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 2

Quantitative real-time PCR analysis, macroscopic analysis, and histology of RAMP2–/– embryos.

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Quantitative real-time PCR analysis, macroscopic analysis, and histology...
(A) Gene expression of AM, CRLR, and RAMPs in E13.5 WT and RAMP2–/– embryos, assessed by real-time PCR of total RNA. No RAMP2 expression was detected in RAMP2–/– mice, confirming RAMP2 was successfully destroyed. Conversely, RAMP3 expression did not differ between RAMP2–/– and WT mice, showing that the absence of RAMP2 did not induce compensatory upregulation of RAMP3 during development. AM expression was upregulated more than 5-fold in RAMP2–/– mice. n = 6 per group. **P < 0.01 vs. WT. (B–L) Development of blood vessels in E13.5 WT and RAMP2–/– mice. Appearance of the yolk sac (B) and vitelline arteries (C and D). (E and F) CD31 immunostaining of sections of yolk sacs. Arrows indicate sections of vitelline arteries. (G and H) Whole-mount immunofluorescence staining of CD31 in yolk sacs. In C–H, vitelline arteries were well developed on the yolk sacs of WT mice but poorly developed on those of RAMP2–/– mice. (I–L) TUNEL staining of sections of vitelline artery (I and J) and umbilical vessel (K and L) in E13.5 WT and RAMP2–/– embryos. Apoptosis was visualized in green fluorescence. Arrows indicate vessel lumens. Some ECs in RAMP2–/– mice were TUNEL positive. (M and N) Severe systemic edema observed in RAMP2–/–. Front (M) and side (N) views of WT and RAMP2–/– embryos at midgestation. Some RAMP2–/– embryos showed severe systemic edema. (O–R) Pericardial effusion in RAMP2–/– mice. (O and P) Magnified side view of embryos at midgestation revealing the appearance of the pericardial space in RAMP2–/– embryos. (Q and R) Sagittal sections showing the pericardial space in embryos at midgestation. The pericardial space was larger in RAMP2–/– than WT embryos and showed the accumulation of pericardial effusion. (S–U) Severe hemorrhagic changes in RAMP2–/– mice. (S) Side view of WT and RAMP2–/– embryos at midgestation. (T and U) Sections of the liver at the same stage. Some RAMP2–/– embryos showed severe hemorrhagic changes that were apparent on their surface and within the liver. Scale bars: 20 μm (E and F); 50 μm (I–L, T, and U); 200 μm (Q and R).