Vascular endothelial growth factor upregulates expression of ADAMTS1 in endothelial cells through protein kinase C signaling.
OBJECTIVE
ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) has been demonstrated to inhibit angiogenesis in vivo and to suppress endothelial cell proliferation in vitro. The purpose of this study was to investigate the expression of ADAMTS1 in endothelial cells and in a mouse model of ischemia-induced retinal neovascularization and to study the regulation of ADAMTS1 expression in endothelial cells by vascular endothelial growth factor (VEGF). In addition, the potential function of endothelial cell-derived ADAMTS1 on cell proliferation was investigated.


METHODS
Expression of ADAMTS1 in human retinal endothelial cells (HRECs), human umbilical vein endothelial cells (HUVECs), and the mouse model of ischemia-induced retinal neovascularization was assayed by real-time PCR and Western blot analysis. The effect of ADAMTS1 on endothelial cell proliferation was evaluated using siRNA knockdown and [3H] thymidine incorporation.


RESULTS
ADAMTS1 mRNA and protein levels were increased in a mouse model of ischemia-induced retinal neovascularization, and VEGF induced time- and dose-dependent increases in ADAMTS1 mRNA and protein expression in endothelial cells. This upregulation was inhibited by the VEGF receptor (VEGFR)2 inhibitor SU1498, anti-VEGFR2 neutralizing antibody, and the phospholipase C (PLC)-gamma inhibitor U73122. VEGF upregulation of ADAMTS1 expression was completely abolished by the inhibition of protein kinase C by calphostin C and largely blocked by the specific inhibition of PKCbeta. Knockdown of endogenous ADAMTS1 resulted in increased proliferation of endothelial cells.


CONCLUSIONS
These results indicate that VEGF significantly induces ADAMTS1 expression in endothelial cells in a PKC-dependent fashion. ADAMTS1 expression is also increased, along with VEGF expression, in vivo in ischemia-induced retinal neovascularization. In addition, ADAMTS1 appears to be an endogenous regulator of endothelial cell proliferation. Therefore, VEGF upregulation of ADAMTS1, a potent angiogenesis inhibitor, may represent a mechanism for feedback inhibition of angiogenesis and retinal neovascularization.
Xu
Zhenhua
Z
Department of Ophthalmology, The Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Yu
Yilin
Y
Duh
Elia J
EJ
D051722:ADAM Proteins / Q000235:genetics* / Q000378:metabolism; D000071097:ADAMTS1 Protein; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen / Q000633:toxicity; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015861:Retinal Neovascularization / Q000378:metabolism; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D012333:RNA, Messenger; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D011493:Protein Kinase C; D051722:ADAM Proteins; D000071097:ADAMTS1 Protein; C103859:ADAMTS1 protein, human; D010100:Oxygen