PKM2 regula la angiogénesis mediante la producción de ATP de forma local en celulas endoteliales

Resumen

Angiogenesis is the formation of new blood vessels from pre-existing ones. This process begins with the reception by the endothelial cells of a stimulus that triggers the angiogenic response. For that purpose, one endothelial cell, called tip cell, is activated and begins to migrate in direction of the stimulus, with the other cells (stalk cells) of the vessel following it. Several cell mechanisms are implicated at this initial step, as the reorganization of endothelial junctions, the migration of the tip cell by generating cytoskeletal protrusions called filopodia, and the proliferation of the stalk cells. The impairment of these processes avoid a suitable angiogenesis process. The activation of the endothelial cells induces an increase of the metabolism that is crucial, as its inhibition blocks angiogenesis. Glycolysis is the main metabolic pathway to obtain energy by the synthesis of ATP in the endothelial cells, and has several steps that regulate the flux to allow the production of energy, and to provide the other metabolic pathways with intermediate metabolites of the glycolysis. Moreover, all the cell mechanisms that allow the growth of the new vessel (endothelial cell junction, migration and proliferation) need an efficient support of ATP to work efficiently. Cell metabolism is not homogenous as several enzymes can be compartmentalized in order to function in a more effective way, as the diffusion of ATP through the cell is not fast enough to provide energy for processes that occur in specific places of the cell. Local ATP production can regulate endothelial cell dynamics, the establishment of new cell-contacts, the movement of vesicles through cytoskeletal structures, or the reorganization of the cytoskeleton. PKM2 is a key regulatory enzyme that catalyzes the last step of the glycolysis. It is a protein with a great implication in several pathologies as cancer or inflammation. For this reason it has been investigated mainly in cancer cells, but there are however scarce studies in endothelial cells or angiogenesis. PKM2 has two conformations, one more active and other more inactive, and thus it can regulate the flux of the glycolysis to allow the production of ATP or the synthesis of macromolecules for the proliferation of the cells. We found that PKM2 is important for angiogenesis as in spheroid sprouting assays the interference of its expression by a siRNA reduces the number and the length of the new vessel structures. Analyzing the phenotype of the interference in endothelial cells in culture, we obtained that PKM2 regulates the migration and the endothelial cell junction dynamics. Indeed the regulation of the barrier function of the endothelial cells was compromised by the interference of PKM2. In contrast, PKM2 expression is dispensable for the proliferation in endothelial cells. A pool of PKM2 localizes at endothelial cell junction, and also at the lamellipodias of migrating cells in culture. We checked the localization in the sprout assay, and confirmed that PKM2 is located at endothelial junction and also at the filopodia. As PKM2 is an enzyme, we study the metabolism state of the interfered cells. The ECAR values that reflect the glycolysis flux was affected, but not the oxygen consumption, reflecting that the function of the mitochondria remained intact. We analyzed the pyruvate kinase activity in cytosolic and in cytoskeletal-membrane fraction, and we discovered that the activity was higher in the cytoskeletal-membrane than in the cytosol. This fact can explain that the proliferation was not affected by PKM2, as the lower activity in the cytosol may allow the accumulation of intermediate metabolites of the glycolysis that provide of substrates for the proliferation, while in the other places is more active, generating ATP for regulating other processes. Indeed we found that the levels of ATP were lower at the cytoskeletal-membrane fraction in endothelial cells by biochemistry and also by the observation of a fluorescence construction, GO-aTeam1, which allows the quantification of ATP at subcellular levels. To test the hypothesis that the activity of PKM2 is the responsible of the phenotype, we used an inhibitor of PKM2, shikonin. We found that the migration and the endothelial cells dynamics were affected, but not the proliferation, in cells in culture, and also in the spheroid sprouting assay. Finally we analyzed the impact of reducing PKM2 in the vascular development in the postnatal mouse retina, by the intravitreal injection of siRNA or with shikonin. We found that the radial vascular growth was reduced in both conditions. The number of filopodia and the endothelial junction dynamics were also affected, but not the number of endothelial cells or the vascular density, reflecting that the proliferation was not compromised. We described the role of PKM2 in angiogenesis, regulating endothelial junctions and cytoskeletal dynamics by local ATP production. Due to the importance of PKM2 in pathologies such as cancer, we provided new features to take in account to develop new strategies for improvement of treatments for these diseases.