Retroviral expression of arginine decarboxylase attenuates oxidative burden in mouse cortical neural stem cells.

Neural stem cells (NSCs) have the potential to integrate seamlessly into the host tissues, and the development of potential stem cells resistant to stress injury is an elusive goal for efficient therapeutic application. Oxidative injury induces cellular and nuclear damages and the balanced regulation of reactive oxygen species is of critical significance for stem cell development, function, and survival. Agmatine, an endogenous primary amine and a novel neuromodulator synthesized from the decarboxylation of l-arginine catalyzed by arginine decarboxylase (ADC), has been reported to possess neuroprotective properties. In the present study, we determined whether the expression of ADC in NSCs can prevent the cells from oxidative injury. Retrovirus expressing human (ADC), (vhADC) was generated using a pLXSN vector. Cortical NSCs were infected with vhADC and subjected to H2O2 injury (200 µM for 15 h). Reverse transcriptase-polymerase chain reaction and immunocytochemical staining revealed that hADC mRNA and protein were highly expressed in the vhADC-infected NSCs (ADC-NSCs). High performance liquid chromatography (HPLC) analysis confirmed high concentration of agmatine in the ADC-NSCs, when exposed to H2O2 injury. Lactate dehydrogenase leakage and intracellular reactive oxygen species formation were about 2-fold reduced in ADC-NSCs when compared with control NSCs and NSCs infected with mock vector (P < 0.05). DNA fragmentation, chromatin condensation, and expression of apoptotic proteins such as p53, bax, and caspase-3 cleavage were significantly decreased in ADC-NSCs (P < 0.05), suggesting the prevention of apoptotic cell death following H2O2 injury. Our study demonstrates that overexpression of ADC is an effective novel approach to protect stem cells from oxidative damage.

Recombinant hexahistidine arginine decarboxylase (hisADC) induced endogenous agmatine synthesis during stress.

The arginine decarboxylase (ADC) is a significant functional enzyme, synthesizes agmatine through arginine metabolism, and agmatine was reported to posses protective properties in various tissues. This study first optimized the conditions for efficient hexahistidine tagged human ADC (hisADC) gene delivery into mouse fibroblast cell line (NIH3T3) using retroviral vector (pLXSN). Later, the functionality of the delivered hisADC gene in synthesizing agmatine during H(2)O(2) injury in NIH3T3 was also elucidated. Amplification of hisADC gene was performed using hisADC specific primers under specified conditions. The hisADC PCR product (1.4 kb) was ligated with pLXSN considering the restriction enzyme sites. The complete hisADC pLXSN clone was transfected into PT67 cell line following CalPhos Mammalian transfection method. RT-PCR and western blot results showed the specific and strong detection of hisADC genes in hisADC PT67 transfected cells compared with normal control and pLXSN transfected PT67 cells. The retrovirus containing hisADC gene (vhisADC) was infected into NIH3T3 (vhisADC NIH) using polybrene reagent. Immunocytochemical results showed hisADC expression in the cytoplasm of vhisADC NIH. HPLC analysis revealed high agmatine concentration in the vhisADC NIH, and the induced agmatine synthesized from the retroviral gene delivery prevented vhisADC NIH from H(2)O(2) injury which is evident by the decrease in lactate dehydrogenase (P < 0.05) leakage into the medium and less number of propidium iodide positive cells during injury compared to control group. The obtained results provide compelling evidence that higher level of hisADC transgene expression completely triggered the endogenous agmatine synthesis during H(2)O(2) injury thus protecting NIH3T3 cells against cytotoxicity.

Endogenous agmatine inhibits cerebral vascular matrix metalloproteinases expression by regulating activating transcription factor 3 and endothelial nitric oxide synthesis.

Earlier investigations from our laboratory demonstrated that the expression of matrix metalloproteinases (MMPs) was down-regulated by exogenously administered agmatine against ischemia-like injuries in the murine brain capillary endothelial (bEnd.3) cells. In our present study, we intended to investigate the mechanism involved in the inhibition of MMPs in bEnd.3 cells infected with retroviral containing human arginine decarboxylase (hADC) gene which can synthesize agmatine endogenously (ADCDeltabEnd.3 cells). The ADCDeltabEnd.3 cells were subjected to oxygen glucose deprivation (OGD, 6 hrs) with reperfusion (18 hrs). High performance liquid chromatography (HPLC) analysis revealed the high levels of agmatine in the ADCDeltabEnd.3 cells compared to other experimental groups. The results demonstrated significant decrease in cell death and increase in the nitric oxide (NO) production in the ADCDeltabEnd.3 cells. The increased expression of MMP-2 and MMP-9, and decreased expression of endothelial nitric oxide synthase (eNOS) by ischemic injury was attenuated in ADCDeltabEnd.3 cells. Moreover, the expression of activating transcription factor 3 (ATF3) was increased significantly in ADCDeltabEnd.3 cells. In addition, the suppression of the MMP-2 and MMP-9 expression in ADCDeltabEnd.3 cells was prevented with ATF3 small interfering RNA (siRNA) treatment. These results suggest that the endogenous agmatine in ADCDeltabEnd.3 cells inhibits the MMPs expression mediated via the regulation of eNOS, NO and ATF3.