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CreER activation transiently disrupts angiogenesis by reducing proliferation and promoting apoptosis in vascular
Elena Ioannou1, Mengmeng Dong1, Victoria S Rashbrook1,2
1UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK.
Abstract:
Tamoxifen-inducible gene targeting in mice with estrogen receptor-dependent Cre (CreER) recombinase has enormously advanced vascular biology research. However, CreER activation under the control of vascular endothelial promoters causes off-target effects that impair the vascularization of the retina, a widely used model to study the molecular and cellular mechanisms of angiogenesis. Although ubiquitously expressed CreER is also used to study retinal angiogenesis, it remains unknown whether it causes similar or more severe off-target effects compared to endothelial-selective CreER activation. Moreover, the molecular and cellular processes disrupted by CreER activation in endothelial cells remain to be identified. Here, we demonstrate that ubiquitous CreER activation in postnatal mice decreases body growth and impairs retinal angiogenesis. We further show that CreER activation slows endothelial cell proliferation and promotes apoptosis concomitant with p21/CDKN1A upregulation in the angiogenic retina, although retinal vasculature progressively normalizes. By contrast, CreER activation in quiescent adult retinal vasculature did not induce endothelial cell apoptosis or p21 activation. Altogether, our findings indicate that ubiquitous promoters should be avoided to drive CreER expression when studying gene function during retinal angiogenesis, and that CreER activation controls, although essential to account for endothelial apoptosis and proliferation defects in short-term studies, may be less critical for adult vascular studies.
Insights
Ubiquitous CreER activation impairs postnatal retinal angiogenesis by slowing endothelial cell proliferation and increasing apoptosis. Avoid ubiquitous promoters for CreER expression in retinal angiogenesis studies.
Area of Science:
- Vascular Biology
- Developmental Biology
- Genetics
Background:
- Tamoxifen-inducible Cre-lox systems using CreER recombinase are vital for studying gene function in vascular biology.
- Endothelial-specific CreER activation can cause off-target effects in retinal angiogenesis models.
- The impact of ubiquitous CreER activation on retinal angiogenesis and its underlying mechanisms are not well understood.
Purpose of the Study:
- To investigate the effects of ubiquitous CreER activation on postnatal retinal angiogenesis.
- To compare off-target effects of ubiquitous versus endothelial-selective CreER activation.
- To identify molecular and cellular processes disrupted by CreER activation in retinal endothelial cells.
Main Methods:
- Tamoxifen administration to induce CreER activation in postnatal mice.
- Assessment of body growth and retinal vascularization.
- Analysis of endothelial cell proliferation and apoptosis in the retina.
- Evaluation of p21/CDKN1A expression.
Main Results:
- Ubiquitous CreER activation decreased body growth and impaired retinal angiogenesis.
- CreER activation slowed endothelial cell proliferation and increased apoptosis in the developing retina.
- p21/CDKN1A was upregulated in the angiogenic retina following CreER activation.
- CreER activation in adult quiescent vasculature did not affect endothelial cells.
Conclusions:
- Ubiquitous promoters are not suitable for driving CreER expression in studies of retinal angiogenesis.
- CreER activation-induced endothelial cell apoptosis and proliferation defects are important considerations for short-term studies.
- CreER activation effects on endothelial cells may be less critical in adult vascular studies.
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