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LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling

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A Corrigendum to this article was published on 25 September 2013

Abstract

Aberrant neovascularization contributes to diseases such as cancer, blindness and atherosclerosis, and is the consequence of inappropriate angiogenic signalling. Although many regulators of pathogenic angiogenesis have been identified, our understanding of this process is incomplete. Here we explore the transcriptome of retinal microvessels isolated from mouse models of retinal disease that exhibit vascular pathology, and uncover an upregulated gene, leucine-rich alpha-2-glycoprotein 1 (Lrg1), of previously unknown function. We show that in the presence of transforming growth factor-β1 (TGF-β1), LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mild retinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly to the TGF-β accessory receptor endoglin, which, in the presence of TGF-β1, results in promotion of the pro-angiogenic Smad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studies reveal a new regulator of angiogenesis that mediates its effect by modulating TGF-β signalling.

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Figure 1: LRG1 is overexpressed in pathogenic retinal vasculature.
Figure 2: LRG1 promotes angiogenesis.
Figure 3: LRG1 contributes to pathogenic neovascularization.
Figure 4: LRG1 modifies the TGF-β receptor complex.
Figure 5: LRG1 promotes angiogenesis via a switch in TGF-β signalling.

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Acknowledgements

This project was supported by grants from the Lowy Medical Research Foundation, the Medical Research Council, The Wellcome Trust, UCL Business (Proof of Concept Grant) and the Rosetrees Trust. J.W.B.B. is supported by a NIHR Research Professorship. H.M.A. is supported by a British Heart Foundation Senior Fellowship. We would also like to thank M. Gillies for his role in initiating the original project, P. Luthert and C. Thaung for human tissue samples and advice on human pathology specimens, S. Perkins and R. Nan for assistance with the surface plasmon resonance analysis, and P. ten Dijke for discussions and advice.

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Authors and Affiliations

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Contributions

The project was conceived by J.G., S.E.M. and X.W. Experiments were designed by J.G., S.E.M., X.W. and S.A. Microarrays were performed by J.A.G.M. and qPCR reactions by X.W. X.W. and S.A. characterized the Lrg1 knockout mice and LRG1 antibody. X.W. performed all the metatarsal assays (except in Fig. 5j, k), aortic ring assays and Matrigel assays, carried out all the biochemical and molecular biology work and analysed the data. S.A. and X.W. undertook the immunohistochemistry and generated the OIR mouse model. U.F.O.L., C.A.K.L., S.A., X.W. and J.W.B.B. performed the CNV experiments, and S.A. and X.W. analysed the data. J.W.B.B. provided human vitreal samples. Z.Z. and H.M.A. generated MLEC;Engfl/fl cells and X.W. performed proliferation assay and biochemical analysis. Z.Z., S.A. and H.M.A. carried out the metatarsal assays on Eng knockout mice. V.B.T. performed the Biacore experiments. N.J. and M.S. provided assistance and technique support. X.W., S.A., J.G. and S.E.M. produced the figures, and J.G. and S.E.M. wrote the text, with all authors contributing to the final manuscript. J.G. and S.E.M. provided leadership throughout the project.

Corresponding authors

Correspondence to Stephen E. Moss or John Greenwood.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-32 and Supplementary Tables 1-2. (PDF 3431 kb)

Three dimensional reconstruction of normal mouse retinal vasculature.

A scanning laser confocal microscopy Z-stack of control 16 week old adult C57/BL6 mouse retina stained for collagen IV (green), PECAM-1 (red) and cell nuclei (blue) was volume rendered to create a three-dimensional reconstruction of the retinal vasculature using Imaris software (Bitplane AG). The inner, intermediate and deep vasculature plexuses can be observed. (MOV 5474 kb)

Three dimensional reconstruction of VLDLR-/- mouse retinal vasculature.

A scanning laser confocal microscopy Z-stack of 18 week old VLDLR-/- mouse retina stained for collagen IV (green), PECAM-1 (red) and cell nuclei (blue) was volume rendered to create a three-dimensional reconstruction of the retinal vasculature using Imaris software (Bitplane AG). Abnormal vascular tufts can be seen penetrating the outer nuclear layer and extending into the sub-retinal space. (MOV 5646 kb)

Three dimensional reconstruction of Grhl3ct/J curly tail mouse retinal vasculature.

A scanning laser confocal microscopy Z-stack of 16 week old Grhl3ct/J curly tail mouse retina stained for collagen IV (green), PECAM-1 (red) and cell nuclei (blue) was volume rendered to create a three-dimensional reconstruction of the retinal vasculature using Imaris software (Bitplane AG). Abnormal vascular projections can be seen penetrating the outer nuclear layer and extending into the sub-retinal space. (MOV 23705 kb)

Three dimensional reconstruction of RD1 mouse retinal vasculature

A scanning laser confocal microscopy Z-stack of 18 week old RD1 mouse retina stained for collagen IV (green), PECAM-1 (red) and cell nuclei (blue) was volume rendered to create a three-dimensional reconstruction of the retinal vasculature using Imaris software (Bitplane AG). Retinal thinning can be seen due to degeneration of photoreceptors (loss of outer nuclear layer compared with WT mice) with a concomitant reduction in retinal vasculature. Abnormal tortuous vessels can be observed. (MOV 4898 kb)

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Wang, X., Abraham, S., McKenzie, J. et al. LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling. Nature 499, 306–311 (2013). https://doi.org/10.1038/nature12345

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