Developmental and tumoral vascularization is regulated by G protein–coupled receptor kinase 2
Verónica Rivas, … , Federico Mayor Jr., Petronila Penela
Verónica Rivas, … , Federico Mayor Jr., Petronila Penela
Published October 25, 2013
Citation Information: J Clin Invest. 2013;123(11):4714-4730. https://doi.org/10.1172/JCI67333.
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Research Article Oncology

Developmental and tumoral vascularization is regulated by G protein–coupled receptor kinase 2

Abstract

Tumor vessel dysfunction is a pivotal event in cancer progression. Using an in vivo neovascularization model, we identified G protein–coupled receptor kinase 2 (GRK2) as a key angiogenesis regulator. An impaired angiogenic response involving immature vessels was observed in mice hemizygous for Grk2 or in animals with endothelium-specific Grk2 silencing. ECs isolated from these animals displayed intrinsic alterations in migration, TGF-β signaling, and formation of tubular networks. Remarkably, an altered pattern of vessel growth and maturation was detected in postnatal retinas from endothelium-specific Grk2 knockout animals. Mouse embryos with systemic or endothelium-selective Grk2 ablation had marked vascular malformations involving impaired recruitment of mural cells. Moreover, decreased endothelial Grk2 dosage accelerated tumor growth in mice, along with reduced pericyte vessel coverage and enhanced macrophage infiltration, and this transformed environment promoted decreased GRK2 in ECs and human breast cancer vessels. Our study suggests that GRK2 downregulation is a relevant event in the tumoral angiogenic switch.

Authors

Verónica Rivas, Rita Carmona, Ramón Muñoz-Chápuli, Marta Mendiola, Laura Nogués, Clara Reglero, María Miguel-Martín, Ramón García-Escudero, Gerald W. Dorn II, David Hardisson, Federico Mayor Jr., Petronila Penela

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Figure 3

Effect of GRK2 downregulation on TGF-β1 endothelial signaling.

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Effect of GRK2 downregulation on TGF-β1 endothelial signaling. (A) ECs w...
(A) ECs with reduced expression of GRK2 are more sensitive to dose-dependent inhibitory effects of TGF-β1 on tube network formation. In vitro tube formation was monitored in the presence of 1 μM S1P supplemented with or without 0.5 or 5 ng/ml TGF-β1 and quantified as in Figure 2, D and E. Data from 3 independent assays performed in duplicate are shown. Scale bar: 500 μm. (B and C) Proper signaling of Smads downstream of ALK1 and ALK5 receptor activation depends on GRK2 expression. (B) Grk2+/– MLECs display enhanced TGF-β1–triggered activation of Smad2/3 paralleled by a decrease in Smad1/5/8 stimulation. The lanes were run on the same gel but, where indicated by the black vertical line, were noncontiguous. (C) BMP9-induced activation of Smad1/5/8 via ALK1 is reduced in Grk2+/– MLECs. (D) Both basal and angiogenic factor–induced PDGF-BB secretion are altered in Grk2+/– MLECs. Serum-starved cells were incubated for 48 hours in 1% FCS supplemented with or without 50 ng/ml VEGF or 5 ng/ml TGF-β1, and PDGF-BB was determined in the cell-conditioned media as described in Methods. (E) The production of PDGF-BB in Grk2+/– MLECs is less sensitive to ALK5 signaling inhibitors. Media of cells treated with 5 ng/ml TGF-β1 for 48 hours in the presence or absence of the ALK5 inhibitor A8301 (5 μM) was processed for PDGF-BB quantification. In B–E data from 3–4 independent experiments are shown.