Aberrant neuronal and paracellular deposition of endostatin in brains of patients with Alzheimer's disease

Abstract

Cerebrovascular pathology is common in Alzheimer's disease (AD) and is considered to contribute to cerebral malfunction. However, distinct antiangiogenic proteins that accumulate in AD brains have not yet been identified. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII that, when added exogenously, inhibits endothelial proliferation and migration in vitro and angiogenesis and tumor growth in vivo by inducing apoptosis in endothelial cells. We produced a monoclonal antibody directed against endostatin and observed significantly more (p < 0.0001) immunoreactive cortical neurons in AD brains compared with age-matched neuropathologically unaltered controls. High numbers of extracellular and frequently perivascular endostatin deposits were detected in the cerebral hemispheres. Double-labeling experiments revealed colocalization of endostatin in amyloid-beta(1-40) (Abeta(1-40)), tau protein, and periodic acid-Schiff stain-positive plaques that were surrounded by focal gliosis. Western blotting revealed more 20 kDa endostatin in an AD patient compared with a control. In unstimulated SKNSH supernatants, endostatin was detected that increased predominantly after hypoxia in supernatants and cellular lysates. Abeta(1-40) (80 microg/ml) supplementation to SKNSH neurons for 24 hr completely abolished the release of endostatin. These data show that endostatin is released by neurons to accumulate in amyloid plaques in Alzheimer's disease. Induction by hypoxia and complete abrogation of endostatin release after Abeta(1-40) challenge reveals intricate interactions between the two proteins and opens new avenues for the development of novel treatment strategies of AD patients.

Fig. 1.

Prominent endostatin deposition (brown) was observed in neurons of all cortical layers in AD brains (A). Counterstain was hemalaun. The bottom inset shows intraneuronal deposition of endostatin (brown). A double-labeling experiment with neurofilament (blue) confirmed the neuronal origin of endostatin⁺ cells (brown). Paracellular endostatin deposition (brown) was detected in the immediate vicinity of blood vessels in an Aβ_(1–40)/endostatin double-labeled section (B). The inset shows a paracellular intraparenchymal endostatin deposit (brown). Extracellular endostatin deposits (brown) were detected in areas of focal glioses (blue) (C), colocalized with Aβ_(1–40) fragments (blue) (inset) and tau protein (blue) (D). No endostatin immunoreactivity (blue) was observed in tau⁺ neurons (brown) with neurofibrillary tangles (bottom inset). Endostatin⁺ deposits (brown), however, colocalized in PAS-positive plaques (top inset).

Fig. 2.

Flow cytometry (top) shows an increase in the number of endostatin-labeled cells after H₂O₂ and CoCl₂ stimulation (A). Western blotting of cellular lysates (middle) and supernatants (bottom) reveals a more accentuated 10 kDa endostatin-immunoreactive band in CoCl₂-stimulated than in unstimulated SKNSH cellular lysates and a more accentuated 20 kDa band in their supernatants (A). Western blotting demonstrates a more accentuated 20 kDa endostatin-immunoreactive band in an AD patient compared with a control (B). Surprisingly, Aβ_(1–40) (80 μg/ml) supplementation to SKNSH neurons for 24 hr completely abolished the release of the 20 kDa band (C).

Fig. 3.

Schematic diagram demonstrating the observed phenomena. Endostatin and Aβ_(1–40) are released by neurons (solid line) and cooperate in the mediation of hypoperfusion that leads to accentuated hypoxia in yet unaffected neurons (dotted line). This in turn accelerates neuronal endostatin release, consequent hypoperfusion, and continuing hypoxia.