Transient coexpression of nestin, GFAP, and vascular endothelial growth factor in mature reactive astroglia following neural grafting or brain wounds

Abstract

The spatial and temporal immunoexpression of the intermediate filament (IF) protein nestin and its relationship to glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and its receptor flt-1 (VEGF-R1) in reactive astroglia was examined following stab wounds or transplants of fetal CNS tissue into the adult brain. Since developmentally regulated proteins and gene transcripts can be reexpressed in reactive astroglia following certain brain injuries, we analyzed the nestin profile in these experimental paradigms in order to more fully understand the nature of the gliotic "scar." Nestin expression was transiently up-regulated in some but not all astrocytes which often had a different morphology than the typical stout, stellate GFAP (+) cells; the processes of the nestin (+) cells tended to be slender and elongated. In reactive astroglia from the mature brain, nestin expression was robust but generally localized to the wound or graft site, peaked at 7-10 days postoperative, and was absent by 28 days, whereas GFAP (+) astrocytes were far more widespread and persisted for many months. Only nestin was strongly expressed immediately adjacent to early stab wounds, whereas GFAP (+) cells were located further from the wound sites. In contrast, there was marked nestin/GFAP colocalization at the graft/host interface. Semiquantitative analysis combined with confocal microscopy revealed a unique compartmentalization of protein expression; processes from single astrocytes could be entirely nestin (+), GFAP (+), or could show coexpression. At 4, 7, and 14 days postoperative, 41, 58, and 32% of the immunoexpression, respectively, was accounted for by nestin at the graft/host interface, and it was essentially undetectable at 28 days postoperative. In situ hybridization studies showed nestin transcripts within GFAP (+) cells primarily between 4 and 10 days postoperative and absent by 28 days. Many nestin (+) astrocytes, as shown by electron microscopy, were closely related to the vasculature. Therefore we further examined the expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen associated with angiogenesis. Nestin colocalized with VEGF in some astrocytes (7%) but far more prominently with the VEGF flt-1 receptor (25%). Early astroglial activation may involve several different IF components and possibly a distinct astrocytic population that shows a rapid, transient nestin expression adjacent to injury sites. Expression of the nestin IF phenotype within affected astrocytes in the surgical vicinity may be indicative of a reversion to an immature phenotype that might be less susceptible to attendant hypoxia after injury. Since injured astrocytes are well known to express many bioactive compounds, such transient reexpression of early, developmentally regulated proteins may be a hallmark for the elaboration of growth factors such as VEGF.