Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function.
OBJECTIVE
This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF165-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology.


METHODS
C57/Bl6 mice were injected with VEGF165 and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF165 and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability.


RESULTS
Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF165. It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF165-mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF165-mediated permeability compared with control (untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF165.


CONCLUSIONS
Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF165-induced paracellular permeability.
Campbell
Matthew
M
UCD School of Biomolecular Biomedical Sciences, Conway Institute, University College Dublin, Dublin, Ireland. campbem2@tcd.ie
Collery
Ross
R
McEvoy
Alice
A
Gardiner
Tom A
TA
Stitt
Alan W
AW
Brankin
Brenda
B
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001813:Blood-Retinal Barrier / Q000187:drug effects*; D015153:Blotting, Western; D002417:Cattle; D043169:Endostatins / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D062793:Occludin; D012171:Retinal Vessels / Q000166:cytology; D019108:Tight Junctions; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D020533:Angiogenesis Inhibitors; D043169:Endostatins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases