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Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity

J. Hoffmann^*,,1, Y. Feng^,1, F. vom Hagen, A. Hillenbrand, J. Lin, R. Erber, P. Vajkoczy, E. Gourzoulidou, H. Waldmann, A. Giannis^||, H. Wolburg^¶, M. Shani^**, V. Jaeger, H. A. Weich, K. T. Preissner, S. Hoffmann^¶¶, U. Deutsch^# and H.-P. Hammes^*,,2

^* Medical Clinic and Policlinic 3, Justus-Liebig University Giessen, Giessen, Germany;
5th Medical Clinic, Faculty of Clinical Medicine, University of Heidelberg, Mannheim, Germany;
Department of Neurosurgery, Medical Faculty of the University of Heidelberg, Mannheim, Germany;
Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany; Fachbereich 3 Organische Chemie, University of Dortmund, Dortmund, Germany;
^|| Institut für Organische Chemie; Universität Leipzig, Leipzig, Germany;
^¶ Institute of Pathology, University of Tuebingen, Tuebingen, Germany;
^** Institute of Animal Science, The Volcani Center, Bet Dagan, Israel;
Department Cell Culture Technology, and
Department Gene Regulation and Differentiation, Gesellschaft für Biotechnologische Forschung GmbH (GBF), Braunschweig, Germany;
Institute for Biochemistry, Faculty of Medicine, Justus-Liebig University, Giessen, Germany;
^¶¶ Medical Research Center, University-Clinic Mannheim, Medical Faculty of the University of Heidelberg, Mannheim, Germany; and
^# Max-Planck-Institute for Vascular Biology, c/o Institute of Cell Biology, University of Muenster, Germany and Theodor Kocher Institute, University of Berne, Berne, Switzerland

²Correspondence: Abt. Fur Nehprologie/Endokrinologie, 5. Medizinische Klinik, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail: hans-peter.hammes{at}med5.ma.uni-heidelberg.de

SPECIFIC AIMS

Since pericyte coverage determines endothelial survival and pericyte loss is a hallmark of early diabetic retinopathy, we determined 1) the susceptibility of the developing retina to vasoregression and consecutive neovascular response in the mouse model of retinopathy of prematurity in relation to quantitative pericyte coverage of retinal capillaries, and 2) the spatial and temporal expression of ligand receptor systems involved in endothelial survival and pericyte recruitment.

PRINCIPAL FINDINGS

1. Evolution of pericyte recruitment in the developing mouse retina
Assessing the dynamics of pericyte recruitment in the developing mouse retina, the largest increment of pericytes was found between postnatal day 7 and 8, due to the formation of the deep capillary layers of the retina. All developing capillaries from both layers were invested by pericytes, as demonstrated by immunohistochemistry of whole mount preparations and electron microscopy.

2. The development of the deep capillary layer reduces the susceptibility of the retina to subsequent neovascularization
The largest neovascular response is shown when mice are exposed to hyperoxia at p7 before the formation of the deep capillary network. As markers of hyperoxia-induced capillary regression, avascular zones are larger at p12, when mice are exposed to high oxygen before the deep capillary layer has formed, than at p13. Preretinal neovascularization as the response to hypoxia is > 80% less in mice at p18 in mice that had already formed part of the deep capillary network before oxygen exposure.

3. Formation of the deep vascular plexus coincides with pericyte recruiting factor up-regulation at postnatal day 8
The progressive maturation of the retinal vasculature was associated with a combined up-regulation of angiopoietin-1 (Ang-1) and PDGF-B, whereas VEGF remained almost unchanged during the transition from a susceptible to a resistant capillary network (Fig. 1) . Inhibition of Ang-1 by soluble Tie-2 (sTie-2) partially restored the propensity to form neovascularizations in the ROP model without affecting capillary coverage with pericytes. Progressive independence from hypoxia-inducible growth factors and simultaneous up-regulation of endothelium-protective and pericyte recruiting factors coincide with the formation of the deep capillary plexus and represents retinal vessel maturation.

View larger version (27K): [in this window] [in a new window]   Figure 1. A) Immunoblot of retinal extracts of mice at p7, p8, and p26 for expression of VEGF, PDGF-B, and PDGF-Rß. B) Densitometric quantitation of the blots shown in panel A, relative to actin expression. C) Real-time quantitative PCR of the three ligand receptor systems VEGF/-R2, PDGF-B/R-ß, and Ang-1-2/Tie-2 at the three relevant time points. D) Nonradioactive in situ hybridization of Ang-1 in the mouse retina at p7 and p8. Negative control is in a retina from p8. Original magnification 200x.

CONCLUSIONS AND SIGNIFICANCE

Our findings demonstrate that pericytes closely follow the formation of all retinal capillary layers, suggesting that the window of plasticity, marked by the failure to resist hyperoxia-induced vasogression, is unrelated to the coverage of capillaries by pericytes. This is consistent with recent reports showing that pericytes are in the proximity of the sprouting endothelial tip in the superficial retinal vasculature. Our experiments using immunohistochemistry and electron microscopy confirm previous studies and extend the findings to the deep capillary layers, which are primarily affected in early diabetic retinopathy. Formation of the deep capillary layers determine the observed reduced susceptibility to preretinal neovascularizations in the ROP model. During the transition from p7 to p8, the retina expresses high levels of the ligands Ang-1 and PDGF-B, which are not only pericyte-recruiting factors but also exhibit endothelial protective signals. TGF-ß was not regulated between p7 and p8 in our experimental setting. Ang-1 is an efficient endothelial survival factor with the additional propensity of permeation inhibition (not studied here). In summary, our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depends on the completion of the multilayer structure, especially the deep capillary layers, and is independent of the coverage by pericytes.

View larger version (13K): [in this window] [in a new window]   Figure 2. Schematic summary of the molecular expression patterns of endothelial survival factors expressed during the retinal transition from a susceptible to a resistant vasculature.

FOOTNOTES

¹ These authors equally contributed to the paper.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2109fje;

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This Article Full Text (PDF) All Versions of this Article: 19/14/2035 04-2109fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoffmann, J. Articles by Hammes, H.-P. Search for Related Content PubMed PubMed Citation Articles by Hoffmann, J. Articles by Hammes, H.-P.