FIG. 2.

Ang-1 is rapidly downregulated after TBI and in in vitro models of neuronal cell death. (A) qPCR for Ang-1 mRNA in cortex and hippocampus after TBI. Data are presented as fold change compared to uninjured controls. Data represent the mean ± SEM (one-way ANOVA, SNK post-hoc analysis); *p < 0.05; **p < 0.01; ***p < 0.001 versus naïve control (N = 5). (B) RCNs were treated with 50 μM of etoposide (Etop). Cell lysates after 3, 6, and 24 h of treatment were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Ang-1 and β-actin. Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. qPCR quantification of Ang-1 expressions in RCNs treated with etoposide. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; *p < 0.05; **p < 0.01; ***p < 0.001 versus cnt (N = 5). (C) RCNs were treated with 50 uM of β-amyloid. qPCR quantification of Ang-1 expressions in RCNs treated with β-amyloid at the times indicated. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; *p < 0.05; **p < 0.01; ***p < 0.001 versus cnt (N = 3). (D) Ang-1 is neuroprotective in etoposide models of neuronal cell death. Neurons were treated with etoposide alone or with etoposide and recombinant Ang-1. Twenty-four hours later, LDH release and cell viability were measured. Data are expressed as percentage of control untreated neurons (cnt). Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; ⁺⁺⁺p < 0.001 versus cnt; **p < 0.01; ***p < 0.001 versus etoposide treated (N = 3). (E) Ang-1 is neuroprotective in β-amyloid models of neuronal cell death. Neurons were treated with β-amyloid alone or with β-amyloid and recombinant Ang-1. Twenty-four hours later, LDH release and cell viability were measured. Data are expressed as percentage of control untreated neurons (cnt). Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; ⁺⁺⁺p < 0.001 versus cnt; **p < 0.01; ***p < 0.001 versus etoposide treated (N = 3). (F) Silencing Ang-1 caused increase neuronal cell death and caspase-3 activation. RCNs were transfected with Ang-1, negative control (-ve Cnt) siRNA, or mock transfected. Cells were harvested 24 h after treatment. Whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Ang-1 and β-actin. (G) RCNs were transfected with Ang-1, negative control (-ve Cnt) siRNA, or mock transfected. Twenty-four hours after transfection, neurons were treated with etoposide. Twenty-four hours later, LDH release, cell viability, and caspase-3–like activity in cell lysates were measured. Data are expressed as percentage of mock transfected cells (mock). Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; ***p < 0.001 versus mock; ^{^^^}p < 0.001 versus etoposide treated; ⁺⁺p < 0.01; ⁺⁺⁺p < 0.001 versus −ve Cnt (N = 3). Ang-1, angiopoietin-1; ANOVA, analysis of variance; LDH, lactate dehydrogenase; mRNA, messengter RNA; qPCR, quantitative real-time polymerase chain reaction; RCN, rat cortical neuron; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; siRNA, small interfering RNA; SNK, Student–Newman–Keuls; TBI, traumatic brain injury.