Developmental and tumoral vascularization is regulated by G protein–coupled receptor kinase 2
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
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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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 8

Absence of endothelial GRK2 expression enhances monocyte migration in vitro and promotes tumor growth in a macrophage-dependent manner.

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Absence of endothelial GRK2 expression enhances monocyte migration in vi...
(A) Enhanced RAW267.4 macrophage recruitment in response to conditioned media from ECs lacking GRK2. 48-hour-conditioned media of MLECs from WT Grk2+/+ or Tie2Cre-Grk2fl/fl mice were used as chemoattractant in Transwell migration assays (data from 3 to 4 independent experiments performed in duplicate). (B) Lack of endothelial GRK2 contributes to enhance intratumoral hypoxia and adrenomedullin expression. B16F10 melanoma cells were subcutaneously injected in mice, and tumors were removed 17 days after and stained with the anti-pimonidazole–based adducts FITC-Mab1 antibody for detection of hypoxia or anti-adrenomedullin antibody, as detailed in Methods, and hematoxylin counterstained. Scale bar: 500 μm (hypoxia, first and third images); 50 μm (hypoxia, second and fourth images); 100 μm (adrenomedullin). (C and D) M2-polarized macrophages enhance tumor growth. B16F10 melanoma cells were subcutaneously injected in WT or Tie2Cre-Grk2fl/fl mice in the presence or absence of IL4-pretreated RAW267.4 cells as described in Methods. Sizes of tumors implanted in WT mice equal those in Tie2Cre-Grk2fl/fl mice in control conditions when tumor cells in the formers are implemented with macrophages. (E and F) Effects of clodronate-promoted depletion of macrophages on tumor growth. Tumor-bearing mice were treated with control liposomes (encapsome) or clodronate-encapsulated liposomes by intraperitoneal injection starting 24 hours before tumor cell inoculation, followed by treatments every 4 days. Macrophages are involved in the higher growth of tumor cells implanted in Tie2Cre-Grk2fl/fl mice compared to WT animals, as such differential growth is abrogated in clodronate-treated Tie2Cre-Grk2fl/fl mice. Five to eight animals were analyzed for each condition.