SUMMARY:Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) belong to a novel family of endothelial growth factors that function as ligands for the endothelial-specific receptor tyrosine kinase, Tie-2. Ang-1 reduces endothelial permeability of noncerebral vessels and has a major role in vascular stabilization and maturation, whereas Ang-2 is thought to be an endogenous antagonist of the action of Ang-1 at Tie-2. Expression of these ligands at the mRNA and protein level were studied during both blood-brain barrier (BBB) breakdown and cerebral angiogenesis occurring in the rat cortical cold-injury model by RT-PCR analysis and immunohistochemistry respectively, during a time course of 6 hours to 6 days. In addition, immunohistochemical detection of fibronectin was used to detect BBB breakdown at the lesion site and dual labeling was used to determine whether the vessels demonstrating BBB breakdown expressed endothelial Ang-1 or Ang-2. Endothelial Ang-1 and Tie-2 proteins were present in all cerebral vessels of normal brain including those of the choroid plexuses, whereas both these proteins as well as Ang-2 were present in choroid plexus epithelium and in ependymal cells, suggesting that angiopoietins have an autocrine effect on these cell types as well. In contrast, in the early phase after injury during the known period of BBB breakdown, increased Ang-2 mRNA and protein and decreased endothelial Ang-1 and Tie-2 proteins were observed. Two to 6 days after injury, the progressive increase in Ang-1 mRNA and protein and the decrease in Ang-2 coincided with cerebrovascular angiogenesis. Confocal microscopy showed colocalization of both Ang-1 and Ang-2 in endothelium of lesion vessels, and our observation of colocalization of Ang-1 and Ang-2 in polymorphonuclear leukocytes and macrophages has not been reported previously. This study demonstrates that Ang-1 is an important factor in maintaining normal homeostasis in the brain. Thus Ang-1 therapy may have therapeutic potential in reducing BBB breakdown and the ensuing edema after massive brain injury. (Lab Invest 2003, 83:1211-1222.

Interaction of vascular endothelial growth factor (VEGF) with the angiopoietins (Ang) is an essential component of angiogenesis. Localization of VEGF in the anterior pituitary raises the possibility that Ang must be present in the pituitary gland as well. In this study Ang expression was detected in the normal rat pituitary gland at the gene level by reverse-transcriptase polymerase chain reaction and at the protein level by immunohistochemistry. The latter was analyzed by both light and confocal microscopy. Constitutive expression of Ang1, Ang2, and their receptor Tie2 was detected at both the mRNA and protein level in all the pituitary glands studied. Of interest was the localization of both Ang1 and Ang2 in scattered PAS positive adenohypophysial cells rather than in endothelial cells. Confocal microscopy showed colocalization of both Ang1 and Ang2 proteins within the same adenohypophysial cells. Dual immunostaining for Ang1 and the anterior pituitary hormones that show PAS positivity demonstrated colocalization of Ang1 with follicle stimulating hormone and luteinizing hormone. In the posterior pituitary, strong Ang1 signal observed in vascular endothelial cells masked the weak Ang2 signal, a pattern that is similar to that reported in brain endothelial cells. The presence of both angiopoietins and VEGF in the pituitary gland suggest that these ligands interact during angiogenesis as they are known to do in other systems to maintain the rich vascular network of the gland. This first report of angiopoietin localization in the rat pituitary gland opens a new line of investigation on angiogenesis in pituitary glands that will impact human endocrinology in the future.