF) pathway may prolong progression-free survival and overall survival in some patients. However, the results are more modest than predicted from pre-clinical studies, reflecting the complexity of tumor angiogenesis, and underlining the requirement of further studies aiming at the identification of the inherent limitations of this treatment as well as new targets of anti-angiogenic therapy. This thesis aims at investigating molecular aspects of hypoxia-dependent regulation of endothelial cell functions, and to identify new, potential targets of anti-angiogenic therapy. In the first part of the thesis, it is demonstrated that polyamines are potent modulators of endothelial cell biology. Accordingly, polyamines regulate both hypoxia-induced apoptosis in endothelial cells, through PI3K/AKT and MCL-1 dependent pathways, and sprouting angiogenesis via endothelial cell migration, partially explaining the anti-angiogenic effects of polyamine depletion in animal tumor models. During the initial steps of hypoxia-driven angiogenesis, endothelial cells respond to hypoxia by induction of a variety of adaptive mechanisms. Results presented in this thesis show that microRNAs seem to be involved in the endothelial cell adaptation to low oxygen levels, since hypoxia profoundly regulates the expression of a subset of miRNAs, and one of them, miRNA-424*, has a functional role in hypoxia-dependent pro-angiogenic activity of endothelial cells. Finally, exosomes derived from aggressive brain tumor cells (glioblastoma) seem to be a novel, intercellular communication route eliciting a hypoxic, pro-angiogenic response in endothelial cells; a defined, hypoxic molecular profile of circulating, plasma-derived exosomes may serve as a non-invasive biomarker to assess the oxygenation status and aggressiveness of glioblastoma tumors. In conclusion, data presented in this thesis identify new players of hypoxia-mediated tumor angiogenesis and implicates them as potential targets of anti-angiogenic therapeutic intervention as well as biomarkers of cancer

Biodistribution PET/CT study of hemoglobin-DFO-89Zr complex in healthy and lung tumor-bearing mice

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells

Global profiling of metabolic adaptation to hypoxic stress in human glioblastoma cells.

Oncogenetic events and unique phenomena of the tumor microenvironment together induce adaptive metabolic responses that may offer new diagnostic tools and therapeutic targets of cancer. Hypoxia, or low oxygen tension, represents a well-established and universal feature of the tumor microenvironment and has been linked to increased tumor aggressiveness as well as resistance to conventional oncological treatments. Previous studies have provided important insights into hypoxia induced changes of the transcriptome and proteome; however, how this translates into changes at the metabolite level remains to be defined. Here, we have investigated dynamic, time-dependent effects of hypoxia on the cancer cell metabolome across all families of macromolecules, i.e., carbohydrate, protein, lipid and nucleic acid, in human glioblastoma cells. Using GC/MS and LC/MS/MS, 345 and 126 metabolites were identified and quantified in cells and corresponding media, respectively, at short (6 h), intermediate (24 h), and prolonged (48 h) incubation at normoxic or hypoxic (1% O2) conditions. In conjunction, we performed gene array studies with hypoxic and normoxic cells following short and prolonged incubation. We found that levels of several key metabolites varied with the duration of hypoxic stress. In some cases, metabolic changes corresponded with hypoxic regulation of key pathways at the transcriptional level. Our results provide new insights into the metabolic response of glioblastoma cells to hypoxia, which should stimulate further work aimed at targeting cancer cell adaptive mechanisms to microenvironmental stress

Establishment of heparan sulphate deficient primary endothelial cells from EXT-1(flox/flox) mouse lungs and sprouting aortas.

Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1(flox/flox) mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field

Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells.

The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by alpha-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo

The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1.

Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization