Abstract P751: Global Analyses of Protein and Microrna Cargo in Neural Stem Cell Derived-Exosomes After Brain Ischemia

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Xianshuang S LIU ◽  
Baoyan Fan ◽  
Chao Li ◽  
Albert Levin ◽  
Rui Lan Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pathway Analysis ◽  
Critical Role ◽  
Neuronal Cell ◽  
Artery Occlusion ◽  
Mirna Array ◽  
Transmission Electron ◽  
Cargo Proteins ◽  
Pathway Analyses ◽  
Neurovascular Remodeling

Background: Adult neurogenesis facilitates brain remodeling after stroke. Exosomes derived from neural stem cells (NSCs) promote ischemic neurovascular remodeling including angiogenesis and axonal outgrowth, potentially by transferring their cargo proteins and miRNAs to recipient cells. However, cargo profiles of proteins and miRNAs in NSC exosomes have not been investigated. Methods: Exosomes were isolated from supernatants of cultured NSCs harvested from the subventricular zone of rats subjected to 7 day middle cerebral artery occlusion (MCAO) and non-MCAO rats, respectively. Mass spectrometry and miRNA array were utilized to determine the protein and miRNA profiles of NSC-derived exosomes (NSC-Exos). Bioinformatic pathway analyses were performed using Ingenuity Pathway Analysis (IPA). Results: Exosome markers and size distribution (50-200nm) were validated with Western blot, transmission electron microscopy and Nanosight measurements, respectively. Proteomics analysis yielded a total of 1,770 proteins in ischemic NSC-Exos. Bioinformatics analysis identified 24, 23 and 23 proteins that were related to neuronal cell proliferation, migration and differentiation, respectively. Intriguingly, enrichment signaling pathway analysis revealed cargo proteins in ischemic NSC-Exos were highly associated with dysfunction, membrane, and permeability of mitochondrion, indicating a critical role of extracellular mitochondrion in stroke-induced neurogenesis. In addition, 318 miRNAs were detected in ischemic NSC-Exos. Gene ontology analysis demonstrated that differentially expressed miRNAs between ischemic and non-ischemic NSC-Exos were highly related to inflammation, cell proliferation, cell cycle, and differentiation. The top 3 upregulated miRNAs including miR-106b, miR-542,miR-125b were validated in ischemic NSC-Exos using RT-PCR. The functions of these miRNAs are related to the induction of angiogenesis. Summary/Conclusion: Our results for the first time demonstrate that ischemic NSC-Exos contain a robust profile of protein and miRNA effectors, which may provide new insights into the function of NSC-Exos in stroke-induced neurogenesis and potentially lead to new therapeutic targets against stroke.

scholarly journals Quercetin attenuates the reduction of parvalbumin in middle cerebral artery occlusion animal model

2021 ◽  
Vol 37 (1) ◽  
Author(s):  
Dong-Ju Park ◽  
Ju-Bin Kang ◽  
Fawad-Ali Shah ◽  
Phil-Ok Koh
Keyword(s):  
Middle Cerebral Artery ◽  
Middle Cerebral Artery Occlusion ◽  
Cerebral Artery ◽  
Neuroprotective Effect ◽  
Neuronal Cell ◽  
Artery Occlusion ◽  
Male Rats ◽  
Neurological Deficits ◽  
Glutamate Toxicity ◽  
Cerebral Artery Occlusion

Abstract Background Calcium is a critical factor involved in modulation of essential cellular functions. Parvalbumin is a calcium buffering protein that regulates intracellular calcium concentrations. It prevents rises in calcium concentrations and inhibits apoptotic processes during ischemic injury. Quercetin exerts potent antioxidant and anti-apoptotic effects during brain ischemia. We investigated whether quercetin can regulate parvalbumin expression in cerebral ischemia and glutamate toxicity-induced neuronal cell death. Adult male rats were treated with vehicle or quercetin (10 mg/kg) 30 min prior to middle cerebral artery occlusion (MCAO) and cerebral cortical tissues were collected 24 h after MCAO. We used various techniques including Western blot, reverse transcription-PCR, and immunohistochemical staining to elucidate the changes of parvalbumin expression. Results Quercetin ameliorated MCAO-induced neurological deficits and behavioral changes. Moreover, quercetin prevented MCAO-induced a decrease in parvalbumin expression. Conclusions These findings suggest that quercetin exerts a neuroprotective effect through regulation of parvalbumin expression.

scholarly journals Sp1 mediates cell proliferation-dependent regulation of rat DNA topoisomerase IIα gene promoter

1999 ◽  
Vol 344 (2) ◽  
pp. 367-374 ◽  
Author(s):  
Jeong Ho YOON ◽  
Jeong Kee KIM ◽  
Geun Bae RHA ◽  
Misook OH ◽  
Se-Ho PARK ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Promoter Activity ◽  
Expression System ◽  
Critical Role ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Specific Regulation ◽  
Dna Topoisomerase Iiα

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIα is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5′ and/or 3′ serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIα. The luciferase activities of the mutated reporter plasmids in G0-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIα in growing cells.

scholarly journals Rha1, an Arabidopsis Rab5 Homolog, Plays a Critical Role in the Vacuolar Trafficking of Soluble Cargo Proteins

2003 ◽  
Vol 15 (5) ◽  
pp. 1057-1070 ◽  
Author(s):  
Eun Ju Sohn ◽  
Eol Sun Kim ◽  
Min Zhao ◽  
Soo Jin Kim ◽  
Hyeran Kim ◽  
...  
Keyword(s):  
Critical Role ◽  
Cargo Proteins ◽  
Vacuolar Trafficking

scholarly journals The Role of EGFR/PI3K/Akt/cyclinD1 Signaling Pathway in Acquired Middle Ear Cholesteatoma

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Wei Liu ◽  
Hongmiao Ren ◽  
Jihao Ren ◽  
Tuanfang Yin ◽  
Bing Hu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
External Auditory Canal ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Immunohistochemical Method ◽  
Cycle Progression ◽  
Middle Ear Cholesteatoma

Cholesteatoma is a benign keratinizing and hyper proliferative squamous epithelial lesion of the temporal bone. Epidermal growth factor (EGF) is one of the most important cytokines which has been shown to play a critical role in cholesteatoma. In this investigation, we studied the effects of EGF on the proliferation of keratinocytes and EGF-mediated signaling pathways underlying the pathogenesis of cholesteatoma. We examined the expressions of phosphorylated EGF receptor (p-EGFR), phosphorylated Akt (p-Akt), cyclinD1, and proliferating cell nuclear antigen (PCNA) in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium by immunohistochemical method. Furthermore,in vitrostudies were performed to investigate EGF-induced downstream signaling pathways in primary external auditory canal keratinocytes (EACKs). The expressions of p-EGFR, p-Akt, cyclinD1, and PCNA in cholesteatoma epithelium were significantly increased when compared with those of control subjects. We also demonstrated that EGF led to the activation of the EGFR/PI3K/Akt/cyclinD1 signaling pathway, which played a critical role in EGF-induced cell proliferation and cell cycle progression of EACKs. Both EGFR inhibitor AG1478 and PI3K inhibitor wortmannin inhibited the EGF-induced EGFR/PI3K/Akt/cyclinD1 signaling pathway concomitantly with inhibition of cell proliferation and cell cycle progression of EACKs. Taken together, our data suggest that the EGFR/PI3K/Akt/cyclinD1 signaling pathway is active in cholesteatoma and may play a crucial role in cholesteatoma epithelial hyper-proliferation. This study will facilitate the development of potential therapeutic targets for intratympanic drug therapy for cholesteatoma.

scholarly journals Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region

2006 ◽  
Vol 398 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Lan Liu ◽  
Xin Guo ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Bernard S. Marasa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Repression ◽  
Critical Role ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Transcriptional Level ◽  
Intestinal Epithelial ◽  
Major Player ◽  
Stimulation Of

Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with α-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation.

Abstract 13287: Dendritic Cells Play a Critical Role in the Initiation of Atherosclerosis

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Mengyao Jin ◽  
Peng Liu
Keyword(s):  
Cell Proliferation ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Critical Role ◽  
Cell Interaction ◽  
Plaque Formation ◽  
T Cell Proliferation ◽  
Control Group

Introduction: Dendritic cells (DCs) that are known as professional antigen-presenting cells have been found to pre-locate in non-inflammatory arterial wall and increasingly accumulate during atherosclerosis progression. Previous findings suggested that residential DCs in the intima are responsible for capturing modified lipids and forming foam cells during the initiation of atherosclerosis. Hypothesis: DC accumulation and enhanced DC-T cell interaction play a critical role in the initiation of atherosclerosis. Methods: We measured plaque formation, vascular DC accumulation and antigen-specific T cell proliferation mediated by isolated aortic cells in ApoE-/- mice, as well as DTR-CD11c/ApoE-/- or DTR-CD11b/ApoE-/- mice for conditional depletion of DCs or macrophages, respectively. A brief high-fat diet for 10 days was used as a model of initial atherosclerosis. Results: In addition to increased intimal DC accumulation and plaque formation in aortic roots, 10 days of HFD induced T cell infiltration in ApoE-/- mice, compared to those without HFD as the control. Isolated aortic cells from mice with 10-day HFD showed stronger capability in inducing antigen-specific T cell proliferation, compare to the control (HFD: 3.14±0.71%; no HFD: 1.56±0.36%; p=0.022). Single diphtheria toxin (DT) injection at day 1 yielded approximately 50% decrease in intimal DC accumulation, as well as 60% attenuation in plaque formation in DTR-CD11c/ApoE-/- mice after 10-day HFD. Capability of stimulating antigen-specific T cell proliferation was also impaired in aortic cells from DC-depleted mice (DT-treated: 1.62±0.30%; PBS-treated: 3.04±0.59%; p= 0.004), along with reduction in indirect conduction of T cell activation. In contrast, no significant changes were found in plaque formation and DC accumulation in DT-injected DTR-CD11b/ApoE-/- mice after 10 days of HFD, compared to control group. Furthermore, depletion of CD11b+ macrophages in either aortas or spleens didn’t alter capability of inducing antigen-specific T cell proliferation in DT-injected mice. Conclusions: These results suggested that vascular DCs rather than macrophages play a more important role in T cell activation and initiation of atherosclerosis.

scholarly journals 8e Protects against Acute Cerebral Ischemia by Inhibition of PI3Kγ-Mediated Superoxide Generation in Microglia

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2828 ◽  
Author(s):  
Linna Wang ◽  
Xiaoli Wang ◽  
Tingting Li ◽  
Yihua Zhang ◽  
Hui Ji
Keyword(s):  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Critical Role ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Potential Candidate ◽  
Promising Target ◽  
Acute Cerebral Ischemia ◽  
Penumbral Region ◽  
Phosphoinositide 3 Kinase

The inflammatory response mediated by microglia plays a critical role in the progression of ischemic stroke. Phosphoinositide 3-kinase gamma (PI3Kγ) has been implicated in multiple inflammatory and autoimmune diseases, making it a promising target for therapeutic intervention. The aim of this study was to evaluate the efficacy of 8e, a hydrogen sulfide (H2S) releasing derivative of 3-n-butylphthalide (NBP), on brain damage and PI3Kγ signaling following cerebral ischemia injury. 8e significantly reduced sensorimotor deficits, focal infarction, brain edema and neural apoptosis at 72 h after transient middle cerebral artery occlusion (tMCAO). The NOX2 isoform of the NADPH oxidase family is considered a major enzymatic source of superoxide. We found that the release of superoxide, together with the expression of NOX2 subunits p47phox, p-p47phox, and the upstream PI3Kγ/AKT signaling were all down-regulated by 8e, both in the penumbral region of the rat brain and in the primary cultured microglia subjected to oxygen-glucose deprivation (OGD). With the use of siRNA and pharmacological inhibitors, we further demonstrated that 8e regulates the formation of superoxide in activated microglia through the PI3Kγ/AKT/NOX2 signaling pathway and subsequently prevents neuronal death in neighboring neurons. Our experimental data indicate that 8e is a potential candidate for the treatment of ischemic stroke and PI3Kγ-mediated neuroinflammation.

scholarly journals Inhibition of Vascular Nitric Oxide after Rat Chronic Brain Hypoperfusion: Spatial Memory and Immunocytochemical Changes

2005 ◽  
Vol 25 (6) ◽  
pp. 663-672 ◽  
Author(s):  
Jack C. de la Torre ◽  
Gjumrakch Aliev
Keyword(s):  
Nitric Oxide ◽  
Spatial Memory ◽  
Memory Function ◽  
Critical Role ◽  
Artery Occlusion ◽  
Carotid Artery Occlusion ◽  
Significant Loss ◽  
Electron Microscopic ◽  
Vessel Occlusion ◽  
Common Carotid Artery Occlusion

An aging rat model of chronic brain hypoperfusion (CBH) that mimics human mild cognitive impairment (MCI) was used to examine the role of nitric oxide synthase (NOS) isoforms on spatial memory function. Rats with CBH underwent bilateral common carotid artery occlusion (2-vessel occlusion (2-VO)) for either 26 or 8 weeks and were compared with nonoccluded sham controls (S-VO). The neuronal and endothelial (nNOS/eNOS) constitutive inhibitor nitro-L-arginine methyl ester (L-NAME) 20 mg/kg was administered after 26 weeks for 3 days to 2-VO and S-VO groups and spatial memory was assessed with a modified Morris watermaze test. Only 2-VO rats worsened their spatial memory ability after L-NAME. Electron microscopic immunocytochemical examination using an antibody against eNOS showed 2-VO rats had significant loss or absence of eNOS-containing positive gold particles in hippocampal endothelium and these changes were associated with endothelial cell compression, mitochondrial damage and heavy amyloid deposition in hippocampal capillaries and perivascular region. In the 8-week study, three groups of 2-VO rats were administered an acute dose of 7-NI, aminoguanidine or L-NIO, the relatively selective inhibitors of nNOS, inducible NOS and eNOS. Only rats administered the eNOS inhibitor L-NIO worsened markedly their watermaze performance ( P=0.009) when compared with S-VO nonoccluded controls. We conclude from these findings that vascular nitric oxide derived from eNOS may play a critical role in spatial memory function during CBH possibly by keeping cerebral perfusion optimal through its regulation of microvessel tone and cerebral blood flow and that disruption of this mechanism can result in spatial memory impairment. These findings may identify therapeutic targets for preventing MCI and treating Alzheimer's disease.

scholarly journals Polymethylmethacrylate (PMMA) Encapsulated Graphene Oxide Nanocomposite: Pre-modification, Synthesis, and Latex Stability

2020 ◽  
Author(s):  
Hui Wang ◽  
Letian Wang ◽  
Shanyu Meng ◽  
Hanxue Lin ◽  
Melanie Correll ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
Critical Role ◽  
Miniemulsion Polymerization ◽  
Sem Analysis ◽  
High Molecular Weight Polymer ◽  
Molecular Weight Polymer ◽  
Transmission Electron ◽  
Composite Production ◽  
Uniform Particle Size

The compatibility of graphene or graphene oxide with its dispersion medium (polymer) plays a critical role in the formation nanocomposite materials with significant property improvements. Environmentally friendly miniemulsion polymerization, which allows a formation of nanoencapsulation in an aqueous phase and high molecular weight polymer/composite production is one promising method. In this study, we screened a series of amphiphilic modifiers and found that the quaternary ammonium (ar-vinyl benzyl) trimethyl ammonium chloride (VBTAC) pending carbon double bonds could effectively modify the graphene oxide (GO) to be compatible with the organophilic monomer. After that, free radical miniemulsion polymerization could successfully synthesize stable latex of exfoliated poly (methyl methacrylate) (PMMA)/ GO nanocomposite. The final latex had an extended storage life and a relatively uniform particle size distribution. X-ray powder diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) analysis of this latex and its films indicated successful encapsulation of exfoliated nano-dimensional graphene oxide inside a polymer matrix.

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