Abstract
RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.
Highlights
Angiogenesis is defined as a process that leads to the formation of new blood vessels from pre-existing ones
MiRNAs appear to be critically involved in regulating the expression of angiogenesisrelated factors and, through them, influence important sub steps of angiogenesis, such as angiogenic sprouting, migration, proliferation, and tube formation of endothelial cells [10]
In addition to altered expression levels of certain miRNAs, it has been demonstrated that the process of new blood vessel formation and the function and angiogenic capacity of human endothelial cells can be modified by RNAi-mediated downregulation of the miRNAs biogenesis enzymes DICER1 and DROSHA, enzymes that may be involved in endothelial cell differentiation and morphogenesis in the embryonic or fetal organism [12,13,14]
Summary
Angiogenesis is defined as a process that leads to the formation of new blood vessels from pre-existing ones. At the beginning of the angiogenic process, activation of the normally quiescent vascular endothelium mediated by pro-angiogenic signals occurs, which initiates and facilitates angiogenic sprouting, proliferation, migration, and tube formation of endothelial cells in the surrounding tissue. This leads to the formation of new vascular structures that are connected to the blood circulation and mature under the mechanical influences of blood flow and through recruitment of mural cells to gain full functionality [1,2]. It plays a role in the regulation of vascular tone and systemic blood pressure, which are thought to be influenced by VEGF-A-VEGFR-2-mediated activation of Akt-eNOS and activation of endothelial survival signaling and nitric oxide release [7]
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