TY - JOUR
T1 - Claudin-5 redistribution induced by inflammation leads to anti-VEGF–resistant diabetic macular edema
AU - Arima, Mitsuru
AU - Nakao, Shintaro
AU - Yamaguchi, Muneo
AU - Feng, Hao
AU - Fujii, Yuya
AU - Shibata, Kensuke
AU - Wada, Iori
AU - Kaizu, Yoshihiro
AU - Ahmadieh, Hamid
AU - Ishibashi, Tatsuro
AU - Stitt, Alan W.
AU - Sonoda, Koh Hei
N1 - Funding Information:
This study was partly supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (17K16997 [M.A.] and 17K11456 [S.N.]), Charitable Trust Fund for Ophthalmic Research in Commemoration of Santen Pharmaceutical’s Founder (S.N.), and the Takeda Science Foundation (S.N.). The powder and ophthalmic solution of ripasudil used in this study were provided free of charge from Kowa Company, Ltd., to Kyushu University.
Funding Information:
Acknowledgments. The authors thank Dr. Elizabeth P. Rakoczy (University of Western Australia) for the gift of Kimba mice. The authors also thank Mitsuhiro Kurata, Takako Iwasaki, Masayo Eto, and Mizuho Oda (Kyushu University) for technical assistance. Funding. This study was partly supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (17K16997 [M.A.] and 17K11456 [S.N.]), Charitable Trust Fund for Ophthalmic Research in Commemoration of Santen Pharmaceutical’s Founder (S.N.), and the Takeda Science Foundation (S.N.). The powder and ophthalmic solution of ripasudil used in this study were provided free of charge from Kowa Company, Ltd., to Kyushu University.
Funding Information:
Duality of Interest. This study was also supported by the Bayer Retina Award (S.N.), Novartis Pharmaceuticals research grants (S.N.), Alcon research grants (S.N.), and grants from Kowa Company, Ltd. This research was conducted in collaboration with Kyushu University and Kowa Company, Ltd. S.N. and K.-H.S. received research support fees from Kowa Company, Ltd. No other potential conflicts of interest relevant to this article were reported. Author Contributions. S.N. conceived the project and designed the experiments with M.A. With critical assistance from A.W.S., T.I., and K.-H.S., M.A., M.Y., Y.F., and K.S. performed the in vivo experiments and M.A., H.F., Y.K., and I.W. conducted the in vitro experiments. H.A. performed the clinical study of intravitreal fasudil injection with DME patients. M.A. and S.N. were involved in data analysis. M.A. and S.N. wrote the manuscript. S.N. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Publisher Copyright:
© 2020 by the American Diabetes Association.
PY - 2020/5/1
Y1 - 2020/5/1
N2 - Approximately 40% of patients with diabetic macular edema (DME) are resistant to anti–vascular endothelial growth factor (VEGF) therapy (rDME). Here, we demonstrate that significant correlations between inflammatory cytokines and VEGF, as observed in naive DME, are lost in patients with rDME. VEGF overexpression in the mouse retina caused delayed inflammatory cytokine upregulation, monocyte/macrophage infiltration (CD11b1 Ly6C1 CCR21 cells), macrophage/microglia activation (CD11b1 CD801 cells), and blood-retinal barrier disruption due to claudin-5 redistribution, which did not recover with VEGF blockade alone. Phosphorylated protein analysis of VEGF-overexpressed retinas revealed rho-associated coiled-coil–containing protein kinase (ROCK) activation. Administration of ripasudil, a selective ROCK inhibitor, attenuated retinal inflammation and claudin-5 redistribution. Ripasudil also contributed to the stability of claudin-5 expression by both transcriptional enhancement and degradation suppression in inflammatory cytokine–stimulated endothelium. Notably, the anti-VEGF agent and the ROCK inhibitor were synergic in suppressing cytokine upregulation, monocyte/macrophage infiltration, macrophage/microglia activation, and claudin-5 redistribution. Furthermore, in vitro analysis confirmed that claudin-5 redistribution depends on ROCK2 but not on ROCK1. This synergistic effect was also confirmed in human rDME cases. Our results suggest that ROCK-mediated claudin-5 redistribution by inflammation is a key mechanism in the anti-VEGF resistance of DME.
AB - Approximately 40% of patients with diabetic macular edema (DME) are resistant to anti–vascular endothelial growth factor (VEGF) therapy (rDME). Here, we demonstrate that significant correlations between inflammatory cytokines and VEGF, as observed in naive DME, are lost in patients with rDME. VEGF overexpression in the mouse retina caused delayed inflammatory cytokine upregulation, monocyte/macrophage infiltration (CD11b1 Ly6C1 CCR21 cells), macrophage/microglia activation (CD11b1 CD801 cells), and blood-retinal barrier disruption due to claudin-5 redistribution, which did not recover with VEGF blockade alone. Phosphorylated protein analysis of VEGF-overexpressed retinas revealed rho-associated coiled-coil–containing protein kinase (ROCK) activation. Administration of ripasudil, a selective ROCK inhibitor, attenuated retinal inflammation and claudin-5 redistribution. Ripasudil also contributed to the stability of claudin-5 expression by both transcriptional enhancement and degradation suppression in inflammatory cytokine–stimulated endothelium. Notably, the anti-VEGF agent and the ROCK inhibitor were synergic in suppressing cytokine upregulation, monocyte/macrophage infiltration, macrophage/microglia activation, and claudin-5 redistribution. Furthermore, in vitro analysis confirmed that claudin-5 redistribution depends on ROCK2 but not on ROCK1. This synergistic effect was also confirmed in human rDME cases. Our results suggest that ROCK-mediated claudin-5 redistribution by inflammation is a key mechanism in the anti-VEGF resistance of DME.
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U2 - 10.2337/db19-1121
DO - 10.2337/db19-1121
M3 - Article
C2 - 32139595
AN - SCOPUS:85083769919
SN - 0012-1797
VL - 69
SP - 981
EP - 999
JO - Diabetes
JF - Diabetes
IS - 5
ER -
