Mechanism of hypoxic precondition in regulating neural stem cell migratory capacity to brain tumor sites in vitro

Neural stem cells (NSCs) are pnmrtive cells Wh1ch are capable to self-renew or differentiate into various matured neuronal cells. These cells res•de in subveotricufar zone of adult mammalian bra1n, a speCialized niche thai mamtains the pluripotent stem cell characteristics. Recently, researchers reported that NSCs showed a preferential mtgration to brain tumor s1tes in vivo. opening the opportunity to use these cells as spec1al dehvery agent to dehver anti-cancer drugs to cancer si1es d1rectly to avoid unne1..-essary s1de effect on adjacent normal health cells. Here. we propose to evaluate the mtgration of NSC to glioma cells 1n vstro carrying amicancer drugs used m d;n1cal (Tamox1den and TemozolomKle) and also the natural anti-cancer drug:; ex1racted from mediCinal plant- Quercus snfedona (QI). Besides. 1n th1s study, we also proposed to optimize the migratiOn capactty vsa hypoxic precond1\Jornng method. To do thiS. NSC lsne will be cultured under physsologic<f Ol<)'9en levels (2% oxygen; lermed hypo>Oa) and the rcsuH1ng changes m NSC gene wsll be Investigated ustng real hme PCR and western blot and compared to the eels cultured under atmosphenc oxygen (21% oxygen; termed normol<ia). We found that hypoxic NSC showed maeased HIF and CXCR4 expression After that. 01 was extracted uslng soxhlet tecl'tnique wtth 100% (QI-100%) or 70% (Qt-70%) methanol solvent ICSO of Ql-1 00% and 01-70% on human NSC line (H9-hNSC) and human glsorna celll!ne (DBTRG-OSMG) were determtned using MTT assay. Both Ql-100~.- and 01-70% sh01o'ied ants-proliferative properties aga1nst OBTRG-OSMG atJCSO, but not on H9-hNSC. Free radiCals scavenging activrty (0°PH solution testlsn the 01-100% and 01-70% were found to be 97 .3±1.4% and 96 4±3 7%, respeclil>ely Concentration of tannic and gatltc acids measured ussng HPLC was 72.56 IJgtml and 43.65 IJg/mltn Ql-100% while 1n Ql-70%, the concentratsons were 72.'11 ~!ml and 43.31 jJg/ml. res[lectlVely Ta~en together, both DPPH and HPLC data indicated that both Ql eKtracts contamed tannic and gallic acids which eKhibrt 1nherent anlioKidant activity. 01-treated H9-hNSC was seeded 1n a modifted Boyden chamber to investigate Its m1grat1on capacsty towards DBTRG-OSMG. Result showed that H9-hNSC migrated towards DBTRG-05MG wsth 4-folds h1gher capacity compared to the control However, there Is no Significant different between the normoxic and hypoxic NSC migration. In additson, the migration of Ql-100% treated H9-hNSC successfully reduced the number of DBTRG-05MG cells, indicatsng the antl-GBM potenbal of these cells In condusion, we successfully denonstrated that the NSCs are able to migrate and dehver Ql extracts towards glioma sn \11110 and reduces the glioma cell number

ASSOCIATION BETWEEN ENDOTHELIAL PROGENITOR CELLS AND VON WILLEBRAND FACTOR IN ASTROCYTIC GLIOMA

Astrocytic gliomas are highly aggressive and lethal brain tumours that depend on angiogenesis for growth. The endothelial progenitor cell (EPCs) and von Willebrand factor (vWF) involved in the formation of new blood vessels in astrocytic glioma. Objectives: This study aimed to investigate the association between circulating and tissue resident EPCs with vWF in astrocytic glioma patients. Methods: Blood specimen and brain tissue biopsy were collected from a total of 22 astrocytic glioma patients admitted to Hospital Universiti Sains Malaysia. Circulating EPCs (blood) and tissue resident EPCs (tissue biopsy) were characterized using EPC-specific markers, CD133 and VEGFR2 and quantified using fluorescenceactivated cell sorting analysis and immunofluorescence microscopy, respectively. The plasma vWF was measured by using commercialized Elisa kit (Cusabio Biotech Co.,Ltd). Results: The mean percentage of circulating EPCs was (0.01 ± 0.01%), brain tumor tissue EPCs (0.48 ± 0.38%) and adjacent normal brain tissue EPCs (0.18 ± 0.23%). The mean plasma vWF was 9.23 ± 7.57%. Positive correlation was found between brain tumor EPCs and plasma vWF (Spearman’s rho r = 0.45, p = 0.035). However no correlation was found between adjacent normal brain EPCs and plasma vWF. About 14 patients had (mild vWF level of&gt; 5%), 8 patients had (moderate vWF level of 1-5%) and no patients had (severe vWF level of &lt; 1%). The mean percentage of patients with mild vWF level was 12.48 ± 7.77% and moderate vWF level was 3.53 ±1.32%. There was a significant correlation between circulating EPCs and patients with mild vWF level(Spearman’s rho r = 0.63, p = 0.015). Conclusion: This study demonstrated that EPCs have significant positive association with vWF suggesting the homing of plasma vWF at the tumor site.&nbsp

The potential of neural stem cell as vehicle to deliver Quercus infectoria extract to glioma cell in vitro

Glioblastoma multiforme (GBM) is the most malignant subtype of brain cancer. However, current clinical treatments for GBM are limited in effectiveness and often impose additional side effects on patients. Here, we developed targeted anti-cancer therapy (TAT) using neural stem cells (NSC) as delivery agent to transport anti-cancer compounds directly to GBM in vitro. Anti-cancer active compounds: Tannic acid (TA) and gallic acid (GA) were extracted from local medicinal plant - Quercus infectoria (QI) using soxhlet technique with 100% methanol (QI-100%) or 70% methanol (QI-70%) solvent. Concentration of TA and GA measured using HPLC were 72.56 and 43.66 μg/mL in QI-100%, while in QI-70%, the concentrations were 72.41 and 43.31 μg/mL, respectively. Cytotoxicity effects of QI-100% and QI-70% on human GBM cell line (DBTRG-05MG), human NSC line (H9-hNSC) and human normal brain glial cell line (SVG-p12) (as negative control) were determined using MTT assay. Both QI-100% and QI-70% showed anti-proliferative properties against DBTRG-05MG at IC50, but not on H9-hNSC and SVG-p12. Taken together, data indicated that both QI extracts contained TA and GA which exhibit anti-proliferative effect specifically on cancerous cells only. Next, QI-treated H9-hNSC was seeded in a modified Boyden chamber for 12 h to investigate its migration capacity towards DBTRG-05MG. The result showed that H9-hNSC migrated towards DBTRG-05MG with 4-folds higher capacity compared to control. In addition, the migration of QI-100% treated H9-hNSC successfully reduced the number of DBTRG-05MG, indicating the anti-GBM potential of these cells after migration. In conclusion, NSC could be a specific anti-cancer compound delivery agent for GBM, reducing unwanted side effects on patients

Effect of gibberellic acid and eggshell on Hylocereus polyrhizus

Dragon fruit (Hylocereus polyrhizus) is a tropical fruit. Recently, it has gained interest from the public due to its potential beneficial effects on health. The acclimatization of micropropagated Hylocereus polyrhizus depends on the application of gibberellic acid (GA3 ) to increase plant growth. Eggshells are waste materials from industrial sectors, and they are composed of calcium source that is vital for the development of plant shoots and root. The objective of this research is to investigate the effect of different concentrations of GA3 and eggshell either added individually or in combination on the growth of shoot length and shoot diameter of H. polyrhizus. The result showed the shoot length of the H. polyrhizus increased by approximately 54.69%, from 0.64 ± 0.13 cm to 0.99 ± 0.26 cm, as the concentration of GA3 increased from 0 ppm to 10 ppm. Furthermore, this finding also reported that with eggshells, GA3 showed an adverse effect on the development of shoot diameter. The growth of shoot length and shoot diameter with the addition of eggshell was different, perhaps due to the gibberellic acid affecting the shoot length but not the shoot diameter. Generally, the growth of shoot length and shoot diameter with eggshells was higher in comparison with those without eggshells. With that, we can prove that eggshell is a good additive to promote the growth of H. polyrhizus

Effect of Gibberellic Acid and Eggshell on Hylocereus polyrhizus

Dragon fruit (Hylocereus polyrhizus) is a tropical fruit. Recently, it has gained interest from the public due to its potential beneficial effects on health. The acclimatization of micropropagated Hylocereus polyrhizus depends on the application of gibberellic acid (GA3) to increase plant growth. Eggshells are waste materials from industrial sectors, and they are composed of calcium source that is vital for the development of plant shoots and root. The objective of this research is to investigate the effect of different concentrations of GA3 and eggshell either added individually or in combination on the growth of shoot length and shoot diameter of H. polyrhizus. The result showed the shoot length of the H. polyrhizus increased by approximately 54.69%, from 0.64 ± 0.13 cm to 0.99 ± 0.26 cm, as the concentration of GA3 increased from 0 ppm to 10 ppm. Furthermore, this finding also reported that with eggshells, GA3 showed an adverse effect on the development of shoot diameter. The growth of shoot length and shoot diameter with the addition of eggshell was different, perhaps due to the gibberellic acid affecting the shoot length but not the shoot diameter. Generally, the growth of shoot length and shoot diameter with eggshells was higher in comparison with those without eggshells. With that, we can prove that eggshell is a good additive to promote the growth of H. polyrhizus

Preconditioning of Cardiosphere-Derived Cells With Hypoxia or Prolyl-4-Hydroxylase Inhibitors Increases Stemness and Decreases Reliance on Oxidative Metabolism

Cardiosphere-derived cells (CDCs), which can be isolated from heart explants, are a promising candidate cell source for infarcted myocardium regeneration. However, current protocols used to expand CDCs require at least 1 month in vitro to obtain sufficient cells for transplantation. We report that CDC culture can be optimized by preconditioning the cells under hypoxia (2% oxygen), which may reflect the physiological oxygen level of the stem cell niche. Under hypoxia, the CDC proliferation rate increased by 1.4-fold, generating 6 × 10(6) CDCs with higher expression of cardiac stem cell and pluripotency gene markers compared to normoxia. Furthermore, telomerase (TERT), cytokines/ligands involved in stem cell trafficking (SDF/CXCR-4), erythropoiesis (EPO), and angiogenesis (VEGF) were increased under hypoxia. Hypoxic preconditioning was mimicked by treatment with two types of hypoxia-inducible factor (HIF) prolyl-4-hydroxylase inhibitors (PHDIs): dimethyloxaloylglycine (DMOG) and 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (BIC). Despite the difference in specificity, both PHDIs significantly increased c-Kit expression and activated HIF, EPO, and CXCR-4. Furthermore, treatment with PHDIs for 24 h increased cell proliferation. Notably, all hypoxic and PHDI-preconditioned CDCs had decreased oxygen consumption and increased glycolytic metabolism. In conclusion, cells cultured under hypoxia could have potentially enhanced therapeutic potential, which can be mimicked, in part, by PHDIs

On the pivotal role of PPARa in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury

The role of peroxisome proliferator activated alpha (PPARα) -mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 weeks before in vivo contractile function was measured using cine magnetic resonance (MR) imaging. In isolated, perfused hearts, energetics were measured using 31P MR spectroscopy and glycolysis and fatty acid oxidation were measured using 3H labelling. Compared with normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation and mitochondrial UCP3 levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations and thereby ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation and UCP3 levels, with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high fat diet, with the result that ATP concentrations and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have already occurred in PPARα-/- mouse hearts, and sustained function in hypoxia despite an inability for further metabolic remodelling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodelling in hypoxia, but is prevented by dietary fat

Cardiosphere-Derived Cells Improve Function in the Infarcted Rat Heart for at Least 16 Weeks – an MRI Study

Aims Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart. Methods and Results CDCs, cultured from neonatal rat heart, comprised a heterogeneous population including cells expressing the mesenchymal markers CD90 and CD105, the stem cell marker c-kit and the pluripotency markers Sox2, Oct3/4 and Klf-4. CDCs (2×106) expressing green fluorescent protein (GFP+) were labelled with fluorescent micron-sized particles of iron oxide (MPIO). Labelled cells were administered to the infarcted rat hearts (n = 7) by intramyocardial injection immediately following reperfusion, then by systemic infusion (4×106) 2 days later. A control group (n = 7) was administered cell medium. MR hypointensities caused by the MPIOs were detected at all times and GFP+ cells containing MPIO particles were identified in tissue slices at 16 weeks. At two days after infarction, cardiac function was similar between groups. By 6 weeks, ejection fractions in control hearts had significantly decreased (47±2%), but this was not evident in CDC-treated hearts (56±3%). The significantly higher ejection fractions in the CDC-treated group were maintained for a further 10 weeks. In addition, CDC-treated rat hearts had significantly increased capillary density in the peri-infarct region and lower infarct sizes. MPIO-labelled cells also expressed cardiac troponin I, von Willebrand factor and smooth muscle actin, suggesting their differentiation along the cardiomyocyte lineage and the formation of new blood vessels. Conclusions CDCs were retained in the infarcted rat heart for 16 weeks and improved cardiac function

Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI