SU5416 induces premature senescence in endothelial progenitor cells from patients with age-related macular degeneration.
OBJECTIVE
We recently demonstrated increased frequency and growth potential of late outgrowth endothelial progenitor cells (OECs) in patients with neovascular age-related macular degeneration (nvAMD). This study investigated the effects of short- and long-term in vitro inhibition of vascular endothelial growth factor (VEGF) Receptor-2 (VEGFR-2) signaling by SU5416 and other inhibitors of the VEGF signaling pathway in OECs.


METHODS
OECs, from the peripheral blood of patients with nvAMD, and human umbilical vein endothelial cells were grown in the presence of SU5416, other VEGFR-2 tyrosine kinase inhibitors (TKIs), and inhibitors of phosphatidylinositol 3'-Kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) in complete angiogenic medium. Apotosis was assessed after 48 h using the fluorescein isothiocyanate Annexin V method. Cell counts were performed for 10 days, and features of senescence were analyzed using senescence-associated β-galactosidase staining, the telomeric repeat amplification protocol for telomerase activity, Southern blot analysis for mean telomere length, flow cytometric analysis for cell-cycle arrest, and western blot for p53 and p21. Control OECs, cells treated for 7 days with inhibitors, as well as naturally senescent OECs were analyzed for expression of different endothelial antigens, including VEGFR-2 and the receptor for stromal cell-derived factor 1, chemokine receptor 4 (CXCR-4). Migration in vitro to VEGF and stromal cell-derived factor 1 of OECs was assessed.


RESULTS
SU5416, other VEGFR-2 TKIs, and inhibitors of PI3K, Akt, and PKC induced apoptosis, inhibited long-term proliferation, reduced telomerase activity, and induced premature senescence and cell-cycle arrest in OECs as well as in human umbilical vein endothelial cells. Naturally senescent cells and cells rendered senescent by VEGFR-2 TKIs had reduced VEGFR-2 and CXCR-4 expression and demonstrated reduced migratory ability to VEGF.


CONCLUSIONS
This study demonstrates apoptosis upon short-term inhibition and inhibition of long-term survival of OECs from patients with nvAMD by SU5416, presumably via PI3K/Akt and/or PKC-mediated reduction in telomerase activity and subsequent induction of premature senescence, which is accompanied by impaired endothelial activity. Therefore, induction of premature senescence in endothelial cells may represent a potential therapeutic target in nvAMD.
University Hospital Hamburg-Eppendorf, Ophthalmology, Martinistrasse 52, Hamburg, Hamburg 20246, Germany. mthill@uke.de
Michelle
M
Thill
Marc J
MJ
Berna
Frank
F
Kunst
Henning
H
Wege
Natalya V
NV
Strunnikova
Natalya
N
Gordiyenko
Rebecca
R
Grierson
Gisbert
G
Richard
Karl G
KG
Csaky
D017209:Apoptosis; D016922:Cellular Senescence / Q000187:drug effects*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008268:Macular Degeneration / Q000188:drug therapy* / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology*; D019718:Receptors, CXCR4 / Q000096:biosynthesis; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects*; D016896:Treatment Outcome; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis / Q000378:metabolism
D007211:Indoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D019718:Receptors, CXCR4; C116890:Semaxinib; D040301:Vascular Endothelial Growth Factor Receptor-2; D011493:Protein Kinase C
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