Astrocytes Modulate Endothelial Cell Growth, Differentiation, and Fibronectin Expression

Abstract

The retinal vascular development in the nerve fiber layer occurs by in situ differentiation of vascular precursor cells, but the mechanisms controlling this process are still unknown. We sought to determine the stimuli regulating. the organization of vascular precursor cells into retinal vessels by analyzing the roles of extracellular matrix and astrocytes. Immunolocalization analyses in the developing rat show that rat retinal vessels form as a polygonal network, beginning at the optic disc and extending to the ora. serrata by pll. Factor VIII-positive endothelial cells and laminin- and fibronectin-positive basement membrane codistribute with lumenized vessels. GFAP-positive astrocytic processes associate closely with the formed vessels, but ~re also present between the vessels. In the avascular area just distal to the forming vessels, GFAP-positive astrocytes form a fibronectin-positive polygonal meshwork pattern that resembles the immature vascular network. Laminin is not expressed in this area. In the area of the astrocyte-fibronectin meshwork, Bandierea Griffonia Simplifolia I B4 lectin labeled vascular precursor cells elongate and align along the astrocyte processes. Double label analysis indicates that both astrocytes and vascular . cells produce fibronectin. In situ hybridization shows that fibronectin mRNA expression is heaviest in the meshwork areas, suggesting that fibronectin synthesis is required in the early stage of retinal vasculogenesis. 0~11 culture experiments confirmed a strong influence of astrocytes on retinal microvascular endothelial cell growth, morphological differentiation, and extracellular matrix expression. Immunolocalization studies of cultured cells suggested and Western blot and Northern blot analyses confirmed a significant increase in fibronectin expression, when endothelial cells were grown in conditioned medium (CM) from astrocytes as compared with controls grown in CM from endothelial cells or pericytes. When retinal microvascular endothelial cells were grown in CM from astrocyte I endothelial cell co-culture, their growth was significantly inhibited as compared with controls grown in normal growth medium or CM from endothelial cells, astrocytes, or a mixture of 50% astrocyte CM/50% endothelial cell CM. In addition, endothelial cells grown in CM from astrocyte I endothelial cell co-culture elongated and became aligned in capillary-like structures. Endothelial cells grown in CM from astrocytes, endothelial cells, or a mixture of 50%astrocyte CM/50% endothelial cell CM alone retained normal· cobblestone morphology. CM from astrocytes alone stimulates retinal microvascular endothelial cell growth as compared with CM from endothelial cells, astrocyte I endothelial cell co-culture, or a mixture of 50% astrocyte CM/50% endothelial cell CM. These data indicate that rat retinal vascular development occurs by in situ differentiation of vascular precursor cells in close contact with astrocytes. Moreover, astrocytes modulate retinal microvascular endothelial cell growth, morphological differentiation, and fibronectin expression by release of growth factor-like substances. At least two factors are involved - one that requires astrocyte/ endothelial cell contact for activity and one that does not.