Astrocytes

The Role of Astrocytes in the Development of the Blood-Retinal Barrier

Recent studies have shown that retinal astrocytes play a significant role in retinal vascular development by angiogenesis and in the induction and maintenance of the blood-retinal barrier (BRB). Glial fibrillary acidic protein (GFAP)-positive astrocytes are found in all vertebrate species that have a retinal vasculature, and the temporal-spatial development of astrocytes precedes the onset of angiogenesis in the retina [2, 5, 8]. In adults, astrocytes ensheathe the superficial retinal blood vessels (Figure 2), and their association with the vasculature is spatially and temporally correlated with the establishment of the BRB [8]. The functional role of astrocytes in establishing a blood-tissue barrier has been more clearly defined in the central nervous system and in culture (Reviewed in [9]). The end feet of astrocytes make an extensive network of cell processes that lie in close apposition to the basal surface of the microvascular endothelium. The intimate association between astrocytes and endothelial cells suggests that astro-cytes may provide an inductive signal to promote the establishment of the blood-brain barrier (BBB) [9], which is analogous to the BRB. Although the exact mechanism for this induction is unknown, it can be mimicked in vitro and has been shown to require close proximity and correct cell polarity between astrocytes and vascular endothelial cells. As with the BBB, the BRB is likely to be induced by glial cells, with astrocytes providing this function in the superficial blood vessels and Müller cells in the deep capillary network. In addition to their role in establishing the BRB, retinal astrocytes also appear to play an essential role in driving the angiogenic phase of retinal vascular development through the expression of VEGF. Differentiated astrocytes are located in the retina in advance of the formation of patent vessels. In the absence of adequate blood circulation, astrocytes experience relative hypoxic conditions and have been shown to upregulate VEGF, which drives retinal vascular development by angiogenesis [2]. The association of astrocytes to retinal vessels also appears to contain their growth and provide vascular stability, and a breech of this association may result in unrestrained neovascularization and hemorrhage [10]. In order to understand the role of astrocytes in retinal vascular development, it is necessary to understand the migration, growth, and development of astrocytes in the retina.

Development of Retinal Astrocytes

Although there are significant differences between humans and mice, astrocytes in both species migrate into the retina through the optic nerve as immature astrocyte progenitors [8]. The progenitors have been defined as cells expressing Pax2 and vimentin, but not GFAP or S100 [8]. The progenitors then migrate through the nerve fiber layer toward the anterior margins of the retina. This creates a spatial and temporal pattern in which the progenitors, which are the least differentiated, are found at the leading edge of migration and the older astrocytes, which have begun to differentiate and mature, are found in the central retina. At least three stages of maturation have been described. The first stage has been defined as immature perinatal astrocytes that express vimentin, Pax2, S100, and GFAP. This is followed by the appearance of mature perinatal astrocytes that lose vimentin expression, but retain Pax2, S100, and GFAP expression. After the final stage of development, adult astro-cytes are found throughout the retina and have robust expression of GFAP and S100, but have lost expression of Pax2 [8]. In mice, the interaction with the vascular endothelium seems to increase the expression of GFAP and S100 in retinal astrocytes, suggesting that this interaction may be important for the final stage of astrocyte maturation [5]. This

Figure 2 Immunocytochemical staining for glial fibrillary acidic protein in a P15 mouse retina decorates the astrocytic processes that encircle the vessels in the inner retina. (see color insert)

Figure 2 Immunocytochemical staining for glial fibrillary acidic protein in a P15 mouse retina decorates the astrocytic processes that encircle the vessels in the inner retina. (see color insert)

is most apparent in flat-mounted retinas from P5 mice that have been immunostained for S100 and griffonia simplici-folia isolectin B4 (GSA; Figure 3). At this stage of mouse development, S100-positive astrocytes have spread throughout the nerve fiber layer of the retina, while the developing GSA-positive vasculature has only spread throughout the central region of the retina. S100 expression is much higher in the vascular-covered central retina than in the avascular periphery.

The Role of PDGF-A in Astrocyte Migration and Proliferation

Although the inductive signals for astrocyte development have not been fully identified, the current data suggest that platelet-derived growth factor A (PDGF-A) plays an important role in their migration and proliferation. Astrocyte progenitors enter the retina well after the differentiation of retinal ganglion cells. This is perhaps a necessary sequence as astrocytes migrate into the retina through the optic nerve along the ganglion cell axons. Once in the retina, astrocyte progenitor migration may be independent of the presence of ganglion cells [11]. However, other studies have shown a direct role for ganglion cells in astrocyte development. Ganglion cells secrete PDGF-A, and astrocytes can respond to PDGF-A through the expression of PDGF receptor a (PDGFRa) [5, 12]. The simplest model suggests that astro-

Figure 3 Immunofluorescent staining using antibodies to S100 (A), which is expressed only in astrocytes in the P5 mouse retina, and fluorescence staining of the developing vasculature using Griffonia simplicifolia isolectin B4 (B). S100 positive astrocytes are located throughout the retina; however, the staining intensity is higher in the central retina (A). At this age, the developing vasculature covers only the central region of the retina (B). The arrow in all panels marks the leading edge of the developing vasculature. In the merged image (C), it is clear that astrocytes in contact with the vasculature have higher expression of S100 than astrocytes in the avascular periphery. (see color insert)

Figure 3 Immunofluorescent staining using antibodies to S100 (A), which is expressed only in astrocytes in the P5 mouse retina, and fluorescence staining of the developing vasculature using Griffonia simplicifolia isolectin B4 (B). S100 positive astrocytes are located throughout the retina; however, the staining intensity is higher in the central retina (A). At this age, the developing vasculature covers only the central region of the retina (B). The arrow in all panels marks the leading edge of the developing vasculature. In the merged image (C), it is clear that astrocytes in contact with the vasculature have higher expression of S100 than astrocytes in the avascular periphery. (see color insert)

cytes and/or their progenitors migrate into and proliferate within the retina following the chemoattractant and mito-genic signal, PDGF-A. In support of this model, studies have shown that overexpression and ectopic expression of PDGF-A from the lens results in an astrocytic hyperplasia known as a hamartoma near the optic nerve head of the retina [12]. PDGF-A has also been overexpressed in transgenic mouse retina using the neuron-specific enolase promoter and the opsin promoter [5, 13]. In both models, there was excessive accumulation of astrocytes in the retina. In each of the overexpression models, the ectopic expression of PDGF-A led to mislocalized astrocytes, suggesting that the site of PDGF-A expression is important for migration cues in normal development. PDGF-B, which can also bind and activate the PDGFRa on astrocytes, can also induce their mislocalization and hyper-proliferation [4]. Further studies have shown that disruption of PDGF-A signaling by neutralizing antibodies to PDGFRa disrupts the normal development of retinal astrocytes [5]. These studies demonstrate that PDGF-A is a potent chemoattractant and mitogen for astrocytes and that its expression is required for normal astrocyte migration in the retina. It is interesting to note that retinal vascular development is dramatically impaired in all of the mouse models overexpressing PDGF-A. These findings clearly demonstrate that growth and development of retinal astrocytes is important for the development of a functional retinal vasculature.

Glossary

Astrocyte: A neuroglial cell of ectodermal origin with processes that ensheathe vessels in the inner retina and brain, helping to establish the blood-retinal or blood-brain barrier; they represent a supporting cell type for neurons as well as the vasculature.

Pericyte: Avascular supporting cell of mesenchymal origin with contractile properties that surrounds the vascular endothelium and helps regulate vascular integrity.

Platelet-derived growth factor A: A chemoattractant and mitogen for astrocytes that is important in retinal vascular development.

Platelet-derived growth factor B: A proangiogenic factor that is necessary for pericyte survival and migration and for normal retinal vascular development.

Vascular endothelial cells: The cells that line the lumen of blood vessels.

References

1. Diaz-Flores, L., Gutierrez, R., Varela, H., Rancel, N., and Valladares, F. (1991). Microvascular pericytes: A review of their morphological and functional characteristics. Histol. Histopathol. 6, 269-286. This is an excellent review describing the morphological and functional properties of pericytes.

2. Provis, J. M. (2001). Development of the primate retinal vasculature.

Progress Retinal Eye Res. 20, 799-821. This review describes the roles of the various cell types in the development of the retinal vasculature in primates and compares the findings to those of other species.

3. Benjamin, L. E., Hemo, I., and Keshet, E. (1998). A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125, 1591-1598. This study describes the role of the pericyte in the development of the retinal vasculature and how PDGF-B and VEGF affect pericyte—endothelial interactions.

4. Vinores, S. A., Seo, M. S., Derevjanik, N. L., and Campochiaro, P. A. (2003). Photoreceptor-specific overexpression of platelet-derived growth factor induces proliferation of endothelial cells, pericytes, and glial cells and aberrant vascular development: An ultrastructural and immunocytochemical study. Dev. Brain Res. 140, 169-183. This study shows the effect of overexpression of PDGF-B in the retinas of transgenic mice on endothelial cells, pericytes, and retinal glia and how this affects retinal vascular development.

5. Fruttiger, M. (2002). Development of the mouse retinal vasculature: Angiogenesis versus vasculogenesis. Invest. Ophthalmol. Vis. Sci. 43, 522-527. This is the most recent study describing the relationship between prior astrocyte and pericyte development and vascular development in the mouse retina.

6. Connolly, S. E., Hores, T. A., Smith, L. E. H., and D'Amore, P. A. (1988). Characterization of vascular development in the mouse retina. Microvasc. Res. 36, 275-290.

7. Lindahl, P., Johansson, B. R., Leveen, P., and Bertsholtz, C. (1997). Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242-245.

8. Chu, Y., Hughes, S., and Chan-Ling, T. (2001). Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: Relevance to optic nerve coloboma. FASEB J. 15, 2013-2015. This study carefully describes four unique stages of astrocyte development in the human retina.

9. Abbott, N. J. (2002). Astrocyte-endothelial interactions and blood-brain barrier permeability. J. Anat. 200, 629-638. This review describes our current understanding of the role of astrocytes in the establishment and maintenance of the blood—brain barrier.

10. Zhang, Y., and Stone, J. (1997). Role of astrocytes in the control of developing retinal vessels. Invest. Ophthalmol. Vis. Sci. 38, 1653-1666.

11. Chan-Ling, T. (1994). Glial, neuronal and vascular interactions in the mammalian retina. Prog. Retin. Eye Res. 13, 357-389. This is an excellent review of the interactions between retinal glial cells, neurons, and vascular endothelium in normal development.

12. Reneker, L. W., and Overbeek, P. A. (1996). Lens-specific expression of PDGF-A in transgenic mice results in retinal astrocytic hamartomas. Invest. Ophthalmol. Vis. Sci. 37, 2455-2466.

13. Yamada, H., Yamada, E., Ando, A., Seo, M. S., Esumi, N., Okamoto, N., Vinores, M., LaRochelle, W., Zack, D. J., and Campochiaro, P. A. (2000). Platelet-derived growth factor-A-induced retinal gliosis protects against ischemic retinopathy. Am. J. Pathol. 156, 477-487.

Capsule Biographies

Dr. Vinores has been a member of the faculty of the Wilmer Eye Institute at the Johns Hopkins University School of Medicine in Baltimore, Maryland, since 1991. His research primarily focuses on the blood-retinal barrier, angiogenesis, and immunocytochemistry. He is supported by funds from the National Institutes of Health (grants EY10017 and EY05951) and Research to Prevent Blindness (Lew R. Wasserman Merit Award).

Dr. Ash has been an affiliate of the Dean A. McGee Eye Institute and on the faculty at the University of Oklahoma Health Sciences Center since 1999. His research is primarily focused on the development of the vascula-ture in the eye and the role of astrocyte-endothelial cell interactions during retinal angiogenesis. He is supported by funds from the National institutes of Health (grants EY14206-01, EY012190, and RR017703-01), Research to Prevent Blindness, and the Oklahoma Center for Science and Technology (HR02-140RS).

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