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CAMKII as a therapeutic target for growth factor–induced retinal and choroidal neovascularization
Sadaf Ashraf, … , J. Graham McGeown, Tim M. Curtis
Sadaf Ashraf, … , J. Graham McGeown, Tim M. Curtis
Published February 5, 2019
Citation Information: JCI Insight. 2019;4(6):e122442. https://doi.org/10.1172/jci.insight.122442.
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Research Article Angiogenesis Ophthalmology

CAMKII as a therapeutic target for growth factor–induced retinal and choroidal neovascularization

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Abstract

While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti‑VEGF therapy include the upregulation of other proangiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signaling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor–induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and -δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularization in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signaling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularization, while enhancing reparative angiogenesis in the ischemic retina.

Authors

Sadaf Ashraf, Samuel Bell, Caitriona O’Leary, Paul Canning, Ileana Micu, Jose A. Fernandez, Michael O’Hare, Peter Barabas, Hannah McCauley, Derek P. Brazil, Alan W. Stitt, J. Graham McGeown, Tim M. Curtis

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Figure 1

Ca2+/calmodulin-dependent kinase II (CAMKII) contributes to growth factor–induced retinal angiogenesis in vitro.

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Ca2+/calmodulin-dependent kinase II (CAMKII) contributes to growth facto...
(A) The time-dependent effects of various growth factors on the phosphorylation level of the CAMKII autoactivation site (T287) was investigated in human retinal microvascular endothelial cells (HRMECs) by Western blot analysis. Left, representative Western blots showing that VEGF, bFGF, HGF, and IGF-1 trigger CAMKII phosphorylation (pCAMKII) in HRMECs in a time-dependent manner. β-Actin was used as a loading control. Right, summary data calculated from the integrated density of the protein bands (normalized to β-actin) and expressed as a fold-change from untreated cells (time 0). Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. time 0 based on ANOVA; n = 4 biological replicates. (B) Tubulogenesis assays were performed on HRMECs that were untreated or treated with various growth factors in the absence or presence of the CAMKII inhibitor KN93 (10 μM) or its inactive analogue KN92 (10 μM). Top, typical images showing the effects of KN93 and KN92 on VEGF-induced tube formation in HRMECs stained with calcein green. Scale bar: 100 μm. Bottom, box-and-whisker plots (min, max, 25th–75th percentile, median) showing that VEGF, bFGF, HGF, and IGF-1 stimulate HRMEC tubulogenesis in a CAMKII-dependent manner. *P < 0.05, ***P < 0.001 based on ANOVA; n = 4 biological and 3 technical replicates.

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