New Insights in ATP Synthesis as Therapeutic Target in Cancer and Angiogenic Ocular Diseases.

Lactate and ATP formation by aerobic glycolysis, the Warburg effect, is considered a hallmark of cancer. During angiogenesis in non-cancerous tissue, proliferating stalk endothelial cells (ECs) also produce lactate and ATP by aerobic glycolysis. In fact, all proliferating cells, both non-cancer and cancer cells, need lactate for the biosynthesis of building blocks for cell growth and tissue expansion. Moreover, both non-proliferating cancer stem cells in tumors and leader tip ECs during angiogenesis rely on glycolysis for pyruvate production, which is used for ATP synthesis in mitochondria through oxidative phosphorylation (OXPHOS). Therefore, aerobic glycolysis is not a specific hallmark of cancer but rather a hallmark of proliferating cells and limits its utility in cancer therapy. However, local treatment of angiogenic eye conditions with inhibitors of glycolysis may be a safe therapeutic option that warrants experimental investigation. Most types of cells in the eye such as photoreceptors and pericytes use OXPHOS for ATP production, whereas proliferating angiogenic stalk ECs rely on glycolysis for lactate and ATP production. (J Histochem Cytochem XX.XXX-XXX, XXXX).

Angiogenesis in gynecological cancers and the options for anti-angiogenesis therapy.

Angiogenesis is required in cancer, including gynecological cancers, for the growth of primary tumors and secondary metastases. Development of anti-angiogenesis therapy in gynecological cancers and improvement of its efficacy have been a major focus of fundamental and clinical research. However, survival benefits of current anti-angiogenic agents, such as bevacizumab, in patients with gynecological cancer, are modest. Therefore, a better understanding of angiogenesis and the tumor microenvironment in gynecological cancers is urgently needed to develop more effective anti-angiogenic therapies, either or not in combination with other therapeutic approaches. We describe the molecular aspects of (tumor) blood vessel formation and the tumor microenvironment and provide an extensive clinical overview of current anti-angiogenic therapies for gynecological cancers. We discuss the different phenotypes of angiogenic endothelial cells as potential therapeutic targets, strategies aimed at intervention in their metabolism, and approaches targeting their (inflammatory) tumor microenvironment.

The role of glycolysis and mitochondrial respiration in the formation and functioning of endothelial tip cells during angiogenesis.

During sprouting angiogenesis, an individual endothelial tip cell grows out from a pre-existing vascular network and guides following and proliferating stalk cells to form a new vessel. Metabolic pathways such as glycolysis and mitochondrial respiration as the major sources of adenosine 5'-triphosphate (ATP) for energy production are differentially activated in these types of endothelial cells (ECs) during angiogenesis. Therefore, we studied energy metabolism during angiogenesis in more detail in tip cell and non-tip cell human umbilical vein ECs. Small interfering RNA was used to inhibit transcription of glycolytic enzymes PFKFB3 or LDHA and mitochondrial enzyme PDHA1 to test whether inhibition of these specific pathways affects tip cell differentiation and sprouting angiogenesis in vitro and in vivo. We show that glycolysis is essential for tip cell differentiation, whereas both glycolysis and mitochondrial respiration occur during proliferation of non-tip cells and in sprouting angiogenesis in vitro and in vivo. Finally, we demonstrate that inhibition of mitochondrial respiration causes adaptation of EC metabolism by increasing glycolysis and vice versa. In conclusion, our studies show a complex but flexible role of the different metabolic pathways to produce ATP in the regulation of tip cell and non-tip cell differentiation and functioning during sprouting angiogenesis.

IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers.

Tip cells, the leading cells of angiogenic sprouts, were identified in cultures of human umbilical vein endothelial cells (HUVECs) by using CD34 as a marker. Here, we show that tip cells are also present in primary human microvascular endothelial cells (hMVECs), a more relevant endothelial cell type for angiogenesis. By means of flow cytometry, immunocytochemistry, and qPCR, it is shown that endothelial cell cultures contain a dynamic population of CD34+ cells with many hallmarks of tip cells, including filopodia-like extensions, elevated mRNA levels of known tip cell genes, and responsiveness to stimulation with VEGF and inhibition by DLL4. Furthermore, we demonstrate that our in vitro tip cell model can be exploited to investigate cellular and molecular mechanisms in tip cells and to discover novel targets for anti-angiogenesis therapy in patients. Small interfering RNA (siRNA) was used to knockdown gene expression of the known tip cell genes angiopoietin 2 (ANGPT2) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), which resulted in similar effects on tip cells and sprouting as compared to inhibition of tip cells in vivo. Finally, we identified two novel tip cell-specific genes in CD34+ tip cells in vitro: insulin-like growth factor 2 (IGF2) and IGF-1-receptor (IGF1R). Knockdown of these genes resulted in a significant decrease in the fraction of tip cells and in the extent of sprouting in vitro and in vivo. In conclusion, this study shows that by using our in vitro tip cell model, two novel essential tip cells genes are identified.

Correction: CD34 Promotes Pathological Epi-Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

[This corrects the article DOI: 10.1371/journal.pone.0157902.].

Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein.

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET(Δ7-8). MET(Δ7-8) is located predominantly in the cytosol and is constitutively active. The auto-activating nature of MET(Δ7-8), in combination with a lack of transmembrane localization, renders MET(Δ7-8) not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.