Significant Increase In Cell Survival Research Articles

Dose-Dependent Effects of Radiation on Mitochondrial Morphology and Clonogenic Cell Survival in Human Microvascular Endothelial Cells.

To better understand radiation-induced organ dysfunction at both high and low doses, it is critical to understand how endothelial cells (ECs) respond to radiation. The impact of irradiation (IR) on ECs varies depending on the dose administered. High doses can directly damage ECs, leading to EC impairment. In contrast, the effects of low doses on ECs are subtle but more complex. Low doses in this study refer to radiation exposure levels that are below those that cause immediate and necrotic damage. Mitochondria are the primary cellular components affected by IR, and this study explored their role in determining the effect of radiation on microvascular endothelial cells. Human dermal microvascular ECs (HMEC-1) were exposed to varying IR doses ranging from 0.1 Gy to 8 Gy (~0.4 Gy/min) in the AFRRI 60-Cobalt facility. Results indicated that high doses led to a dose-dependent reduction in cell survival, which can be attributed to factors such as DNA damage, oxidative stress, cell senescence, and mitochondrial dysfunction. However, low doses induced a small but significant increase in cell survival, and this was achieved without detectable DNA damage, oxidative stress, cell senescence, or mitochondrial dysfunction in HMEC-1. Moreover, the mitochondrial morphology was assessed, revealing that all doses increased the percentage of elongated mitochondria, with low doses (0.25 Gy and 0.5 Gy) having a greater effect than high doses. However, only high doses caused an increase in mitochondrial fragmentation/swelling. The study further revealed that low doses induced mitochondrial elongation, likely via an increase in mitochondrial fusion protein 1 (Mfn1), while high doses caused mitochondrial fragmentation via a decrease in optic atrophy protein 1 (Opa1). In conclusion, the study suggests, for the first time, that changes in mitochondrial morphology are likely involved in the mechanism for the radiation dose-dependent effect on the survival of microvascular endothelial cells. This research, by delineating the specific mechanisms through which radiation affects endothelial cells, offers invaluable insights into the potential impact of radiation exposure on cardiovascular health.

Improvement of Endothelial Cell-Polycaprolactone Interaction through Surface Modification via Aminolysis, Hydrolysis, and a Combined Approach

Polycaprolactone (PCL) is a promising material for the fabrication of alternatives to autologous grafts used in coronary bypass surgery. PCL biodegrades over time, allowing cells to infiltrate the polymeric matrix, replacing the biodegrading graft, and creating a fully functional vessel constituted of autologous tissue. However, the high hydrophobicity of PCL is associated with poor cell affinity. Surface modification of PCL can increase this cell affinity, making PCL an improved scaffold material for acellular vascular grafts. In this study, the surface of PCL films was modified by hydrolysis, aminolysis, and the combination thereof to introduce carboxyl, hydroxyl, and amino groups on the surface. Only the hydrolyzed films exhibited a significant increase in their hydrophilicity, although further testing showed that all aminolysis conditions had amino groups on the surface. Furthermore, in vitro experiments with human umbilical endothelial cells (HUVECs) were performed to assess changes in cell affinity for PCL due to the surface treatments. PCL treated with sodium hydroxide (NaOH), a hydrolysis reaction, showed a significant increase in endothelial cell adhesion after 24 hours with a significant increase in cell survival after 72 hours. Thus, NaOH treatment improves the biocompatibility and endothelialization of PCL, creating a competent candidate for artificial, acellular, biodegradable vascular grafts.

Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

BackgroundNeural stem cells (NSCs) derived from the embryonic spinal cord are excellent candidates for the cellular regeneration of lost neural cells after spinal cord injury (SCI). Semaphorin 3 A (Sema3A) is well known as being implicated in the major axon guidance of the growth cone as a repulsive function during the development of the central nervous system, yet its function in NSC transplantation therapy for SCI has not been investigated. Here, we report for the first time that embryonic spinal cord-derived NSCs significantly express Sema3A in the SCI environment, potentially facilitating inhibition of cell proliferation after transplantation.MethodssiRNA-Sema3A was conjugated with poly-l-lysin-coated gold nanoparticles (AuNPs) through a charge interaction process. NSCs were isolated from embryonic spinal cords of rats. Then, the cells were embedded into a dual-degradable hydrogel with the siRNA- Sema3A loaded-AuNPs and transplanted after complete SCI in rats.ResultsThe knockdown of Sema3A by delivering siRNA nanoparticles via dual-degradable hydrogels led to a significant increase in cell survival and neuronal differentiation of the transplanted NSCs after SCI. Of note, the knockdown of Sema3A increased the synaptic connectivity of transplanted NSC in the injured spinal cord. Moreover, extracellular matrix molecule and functional recovery were significantly improved in Sema3A-inhibited rats compared to those in rats with only NSCs transplanted.ConclusionsThese findings demonstrate the important role of Sema3A in NSC transplantation therapy, which may be considered as a future cell transplantation therapy for SCI cases.Graphical

Radioprotective Effect of Rosmarinus officinalis L (Rosemary) Essential Oil on Apoptosis, Necrosis and Mitotic Death of Human Peripheral Lymphocytes (PBMCs).

Background: Rosemary plant, with phenolic compounds, is known as an antioxidant herb and able to scavenge free radical agents in the biological environment; therefore, it is expected that the rosemary essential oil (R-EO) shows the radioprotective effect to protect individuals who are physically in contact with ionizing radiation.Objective: This study aimed to assess the radioprotective effect of R-EO on human peripheral blood mononuclear cells (PBMCs).Material and Methods: In this experimental study, the toxicity of the rosemary essential oil on PBMCs was assessed by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cells were irradiated at 0. 25 and 200 cGy using a 6 MV X-ray linear accelerator. The survival, apoptosis, necrosis, and survival enhancement factors of cells were analyzed by MTT and flow cytometry analyses with a non-toxic concentration of the rosemary essential oil (IC10).Results: Irradiation of cells in the presence of R-EO caused a significant increase in cell survival compared with the control in both 25 and 200 cGy radiation doses. Also, the percentages of apoptosis and necrosis of cells showed a significant decrease compared with the control.Conclusion: Rosemary essential oil as a natural and non-toxic compound could show favorable radioprotective effects in such a way that significantly increases the survival rate and decreases the percentage of apoptosis and necrosis of PBMCs.

Abstract 3833: The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells

Abstract Around 80 percent of high-grade serous ovarian cancer patients will relapse with chemotherapy resistant disease. Despite extensive research and developments in immunotherapy, there are limited improvements in overall survival for these patients. Recent research has found that the tumor microenvironment (TME) plays a crucial role in tumor initiation, therapy resistance, and tumor relapse. The TME, which consists of immune cells, blood vessels, and an extracellular matrix (ECM) built primarily by fibroblasts, may enhance the survival and growth of tumor-initiating cells (TICs), chemoresistant stem-like cells thought to be responsible for tumor relapse. Cytotoxic chemotherapies have been shown to modify the TME and this modulation may favor the survival of TICs. For example, carboplatin can activate stromal cells such as fibroblasts to secrete an altered ECM profile, including increases in collagen 1a1 and fibronectin production, which can bind specific integrins upregulated in TICs. Using ex-vivo decellularized peritoneum tissues from mice exposed to chemotherapy or vehicle treatment, we observed a 2-fold increase in growth in peritoneums pre-exposed to chemotherapy relative to those pre-exposed to vehicle. We hypothesize that alterations in the ECM following chemotherapy treatment permit TIC adhesion and growth. To investigate this, mouse embryonic fibroblasts (3T3s) were exposed to chemotherapy during ECM production, decellularized, and reseeded with a panel of ovarian cancer cell lines. Our results suggest a significant increase in cell survival in matrices pre-exposed to chemotherapy compared to control matrices pre-exposed to vehicle. Using this model, we are examining the features of cells that preferentially survive on these matrices. In addition to drug resistance, we are using flow cytometry to assess TIC markers, such as CD117, and CD133, and qRT-PCR to measure stemness genes such as SOX2, OCT4, and NANOG. These studies highlight the need for further investigation into the systemic effects of chemotherapies on the TME and how these changes may be fostering cancer cell adhesion, growth, and survival. Understanding these mechanisms will allow us to develop therapies to achieve better clinical outcomes. Citation Format: Omar Lujano-Olazaba, Mikella Robinson, Samuel F. Gilbert, Emily Kogan, Carrie D. House. The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3833.

Role of Myostatin in Muscle Degeneration by Random Positioning Machine Exposure: An in vitro Study for the Treatment of Sarcopenia.

Several scientific evidence have shown that exposure to microgravity has a significant impact on the health of the musculoskeletal system by altering the expression of proteins and molecules involved in bone–muscle crosstalk, which is also observed in the research of microgravity effect simulation. Among these, the expression pattern of myostatin appears to play a key role in both load-free muscle damage and the progression of age-related musculoskeletal disorders, such as osteoporosis and sarcopenia. Based on this evidence, we here investigated the efficacy of treatment with anti-myostatin (anti-MSTN) antibodies on primary cultures of human satellite cells exposed to 72 h of random positioning machine (RPM). Cell cultures were obtained from muscle biopsies taken from a total of 30 patients (controls, osteoarthritic, and osteoporotic) during hip arthroplasty. The Pax7 expression by immunofluorescence was carried out for the characterization of satellite cells. We then performed morphological evaluation by light microscopy and immunocytochemical analysis to assess myostatin expression. Our results showed that prolonged RPM exposure not only caused satellite cell death, but also induced changes in myostatin expression levels with group-dependent variations. Surprisingly, we observed that the use of anti-MSTN antibodies induced a significant increase in cell survival after RPM exposure under all experimental conditions. Noteworthy, we found that the negative effect of RPM exposure was counteracted by treatment with anti-MSTN antibodies, which allowed the formation of numerous myotubes. Our results highlight the role of myostatin as a major effector of the cellular degeneration observed with RPM exposure, suggesting it as a potential therapeutic target to slow the muscle mass loss that occurs in the absence of loading.

Growth Hormone (GH) Enhances Endogenous Mechanisms of Neuroprotection and Neuroplasticity after Oxygen and Glucose Deprivation Injury (OGD) and Reoxygenation (OGD/R) in Chicken Hippocampal Cell Cultures.

As a classical growth promoter and metabolic regulator, growth hormone (GH) is involved in development of the central nervous system (CNS). This hormone might also act as a neurotrophin, since GH is able to induce neuroprotection, neurite growth, and synaptogenesis during the repair process that occurs in response to neural injury. After an ischemic insult, the neural tissue activates endogenous neuroprotective mechanisms regulated by local neurotrophins that promote tissue recovery. In this work, we investigated the neuroprotective effects of GH in cultured hippocampal neurons exposed to hypoxia-ischemia injury and further reoxygenation. Hippocampal cell cultures obtained from chick embryos were incubated under oxygen-glucose deprivation (OGD, <5% O2, 1 g/L glucose) conditions for 24 h and simultaneously treated with GH. Then, cells were either collected for analysis or submitted to reoxygenation and normal glucose incubation conditions (OGD/R) for another 24 h, in the presence of GH. Results showed that OGD injury significantly reduced cell survival, the number of cells, dendritic length, and number of neurites, whereas OGD/R stage restored most of those adverse effects. Also, OGD/R increased the mRNA expression of several synaptogenic markers (i.e., NRXN1, NRXN3, NLG1, and GAP43), as well as the growth hormone receptor (GHR). The expression of BDNF, IGF-1, and BMP4 mRNAs was augmented in response to OGD injury, and exposure to OGD/R returned it to normoxic control levels, while the expression of NT-3 increased in both conditions. The addition of GH (10 nM) to hippocampal cultures during OGD reduced apoptosis and induced a significant increase in cell survival, number of cells, and doublecortin immunoreactivity (DCX-IR), above that observed in the OGD/R stage. GH treatment also protected dendrites and neurites during OGD, inducing plastic changes reflected in an increase and complexity of their outgrowths during OGD/R. Furthermore, GH increased the expression of NRXN1, NRXN3, NLG1, and GAP43 after OGD injury. GH also increased the BDNF expression after OGD, but reduced it after OGD/R. Conversely, BMP4 was upregulated by GH after OGD/R. Overall, these results indicate that GH protective actions in the neural tissue may be explained by a synergic combination between its own effect and that of other local neurotrophins regulated by autocrine/paracrine mechanisms, which together accelerate the recovery of tissue damaged by hypoxia-ischemia.

Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9nM) and MTV-LRP1 (Kd 399 ± 50.5nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

Extracts of Digested Berries Increase the Survival of Saccharomyces cerevisiae during H2O2 Induced Oxidative Stress

Many studies suggest anthocyanins may prevent the development of several diseases. However, anthocyanin bioactivity against cellular stress is not fully understood. This study aimed to evaluate the protective effect of berry anthocyanins on stressed cells using Saccharomyces cerevisiae. The impact of in vitro gastrointestinal digestion on anthocyanin profiles was also assessed. Bilberry and blackcurrant had higher anthocyanin levels than raspberry and strawberry, but digestion reduced the detected anthocyanins by approximately 90%. Yeast cells with and without digested or nondigested anthocyanin extracts were exposed to H2O2 and examined for survival. In the presence of anthocyanins, particularly from digested strawberry, a significant increase in cell survival was observed, suggesting that the type and levels of anthocyanins are important factors, but they also need to undergo gastrointestinal (GI) structural modifications to induce cell defence. Results also showed that cells need to be exposed to anthocyanins before the stress was applied, suggesting induction of a cellular defence system by anthocyanins or their derivatives rather than by a direct antioxidative effect on H2O2. Overall, data showed that exposure of severely stressed yeast cells to digested berry extracts improved cell survival. The findings also showed the importance of considering gastrointestinal digestion when evaluating anthocyanins’ biological activity.

Therapeutic Effects of Conditioned Medium of Neural Differentiated Human Bone Marrow-Derived Stem Cells on Rotenone-Induced Alpha-Synuclein Aggregation and Apoptosis.

Mesenchymal stem cells (MSCs) have been used against several diseases. Their potential mainly appears from its secreted biomolecules. Human bone marrow-derived stem cells (hBMSC) displayed neuronal functional characteristics after differentiation by basic fibroblast growth factor (bFGF) and forskolin. PD is a chronic age-related neurodegenerative disease (NDD) characterized by loss of dopaminergic neurons in the substantia nigra (SN) and abnormal accumulation of α-synuclein (α-syn) aggregations. In this present study, we evaluated the therapeutic effects of neural differentiated hBMSC (NI-hBMSC) conditioned medium (NI-hBMSC-CM) to a rotenone- (ROT-) induced Parkinson's disease (PD) model in SH-SY5Y cells. NI-hBMSC-CM treatment (50% diluted) in the last 24 h of 48 h ROT (0.5 μM) toxicity showed a significant increase in cell survival. The decreased tyrosine hydroxylase (TH) expression as a hallmark of PD was increased by NI-hBMSC-CM. The Triton X-100-soluble and Triton X-100-insoluble cell lysate fractions were used in Western blotting. The oligomeric, dimeric, and monomeric phosphorylated serine129 (p-S129) α-syn and total monomeric α-syn were decreased during ROT toxicity in the Triton X-100-soluble fraction. The Triton X-100-insoluble fraction revealed that ROT toxicity significantly increased the oligomeric but decreased the dimeric and monomeric p-S129 α-syn expressions while all forms of total α-syn were increased in SH-SY5Y cells. NI-hBMSC-CM stabilized the physiological α-syn monomers and reduced aggregated insoluble p-S129 α-syn against ROT. The cytoskeletal proteins, neurofilament-H (NF-H), β3-tubulin (Tuj1), neuronal nuclei (NeuN), and synaptophysin (SYP) were significantly decreased during ROT toxicity. In addition, proapoptotic Bax was increased by ROT with decreased antiapoptotic Bcl-2 and Mcl-1 as well as proforms of caspase-9, caspase-3, caspase-7, and PARP-1. NI-hBMSC-CM ameliorated the neurotrophic protein expressions, controlled the Bax/Bcl-2 ratio, upregulated procaspases, and inactivated PARP-1. From our results, we conclude that NI-hBMSC-CM containing released biomolecules during neural differentiation employs regenerative effects on the ROT model of PD in SH-SY5Y cells.

Wound Healing Activity of a Novel Formulation SKRIN via Induction of Cell Cycle Progression and Inhibition of PCNA-p21 Complex Interaction Leading to Cell Survival and Proliferation.

The process of wound healing is a dynamic event that starts with inflammation, proliferation, and cell migration of various types of fibroblast cells. Therefore, identification of potential molecules which may increase the wound healing capacity of fibroblast cells is crucial. A novel hydroalcoholic formulation of belladonna (SKRIN), was developed and characterized by GC-MS/MS, DLS, TEM, and AFM and was found to contain atropine and scopolamine exhibit in aggregated nanosized particles. SKRIN-mediated fibroblast cell survival was elucidated in the presence of H2O2 by MTT and flow cytometry based assays. With an EC50 of 4.41 μg/mL, SKRIN treatment showed significant increase in cell survival that was evident from a 1.11-fold increase (p < 0.0122) in the live cell population and 4.21-fold (p < 0.0001) and 2.59-fold (p < 0.0001) reductions in the early and late apoptotic cell populations, respectively. SKRIN-mediated wound healing was measured by cell scratch assay and cell cycle analysis. During the wound closure phenomenon, SKRIN increases repairing fibroblast cell proliferation by 1.24-fold (p = 0.0481) and increases the count of G2/M phase cells by 1.76-fold (p = 0.0002) which was confirmed by increased PCNA and reduced p21 protein expressions probably mediated by molecular interactions of PCNA-p21 complex with alkaloids present in SKRIN. Relative gene expression analysis further showed that SKRIN increases the PI3K, Akt, and NF-κB expression. Our data suggests that SKRIN exhibits wound healing property by increasing cell survival and repairing fibroblast proliferation via activation of the PI3K-Akt-NF-κB pathway probably mediated by inhibition of PCNA-p21 complex interaction.

Treating Senescence like Cancer: Novel Perspectives in Senotherapy of Chronic Diseases.

The WHO estimated around 41 million deaths worldwide each year for age-related non-communicable chronic diseases. Hence, developing strategies to control the accumulation of cell senescence in living organisms and the overall aging process is an urgently needed problem of social relevance. During aging, many biological processes are altered, which globally induce the dysfunction of the whole organism. Cell senescence is one of the causes of this modification. Nowadays, several drugs approved for anticancer therapy have been repurposed to treat senescence, and others are under scrutiny in vitro and in vivo to establish their senomorphic or senolytic properties. In some cases, this research led to a significant increase in cell survival or to a prolonged lifespan in animal models, at least. Senomorphics can act to interfere with a specific pathway in order to restore the appropriate cellular function, preserve viability, and to prolong the lifespan. On the other hand, senolytics induce apoptosis in senescent cells allowing the remaining non–senescent population to preserve or restore tissue function. A large number of research articles and reviews recently addressed this topic. Herein, we would like to focus attention on those chemical agents with senomorphic or senolytic properties that perspectively, according to literature, suggest a potential application as senotherapeutics for chronic diseases.

P.089 AMPA receptor modulation as a therapeutic strategy to enhance survival of spinal cord neural stem cells

Background: Transplantation of neural stem/progenitor cells (NSPCs) following spinal cord injury (SCI) is a promising strategy to enhance regeneration but is limited by poor survival of grafted cells. Recently, we demonstrated for the first time that the excitatory neurotransmitter glutamate, which is released after SCI, promotes survival/proliferation of spinal cord NSPCs via the AMPA subtype of glutamate receptors. Here, we examine the therapeutic potential of selective AMPA receptor modulation on NSPC survival using allosteric AMPA receptor modulators known as ampakines. Methods: NSPCs from the periventricular region of the adult rat spinal cord were treated with ampakines CX614 and CX546 for 72h either alone or in the presence of low-dose glutamate (50uM). Results: Treatment with CX-546 or CX-614 in the presence of glutamate led to a significant increase in live cell numbers. This was due to both a reduction in cell death and increase in cellular proliferation. Ampakine/glutamate treatment led to a significant increase in cell survival compared to controls in the setting of oxidative stress. Conclusions: We present the first examination of the effect of allosteric AMPA receptor modulators on adult spinal cord--derived NSPCs. Positive modulation of AMPA receptors may be a promising therapeutic strategy in the subacute/chronic phases after SCI to increase survival of endogenous or transplanted NSPCs.

MiR‐486a‐5p Alters Cell Survival in Ischemia‐Reperfusion Induced Myocardial Injury

Previous work in our lab has shown that mesenchymal stem cells (MSC)‐derived exosomes protect cardiomyocytes from ischemia‐reperfusion (I/R) induced myocardial injury both in vitro and in vivo. We found that microRNA‐486a‐5p (miR‐486) was highly and selectively expressed in MSC‐derived exosomes (1686‐fold relative to MSC (p&lt;0.001)). The role of miR‐486 in cell survival is still debated and miR‐486 has been reported to act as either a tumor suppressor or an oncogene. We hypothesized that miR‐486 upregulation will protect cardiomyocytes from I/R injury. We searched for miR‐486 gene targets using miRanda and Targetscan and found 1687 gene targets. We focused upon targets involving in the cell death and survival pathways, leaving 104 gene targets. Of these 104 targets, program cell death 4 (PDCD4), a tumor suppressor and PTEN, which negatively regulates the PI3K/Akt survival pathway, were chosen for further study based upon considerations of scores for mRNA‐miRNA interaction and the predicted targets. Luciferase reporter gene assays indicated that the 3′‐UTR of PDCD4 and PTEN are both targeted by miR‐486. A mimic and inhibitor (antagomir) of miR‐486 were used to assess its ability to modulate PDCD4, and PTEN expression and Akt activity. Although the mRNA levels of PDCD4 and PTEN were not altered by treatments, immunoblotting results showed a 0.5‐fold reduction in PDCD4 protein expression with miR‐486 mimic and no change in expression with antagomir (n=3). We did not observe a change in PTEN protein levels, and we also saw no change in pAkt/Akt ratio which is regulated downstream of PTEN. Simulated ischemia/reperfusion (sim I/R) was performed on H9c2 cells treated with miR‐486 mimic or scrambled control to measure the cytoprotective effect of miR‐486. H9c2 cells treated with the mimic of miR‐486 showed a significant increase in cell survival by 28–43% (p&lt;0.001, n=4–6) and a reduction of 20% in LDH release compared to control cells (p&lt;0.05, n=6). Western blot results with cell lysate after sim I/R showed a marked decrease in levels of PDCD4, cleaved‐caspase 3 and Bax in the group treated with miR‐486 mimic compared to control cells (p&lt;0.05, n=3). These findings support that miR‐486 upregulation is anti‐apoptotic. Additional studies are needed to discern whether miR‐486 upregulation will reduce cell injury using rodent models, and how much exosomal miR‐486a‐5p contributes to the effects of exosomes and stem cells. Such efforts will lead to a new therapeutic approach for prevention damage of I/R injury.Support or Funding InformationNIH 5R01HL091478 (WKJ)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Urocortin protects chondrocytes from acute trauma in articular cartilage

Purpose: Single injurious mechanical impact to joints, as seen in sports injury, is known to be associated with a high risk of later development of osteoarthritis (OA). Irreversible chondrocyte death is initiated following impact and results in progressive breakdown of articular cartilage. Promotion of chondrocyte survival is therefore a necessary target in the prevention of development of further OA. Urocortin1 (Ucn) has recently been shown to have a pro-survival effect on chondrocytes in both the presence and absence of pro-apoptotic stimuli. The aim of this study was to investigate the role of Ucn in response to mechanical impact. Methods: Full thickness porcine cartilage explants from lateral and medial femoral condyles were impacted with a 500g weight using a custom-made drop tower device. Impacted explants were treated with human Ucn (10-7M, 30 minutes prior to or following impact), non-selective cation channel blocker Gadolinium (500μM, 30 minutes following impact), or mechanosensitive ion channel blocker GsMTx4 (40μM, 2 hours prior to impact) and cultured for 72hrs. Cell viability was assessed with a CMFDA:PI live dead assay, using both macro confocal microscopy, as a means of avoiding bias, and high magnification confocal microscopy techniques. Lactate dehydrogenase (LDH) assays were conducted at 72hrs to provide a quantitative read out of cell death for each explant. Calcium influx was assessed 1hr post impact by Fluo-4 AM staining and visualised by multiphoton microscopy. Results: Porcine explants were impacted from a range of heights (10-70mm) in order to establish the correct energy of impact for inducing cell death without excess damage and fracture of the cartilage explant. Cell death increased in a dose dependent manner with height. 50mm was chosen as optimal height for all further experiments. Pre-treatment of explants with Ucn had a significant increase on cell viability when compared to impacted alone, as seen by quantification of the CMFDA:PI ratio on macro confocal (p=0.0042) and by high magnification confocal microscopy (p=0.0015). This increase in cell viability was confirmed by LDH assay (p=0.0063). Post-impact treatment of the explants with Ucn also resulted in a significant increase in cell survival, seen in both CMFDA:PI ratio (p=0.0009) and LDH assay (p=0.0053). We have previously found in primary human chondrocytes that Ucn exerts its protective effect on chondrocytes by the inhibition of excessive calcium influx. Calcium influx was therefore visualised in impacted explants treated pre- and post-impact with Ucn. A significant decrease in the levels of calcium staining was seen in both of these conditions when compared to explants impacted in the absence of Ucn (p=0.0073, p=0.05). In vitro work also highlighted a role for the mechanosensitive ion channel Piezo1 in cell death associated with the absence of Ucn. Explants were therefore treated with both Gadolinium and GsMTx4 post-impact and cell viability assessed by multiphoton microscopy. An increase in cell viability was observed in both conditions, comparable to those seen with addition of Ucn. Conclusions: A novel role for Ucn in the protection of articular chondrocytes maintained in cartilage explants has been shown and validated by two microscopy techniques and LDH assay. Ucn has also been shown to reduce calcium influx in impacted explants at 60 minutes post impact, and increased cell viability was observed by blocking mechanosensitive ion channels. These data suggest the importance of regulation of calcium influx in Ucn-mediated cell protection. The pro-survival effect of Ucn was seen both pre- and post-impact, highlighting the potential prophylactic and therapeutic use of Ucn for treating sudden injury to cartilage, and subsequent progression to post-traumatic OA.

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

Atypical protein kinase C zeta: potential player in cell survival and cell migration of ovarian cancer.

Ovarian cancer is one of the most aggressive gynaecological cancers, thus understanding the different biological pathways involved in ovarian cancer progression is important in identifying potential therapeutic targets for the disease. The aim of this study was to investigate the potential roles of Protein Kinase C Zeta (PRKCZ) in ovarian cancer. The atypical protein kinase C isoform, PRKCZ, is involved in the control of various signalling processes including cell proliferation, cell survival, and cell motility, all of which are important for cancer development and progression. Herein, we observe a significant increase in cell survival upon PRKCZ over-expression in SKOV3 ovarian cancer cells; additionally, when the cells are treated with small interference RNA (siRNA) targeting PRKCZ, the motility of SKOV3 cells decreased. Furthermore, we demonstrate that over-expression of PRKCZ results in gene and/or protein expression alterations of insulin-like growth factor 1 receptor (IGF1R) and integrin beta 3 (ITGB3) in SKOV3 and OVCAR3 cells. Collectively, our study describes PRKCZ as a potential regulatory component of the IGF1R and ITGB3 pathways and suggests that it may play critical roles in ovarian tumourigenesis.

AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4–7 Sprague–Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 μM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 μM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 μM) or the combination of s-AMPA and cyclothiazide (50 μM) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival.

Dose and time dependence of box jellyfish antivenom.

BackgroundThe effectiveness of the currently available box jellyfish (Chironex fleckeri) antivenom has been subject of debate for many years. To assess whether the box jellyfish antivenom has the ability to attenuate venom-induced damage at cellular level, the present study analyzed the dose and time dependence of the antivenom in a cell-based assay.MethodsDifferent doses of antivenom were added to venom and subsequently administered to cells and the cell index was measured using xCelligence Technology (ACEA Biosciences). Similarly, antivenom and venom were incubated over different time periods and cell survival measured as stated above. For both experiments, the cell index was plotted as a measure of cell survival against the dose or incubation time and significance was determined with the use of a one-way ANOVA with a LSD post hoc test.ResultsIncreasing concentrations of antivenom significantly augmented cell survival, with a concentration of approximately five times the currently recommended dose for human envenomation, causing the first significant increase in cell survival compared venom alone. Further, cell survival improved with increasing incubation time of venom and antivenom prior to addition to the cells, indicating that box jellyfish antivenom requires approximately 70 minutes to neutralize C. fleckeri venom.ConclusionThe presented results suggest that the currently recommended dose of antivenom requires adjustment, and more importantly, a human trial to test the effects of higher concentrations is also necessary. Further, antivenom has delayed neutralizing effects (i.e. after 70 minutes) which underlines the eminence of immediate and prolonged cardiopulmonary resuscitation in victims suffering from a C. fleckeri venom-induced cardiovascular collapse.

MiR-210 expression reverses radioresistance of stem-like cells of oesophageal squamous cell carcinoma

To investigate the expression of miR-210 and the role it plays in the cell cycle to regulate radioresistance in oesophageal squamous cell carcinoma (ESCC). MiR-210 expression was evaluated in 37 pairs of ESCC tissues and matched para-tumorous normal oesophageal tissues from surgical patients who had not received neoadjuvant therapy, and in the cells of two novel radioresistant cell lines, TE-1R and Eca-109R, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The transient up-regulation of miR-210 expression in TE-1R and Eca-109R cells was studied using liposomes and was confirmed using qRT-PCR. The rate of cell survival after a series of radio-treatment doses was evaluated using the clone formation assay. Flow cytometry was used to detect the changes to the cell cycle patterns due to radiation treatment. RT-PCR and Western blot were used to detect the expression of ataxia telangiectasia mutated (ATM) and DNA dependent protein kinase (DNA-PKcs) after irradiation, and the cell sphere formation assay was used to evaluate the proliferative ability of the cancer stem-like cells. The level of miR-210 expression was significantly decreased, by 21.3% to 97.2%, with the average being 39.2% ± 16.1%, in the ESCC tissues of most patients (81.1%, 30 of 37 vs patients with high miR-210 expression, P < 0.05). A low level of expression of miR-210 was correlated with a poorly differentiated pathological type (P < 0.01) but was not correlated with the T-stage or lymph node infiltration (both P > 0.05). Early local recurrences (< 18 mo, n = 19) after radiotherapy were significantly related with low miR-210 expression (n = 13, P < 0.05). The level of miR-210 was decreased by approximately 73% (vs TE-1, 0.27 ± 0.10, P < 0.01) in the established radioresistant TE-IR cell line and by 52% (vs Eca-109, 0.48 ± 0.17, P < 0.05) in the corresponding Eca-109R line. Transient transfection with a miR-210 precursor increased the level of miR-210 expression, leading to a significant increase in cell survival after radiotherapy (P < 0.05). Twenty-four hours after radiation, the proportion of pmiR-210 cells in S phase was increased (vs control cells, 30.4% ± 0.4%, and vs untreated TE-1R cells, 23.3% ± 0.7%, P < 0.05 for both). The levels of DNA-PKcs (0.21 ± 0.07) and ATM (0.12 ± 0.03, P < 0.05) proteins were significantly lower in the PmiR-210 cells than in control cells, but no differences were found in the levels of the corresponding mRNAs in the two cell types (P > 0.05 for all). Exogenous miR-210 expression decreased the diameter of pmiR-210 cell spheres (vs control cells, 0.60 ± 0.14, P < 0.05). MiR-210 expression is negatively correlated with the pathological type and the local survival rate after radiotherapy, and high expression of miR-210 may reverse the radioresistance of ESCC stem-like cells.