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 2

Endothelial GRK2 protein levels regulate vascular morphogenesis.

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Endothelial GRK2 protein levels regulate vascular morphogenesis. (A–C) A...
(A–C) Angiogenesis in mice with global or endothelial-specific reduction of GRK2 expression is characterized by a lack of functional vessels, concomitant with the occurrence of aberrant vessel-like structures with a poor mural coverage. Sections of FGF2-embeded Matrigel plugs excised (A) from WT Grk2+/+ and global Grk2+/– or (C) from Tie2Cre-Grk2fl/fl mice were analyzed by immunohistochemistry, and the density of invasion by either endothelial or mural cells was determined with (A) anti-CD31 or anti-NG2 or (C) SMA antibodies, respectively, as detailed in Methods. Ratios of CD31-positive cells to total DAPI-stained cells in FGF2-stimulated WT Grk2+/+ and Tie2Cre-Grk2fl/fl mice were 0.22 ± 0.06 and 0.58 ± 0.28, respectively. (B) Endothelial GRK2 downmodulation is sufficient to impair the functionality of angiogenic vessels. The angiogenic response in WT or Tie2Cre-GRK2 f/+ mice was determined as in Figure 1A. Data from 12 animals for each condition in 4 independent experiments are shown. (D and E) ECs with deficient GRK2 expression are less competent to form capillary-like networks in vitro. MLECs from WT (Grk2+/+), global hemizygous (Grk2+/–), or endothelial-specific (Tie2Cre-Grk2fl/fl) GRK2 knockout mice were seeded on Matrigel-coated wells in the presence of (D) 1% FCS or (E) 5% FCS. Photographs were taken after 16 to 18 hours in culture (original magnification, ×5), and formation of tubular networks was quantified as described in Methods. Data were obtained from 2 to 3 independent assays performed in duplicate. Scale bar: 100 μm (A); 25 μm (C, Tie2Cre-Grk2fl/fl and vehicle-treated Grk2+/+); 50 μm (C, FGF2-treated Grk2+/+); 500 μm (D and E).