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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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 4

Loss of endothelial GRK2 impairs developmental outgrowth and maturation of the retinal vasculature.

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Loss of endothelial GRK2 impairs developmental outgrowth and maturation ...
(A and B) Expansion of the primary vascular plexus and endothelial filopodia was reduced in the absence of GRK2. (A) Whole-mount P9 retinas from WT (Grk2fl/fl; n = 5) or Tie2Cre-Grk2fl/fl (n = 5) pups were stained with endothelial FITC-ILB4 marker. The ILB4-positive area was quantified in the entire retinal surface (low magnification) or in proximal regions of the vascular plexus (high magnification) (WT, n = 15; mutant, n = 12) as detailed in Methods. (B) Filopodia of tip cells were counted in high-power fields of 5 WT retinas (n = 17) and 6 Tie2Cre-Grk2fl/fl retinas (n = 18). (C and D) Delayed recruitment of pericytes to retinal ECs and perturbed cell-cell interactions in mutant mice. Whole-mount (C) P9 and (D) P14 retinas of WT (n = 5) and Tie2Cre-Grk2fl/fl (n = 5) mice were double stained with ILB4-FITC and anti-NG2 antibodies, and the corresponding positive areas were quantified (n = 14 and n = 11 images, respectively) as detailed in Methods. (C) Pericyte coverage was expressed as the percentage fraction of colocalizing pericyte- and endothelial-positive areas. (D) Abnormal association of pericytes with endothelial capillaries in the primary vascular plexus of P14 Tie2Cre-Grk2fl/fl retinas. Some pericytes remain dissociated from ECs and make irregular connections between endothelial capillaries (zoomed images). High-magnification fields (n = 12 from retinas of 5 Grk2fl/fl; n = 20 from retinas of 5 Tie2Cre-Grk2fl/fl) were inspected. m, macrophages. Scale bar: 100 μm (A, C, and D); 25 μm (B).