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Retroviral Expression of Human Arginine Decarboxylase Reduces Oxidative Stress Injury in Mouse Cortical Astrocytes

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Retroviral Expression of Human Arginine Decarboxylase Reduces Oxidative Stress Injury in Mouse Cortical Astrocytes

Samin Hong et al. BMC Neurosci.

Abstract

Background: In physiologic and pathologic conditions of the central nervous system (CNS), astrocytes are a double-edged sword. They not only support neuronal homeostasis but also contribute to increases in neuronal demise. A large body of experimental evidence has shown that impaired astrocytes play crucial roles in the pathologic process of cerebral ischemia; therefore, astrocytes may represent a breakthrough target for neuroprotective therapeutic strategies. Agmatine, an endogenous polyamine catalyzed from L-arginine by arginine decarboxylase (ADC), is a neuromodulator and it protects neurons/glia against various injuries.

Results: In this investigation, agmatine-producing mouse cortical astrocytes were developed through transduction of the human ADC gene. Cells were exposed to oxygen-glucose deprivation (OGD) and restored to a normoxic glucose-supplied condition. Intracellular levels of agmatine were measured by high performance liquid chromatography. Cell viability was evaluated by Hoechest/propidium iodide nuclear staining and lactate dehydrogenase assay. Expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase s (MMPs) were assessed by a reverse transcription polymerase chain reaction, Western immunoblots, and immunofluorescence. We confirmed that ADC gene-expressed astrocytes produce a great amount of agmatine. These cells were highly resistant to not only OGD but also restoration, which mimicked ischemia-reperfusion injury in vivo. The neuroprotective effects of ADC seemed to be related to its ability to attenuate expression of iNOS and MMPs.

Conclusion: Our findings imply that astrocytes can be reinforced against oxidative stress by endogenous agmatine production through ADC gene transduction. The results of this study provide new insights that may lead to novel therapeutic approaches to reduce cerebral ischemic injuries.

Figures

Figure 1
Figure 1
Construction and infection of recombinant retroviral vector containing the human arginine decarboxylase (hADC) gene. (A) hADC pLXSN vector map: hADC pLXSN includes the Col E1 origin of replication and E.coli Ampr gene for propagation and antibiotic selection. The 5' viral LTR in this vector contains promoter/enhancer sequences that control expression of the gene of interest in the multiple cloning site. The SV40 early promoter (PSV40e) controls expression of the neomycin resistance gene (Neor), which allows antibiotic selection in eukaryotic cells. (B) Genetic confirmation by restriction analysis: Lane 1, size marker; Lane 2, restriction enzyme digestion using EcoRI and XhoI; Lane 3, control hADC pLXSN. (C) RNA levels of hADC were verified by RT-PCR in PT67 cells and mouse cortical astrocytes (Astro) after hADC pLXSN infection. (D) Expression of hADC was validated by immunocytochemistry in hADC pLXSN-infected astrocytes. Scale bar = 50 μm.
Figure 2
Figure 2
Endogenous agmatine measurement using high performance liquid chromatography (HPLC). The level of amines related to the agmatine pathway was determined in mouse cortical astrocytes infected with retrovirus containing the human arginine decarboxylase (hADC) gene. Cells underwent oxygen-glucose deprivation (OGD) for 4 hrs. Data are expressed as a mean ± SD of three different experiments performed from separate cell preparation, with triplicate determinations performed in each experiment. Asterisks indicate a p < 0.001 when the samples were compared to no treatment control.
Figure 3
Figure 3
Astrocyte-protective effect of human arginine decarboxylase (hADC) transduction against oxygen-glucose deprivation (OGD). Cytotoxicity was assessed by Hoechst 33258 and propidium iodide (PI) nuclear staining; (A) No treatment control astrocytes, (B) astrocytes after 4 hrs OGD, (C) hADC-overexpressing astrocytes (hADC-astrocytes), (D) hADC-astrocytes after 4 hrs OGD. Scale bars = 100 μm. (E) Proportion of PI-labeled damaged cells with red fluorescence. Asterisks indicate a p < 0.05 when the samples were compared to no treatment control. Double asterisk indicate a p < 0.05 that that the two samples were significantly different which was confirmed by a post-hoc analysis.
Figure 4
Figure 4
Neuroprotective effect of human arginine decarboxylase (hADC) transduction against oxygen-glucose deprivation (OGD) and restoration injury. Astrocytes restored to normoxic glucose-supplied conditions after 4 hrs OGD. Cytotoxicity was assessed by the lactate dehydrogenase (LDH) assay. Data are expressed as mean of ± SD of the three different experiments performed from separate cell preparations, with triplicate determinations performed in each experiment. Asterisks indicate a p < 0.001.
Figure 5
Figure 5
Inducible nitric oxide synthase (iNOS) and matrix metalloproteinases (MMPs) expression in human arginine decarboxylase (hADC)-introduced astrocytes after oxygen-glucose deprivation (OGD). The mRNA levels were determined using reverse transcription polymerase chain reaction (RT-PCR) (A) and protein expression was evaluated using Western immunoblots (B). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin were used as internal controls, respectively. Asterisks indicate a p < 0.05 when the samples were compared to no treatment control. Double asterisk indicate a p < 0.05 that two samples were significantly different which was confirmed by a post-hoc analysis.
Figure 6
Figure 6
Immunofluorescence staining of human arginine decarboxylase (hADC)-introduced astrocytes after oxygen-glucose deprivation (OGD). (A, D, G) No treatment control astrocytes; (B, E, H) astrocytes after 4 hrs OGD; (C, F, I) hADC-astrocytes after 4 hrs OGD. Cells reacted to inducible nitric oxide synthase (iNOS) (A-C), matrix metalloproteinase (MMP) 2 (D-F), and MMP 9 (G-I) showed green fluorescence. Cells were counter-stained with astrocyte marker glial fibrillary acidic protein (GFAP; red fluorescence) and nuclear marker 4',6-diamidino-2-phenylindole (DAPI; blue fluorescence). Scale bar = 50 μm.

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