The effects of silencing EDF-1 in human endothelial cells.
OBJECTIVE
EDF-1, a 16 kDa highly conserved intracellular protein, serves as a calmodulin binding protein and, upon nuclear translocation, functions as a coactivator of several transcription factors. To understand whether EDF-1 is implicated in regulating endothelial function, we silenced EDF-1 expression using small hairpin (sh) RNA.


METHODS
Human umbilical vein endothelial cells (HUVEC) were utilized and EDF-1 levels were detected by western blot. Cell proliferation, cell organization in fibrin gel and nitric oxide release were evaluated in cells silencing EDF-1 after transfection with shRNA.


RESULTS
EDF-1 was downregulated in quiescent and senescent HUVEC, whereas it was upregulated in proliferating cells. Knocking down EDF-1 promoted the acquisition of a spindle phenotype, inhibited cell proliferation, accelerated the organization into capillary-like networks on fibrin gels and induced the production of nitric oxide (NO). While the total amounts and the degree of phosphorylation of endothelial NO synthase are not altered in cells silencing EDF-1, we found an increased interaction between calmodulin and endothelial NO synthase. Accordingly, the calmodulin inhibitor calmidazolium significantly decreased NO release in cells silencing EDF-1. These results suggest that knocking down EDF-1 might increase free calmodulin which ultimately activates endothelial NO synthase.


CONCLUSIONS
Since EDF-1 (i) is involved in the control of endothelial proliferation and organization, events which are crucial to repair damages to the vessel wall, and (ii) increases NO, which exerts anti-atherogenic and anti-thrombotic effects, we conclude that EDF-1 is implicated in molecular events that are pivotal to endothelial function and, therefore, to vascular integrity.
Leidi
Marzia
M
Department of Clinical Sciences, Università di Milano Medical School, Via GB Grassi 74, 20157 Milano, Italy.
Mariotti
Massimo
M
Maier
Jeanette A M
JA
D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D014471:Umbilical Veins / Q000378:metabolism; D015854:Up-Regulation
D002148:Calmodulin-Binding Proteins; C115911:EDF1 protein, human; D007093:Imidazoles; C031938:calmidazolium; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III