The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity
Yuka Ichikawa-Shindo, … , Ryozo Nagai, Takayuki Shindo
Yuka Ichikawa-Shindo, … , Ryozo Nagai, Takayuki Shindo
Published December 20, 2007
Citation Information: J Clin Invest. 2008;118(1):29-39. https://doi.org/10.1172/JCI33022.
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Research Article

The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity

Abstract

Adrenomedullin (AM) is a peptide involved both in the pathogenesis of cardiovascular diseases and in circulatory homeostasis. The high-affinity AM receptor is composed of receptor activity–modifying protein 2 or 3 (RAMP2 or -3) and the GPCR calcitonin receptor–like receptor. Testing our hypothesis that RAMP2 is a key determinant of the effects of AM on the vasculature, we generated and analyzed mice lacking RAMP2. Similar to AM–/– embryos, RAMP2–/– embryos died in utero at midgestation due to vascular fragility that led to severe edema and hemorrhage. Vascular ECs in RAMP2–/– embryos were severely deformed and detached from the basement membrane. In addition, the abnormally thin arterial walls of these mice had a severe disruption of their typically multilayer structure. Expression of tight junction, adherence junction, and basement membrane molecules by ECs was diminished in RAMP2–/– embryos, leading to paracellular leakage and likely contributing to the severe edema observed. In adult RAMP2+/– mice, reduced RAMP2 expression led to vascular hyperpermeability and impaired neovascularization. Conversely, ECs overexpressing RAMP2 had enhanced capillary formation, firmer tight junctions, and reduced vascular permeability. Our findings in human cells and in mice demonstrate that RAMP2 is a key determinant of the effects of AM on the vasculature and is essential for angiogenesis and vascular integrity.

Authors

Yuka Ichikawa-Shindo, Takayuki Sakurai, Akiko Kamiyoshi, Hisaka Kawate, Nobuyoshi Iinuma, Takahiro Yoshizawa, Teruhide Koyama, Junichi Fukuchi, Satoshi Iimuro, Nobuo Moriyama, Hayato Kawakami, Toshinori Murata, Kenji Kangawa, Ryozo Nagai, Takayuki Shindo

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Figure 4

Establishment and functional analysis of the RAMP2O/E line.

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Establishment and functional analysis of the RAMP2O/E line. (A) Plasmid ...
(A) Plasmid vector used to overexpress RAMP2 (see Methods). (B) Western blot analysis of the membrane protein fraction from RAMP2O/E cells showing expression of the transfected gene. (C–E) Capillary formation by EAhy926 cells on Matrigel. RAMP2O/E cells or control ECs were cultured in 24-well culture plates coated with Matrigel in medium containing 10–7 M AM, and capillary formation was monitored microscopically. (C) Capillary area relative to day-1 cell surface area. RAMP2O/E cells exhibited greater angiogenesis than control. n = 8 per group. (D and E) Representative photomicrographs of RAMP2O/E and control cells. (F) In vitro vascular permeability assay (see Methods). The permeability of the monolayer, assessed using a fluorescence microplate reader, is expressed relative to control at 5 min. RAMP2O/E cells showed significantly lower permeability than control ECs. n = 10 per group. *P < 0.05, **P < 0.01 vs. control. (G–J) Immunostaining of ZO-1. ECs were cultured until confluent on chamber slides in the presence of 10-7 M AM. Two hours after treatment with 0.5 mM H2O2, the cells were immunostained using anti–ZO-1 antibody and Hoechst 33342. (K) Comparison of the tight junctions illustrated by the immunostaining in G–J. Tight junctions were better preserved after H2O2 treatment in RAMP2O/E cells than control ECs. **P < 0.01 vs. H2O2-treated control; comparison in 4 microscopic fields each from 3 independent experiments. (L) Quantitative real-time PCR analysis of gene expression in ECs cultured on Matrigel. Values are relative to control ECs treated with 10–7 M AM. RAMP2O/E cells showed stronger expression of VEGF, eNOS, and CDN5 than control cells; this effect was blocked by LY294002 (10-6 M) or a PKA inhibitor (10-6 M). n = 6 per group. ##P < 0.01 and #P < 0.05 vs. AM-treated control. **P < 0.01 and *P < 0.05 vs. AM-treated RAMP2O/E. Scale bars: 50 μm (D and E); 10 μm (G–J).