Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting
Peter D. Westenskow, Toshihide Kurihara, Edith Aguilar, Elizabeth L. Scheppke, Stacey K. Moreno, Carli Wittgrove, Valentina Marchetti, Iacovos P. Michael, Sudarshan Anand, Andras Nagy, David Cheresh, Martin Friedlander
Peter D. Westenskow, Toshihide Kurihara, Edith Aguilar, Elizabeth L. Scheppke, Stacey K. Moreno, Carli Wittgrove, Valentina Marchetti, Iacovos P. Michael, Sudarshan Anand, Andras Nagy, David Cheresh, Martin Friedlander
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Research Article Vascular biology

Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting

Abstract

Vascular networks develop from a growing vascular front that responds to VEGF and other guidance cues. Angiogenesis is required for normal tissue function, but, under conditions of stress, inappropriate vascularization can lead to disease. Therefore, inhibition of angiogenic sprouting may prevent neovascularization in patients with blinding neovascular eye diseases, including macular degeneration. VEGF antagonists have therapeutic benefits but also can elicit off-target effects. Here, we found that the Ras pathway, which functions downstream of a wide range of cytokines including VEGF, is active in the growing vascular front of developing and pathological vascular networks. The endogenous Ras inhibitor p120RasGAP was expressed predominately in quiescent VEGF-insensitive endothelial cells and was ectopically downregulated in multiple neovascular models. MicroRNA-132 negatively regulated p120RasGAP expression. Experimental delivery of α-miR-132 to developing mouse eyes disrupted tip cell Ras activity and prevented angiogenic sprouting. This strategy prevented ocular neovascularization in multiple rodent models even more potently than the VEGF antagonist, VEGF-trap. Targeting microRNA-132 as a therapeutic strategy may prove useful for treating multiple neovascular diseases of the eye and for preventing vision loss regardless of the neovascular stimulus.

Authors

Peter D. Westenskow, Toshihide Kurihara, Edith Aguilar, Elizabeth L. Scheppke, Stacey K. Moreno, Carli Wittgrove, Valentina Marchetti, Iacovos P. Michael, Sudarshan Anand, Andras Nagy, David Cheresh, Martin Friedlander

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Figure 3

α-miR-132 injections enhance p120RasGAP expression and prevent neovascularization in OIR mice.

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α-miR-132 injections enhance p120RasGAP expression and prevent neovascul...
(A) Rasa1 expression during normal development was quantified using qPCR and plotted in relative values (normalized to Rasa1 expression levels measured at P9). Note the linear increase until P16 when expression levels saturate. (B) miR-132 expression in the same retinas was plotted similarly. Expression of miR-132 is inverted compared with that of Rasa1, suggesting that miR-132 negatively regulates p120RasGAP to promote angiogenesis until the vascular networks are constructed. (C) Quantification of Rasa1 and miR-132 in OIR mice. The fold change data were normalized to fit on a 1.0- to 2.3-fold change scale (the actual highest fold change value for both was 2.3 for miR-132 and 2.2 for Rasa1). (D and E) Fluorophore-conjugated α-miR-132 constructs (green) were injected at P12, and the constructs were largely taken up by activated endothelial cells (red) at P14 (D) and P16 (E). (F) qPCR analyses confirm that Rasa1 is significantly upregulated and miR-132 is downregulated in eyes injected with α-miR-132 (n = 4; *P = 0.04). GS lectin–stained neural retina flat-mount preparations of (G) vehicle- and (H) α-miR-132–injected retinas isolated from P17 OIR mice. Neovascular tufts and vaso-obliterated regions are labeled red and yellow, respectively. (I) Total area of tufts and vaso-obliterated regions in vehicle- and α-miR-132–injected eyes was calculated and plotted (OIR experiments were repeated 6 times with n = 8–13 mice; a representative experiment with n = 12 is shown; *P = 0.001). Scale bars: 50 μm (D and E); 1 mm (G and H). Error bars represent SEM.