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Titre
PKM2 regula la angiogénesis mediante la producción de ATP de forma local en celulas endoteliales
Otros títulos
PKM2 regulates angiogenesis by local ATP production in endothelial cells
Autor(es)
Director(es)
Sujet
Centro Nacional de Investigaciones Cardiovasculares (Madrid, España)
PHARMACY
Tesis y disertaciones académicas
Universidad de Salamanca (España)
Academic dissertations
Metabolismo energético
Angiología
Biología molecular
Fecha de publicación
2017-09-15
Resumen
[EN] 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.
URI
DOI
10.14201/gredos.136877
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