Cell-Driven Angiogenesis and Neurogenesis after Stroke Is Regulated by Platelet’s Microparticles.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1899-1899 ◽  
Author(s):  
David Varon ◽  
Yael Haion ◽  
Alexander Brill ◽  
Ronen Leker
Keyword(s):  
Survival Rates ◽  
Brain Regions ◽  
Artery Occlusion ◽  
Survival Times ◽  
Differentiation Potential ◽  
Ischemic Brain ◽  
Spontaneously Hypertensive ◽  
Repair Mechanisms

Abstract Repair mechanisms based on the proliferation of endogenous cells have been identified in ischemic brain regions suggesting that precursors at the ventricular surface proliferate, migrate to sites of injury and differentiate into neurons. However, this response is significantly limited by short survival times and poor differentiation capacity of these cells. External factors can boost endogenous neural stem cells (eNSC)-dependent repair mechanisms in the cerebral cortex. Upon activation, platelets shed platelet derived microparticles (PMP) which contain a variety of growth factors central to angiogenesis and neurogenesis. We have previously reported a pro angiogenic effect of PMPs, both in vitro and in vivo. We thus hypothesized that the functional benefits obtained from the presence of newborn cells can be further enhanced by increasing the survival rates, migration and differentiation potential of these cells, as well as by promoting angiogenic response, applying PMPs at the site of a stroke. Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO) and were given BrdU to label newborn cells. Animals were treated with vehicle or PMP delivered via a biodegradable polymer applied to the brain surface. Rats were tested with a neurological severity score and infarct volumes were measured at 90 days post –PMCAO. We used immunohistochemistry to determine the fate of newborn cells and to count blood vessels in the ischemic brain. The results show that PMP treatment led to a dose dependent increase in neurogenesis and angiogenesis at the infarct boundary zone. PMP also significantly improved behavioral deficits without affecting infarct volumes.

Abstract W P248: miRNA Profiling of the Ischaemic Penumbra

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Emily N Ord ◽  
Chris McCabe ◽  
Catriona McDonald ◽  
John D McClure ◽  
I M Macrae ◽  
...  
Keyword(s):  
Cell Line ◽  
Neuronal Cell ◽  
Mrna Translation ◽  
In Vitro Study ◽  
Brain Regions ◽  
Artery Occlusion ◽  
Paramagnetic Resonance ◽  
Spontaneously Hypertensive

MicroRNAs (miRNAs) are small non-coding RNA molecules (20 - 24 nucleotides) that inhibit mRNA translation. Demonstrated to have key roles in normal CNS development & function, they have also emerged to have effecter roles in the pathogenesis & endogenous repair mechanisms following stroke. To select 2 miRNAs to modulate therapeutically we profiled miRNA expression of the evolving (24h) & final (72h) peri-infarct tissue of adult spontaneously hypertensive stroke-prone rats (SHRSP) following 45 min transient middle cerebral artery occlusion (tMCAO). T2-weighted magnetic resonance imaging (MRI) was used for accurate dissection of the peri-infarct tissue, with equivalent brain regions taken from time-matched shams (n=6/group). Of the 754 miRNAs evaluated (TaqMan® human miRNA microarray card v3.0 Applied Biosystems) 89 were determined as differently regulated following tMCAO. 22 of these miRNAs were relevant in stroke & were thus validated by Taqman® qRT-PCR using specific probes (n=9 /group). 5 miRNAs were successfully validated; miR-34b & miR-520b were selected as miRNAs of interest due to their novelty, time of endogenous regulation & targets. An in vitro study to determine whether upregulation/knock-down of these miRNAs would demonstrate functional effects in classical hypoxic pathways was performed. A rat neuronal cell line (B50) & glial cell line (B92) were subjected to 9hr hypoxia (1% O2 -serum) & 24h reoxygenation (+serum) +/- miR-34b or miR-520b regulation. Upregulation of either miRNA in B50 cells demonstrated a reduction in apoptosis, assessed qualitatively by Caspase-3 immunocytochemistry & quantitatively by cell death detection ELISA (p<0.01 vs hypoxic non-treated cells (NTC)). Upregulation of either miRNA in B92 cells significantly reduced superoxide production, assessed by electron paramagnetic resonance (p<0.001 vs hypoxic NTC). MiR-520b significantly lowered levels of lipid peroxidation in B92 cells, assessed by malondialdehyde assay, & both were significantly effective in B50 cells (p<0.01 vs hypoxic NTC). These data suggest miR-34b & -520b upregulation ameliorates damage following hypoxia/reperfusion in cerebral cell lines. Future studies will assess the effect of modulating these miRNAs in vivo.

scholarly journals Cell heterogeneity, rather than the cell storage solution, affects the behavior of mesenchymal stem cells in vitro and in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yong-Hong Wang ◽  
Ya-Chao Tao ◽  
Dong-Bo Wu ◽  
Meng-Lan Wang ◽  
Hong Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Survival Rates ◽  
Phosphate Buffer Solution ◽  
Hepatic Differentiation ◽  
Differentiation Potential ◽  
Normal Ranges ◽  
Storage Solutions

Abstract Background Mesenchymal stem cells (MSCs) have to be expanded in vitro to reach a sufficient cell dose for the treatment of various diseases. During the process of expansion, some obstacles remain to be overcome. The purpose of this study was to investigate the effects of storage solutions and heterogeneity on the behavior of MSCs in vitro and in vivo. Methods Umbilical cord MSCs (UC-MSCs) of similar sizes within normal ranges were suspended in three different storage solutions, phosphate buffer solution, normal saline, and Dulbecco’s modified Eagle medium. Then, the ultrastructure, viability, and safety of these cells were compared. Other two UC-MSC populations of different sizes were categorized based on their mean diameters. The ultrastructure, proliferation, immunosuppression, hepatic differentiation potential, and number of senescent cells were investigated and compared. The survival rates of mice after the infusion of UC-MSCs of different sizes were compared. Results For UC-MSCs suspended in different storage solutions, the cell apoptosis rates, ultrastructure, and survival rates of mice were similar, and no differences were observed. Cells with a diameter of 19.14 ± 4.89 μm were categorized as the larger UC-MSC population, and cells with a diameter of 15.58 ± 3.81 μm were categorized as the smaller population. The mean diameter of the larger UC-MSC population was significantly larger than that of the smaller UC-MSC population (p < 0.01). Smaller UC-MSCs had more powerful proliferation and immunosuppressive potential and a higher nucleus-cytoplasm ratio than those of large UC-MSCs. The number of cells positive for β-galactosidase staining was higher in the larger UC-MSC population than in the smaller UC-MSC population. The survival rates of mice receiving 1 × 106 or 2 × 106 smaller UC-MSCs were 100%, both of which were higher than those of mice receiving the same amounts of larger UC-MSCs (p < 0.01). The cause of mouse death was explored and it was found that some larger UC-MSCs accumulated in the pulmonary capillary in dead mice. Conclusion Different storage solutions showed no significant effects on cell behavior, whereas heterogeneity was quite prevalent in MSC populations and might limit cells application. Hence, it is necessary to establish a more precise standardization for culture-expanded MSCs.

scholarly journals LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽  
2021 ◽  
Author(s):  
Jiaying Zhu ◽  
Zhu Zhu ◽  
Yipin Ren ◽  
Yukang Dong ◽  
Yaqi Li ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Mice Model ◽  
Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

Physiology, Pharmacology, and Topography of Cholinergic Neocortical Oscillations In Vitro

1997 ◽  
Vol 77 (5) ◽  
pp. 2427-2445 ◽  
Author(s):  
Heath S. Lukatch ◽  
M. Bruce Maciver
Keyword(s):  
Current Source ◽  
Brain Slices ◽  
Receptor Activation ◽  
Brain Regions ◽  
Oscillatory Activity ◽  
Cortical Layers ◽  
Eeg Activity ◽  
Layer 2

Lukatch, Heath S. and M. Bruce MacIver. Physiology, pharmacology, and topography of cholinergic neocortical oscillations in vitro. J. Neurophysiol. 77: 2427–2445, 1997. Rat neocortical brain slices generated rhythmic extracellular field [microelectroencephalogram (micro-EEG)] oscillations at theta frequencies (3–12 Hz) when exposed to pharmacological conditions that mimicked endogenous ascending cholinergic and GABAergic inputs. Use of the specific receptor agonist and antagonist carbachol and bicuculline revealed that simultaneous muscarinic receptor activation and γ-aminobutyric acid-A (GABAA)-mediated disinhibition werenecessary to elicit neocortical oscillations. Rhythmic activity was independent of GABAB receptor activation, but required intact glutamatergic transmission, evidenced by blockade or disruption of oscillations by 6-cyano-7-nitroquinoxaline-2,3-dione and (±)-2-amino-5-phosphonovaleric acid, respectively. Multisite mapping studies showed that oscillations were localized to areas 29d and 18b (Oc2MM) and parts of areas 18a and 17. Peak oscillation amplitudes occurred in layer 2/3, and phase reversals were observed in layers 1 and 5. Current source density analysis revealed large-amplitude current sinks and sources in layers 2/3 and 5, respectively. An initial shift in peak inward current density from layer 1 to layer 2/3 indicated that two processes underlie an initial depolarization followed by oscillatory activity. Laminar transections localized oscillation-generating circuitry to superficial cortical layers and sharp-spike-generating circuitry to deep cortical layers. Whole cell recordings identified three distinct cell types based on response properties during rhythmic micro-EEG activity: oscillation-on (theta-on) and -off (theta-off) neurons, and transiently depolarizing glial cells. Theta-on neurons displayed membrane potential oscillations that increased in amplitude with hyperpolarization (from −30 to −90 mV). This, taken together with a glutamate antagonist-induced depression of rhythmic micro-EEG activity, indicated that cholinergically driven neocortical oscillations require excitatory synaptic transmission. We conclude that under the appropriate pharmacological conditions, neocortical brain slices were capable of producing localized theta frequency oscillations. Experiments examining oscillation physiology, pharmacology, and topography demonstrated that neocortical brain slice oscillations share many similarities with the in vivo and in vitro theta EEG activity recorded in other brain regions.

MST3 is involved in ENaC-mediated hypertension

2019 ◽  
Vol 317 (1) ◽  
pp. F30-F42
Author(s):  
Te-Jung Lu ◽  
Wei-Chih Kan ◽  
Sung-Sen Yang ◽  
Si-Tse Jiang ◽  
Sheng-Nan Wu ◽  
...  
Keyword(s):  
Kidney Cells ◽  
Hypertensive Rats ◽  
Madin Darby Canine Kidney ◽  
Spontaneously Hypertensive ◽  
Hypomorphic Mutation ◽  
And Oxidative Stress ◽  
Darby Canine Kidney ◽  
Canine Kidney

Liddle syndrome is an inherited form of human hypertension caused by increasing epithelial Na+ channel (ENaC) expression. Increased Na+ retention through ENaC with subsequent volume expansion causes hypertension. In addition to ENaC, the Na+-K+-Cl− cotransporter (NKCC) and Na+-Cl− symporter (NCC) are responsible for Na+ reabsorption in the kidneys. Several Na+ transporters are evolutionarily regulated by the Ste20 kinase family. Ste20-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 phosphorylate downstream NKCC2 and NCC to maintain Na+ and blood pressure (BP) homeostasis. Mammalian Ste20 kinase 3 (MST3) is another member of the Ste20 family. We previously reported that reduced MST3 levels were found in the kidneys in spontaneously hypertensive rats and that MST3 was involved in Na+ regulation. To determine whether MST3 is involved in BP stability through Na+ regulation, we generated a MST3 hypomorphic mutation and designated MST3+/− and MST3−/− mice to examine BP and serum Na+ and K+ concentrations. MST3−/− mice exhibited hypernatremia, hypokalemia, and hypertension. The increased ENaC in the kidney played roles in hypernatremia. The reabsorption of more Na+ promoted more K+ secretion in the kidney and caused hypokalemia. The hypernatremia and hypokalemia in MST3−/− mice were significantly reversed by the ENaC inhibitor amiloride, indicating that MST3−/− mice reabsorbed more Na+ through ENaC. Furthermore, Madin-Darby canine kidney cells stably expressing kinase-dead MST3 displayed elevated ENaC currents. Both the in vivo and in vitro results indicated that MST3 maintained Na+ homeostasis through ENaC regulation. We are the first to report that MST3 maintains BP stability through ENaC regulation.

Abstract W MP87: PDGF Contributes to BMSC Treatment Induced Neurogenesis and White Matter and Axonal Remodeling in T2DM Stroke Rats

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Alex Zacharek ◽  
Tao Yan ◽  
Michael Chopp` ◽  
Poornima Venkat ◽  
Ruizhou Ning ◽  
...  
Keyword(s):  
Cell Migration ◽  
White Matter ◽  
Artery Occlusion ◽  
Border Zone ◽  
Axonal Outgrowth ◽  
Ischemic Brain ◽  
Neuroblast Migration ◽  
Gene And Protein Expression ◽  
Underlying Mechanisms

Objective: Our previous studies have found that bone-marrow-stromal cell (BMSC) treatment of stroke in Type two DM (T2DM) rats, initiated at 3 days after stroke, improved functional recovery. Neurogenesis and white matter (WM) remodeling play an important role in neurorestorative effects after stroke. In this study, we tested whether BMSCs regulate neurogenesis and WM remodeling and the underlying mechanisms of BMSC induced neurorestorative effects in T2DM stroke rats. Methods: T2DM was induced with streptozotocin injection in addition to a high fat diet. T2DM rats were subjected to 2h of middle cerebral artery occlusion (MCAo), then treated with human BMSCs (5X106) or vehicle control (n=8/group) initiated at 3 days after MCAo and rats were monitored for 28 days. Neuroblast migration, WM changes, and gene and protein expression were measured in the ischemic brain. Subventricular zone (SVZ) explant cell migration and primary cortical neuron (PCN) axonal outgrowth measurements were performed in vitro. Results: BMSC treatment in T2DM rats significantly improves functional outcome and increases WM remodeling identified by increased myelin and axonal density. BMSCs also increase the neuroblast migration protein doublecortin (DCX, 25.0±4.3% vs control: 4.5±1.1%), platelet-derived growth factor (PDGF)-AA, and bFGF expression in the ischemic border zone. Angiogenic ELISA array data are consistent with the immunostaining data, showing that BMSC treatment increases PDGF-AA (2.1 fold), PDGF-BB (2.5 fold) and bFGF (1.8 fold) in the ischemic brain. Using an in vitro cell culture model, we found that BMSCs secrete high levels of PDGF. PDGF treatment significantly increases SVZ explant cell migration (1.7 fold) and PCN axonal outgrowth (1.9 fold) compared to non-treatment control. Inhibition of PDGF with neutralized anti-PDGF antibody significantly attenuates BMSC conditioned medium induced SVZ cell migration and PCN axon outgrowth. Conclusion: BMSC treatment of stroke in T2DM increases WM remodeling and neurogenesis as well as increases PDGF expression. PDGF not only promotes neuronal migration, but also increases axonal outgrowth. Therefore, increasing PDGF likely contributes to BMSC induced neurogenesis and WM remodeling in T2DM stroke rats.

scholarly journals High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

2019 ◽  
Vol 6 (6) ◽  
pp. 3213-3221
Author(s):  
Hieu Liem Pham ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Culture Medium ◽  
Diabetic Patients ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.  

scholarly journals Hypermethylation of DHRS3 as a Novel Tumor Suppressor Involved in Tumor Growth and Prognosis in Gastric Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Sha Sumei ◽  
Kong Xiangyun ◽  
Chen Fenrong ◽  
Sun Xueguang ◽  
Hu Sijun ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
Tumor Growth ◽  
Human Cancer ◽  
Methylation Status ◽  
Ectopic Expression ◽  
Survival Times

Background/AimsThe role of DHRS3 in human cancer remains unclear. Our study explored the role of DHRS3 in gastric cancer (GC) and its clinicopathological significance and associated mechanisms.MaterialsBisulfite-assisted genomic sequencing PCR and a Mass-Array system were used to evaluate and quantify the methylation levels of the promoter. The expression levels and biological function of DHRS3 was examined by both in vitro and in vivo assays. A two-way hierarchical cluster analysis was used to classify the methylation profiles, and the correlation between the methylation status of the DHRS3 promoter and the clinicopathological characteristics of GC were then assessed.ResultsThe DHRS3 promoter was hypermethylated in GC samples, while the mRNA and protein levels of DHRS3 were significantly downregulated. Ectopic expression of DHRS3 in GC cells inhibited cell proliferation and migration in vitro, decreased tumor growth in vivo. DHRS3 methylation was correlated with histological type and poor differentiation of tumors. GC patients with high degrees of CpG 9.10 methylation had shorter survival times than those with lower methylation.ConclusionDHRS3 was hypermethylated and downregulated in GC patients. Reduced expression of DHRS3 is implicated in gastric carcinogenesis, which suggests DHRS3 is a tumor suppressor.

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