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 5

Reduced responses to angiogenic stimuli in adult RAMP2+/– mice.

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Reduced responses to angiogenic stimuli in adult RAMP2+/– mice. ...
(A) Gene expression in aortas from 8-week-old WT and RAMP2+/– mice. Quantitative real-time PCR analysis of total RNA extracted. In RAMP2+/– mice, RAMP2 expression was about half that in WT mice, while AM expression was significantly upregulated. n = 6 per group. (B–E) Aortic ring assay. Representative photomicrographs of 7-day collagen gel cultures of aortas from 8-week-old mice (see Methods). Aortic explants from WT and RAMP2+/– mice were cultured in the absence or presence of VEGF (50 ng/ml), and capillaries sprouting from the edges of the rings were analyzed. The aortic explants from RAMP2+/– mice showed diminished angiogenesis. n = 6 per group. (F and G) Matrigel angiogenesis assay (see Methods). Representative photomicrographs showing angiogenesis in response to injected Matrigel. Capillary formation toward the Matrigel was greatly reduced in RAMP2+/– (G) compared with WT (F). n = 6 per group. (H and I) In vitro culture of AGM explant (see Methods). Representative photomicrographs showing the vascular network formation from the tissue cultured AGM region of E10.5 WT (H) and RAMP2–/– embryos (I). Vascular network formation was diminished in RAMP2–/– mice. n = 4 per group.