Cerebral Capillary Endothelial Cell Mitogenesis and Morphogenesis Induced by Astrocytic Epoxyeicosatrienoic Acid

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Abstract
Background and Purpose— Epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenation of arachidonic acid. We have previously demonstrated that astrocyte-conditioned medium induced mitogenesis in brain capillary endothelial cells. The goals of the present studies are to further define the mechanism through which this can occur and to confirm that EETs are derived from astrocytes, through which astrocytic activity can regulate cerebral angiogenesis in response to neuronal activation. Methods— Astrocytes and cerebral capillary endothelial cells in primary cultures were cocultured to examine the interaction of the 2 cell types. We used multiple immunohistochemical techniques to characterize the multicellular nature of the capillaries, which is not simply an artifact related to the culture conditions. The mitogenic effect of EETs was determined by 3 H-thymidine incorporation and cell proliferation assay. Endothelial tube formation was examined in vitro and in vivo with the use of a reconstituted basement membrane (Matrigel) assay. Results— In cocultures of astrocytes and capillary endothelium, we observed morphological changes in both cell types such that each assumed certain physiological characteristics, ie, endothelial networks and astrocytes with “footlike” projections as well as intermittent gap junctions forming within the endothelial cells. EETs from astrocytes as well as synthetic EETs promoted mitogenesis of endothelial cells, a process sensitive to inhibition of tyrosine kinase with genistein. Treatments with exogenous EETs were sufficient for endothelial cells to differentiate into capillary-like structures in culture as well as in vivo in a Matrigel matrix. Conclusions— The 2 major conclusions from these data are that astrocytes may play an important role in regulating angiogenesis in the brain and that cytochrome P450–derived EETs from astrocytes are mitogenic and angiogenic.