Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses

The capacity of mesenchymal stem cells (MSCs) to survive and engraft in the target tissue may lead to promising therapeutic effects. However, the fact that the majority of MSCs die during the first few days following transplantation complicates cell therapy. Hence, it is necessary to strengthen the stem cells to withstand the rigors of the microenvironment to improve the efficacy of cell therapy. In this study, we manipulated MSCs to express a cytoprotective factor, heme oxygenase-1 (HO-1), to address this issue. Full-length cDNA of human HO-1 was isolated and cloned into TOPO vector by TOPO cloning reaction. Then, the construct was ligated to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the recombinant virus expressing HO-1 was produced in appropriate mammalian cell line and used to infect MSCs. The HO-1 engineered MSCs were exposed to hypoxic and oxidative stress conditions followed by evaluation of the cells’ viability and apoptosis. Transient expression of HO-1 was detected within MSCs. It was observed that HO-1 expression could protect MSCs against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. The MSCs-HO-1 retained their ability to differentiate into adipogenic, chondrogenic, or osteogenic lineages. These findings could be applied as a strategy for prevention of graft cell death in MSCs-based cell therapy and is a good demonstration of how an understanding of cellular stress responses can be used for practical applications.     

Lipocalin-2-mediated upregulation of various antioxidants and growth factors protects bone marrow-derived mesenchymal stem cells against unfavorable microenvironments

Despite many advantages of mesenchymal stem cells (MSCs) that make them suitable for cell therapy purposes, their therapeutic application has been limited due to their susceptibility to several stresses (e.g., nutrient-poor environment, oxidative stress, and hypoxic and masses of cytotoxic factors) to which they are exposed during their preparation and following transplantation. Hence, reinforcing MSCs against these stresses is a challenge for both basic and clinician scientists. Recently, much attention has been directed toward equipping MSCs with cytoprotective factors to strengthen them against unfavorable microenvironments. Here, we engineered MSCs with lipocalin 2 (Lcn2), a cytoprotective factor that is naturally induced following exposure of cells to stresses imposed by the microenvironment. Lcn2 overexpression not only did not interfere with the multidifferentiation capacity of the MSCs but also granted many protective properties to them. Lcn2 potentiated MSCs to withstand oxidative, hypoxia, and serum deprivation (SD) conditions via antagonizing their induced cytotoxicity and apoptosis. Adhesion rate of MSCs to coated culture plates was also enhanced by Lcn2 overexpression. In addition, Lcn2 induced antioxidants and upregulated some growth factors in MSCs. Our findings suggested a new strategy for prevention of graft cell death in MSC-based cell therapy.     

Mitochondria-targeted antioxidant mito-TEMPO alleviate oxidative stress induced by antimycin A in human mesenchymal stem cells

The cell therapy of damaged tissue, which is linked to hypoxia condition might fail, in large part due to the emergence of oxidative stress (OS) and/or mitochondrial dysfunctions. Thus, the invigoration of stem cells against oxidative stress could be a reliable strategy to improve the cell therapy outcome. Of various antioxidants, mito-Tempo (mito-T) is one of the potent antioxidants that could target and neutralize the mitochondrial oxidative stress. In this study, for the induction of hypoxia and oxidative stress in mitochondria of the mesenchymal stem cells (MSCs) isolated from human adipose tissue, antimycin A (AMA) was used and then several parameters were analyzed, including cell viability and cell cycle arrest of MSCs exposed to AMA, mito-T, antioxidant potential, redox homeostasis, and signaling pathways in MSCs under oxidative stress. Based on our findings, the treated MSCs were found to impose a high resistance to the OS-induced apoptosis, which correlated with the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway required to manage OS. Upon exposure of the MSCs to high oxidative stress conditions using AMA, the cells failed to scavenge. The use of mito-T was found to alleviate the damage induced by oxidative stress through both direct functions of the free radical scavenging and the interplay in terms of cell signaling pathways including the upregulation of the Nrf2 pathway. These findings may pave the way in the stem cell therapy for the hypoxia-mediated tissue damage.     

Hypoxia. 1. Intracellular sensors for oxygen and oxidative stress: novel therapeutic targets

A variety of human disorders, e.g., ischemic heart disease, stroke, kidney disease, eventually share the deleterious consequences of a common, hypoxic and oxidative stress pathway. In this review, we utilize recent information on the cellular defense mechanisms against hypoxia and oxidative stress with the hope to propose new therapeutic tools. The hypoxia-inducible factor (HIF) is a key player as it activates a broad range of genes protecting cells against hypoxia. Its level is determined by its degradation rate by intracellular oxygen sensors prolyl hydroxylases (PHDs). There are three different PHD isoforms (PHD1-3). Small molecule PHD inhibitors improve hypoxic injury in experimental animals but, unfortunately, may induce adverse effects associated with PHD2 inhibition, e.g., angiogenesis. As yet, no inhibitor specific for a distinct PHD isoform is currently available. Still, the specific disruption of the PHD1 gene is known to induce hypoxic tolerance, without angiogenesis and erythrocytosis, by reprogramming basal oxygen metabolism with an attendant decreased oxidative stress in hypoxic mitochondria. A specific PHD1 inhibitor might therefore offer a novel therapy against hypoxia. The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and inducible expression of numerous antioxidant stress genes. Disruption of its gene exacerbates oxidative tissue injury. Nrf2 activity is modulated by Kelch-like ECH-associated protein 1 (Keap1), an intracellular sensor for oxidative stress. Inhibitors of Keap 1 may prove therapeutic against oxidative tissue injury.     

Sinomenine Attenuates Traumatic Spinal Cord Injury by Suppressing Oxidative Stress and Inflammation via Nrf2 Pathway

Traumatic spinal cord injury (SCI) is a devastating condition with few efficacious drugs. Sinomenine, a bioactive alkaloid extracted from medicinal herb, has been used as a treatment of rheumatoid diseases. This present study explored the therapeutic effects of sinomenine on locomotor dysfunction and neuropathology in SCI. Our findings revealed that sinomenine mitigated neurological deficits and enhanced neuronal preservation, paralleled with a reduction of apoptosis. Also, sinomenine significantly reduced inflammatory cytokines and oxidative stress factors. We further examined erythroid-2-related factor 2 (Nrf2) nuclear translocation, which mainly controls the coordinated expression of important antioxidant and detoxification genes. An increase in Nrf2 translocation from cytoplasm to nucleus and Nrf2-mediated transactivation was observed after sinomenine administration. Knocking down Nrf2 by siRNA could counteract sinomenine-mediated anti-oxidant stress and anti-inflammation following H₂O₂-stimulated and LPS-stimulated PC12 cells. Together, our findings indicated that sinomenine has the potential to be an effective therapeutic agent for SCI by inhibiting inflammation and oxidative stress via Nrf2 activation.

     

Fructose-1,6-bisphosphatase aggravates oxidative stress-induced apoptosis in asthma by suppressing the Nrf2 pathway

Asthma is a chronic airway disease that causes excessive inflammation, oxidative stress, mucus production and bronchial epithelial cell apoptosis. Fructose-1,6-bisphosphatase (Fbp1) is one of the rate-limiting enzymes in gluconeogenesis and plays a critical role in several cancers. However, its role in inflammatory diseases, such as asthma, is unclear. Here, we examined the expression, function and mechanism of action of Fbp1 in asthma. Gene Expression Omnibus (GEO) data sets revealed that Fbp1 was overexpressed in a murine model of asthma and in interleukin (IL)-4- or IL-13-stimulated bronchial epithelial cells. We confirmed the findings in an animal model as well as Beas-2B and 16HBE cells. In vitro investigations revealed that silencing of Fbp1 reduced apoptosis and the proportion of cells in the G2/M phase, whereas overexpression led to increases. Fbp1 knock-down inhibited oxidative stress by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, whereas Fbp1 overexpression aggravated oxidative stress by suppressingthe Nrf2 pathway. Moreover, the Nrf2 pathway inhibitor ML385 reversed the changes caused by Fbp1 inhibition in Beas-2B and 16HBE cells. Collectively, our data indicate that Fbp1 aggravates oxidative stress-induced apoptosis by suppressing Nrf2 signalling, substantiating its potential as a novel therapeutic target in asthma.     

Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases,p38 and JNK

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H₂O₂ induced apoptosis were analyzed with Western blot. H₂O₂ induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H₂O₂ induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H₂O₂ caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H₂O₂ activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.     

Bone marrow‐derived mesenchymal stem cells mitigate caspase‐3 and 8‐hydroxy proline induced via β‐adrenergic agonist in pulmonary injured rats

Estimating the ability of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to alleviate pulmonary injury induced via isoproterenol (ISP). ISP was injected in a dose of (100 mg/kg, subcutaneously twice at an interval of 24 h). One month post BM‐MSCs transplantation by intravenous injection, pulmonary oxidative stress was assessed, and Western blot analyses and histopathological investigations were conducted. Compared with the normal control group, BM‐MSCs transplantation significantly decreased the expression of pulmonary anti‐oxidative stress marker. Western blot analysis revealed that ISP significantly reduced the protein expression of the anti‐oxidative stress marker nuclear related factor‐2 (Nrf2). However, the apoptotic marker (caspase‐3) and collagen content marker (8‐hydroxyproline) were markedly elevated. These biochemical markers were confirmed by histopathological investigations. Finally, it was demonstrated that BM‐MSCs transplantation showed a superior effect in improving pulmonary function through alleviating oxidative stress, apoptosis, and collagen content.     

Klotho protects the heart from hyperglycemia-induced injury by inactivating ROS and NF-κB-mediated inflammation both in vitro and in vivo

Cardiac inflammation and oxidative stress play a key role in the pathogenesis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho has been found to protect cells from inflammation and oxidative stress. The current study aimed to explore the cardioprotective effects of Klotho on DCM and the underlying mechanisms. H9c2 cells and neonatal cardiomyocytes were incubated with 33 mM glucose in the presence or absence of Klotho. Klotho pretreatment effectively inhibited high glucose-induced inflammation, ROS generation, apoptosis, mitochondrial dysfunction, fibrosis and hypertrophy in both H9c2 cells and neonatal cardiomyocytes. In STZ-induced type 1 diabetic mice, intraperitoneal injection of Klotho at 0.01 mg/kg per 48 h for 3 months completely suppressed cardiac inflammatory cytokines and oxidative stress and prevented cardiac cell death and remodeling, which subsequently improved cardiac dysfunction without affecting hyperglycemia. This study revealed that Klotho may exert its protective effects by augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression and inactivating nuclear factor κB (NF-κB) activation both in vitro and in vivo. Thus, this work demonstrated for the first time that the anti-aging protein Klotho may be a potential therapeutic agent to treat DCM by inhibiting oxidative stress and inflammation. We also demonstrated the critical roles of the Nrf2 and NF-κB pathways in diabetes-stimulated cardiac injuries and indicated that they may be key therapeutic targets for diabetic complications.     

Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre‐treatment with melatonin, an endogenous hormone with newly found anti‐oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre‐treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD‐1), as well as pro‐angiogenic and mitogenic factors like insulin‐like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre‐treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti‐apoptosis kinases like p‐Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre‐treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.     

Proanthocyanidins alleviate pentylenetetrazole-induced epileptic seizures in mice via the antioxidant activity

The role of oxidative stress in the initiation and progress of epilepsy is well established. Proanthocyanidins (PACs), a naturally occurring polyphenolic compound, have been reported to possess a broad spectrum of pharmacological and therapeutic properties against oxidative stress. However, the protective effects of proanthocyanidins against epilepsy have not been clarified. In the present study, we used the pentylenetetrazole (PTZ)-induced epilepsy mouse model to explore whether proanthocyanidins could help to reduce oxidative stress and protect against epilepsy. Mice were allocated into four groups (n?=?14 per each group): control, PTZ (60 mg/kg, intraperitoneally), PACs?+?PTZ (200 mg/kg, p.o.) and sodium valproate (VPA)?+?PTZ (200 mg/kg, p.o.). PTZ injection caused oxidative stress in the hippocampal tissue as represented by the elevated lipid peroxidation and NO synthesis and increased expression of iNOS. Furthermore, depleted levels of anti-oxidants, GSH, GR, GPx, SOD, and CAT also indicate that oxidative stress was induced in mice exposed to PTZ. Additionally, a state of neuroinflammation was recorded following the developed seizures. Moreover, neuronal apoptosis was recorded following the development of epileptic convulsions as confirmed by the elevated Bax and caspase-3 and the decreased Bcl2 protein. Moreover, AChE activity, DA, NE, 5-HT, brain-derived neurotrophic factor levels, and gene expression of Nrf2 have decreased in the hippocampal tissue of PTZ exposed mice. However, pre-treatment of mice with PACs protected against the generation of oxidative stress, apoptosis, and neuroinflammation in the PTZ exposed mice brain as the biomarkers for all these conditions was bought to control levels. In addition, the gene expression of Nrf2 was significantly upregulated following PACs treatment. These results suggest that PACs can ameliorate oxidative stress, neuroinflammation, and neuronal apoptosis by activating the Nrf2 signaling pathway in PTZ induced seizures in mice.

     

Intermittent Hypoxia-Induced NF-κB and HO-1 Regulation in Human Endothelial EA.hy926 Cells

Intermittent hypoxia (IH) is a hallmark feature in obstructive sleep apnea (OSA) which is increasingly recognized as an independent risk factor for atherosclerosis. Oxidative stress, inflammation, and cell apoptosis are major pathological events initiating or accelerating atherogenesis. This study addressed whether IH would affect these proatherogenic factors in endothelial cells and the mechanistic pathways involved. EA.hy926 cells were exposed to intermittent normoxia or IH for different numbers of cycles (32, 64, or 96). IH exposure time-dependently raised cellular GSSG/GSH ratio, increased production of IL-6 and IL-8, and accelerated cell apoptosis and death, concurrent with activation of NF-κB and inhibition of Nrf2/HO-1 pathways. At 64 cycles, inhibition of NF-κB attenuated IH-induced cellular oxidative stress and accumulation of inflammatory cytokines in cell culture medium but aggravated IH-induced cell apoptosis, while stimulation of HO-1 suppressed IH-induced cellular oxidative stress and cell apoptosis without affecting accumulation of inflammatory cytokines in cell culture medium. We demonstrated that early stage of exposure to IH-induced oxidative and inflammatory stresses leading to acceleration of cell apoptosis via NF-κB and Nrf2/HO-1 pathways in endothelial cells, suggesting the potential mechanisms for IH-induced vascular pathogenesis, in resemblance to OSA.     

Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over‐activation and consequently attenuate DCM. Streptozotocin‐induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure‐volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15‐F2t‐Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme‐linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT‐PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end‐systolic volume (LVVs) as compared to non‐diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15‐F2t‐Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase‐1 (HO‐1) and Keap1. ALP reverted all the above‐mentioned diabetes‐induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia‐induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over‐activation may represent major mechanisms whereby ALP attenuates DCM.