Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice
Yoko Fukushima, … , Shin-Ichi Nishikawa, Akiyoshi Uemura
Yoko Fukushima, … , Shin-Ichi Nishikawa, Akiyoshi Uemura
Published April 18, 2011
Citation Information: J Clin Invest. 2011;121(5):1974-1985. https://doi.org/10.1172/JCI44900.
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Research Article Ophthalmology

Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice

Abstract

During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuron-derived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGF-induced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.

Authors

Yoko Fukushima, Mitsuhiro Okada, Hiroshi Kataoka, Masanori Hirashima, Yutaka Yoshida, Fanny Mann, Fumi Gomi, Kohji Nishida, Shin-Ichi Nishikawa, Akiyoshi Uemura

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Figure 4

Sema3E-induced activation of the small GTPase RhoJ retracts endothelial filopodia.

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Sema3E-induced activation of the small GTPase RhoJ retracts endothelial ...
(A) Whole-mount ISH for Rhoj and IHC for Col IV in the P4 retina. (B) Contraction of cultured HUVECs by ectopic RhoJ expression. HUVECs were transfected with a vector expressing the Myc-tagged RhoJ protein, together with a GFP-expressing vector to identify transfected cells. Note the localization of RhoJ-WT proteins in the membrane blebs. (C) Abrogation of Sema3E-induced contraction of cultured HUVECs by knockdown of RhoJ. Note that targeted, but not control (Ctrl), siRNA led to the disappearance of immunoreactivity for RhoJ (red). (D) Proportions of collapsing cells relative to the GFP-expressing HUVECs in B (left) and to the whole siRNA-pretreated HUVECs in C (right). (E) Measurements of RhoJ activation in cultured HUVECs. RhoJ-GTP levels were diminished by VEGF but restored by Sema3E (3E). The values 10, 50, and 500 indicate the protein concentrations (ng/ml). Error bars represent SEM; **P < 0.01, *P < 0.05. (F) Schematic diagram representing RhoJ as an intracellular molecular switch mediating endothelial filopodia retraction. VEGF and Sema3E inversely regulate the activation status of RhoJ. (G) Scheme of the R26-Rhoj Tg. (H and I) Whole-mount IHC for PECAM-1 (red) and GFP (green) in P5 (H) and P7 (I) retinas of R26-Rhoj conditional Tg mice. In I, 4OHT was systemically administered to the CAG-MerCreMer;R26-Rhoj double Tg mice at P3. Note the membrane blebbing (arrowheads) in RhoJ-overexpressing ECs revealed by GFP expression at the sprouting vascular fronts. Scale bars: 100 μm (A); 20 μm (B, C, H, and I).