Limits...
Retroviral expression of human arginine decarboxylase reduces oxidative stress injury in mouse cortical astrocytes.

Hong S, Son MR, Yun K, Lee WT, Park KA, Lee JE - BMC Neurosci (2014)

Bottom Line: The neuroprotective effects of ADC seemed to be related to its ability to attenuate expression of iNOS and MMPs.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.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Project for Medical Science, and Brain Research Institute, Department of Anatomy, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea. jelee@yuhs.ac.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4150973&req=5

Fig1: 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.

Mentions: A recombinant retroviral vector containing the hADC gene was constructed as described previously (Figure 1) [23–25]. Briefly, the full-length cDNA of hADC (GenBank accession no. AY325129) was amplified by a polymerase chain reaction (PCR) and ligated to the retroviral expression vector pLXSN (Clontech Laboratories, Mountain View, CA) that also contained a neomycin resistance gene. They were then amplified in E.coli DH5a and identified by restriction analysis. The hADC-expressing pLXSN vector was transfected into the retroviral packaging cell line PT67 using Lipofectamine 2000 (Sigma-Aldrich, St. Louis, MO). A stable clone selection was carried out by adding G418 (200 μg/mL; Sigma-Aldrich) to the culture medium of Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% FBS. High-titer clones were selected, and virus-containing supernatant was filtered through a 0.45-mm polysulfonic filter. Primary cultured mouse cortical astrocytes were infected with hADC pLXSN. The cells were incubated with the virus-containing media for 24 hrs and then maintained in normal culture medium for a week before being used for the experiments.Figure 1


Retroviral expression of human arginine decarboxylase reduces oxidative stress injury in mouse cortical astrocytes.

Hong S, Son MR, Yun K, Lee WT, Park KA, Lee JE - BMC Neurosci (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150973&req=5

Fig1: 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.
Mentions: A recombinant retroviral vector containing the hADC gene was constructed as described previously (Figure 1) [23–25]. Briefly, the full-length cDNA of hADC (GenBank accession no. AY325129) was amplified by a polymerase chain reaction (PCR) and ligated to the retroviral expression vector pLXSN (Clontech Laboratories, Mountain View, CA) that also contained a neomycin resistance gene. They were then amplified in E.coli DH5a and identified by restriction analysis. The hADC-expressing pLXSN vector was transfected into the retroviral packaging cell line PT67 using Lipofectamine 2000 (Sigma-Aldrich, St. Louis, MO). A stable clone selection was carried out by adding G418 (200 μg/mL; Sigma-Aldrich) to the culture medium of Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% FBS. High-titer clones were selected, and virus-containing supernatant was filtered through a 0.45-mm polysulfonic filter. Primary cultured mouse cortical astrocytes were infected with hADC pLXSN. The cells were incubated with the virus-containing media for 24 hrs and then maintained in normal culture medium for a week before being used for the experiments.Figure 1

Bottom Line: The neuroprotective effects of ADC seemed to be related to its ability to attenuate expression of iNOS and MMPs.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.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Project for Medical Science, and Brain Research Institute, Department of Anatomy, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea. jelee@yuhs.ac.

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.

Show MeSH
Related in: MedlinePlus