Apln-CreERT:mT/mG reporter mice as a tool for sprouting angiogenesis study

Jingjiang Pi,Lin Zhang,Xiaoli Chen,Yu Cheng,Huan Chang,Yuzhen Zhang,Huimin Sun,Ting Tao,Tao Zhuang,Jie Liu,Yixi Li

doi:10.1186/s12886-017-0556-6

Abstract

BackgroundAngiogenesis is defined as a new blood vessel sprouting from pre-existing vessels, and the sprouting angiogenesis is the start phase of angiogenesis, which is critical for both physiological and pathological processes, such as embryonic development, organ growth, wound healing, tumor growth, diabetic retinopathy and age-related macular degeneration. Better understanding of the mechanisms of sprout angiogenesis will provide a rationale for the treatments of these angiogenesis related diseases.MethodsmT/mG tool mice are crossed with Apln-CreERT mice to generate Apln-CreERT: mT/mG mice, then we used neonatal retinal angiogenesis model to observe the angiogenic pattern of Apln-CreERT:mT/mG mice compared with Cdh5-CreERT:mT/mG mice. FACS analysis was used to sort eGFP and tdTomato endothelial cells (ECs) for measuring Apelin and Cdh5 expression. Retinal sprouting angiogenesis pattern was also observed at different neonatal time when induced by tamoxifen and at hypoxia condition, as well as in vivo tumor in real-time angiogenesis in a dorsal skinfold window chamber in Apln-CreERT:mT/mG mice.ResultsApln-CreERT:mT/mG mice exhibited eGFP signal only in the sprouting angiogenesis, with less eGFP expression in the retinal “optic nerve” area than in that of Cdh5-CreERT: mT/mG mice, which might be due to relative mature vessels in the “optic nerve” area. The ECs sorted by FACS confirmed that the Apelin expression level was higher in eGFP ECs than tdTomato ECs of “optic nerve” area. Further we found that GFP-labeled sprouting angiogenesis decreased gradually following tamoxifen administration from P5-P7, but increased significantly during hypoxia in Apln-CreERT:mT/mG mice. At last, using Apln-CreERT:mT/mG mice we found tumor sprouting angiogenesis in dorsal skinfold, but not in the normal skinfold tissue.ConclusionsApln-CreERT:mT/mG mouse line is a useful tool to differentiate sprouting angiogenesis from whole blood vessels in the investigation of retinal and tumor sprouting angiogenesis in vivo.

Highlights

Angiogenesis is defined as a new blood vessel sprouting from pre-existing vessels, and the sprouting angiogenesis is the start phase of angiogenesis, which is critical for both physiological and pathological processes, such as embryonic development, organ growth, wound healing, tumor growth, diabetic retinopathy and age-related macular degeneration
Sprout angiogenesis was examined in the xenograft tumor, as well as in Results In Membrane tomato red (mT)/Membrane GFP (mG) mice, tdTomato-eGFP was inserted in the promoter area of β-actin, and two LoxP site were inserted in both ends of tdTomato, without Cre recombinase expression the cells expressed tdTomato protein followed by the stop codon (Fig. 1b, blue oval) and the eGFP protein was not expressed
These results suggested that Cdh5-CreERT:mT/mG mice could display both mature and sprouting vasculature, while Apln-CreERT:mT/mG display sprouting angiogenesis which was confirmed by increased retinal sprouting angiogenesis during hypoxia condition when Apelin expression level increased as described below

Summary

Introduction

Angiogenesis is defined as a new blood vessel sprouting from pre-existing vessels, and the sprouting angiogenesis is the start phase of angiogenesis, which is critical for both physiological and pathological processes, such as embryonic development, organ growth, wound healing, tumor growth, diabetic retinopathy and age-related macular degeneration. Sprouting angiogenesis is a critical process for both physiological and pathological processes, such as embryonic development, organ growth, wound healing, tumor growth, diabetic retinopathy and age-related macular degeneration and rheumatoid arthritis [1, 2]. This highly regulated process takes place through two non-exclusive events, the so-called. Cre-loxP-mediated recombination enables in vivo lineage tracing when used in conjunction with a reporter allele that expresses an indelible marker following excision of STOP cassette, and Cre-loxP system have been invented to study gene function of specific tissue or cell depending on the promoter to drive the Cre expression [7,8,9]

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