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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 23, 2005 as doi:10.1096/fj.04-3101fje. |
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* Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland;
Department of Ophthalmology, University of Oulu, Oulu, Finland;
Wolfson Institute for Biomedical Research, University College London, London, UK;
Biocenter Oulu and Department of Pathology, University of Oulu, Oulu, Finland; and
|| Biocenter Oulu and Department of Physiology, University of Oulu, Oulu, Finland
1 Correspondence: Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, P.O. Box 5000, University of Oulu, Oulu 90014, Finland. E-mail: taina.pihlajaniemi{at}oulu.fi
SPECIFIC AIMS
Lack of collagen XVIII, containing the antiangiogenic endostatin-fragment, results in delayed regression of the hyaloid vessels and poor outgrowth of the retinal vessels. This study was originated to assess the roles of the homologous collagens XV and XVIII in the development of eye vasculature. Mice lacking both collagens XV and XVIII were studied to gain insight on their distinct and possible compensatory functions.
PRINCIPAL FINDINGS
1. Lack of type XVIII collagen induces an increased number of astrocytes in the retina
Astrocytes are known to form a template for growing vessels in the retina. However, the poor outgrowth of retinal vessels in type XVIII collagen null mice was not associated with defective proliferation and migration of astrocytes. Instead, retinal whole mounts on postnatal day 10 revealed an increase in the amount of astrocytes.
2. The retina in Col18a1–/– mice is vascularized by anomalous anastomoses from a persistent fetal vasculature in the vitreous body
The retinal angiogenesis is highly impaired in Col18a1–/– mice during the first postnatal days. Nevertheless, we found that both the Col18a1–/– and wild-type mice had developed a retinal vasculature by day 10 (Fig. 1
). The vasculature in the Col18a1–/– mouse eyes was abnormal, however, as some hyaloid vessels had not undergone proper regression but had submerged into the retina to give rise to a vasculature that supported a major part of the retinal blood flow. In whole mount preparations of retinas the wild-type samples showed a "starfish-like" pattern of major veins and arteries whereas the regressing hyaloid vessels had a thin, rudimentary appearance (Fig. 1A
). In contrast, the pattern of veins and arteries in the Col18a1–/– mice was varied, and the retina was supported only by a few blood vessels originating from the optic nerve head. Persistent hyaloid vessels were seen to penetrate through the inner limiting membrane in the peripheral retina (Fig. 1B
). The incomplete regression of the fetal vasculature in the vitreous bodies of the Col18a1–/– mice was also observed in ophthalmoscopic examinations of adult mice.
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3. VEGF mRNA expression is appropriately regulated in Col18a1–/– mice
We found no indication in the Col18a1–/– mice that the hyaloid vessels in close apposition to retina affect the expression pattern of VEGF in the retinal astrocytes. VEGF was down-regulated in the central areas where patent vessels were present, whereas it remained highly expressed in the peripheral retina. However, at P10, the total retinal VEGF mRNA was 
20% lower in Col18a1–/– mice compared with wild-type.
4. Type XV collagen regulates the recruitment of glial cells around blood vessels in double deficient Col15a1–/–; Col18a1–/– mice
The persistent vessels penetrating into the retina in the Col18a1–/– mice gave rise to retinal vessels covered with astrocytes; parts of the vessels in the vitreal space were lacking in astrocytes (Fig. 1B
). No vessels lacking in astrocytes were visible in the vitreous bodies of the double null Col15a1–/–; Col18a1–/– mice at or after day 10, although persistent hyaloid vessels were present. Instead, the persistent hyaloid vessels in the Col15a1–/–; Col18a1–/– samples were fully covered by astrocytes, including the part located in the vitreous body (Fig. 1C, D
).
5. Type XVIII collagen null mice show reduced susceptibility to high oxygen-induced neovascularization
In contrast to their wild-type littermates, the Col18a1–/– mice showed reduced development of neovascular tufts in the mouse model of hypoxia-induced neovascularization (Fig. 2A-D
). Moreover, the response of the astrocyte population to hypoxia was altered in the Col18a1–/– mouse retinas. The astrocytes around the optic nerve head in the wild-type mice expressed very little GFAP after oxygen treatment (Fig. 2E)
, whereas the Müller cell end feet were clearly visible due to GFAP expression in the Müller cells after damage to the retina by hypoxia (Fig. 2E
, insert). The astrocytes around the optic nerve head in the Col18a1–/– mice were not influenced by hypoxia, as the GFAP staining resembled that of an untreated animal (Fig. 2F
). The Müller cells around the optic nerve head in the Col18a1–/– mice also expressed GFAP, however, due to damage to the retina by hypoxia (Fig. 2F
, insert) after vascular pruning.
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CONCLUSIONS AND SIGNIFICANCE
We show here that persistent hyaloid vessels support the retinal blood flow by sending sprouts into the retina in Col18a1–/– mice. Patients with Knobloch syndrome caused by mutations in type XVIII collagen have been reported to have various eye abnormalities, which coincides well with our findings on collagen XVIII-deficient mice. Despite much research on the activities and mechanism of action of the antiangiogenic endostatin fragment of collagen XVIII, its physiological significance is unclear. This study shows that type XVIII collagen is indispensable for proper angiogenesis in the eye.
The phenotype of Col18a1–/– mice shows similarities with mice expressing single isoforms of VEGF, namely VEGF120 and VEGF188 which have a dramatic impairment of retinal vascular outgrowth associated with persistent hyaloid vessels. Our results show that the total retinal VEGF mRNA level in Col18a1–/– mice was 80% of that in wild-type mice, although in retinal whole mount in situ no indication about the influence of hyaloid vessels to the expression pattern of VEGF mRNA could be seen. VEGF mRNA was up-regulated in the hypoxic areas of the retina ahead of the growing vessels, but nevertheless, angiogenesis from the optic nerve head was impaired in Col18a1–/– mice. The different isoforms of VEGF have differences in solubility and certain VEGF isoforms bind to heparin and heparan-sulfate proteoglycans. Since collagen XVIII is one of the major basement membrane heparan sulfate proteoglycans, it is tempting to speculate that type XVIII collagen may be crucial for the proper signaling of VEGF in the developing retina, possibly involving receptor binding.
Although PDGF-A overexpression has been shown to cause hyperproliferation of astrocytes in the retina, Col18a1–/– mice have increased amounts of retinal astrocytes but no sign of PDGF-A overexpression. Where PDGF-A overexpression and the resulting hyperproliferation of astrocytes is associated with excessive angiogenesis, retinal vessel formation was delayed in the Col18a1–/– mice and not excessive despite the overproliferation of astrocytes and normal distribution of VEGF mRNA expression. Our findings are consistent with the idea that many factors regulating retinal angiogenesis have a major role in physiologic conditions.
The astrocytes in the mouse retina are under the inner limiting membrane and in direct contact with it. The inner limiting membrane in the mouse eye is known to contain type XVIII collagen and a change in its composition could lead to the increase in the amount of astrocytes. It could also cause attachment of hyaloid vessels to the retina and lead to the observed abnormal angiogenesis of the retina through persistent hyaloid vessels. The fact that the hyaloid vessels are covered by astrocytes in Col15a1–/–; Col18a1–/– mice but not in Col18a1–/– mice leads us to suggest that it is type XV collagen that regulates the migration of astrocytes, while type XVIII collagen regulates their proliferation. Previous studies with normal mice revealed a lack of type XV collagen in the capillaries of the central nervous system forming the blood-brain barrier. We thus hypothesize that XV could be a negative regulator of the recruitment of glial cells around vessels in the CNS.
Hyperproliferation of astrocytes has been shown to provide protection from oxygen-induced neovascularization in mice. In rats, cyclic hyperoxia induces apoptotic death of astrocytes between and around vessels, resulting in defects in the glia limitans of capillaries through which neovascular preretinal vessels protrude. Although the capillary network around the optic nerve head was pruned in an indistinguishable manner in wild-type, Col18a1–/– and Col15a1–/–; Col18a1–/– mice after oxygen treatment and return to normal room air, the astrocyte population in the Col18a1–/– and Col15a1–/–; Col18a1–/– mice showed no obvious sign of depletion, whereas there were only a few astrocytes around the optic nerve head in the wild-type mice. Col18a1–/– mice also developed fewer neovascular tufts than their wild-type siblings. As a conclusion, lack of type XVIII collagen causes overproliferation and altered properties of astrocytes, possibly due to changes in the inner limiting membrane and capillary basement membranes. This leads to impaired angiogenesis under conditions of normal development and impaired neovascular vessel formation under the extreme hypoxia-driven conditions involved in the experimental neovascularization model.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3101fje;
This article has been cited by other articles:
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  M. N. Rudzinski, L. Chen, and M. R. Hernandez Antiangiogenic Characteristics of Astrocytes From Optic Nerve Heads With Primary Open-angle Glaucoma Arch Ophthalmol, May 1, 2008; 126(5): 679 - 685. [Abstract] [Full Text] [PDF]
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