Abstract
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.
Highlights
Blood vessel sprouts are characterized by leading tip cells that grow out from a pre-existing vascular network, and by trailing stalk cells
To study these metabolic aspects of endothelial cells (ECs) during angiogenesis further, we inhibited mRNA levels of the genes PFKFB3, lactate dehydrogenase A (LDHA), or pyruvate dehydrogenase E1 alpha 1 subunit (PDHA1) to determine whether inhibition of glycolysis and/or mitochondrial respiration affects tip cell and/or non-tip cell differentiation (Fig. 2a)
Silencing of expression of the glycolysis gene PFKFB3 did not have an effect on the percentage of tip cells (Fig. 2b), nor did it result in a consistent directional change of tip cell-specific mRNA levels. mRNA levels of 2 out of 8 tip cell-specific genes (IGF236 and VEGFR3) were increased, whereas mRNA levels of 3 out of 8 tip cell-specific genes (CD34 (1.1-fold), CXCR4, and DLL4) were decreased (Fig. 2d)
Summary
Blood vessel sprouts are characterized by leading tip cells that grow out from a pre-existing vascular network, and by trailing stalk cells. This has the following rationale: PFKFB enzymes generate fructose-2,6-biphosphate (F2,6P2), an allosteric activator of 6-phosphofructo1-kinase (PFK-1) that is involved in one of the rate-limiting steps of glycolysis by the conversion of fructose-6-phosphate (F6P) to fructose-1,6-biphosphate (F1,6P2)[20]. LDHA has been shown to be essential for microvascular ECs by enhancing VEGF production in these cells during angiogenesis[27,28]
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