Astrocyte Regulation of Microvascular Endothelial Cell Growth, Differentiation, and Programmed Cell Death

Abstract

Growth or regression of microvessels in response to long term changes in tissue needs for blood supply is critical for normal development and recovery from injury or disease. Our analyses are focused on determining the cellular mechanisms that control this process. Since astroglial cells are known to be in physiological communication with both neurons and microvessels, we hypothesized that they have a role in microvascular growth control. Our previous analyses using retinal models have supported this hypothesis and suggest that astrocytes regulate microvascular development by generating soluble factors that inhibit endothelial cell growth and induce their morphogenic differentiation (Jiang et al., 1993; Jiang et al., in press; Behzadian et al., 1994). Here we report on experients designed to identify these factors and determine the mechanisms of their actions. Two different endothelial cells, bovine retinal microvascular (BRE) and aortic (BAE) cells were used for these experiments. Mink lung epithelial CCL-64 cell line was used as a control for assaying the activity of transforming growth factor β (TGF-β). Primary cultures of rat brain astrocytes prepared as confluent monolayers were maintained in serum-free medium for 72 hours. This astrocyte- conditioned medium (ACM) was then added to exponentially growing target cells for 3 days and effects on cell growth were tested. ACM was found to inhibit the growth of all three cell types in a dose dependent manner. Furthermore, the inhibitory activity of ACM was significantly increased by transient acidification (pH=2, 10–20 min). This treatment is known to activate the latent form of TGF-β. Adsorption of the ACM using an anti-TGF-P polyclonal antibody column reduced its growth inhibiting activity for CCL cells by 96%.