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Akiyoshi Uemura, Yoko Fukushima, Kohji Nishida, Shin-Ichi Nishikawa; Sema3E-Induced Activation of RhoJ Small GTPase Retracts Endothelial Filopodia in Retinal Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4841.
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© ARVO (1962-2015); The Authors (2016-present)
In retinal angiogenesis, VEGF induces projections of endothelial filopodia, which retract in response to the binding of Sema3E to the endothelial PlexinD1 receptor. To determine intracellular signaling machineries that regulate endothelial filopodia formation, we investigated roles of RhoJ small GTPase in cultured endothelial cells (ECs) and in murine retinal angiogenesis.
We performed whole-mount in situ hybridization in retinas of postnatal mice and of oxygen-induced retinopathy (OIR) mouse models with CD1 or C57BL/6 backgrounds. Cultured human umbilical vein endothelial cells (HUVECs) were transfected with plasmid vectors or Stealth RNAiTM siRNAs (Invitrogen) using TransIT-LT1 (Mirus) or Lipofectamine RNAiMAX (Invitrogen), respectively. RhoJ-GTP levels in cultured HUVECs were measured using the colorimetric G-LISA Cdc42 activation assay (Cytoskeleton Inc) in conjunction with anti-RhoJ antibody (Abnova). R26-RhoJ conditional transgenic mice, in which RhoJ is overexpressed after Cre-loxP-mediated genetic recombination, were crossed to the Tie2-Cre mice.
In postnatal mouse retinas, the RhoJ gene is specifically expressed in ECs of growing vessels. Endothelial RhoJ expression is downregulated in accordance with the maturation of retinal vasculature, but is re-upregulated in the OIR model. In cultured HUVECs, while ectopic RhoJ expression induced cell contraction, knockdown of RhoJ abolished Sema3E-induced cell contraction. Intriguingly, RhoJ was activated by Sema3E, but was inactivated by VEGF. In retinas of Tie2-Cre:R26-RhoJ double transgenic mice, we observed membrane blebbing in ECs of sprouting vessels, which implied the accelerated retraction of endothelial filopodia.
We showed for the first time that RhoJ is an endothelial-specific small GTPase which mediates Sema3E-induced filopodia retraction. Our results indicate the possibility of RhoJ as the therapeutic target to treat aberrant angiogenesis in human retinal diseases.
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