Overexpression of lentivirus-mediated glial cell line-derived neurotrophic factor in bone marrow stromal cells and its neuroprotection for the PC12 cells damaged by lactacystin.
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ABSTRACT:
SUBMITTER: Su YR
PROVIDER: S-EPMC5550589 | biostudies-other | 2007 Mar
REPOSITORIES: biostudies-other
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Project description:Infected or damaged tissues release multiple "alert" molecules such as alarmins and damage-associated molecular patterns (DAMPs) that are recognized by innate immune receptors, and induce tissue inflammation, regeneration, and repair. Recently, an extract from inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®, NTP) was found to induce infarct tolerance in mice receiving permanent ischemic attack to the middle cerebral artery. Likewise, we report herein that NTP prevented the neurite retraction in PC12 cells by nerve growth factor (NGF) deprivation. This effect was accompanied by interaction of Fyn with high-affinity NGF receptor TrkA. Sucrose density gradient subcellular fractionation of NTP-treated cells showed heretofore unidentified membrane fractions with a high-buoyant density containing Trk, B subunit of cholera toxin-bound ganglioside, flotillin-1 and Fyn. Additionally, these new membrane fractions also contained Toll-like receptor 4 (TLR4). Inhibition of TLR4 function by TAK-242 prevented the formation of these unidentified membrane fractions and suppressed neuroprotection by NTP. These observations indicate that NTP controls TrkA-mediated signaling through the formation of clusters of new membrane microdomains, thus providing a platform for crosstalk between neurotrophic and innate immune receptors. Neuroprotective mechanisms through the interaction with innate immune systems may provide novel mechanism for neuroprotection.
| S-EPMC7555716 | biostudies-literature
Project description:Several studies have reported that human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) restore neurological damage in vivo through their secretion of paracrine factors. We previously found that UC-MSCs attenuate brain injury by secreting neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF). However, how these factors contribute to neuroprotection remains unknown. In this study, we aimed to investigate to what extent UC-MSC-derived HGF and BDNF contribute to neuroprotection using a Transwell co-culture system of neonatal cortical neurons damaged by oxygen-glucose deprivation. The influence of HGF and BDNF were determined by investigating neurons in both the presence and absence of UC-MSCs as these cells consistently secrete both factors and can be blocked by neutralizing antibodies. In the co-culture, UC-MSCs significantly improved neuronal injury, as indicated by an increase in immature neuron number, neurite outgrowth, and cell proliferation. Co-culture of damaged neurons with UC-MSCs also exhibited a reduction in the number of neurons displaying signs of apoptosis/necrosis. The neuroprotective actions of UC-MSCs were partially reverted by neutralizing antibodies. Together, our findings reveal that UC-MSC-secreted HGF and BDNF have neuroprotective effects on damaged neurons. Further studies should address the existence of other potential neurotrophic paracrine factors.
| S-EPMC6013549 | biostudies-literature
Project description:ObjectiveThe actin-sequestering proteins, thymosin beta-4 (Tβ4) and hypoxia-inducible factor (HIF)-1α, are known to be associated with angiogenesis after myocardial infarction (MI). Herein, we aimed to identify the mechanism of HIF-1α induction by Tβ4 and investigate the effects of bone marrow mesenchymal stromal cells (BMMSCs) transfected with the Tβ4 gene (TMSB4) in a rat model of MI.MethodsRat BMMSCs were isolated, cultured, and transfected with the TMSB4 gene by using the lentivirus-mediated method. Rats with surgically induced MI were randomly divided into three groups (n = 9/group); after 1 week, the rats were injected at the heart infarcted border zone with TMSB4-overexpressed BMMSCs (BMMSC-TMSB4 O E ), wild-type BMMSCs that expressed normal levels of TMSB4 (BMMSC-TMSB4 W T ), or medium (MI). The fourth group of animals (n = 9) underwent all surgical procedures necessary for MI induction except for the ligation step (Sham). Four weeks after the injection, heart function was measured using transthoracic echocardiography. Infarct size was calculated by TTC staining, and collagen volume was measured by Masson staining. Angiogenesis in the infarcted heart area was evaluated by CD31 immunofluorescence histochemistry. In vitro experiments were carried out to observe the effect of exogenous Tβ4 on HIF-1α and explore the various possible mechanism(s).ResultsIn vivo experiments showed that vascular density 4 weeks after treatment was about twofold higher in BMMSC-TMSB4 O E -treated animals than in BMMSC-TMSB4 W T -treated animals (p < 0.05). The cardiac function and infarct size significantly improved in both cell-treatment groups compared to controls. Notably, the cardiac function and infarct size were most prominent in BMMSC-TMSB4 O E -treated animals (both p < 0.05). HIF-1α and phosphorylated HIF-1α (p-HIF-1α) in vitro were significantly enhanced by exogenous Tβ4, which was nonetheless blocked by the factor-inhibiting HIF (FIH) promoter (YC-1). The expression of prolyl hydroxylase domain proteins (PHD) was decreased upon treatment with Tβ4 and further decreased with the combined treatment of Tβ4 and FG-4497 (a specific PHD inhibitor).ConclusionTMSB4-transfected BMMSCs might significantly improve recovery from myocardial ischemia and promote the generation of HIF-1α and p-HIF-1α via the AKT pathway, and inhibit the degradation of HIF-1α via the PHD and FIH pathways.
| S-EPMC8221609 | biostudies-literature
Project description:Neural stem cells (NSCs) have been proposed as tools for treating neurodegeneration because of their capacity to give rise to cell types appropriate to the structure in which they are grafted. In the present work, we explore the ability of NSCs to stably express transgenes and locally deliver soluble molecules with neuroprotective activity, such as glial cell line-derived neurotrophic factor (GDNF). NSCs engineered to release GDNF engrafted well in the host striatum, integrated and gave rise to neurons, astrocytes, and oligodendrocytes, and maintained stable high levels of GDNF expression for at least 4 months. The therapeutic potential of intrastriatal GDNF-NSCs grafts was tested in a mouse 6-hydroxydopamine model of Parkinson's disease. We found that GDNF-NSCs prevented the degeneration of dopaminergic neurons in the substantia nigra and reduced behavioral impairment in these animals. Thus, our results demonstrate that NSCs efficiently express therapeutic levels of GDNF in vivo, suggesting a use for NSCs engineered to release neuroprotective molecules in the treatment of neurodegenerative disorders, including Parkinson's disease.
| S-EPMC6763865 | biostudies-literature
Project description:Mesenchymal stromal cell (MSC) therapy to treat neurodegenerative diseases has not been as successful as expected in some preclinical studies. Because preclinical research is so diverse, it is difficult to know whether the therapeutic outcome is due to the cell type, the type of transplant or the model of disease. Our aim here was to analyze the effect of the type of transplant on neuroprotection and axonal regeneration, so we tested MSCs from the same niche in the same model of neurodegeneration in the three transplantation settings: xenogeneic, syngeneic and allogeneic. For this, bone marrow mesenchymal stromal cells (BM-MSCs) isolated from healthy human volunteers or C57/BL6 mice were injected into the vitreous body of C57/BL6 mice (xenograft and syngraft) or BALB/c mice (allograft) right after optic nerve axotomy. As controls, vehicle matched groups were done. Retinal anatomy and function were analyzed in vivo by optical coherence tomography and electroretinogram, respectively. Survival of vision forming (Brn3a+) and non-vision forming (melanopsin+) retinal ganglion cells (RGCs) was assessed at 3, 5 and 90 days after the lesion. Regenerative axons were visualized by cholera toxin β anterograde transport. Our data show that grafted BM-MSCs did not integrate in the retina but formed a mesh on top of the ganglion cell layer. The xenotransplant caused retinal edema, detachment and folding, and a significant decrease of functionality compared to the murine transplants. RGC survival and axonal regeneration were significantly higher in the syngrafted retinas than in the other two groups or vehicle controls. Melanopsin+RGCs, but not Brn3a+RGCs, were also neuroprotected by the xenograft. In conclusion, the type of transplant has an impact on the therapeutic effect of BM-MSCs affecting not only neuronal survival but also the host tissue response. Our data indicate that syngrafts may be more beneficial than allografts and, interestingly, that the type of neuron that is rescued also plays a significant role in the successfulness of the cell therapy.
| S-EPMC8600074 | biostudies-literature
Project description:Treatment of rodents after stroke with bone marrow stromal cells (BMSCs) improves functional outcome. However, the mechanisms underlying this benefit have not been ascertained. This study focused on the contribution of neurotrophic and growth factors produced by BMSCs to therapeutic benefit. Rats were subjected to middle cerebral artery occlusion and the ischemic brain extract supernatant was collected to prepare the conditioned medium. The counterpart normal brain extract from non-ischemic rats was employed as the experimental control. Using microarray assay, we measured the changes of the neurotrophin associated gene expression profile in BMSCs cultured in different media. Furthermore, real-time RT-PCR and fluorescent immunocytochemistry were utilized to validate the gene changes. The morphology of BMSCs, cultured in the ischemic brain-conditioned medium for 12 h, was dramatically altered from a polygonal and flat appearance to a fibroblast-like long and thin cell appearance, compared to those in the normal brain-conditioned medium and the serum replacement medium. Forty-four neurotrophin-associated genes in BMSCs were identified by microarray assay under all three culture media. Twelve out of the 44 genes (7 neurotrophic and growth factor genes, 5 receptor genes) increased in BMSCs cultured in the ischemic brain-conditioned medium compared to the normal brain-conditioned medium. Real time RT-PCR and immunocytochemistry validated that the ischemic brain-conditioned medium significantly increased 6/7 neurotrophic and growth factor genes, compared with the normal brain-conditioned medium. These six genes consisted of fibroblast growth factor 2, insulin-like growth factor 1, vascular endothelial growth factor A, nerve growth factor beta, brain-derived neurotrophic factor and epidermal growth factor. Our results indicate that transplanted BMSCs may work as 'small molecular factories' by secreting neurotrophins, growth factors and other supportive substances after stroke, which may produce therapeutic benefits in the ischemic brain.
| S-EPMC2593420 | biostudies-literature
Project description:BackgroundSB623 cells are expanded from marrow stromal cells (MSCs) transfected with a Notch intracellular domain (NICD)-expressing plasmid. In stroke-induced animals, these cells reduce infarct size and promote functional recovery. SB623 cells resemble the parental MSCs with respect to morphology and cell surface markers despite having been in extended culture. MSCs are known to have immunosuppressive properties; whether long-term culture of MSCs impact their immunomodulatory activity has not been addressed.MethodsTo assess the possible senescent properties of SB623 cells, we performed cell cycle related assays and beta-galactosidase staining. To assess the immunomodulatory activity of these expanded NICD-transfected MSCs, we performed co-cultures of SB623 cells or MSCs with either enriched human T cells or monocytes and assessed cytokine production by flow cytometry. In addition, we monitored the immunosuppressive activity of SB623 cells in both allogenic and xenogenic mixed lymphocyte reaction (MLR).ResultsCompared to MSCs, we showed that a small number of senescent-like cells appear in each lot of SB623 cells. Nevertheless, we demonstrated that these cells suppress human T cell proliferation in both the allogeneic and xenogeneic mixed lymphocyte reaction (MLR) in a manner comparable to MSCs. IL-10 producing T cells were generated and monocyte-dendritic cell differentiation was dampened by co-culture with SB623 cells. Compared to the parental MSCs, SB623 cells appear to exert a greater inhibitory impact on the maturation of dendritic cells as demonstrated by a greater reduction in the surface expression of the co-stimulatory molecule, CD86.ConclusionThe results demonstrated that the immunosuppressive activity of the expanded NICD-transfected MSCs is comparable to the parental MSCs, in spite of the appearance of a small number of senescent-like cells.
| S-EPMC3228829 | biostudies-literature
Project description:BackgroundAngiogenesis is a critical part of the endogenous repair process in brain injury and disease, and requires at least two sequential steps. First, angiogenic sprouting of endothelial cells occurs, which entails the initial proliferation of endothelial cells and remodeling of the surrounding extracellular matrix. Second, vessel stabilization is necessary to prevent vascular regression, which relies on vascular smooth muscle recruitment to surround the young vessels. Marrow stromal cells (MSCs) have been shown to promote revascularization after hindlimb ischemia, cardiac ischemia, and stroke. SB623 cells are derived from marrow stromal cells by transfection with a Notch1 intracellular domain (NICD)-expressing plasmid and are known to elicit functional improvement in experimental stroke. These cells are currently used in human clinical testing for treatment of chronic stroke. In the current study, the angiogenic property of SB623 cells was investigated using cell-based assays.MethodsAngiogenic paracrine factors secreted by SB623 cells and the parental MSCs were identified using the Qantibody Human Angiogenesis Array. To measure the angiogenic activity of conditioned medium from SB623 cells and MSCs, endothelial tube formation in the human umbilical vein endothelial cell (HUVEC) assay and endothelial cell sprouting and branching in the rodent aortic ring assay were quantified. To validate the angiogenic contribution of VEGF in conditioned medium, endothelial cells and aortic rings were treated with SU5416, which inhibits VEGFR2 at low dose.ResultsConditioned medium from SB623 cells promoted survival and proliferation of endothelial cells under serum-deprived conditions and supports HUVEC vascular tube formation. In a rodent aortic ring assay, there was enhanced endothelial sprouting and branching in response to SB623-derived conditioned medium. SU5416 treatment partially reversed the effect of conditioned medium on endothelial cell survival and proliferation while completely abrogate HUVEC tube formation and endothelial cell sprouting and branching in aortic ring assays.ConclusionsThese data indicate that SB623 cell-secreted angiogenic factors promoted several aspects of angiogenesis, which likely contribute to promoting recovery in the injured brain.
| S-EPMC3615967 | biostudies-literature
Project description:BackgroundHuman mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity.MethodsIn order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid.ResultsTransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24-36% GFP-expressing cells with a viability of 85-96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system.DiscussionWe report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.
| S-EPMC6323439 | biostudies-literature
Project description:Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.
| S-EPMC4409887 | biostudies-other