Hypoxia activates matrix metalloproteinase expression and the VEGF system in monkey choroid-retinal endothelial cells: Involvement of cytosolic phospholipase A2 activity.
OBJECTIVE
To determine whether the gene expression of matrix metalloproteinases (MMPs) as well as that of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and its receptors change in response to hypoxic exposure in a primate choroid-retinal endothelial cell line, and furthermore, whether cytosolic phospholipase A2 (cPLA2) plays a role in this process.


METHODS
Rhesus macaque choroid-retinal endothelial (RF/6A) cells were incubated under hypoxic conditions for 1, 2, 4, or 8 h prior to RNA extraction. In some experiments cells were pretreated with the cPLA2 inhibitor AACOCF3 (10 microM) for 30 min prior to hypoxia. Changes in gene expression were determined by RT-PCR and quantified by real-time PCR for urokinase plasminogen activator (uPA), collagenase-1 (MMP-1), membrane type-1 metalloproteinase (MT1-MMP), gelatinases A and B (MMP-2, MMP-9), tissue inhibitor-2 (TIMP-2), VEGF and its receptors, Flt-1 (VEGFR-1), KDR (VEGFR-2), and neuropilin-1 (NP-1). MMP-2 secreted by the cells was evaluated by zymography. VEGF release was measured by ELISA. In tube-formation studies, endothelial cells (EC) were seeded into collagen gel, exposed to hypoxia for 4 h, then incubated under normoxic conditions for 72 h.


RESULTS
Hypoxia triggered a three fold increase in the gene expression of MT1-MMP, MMP-2, and TIMP-2, and a ten fold increase in MMP-2 levels. Moreover it also induced tube formation in EC. Expression of uPA, MMP-1, and MMP-9 mRNA was not detected. Pretreatment with AACOCF3 abolished hypoxia-induced tube formation and MT1-MMP, MMP-2, and TIMP-2 transcription. Furthermore, hypoxia produced a significant, sustained increase in the gene expression and release of VEGF-165, the only VEGF-A isoform detected in these cells. AACOCF3 reduced the hypoxia-induced VEGF release at 8 h of hypoxia. VEGF receptors KDR and NP-1 were constitutively expressed in EC and up-regulated under hypoxic conditions.


CONCLUSIONS
In monkey choroid-retinal EC, hypoxia selectively induces MMP-2 activity. This induction is preceded by MT1-MMP, MMP-2, and TIMP-2 mRNA expression, as well as that of the VEGF-165 isoform and its receptors KDR and NP1. These increases possibly result from hypoxia-induced activation of cPLA2 and subsequent release of arachidonic acid and its conversion to prostaglandins. These molecular changes in EC could, in part, contribute to the angiogenic response that occurs in the development of ischemic retinopathies and choroidal neovascularization.
Ottino
Paulo
P
Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Finley
Joelle
J
Rojo
Eileen
E
Ottlecz
Anna
A
Lambrou
George N
GN
Bazan
Haydee E P
HE
Bazan
Nicolas G
NG
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D015687:Cell Hypoxia; D002460:Cell Line; D002829:Choroid / Q000098:blood supply; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D000860:Hypoxia / Q000378:metabolism*; D008253:Macaca mulatta; D020782:Matrix Metalloproteinases / Q000235:genetics* / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000473:pathology; D039942:Neuropilin-1 / Q000235:genetics / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D012333:RNA, Messenger / Q000378:metabolism; D012171:Retinal Vessels; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C081565:arachidonyltrifluoromethane; D039942:Neuropilin-1; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D010741:Phospholipases A; D054467:Phospholipases A2; D020782:Matrix Metalloproteinases