Abstract 4579: Endogenous Brain Natriuretic Peptide (BNP) Protects Against Apoptosis of Ischemic Human Astrocytes in an Autocrine Fashion

Abstract

The plasma brain natriuretic peptide (BNP) level is increased in the acute phase of human stroke, but its source and function are unclear. Recently, we showed that the BNP level was higher in atherothrombotic cerebral infarction (69.1±9.4 pg/ml) than in control subjects (31.1±5.4 pg/ml), and that the BNP level in ischemic stroke was positively correlated with the NIH Stroke Scale (r=0.41, p<0.05) and infarct volume (r=0.34, p<0.05). Astrocytes provide metabolic and trophic support to neurons and modulate synaptic activities. At the early stage of brain ischemia, astrocytes are swollen, and their damage may compromise postischemic neuronal survival. We tested the hypothesis that human astrocytes produce BNP under hypoxia, and this endogenous BNP protects against apoptosis in an autocrine fashion. The human astrocyte cell line, U373MG, was exposed to hypoxia (O 2 ≤1%) for 24 hours. The ratio of BNP to GAPDH mRNA was increased by 7.7±.0 fold after 12-hour hypoxia and further increased by 8.6±1.6 fold after 24-hour hypoxia compared with that in 3-hour normoxia (both, p<0.01). The protein expression assessed by Western blot was increased by 2.0±0.4 fold at 24 hours (n=5, p<0.05). Tyrosine phosphorylation of c-Src was observed by 2.0±0.2-fold increase at 30 minutes. These responses to hypoxia were all blocked by pretreatment with PP1 at 50μM, an inhibitor of c-Src. Apoptosis was measured by detecting caspase activation by flow cytometry, and it was increased by 2.5±0.1 fold after 24-hour hypoxia compared with that in normoxia. To investigate the role of up-regulated BNP in apoptosis, we performed the loss of function test by transfecting a specific siRNA for NPPB that suppressed BNP by more than 80%. The activity of caspases in the BNP knockdown cells was increased by 3.2±0.2 fold after 24-hour hypoxia compared with that in normoxia (n=5, p<0.001), and it was greater than that in the cells transfected with non-targeting siRNA. These results indicate that hypoxia increases BNP gene expression through the c-Src-dependent signaling cascade in the human astrocytes. Endogenous BNP shows brain protection via the anti-apoptotic effect. BNP may be useful in the treatment of ischemic brain diseases.