Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting
Peter D. Westenskow, … , David Cheresh, Martin Friedlander
Peter D. Westenskow, … , David Cheresh, Martin Friedlander
Published October 1, 2013
Citation Information: J Clin Invest. 2013;123(11):4900-4908. https://doi.org/10.1172/JCI70230.
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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 5

α-miR-132 is more effective than VEGF-trap at reducing neovascularization.

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α-miR-132 is more effective than VEGF-trap at reducing neovascularizatio...
Images of superficial plexus from (A) vehicle, (B) VEGF-trap–, and (C) α-miR-132–injected OIR retinas. (D) The area of neovascularization for all 3 conditions was plotted (n = 6–8; PBS vs. α-miR-132, *P = 0.03; VEGF-trap vs. α-miR-132, **P = 0.05). Images of the outer retina from (E) vehicle-, (F) VEGF-trap–, or (G) α-miR-132–injected Vldlr–/– mice. (H) Percentages of Vldlr–/– tufts were calculated from vehicle-, VEGF-trap–, or α-miR-132–injected eyes versus contralateral uninjected eyes (n = 6, *P = 0.02). Laser-induced lesions in wild-type B6 mice in (I) vehicle-, (J) VEGF-trap–, or (K) α-miR-132–injected eyes were imaged using confocal microscopy (Z stacks = 5 μm). The pixels in the lesions were pseudocolored a different color for distinction. 3D renditions of the lesions were generated to emphasize morphology and depth of the injuries. (L) Quantification of the volume of the lesions (PBS vs. α-miR-132 and VEGF-trap vs. α-miR-132, n = 8–20, *P = 0.04). (M) Gene-profiling analyses reveal that most of the genes compensatorily upregulated by VEGF-trap in wild-type B6 retinas are not similarly activated by α-miR-132. (N and O) Proteome profiling arrays involving various markers of angiogenesis reveal that several angiogenic and proinflammatory proteins (separated with a gap in both plots) are dysregulated in a compensatory manner by VEGF-trap (black columns) but not α-miR-132 injections (gray bars) at (N) P15 and (O) P17 in OIR mice. Proteins dysregulated by >1.5 fold with P values of less than 0.05 were plotted. Scale bars: 1 mm (A–C and E–G); 50 μm (I–K). Error bars represent SEM.