The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity
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
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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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 6

In vivo vascular permeability assay.

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In vivo vascular permeability assay. (A) Footpad edema model. λ-Carragee...
(A) Footpad edema model. λ-Carrageenan was injected into the footpad of 8-week-old RAMP2+/– and WT mice to induce edema for the evaluation of vascular permeability in adult mice; swelling of the footpad was measured hourly using a thickness gauge. RAMP2+/– mice showed significantly greater swelling than WT mice. n = 12 per group. **P < 0.01, *P < 0.05 vs. WT. (B) Skin edema model (see Methods). Fluorescence intensity was measured using a fluorescence microplate reader. Permeability levels are presented relative to WT. RAMP2+/– mice (n = 8) showed significantly greater vascular permeability than WT mice (n = 13). **P < 0.01 vs. WT. (C) Brain edema model (see Methods). Vascular permeability in RAMP2+/– mice (n = 12) is presented relative to that in WT mice (n = 10). RAMP2+/– mice showed significantly greater vascular permeability than WT mice. *P < 0.05 vs. WT.