Publications by authors named "Augustas Pivoriūnas"

25 Publications

  • Page 1 of 1

Astrocyte-Endotheliocyte Axis in the Regulation of the Blood-Brain Barrier.

Neurochem Res 2021 May 7. Epub 2021 May 7.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, 01102, Vilnius, Lithuania.

The evolution of blood-brain barrier paralleled centralisation of the nervous system: emergence of neuronal masses required control over composition of the interstitial fluids. The barriers were initially created by glial cells, which employed septate junctions to restrict paracellular diffusion in the invertebrates and tight junctions in some early vertebrates. The endothelial barrier, secured by tight and adherent junctions emerged in vertebrates and is common in mammals. Astrocytes form the parenchymal part of the blood-brain barrier and commutate with endothelial cells through secretion of growth factors, morphogens and extracellular vesicles. These secreted factors control the integrity of the blood-brain barrier through regulation of expression of tight junction proteins. The astrocyte-endotheliocyte communications are particularly important in various neurological diseases associated with impairments to the blood-brain barrier. Molecular mechanisms supporting astrocyte-endotheliocyte axis in health and disease are in need of detailed characterisation.
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http://dx.doi.org/10.1007/s11064-021-03338-6DOI Listing
May 2021

Concentration-dependent duality of bFGF in regulation of barrier properties of human brain endothelial cells.

J Cell Physiol 2021 May 7. Epub 2021 May 7.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.

Multiple paracrine factors regulate the barrier properties of human brain capillary endothelial cells (BCECs). Understanding the precise mode of action of these factors remains a challenging task, because of the limited availability of functionally competent BCECs and the use of serum-containing medium. In the present study, we employed a defined protocol for producing BCECs from human inducible pluripotent stem cells. We found that autocrine secretion of basic fibroblast growth factor (bFGF) is necessary for the establishment a tight BCECs barrier, as revealed by measurements of transendothelial electric resistance (TEER). In contrast, addition of exogenous bFGF in concentrations higher than 4 ng/ml inhibited TEER in a concentration-dependent manner. Exogenous bFGF did not significantly affect expression and distribution of tight junction proteins claudin-5, occludin and zonula occludens (ZO)-1. Treatment with FGF receptor blocker PD173074 (15 µM) suppressed inhibitory effects of bFGF and induced nuclear translocation of protein ZO-1. Inhibition of phosphoinositide 3-Kinase (PI-3K) with LY294002 (25 µM) significantly potentiated an inhibitory effect of bFGF on TEER indicating that PI-3K signalling pathway counteracts bFGF modulation of TEER. In conclusion, we show that autocrine bFGF secretion is necessary for the proper barrier function of BCECs, whereas exogenous bFGF in higher doses suppresses barrier resistance. Our findings demonstrate a dual role for bFGF in the regulation of BCEC barrier function.
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http://dx.doi.org/10.1002/jcp.30410DOI Listing
May 2021

Astrocyte-derived extracellular vesicles mediate intercellular communications of the neurogliovascular unit.

Neural Regen Res 2021 Jul;16(7):1421-1422

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania; Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK; Achucarro Centre for Neuroscience, IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.

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http://dx.doi.org/10.4103/1673-5374.300994DOI Listing
July 2021

Astroglial asthenia and loss of function, rather than reactivity, contribute to the ageing of the brain.

Pflugers Arch 2021 May 26;473(5):753-774. Epub 2020 Sep 26.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya street 16/10, Moscow, Russia, 117997.

Astroglia represent a class of heterogeneous, in form and function, cells known as astrocytes, which provide for homoeostasis and defence of the central nervous system (CNS). Ageing is associated with morphological and functional remodelling of astrocytes with a prevalence of morphological atrophy and loss of function. In particular, ageing is associated with (i) decrease in astroglial synaptic coverage, (ii) deficits in glutamate and potassium clearance, (iii) reduced astroglial synthesis of synaptogenic factors such as cholesterol, (iv) decrease in aquaporin 4 channels in astroglial endfeet with subsequent decline in the glymphatic clearance, (v) decrease in astroglial metabolic support through the lactate shuttle, (vi) dwindling adult neurogenesis resulting from diminished proliferative capacity of radial stem astrocytes, (vii) decline in the astroglial-vascular coupling and deficient blood-brain barrier and (viii) decrease in astroglial ability to mount reactive astrogliosis. Decrease in reactive capabilities of astroglia are associated with rise of age-dependent neurodegenerative diseases. Astroglial morphology and function can be influenced and improved by lifestyle interventions such as intellectual engagement, social interactions, physical exercise, caloric restriction and healthy diet. These modifications of lifestyle are paramount for cognitive longevity.
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http://dx.doi.org/10.1007/s00424-020-02465-3DOI Listing
May 2021

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage.

Int J Mol Sci 2020 Sep 18;21(18). Epub 2020 Sep 18.

Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, 44121 Ferrara, Italy.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.
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http://dx.doi.org/10.3390/ijms21186859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555813PMC
September 2020

Immortalised Hippocampal Astrocytes from 3xTG-AD Mice Fail to Support BBB Integrity In Vitro: Role of Extracellular Vesicles in Glial-Endothelial Communication.

Cell Mol Neurobiol 2021 Apr 22;41(3):551-562. Epub 2020 May 22.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, 01102, Vilnius, Lithuania.

Impairments of the blood-brain barrier (BBB) and vascular dysfunction contribute to Alzheimer's disease (AD) from the earliest stages. However, the influence of AD-affected astrocytes on the BBB remain largely unexplored. In the present study, we created an in vitro BBB using human-immortalized endothelial cells in combination with immortalized astroglial cell lines from the hippocampus of 3xTG-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively). We found that co-culturing endothelial monolayers with WT-iAstro upregulates expression of endothelial tight junction proteins (claudin-5, occludin, ZO-1) and increases the trans-endothelial electrical resistance (TEER). In contrast, co-culturing with 3Tg-iAstro does not affect expression of tight junction proteins and does not change the TEER of endothelial monolayers. The same in vitro model has been used to evaluate the effects of extracellular vesicles (EVs) derived from the WT-iAstro and 3Tg-iAstro. The EVs derived from WT-iAstro increased TEER and upregulated expression of tight junction proteins, whereas EVs from 3Tg-iAstro were ineffective. In conclusion, we show for the first time that immortalized hippocampal astrocytes from 3xTG-AD mice exhibit impaired capacity to support BBB integrity in vitro through paracrine mechanisms and may represent an important factor underlying vascular abnormalities during development of AD.
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http://dx.doi.org/10.1007/s10571-020-00871-wDOI Listing
April 2021

Time-Dependent Memory and Gait Improvement by Intranasally-Administered Extracellular Vesicles in Parkinson's Disease Model Rats.

Cell Mol Neurobiol 2021 Apr 14;41(3):605-613. Epub 2020 May 14.

Department of Pharmacology, Faculty of Medicine, University of Latvia, 3 Jelgavas St, Riga, 1004, Latvia.

We have recently demonstrated that extracellular vesicles (EVs) derived from the human teeth stem cells improve motor symptoms and normalize tyrosine hydroxylase (TH) expression in the nigrostriatal structures of Parkinson's disease (PD) model rats obtained by 6-hydroxydopamine (6-OHDA) unilateral injection into the medial forebrain bundle (MFB). The aim of this study was to clarify: (1) how long therapeutic effects persist after discontinuation of 17-day intranasal administration of EVs in 6-OHDA rats; (2) may EVs reverse cognitive (learning/memory) dysfunction in these PD model rats; (3) whether and how the behavioral improvement may be related to the expression of TH and Nissl bodies count in the nigrostriatal structures. Our results demonstrated that in 6-OHDA rats, gait was normalized even ten days after discontinuation of EVs administration. EVs successfully reversed 6-OHDA-induced impairment in spatial learning/memory performance; however, the beneficial effect was shorter (up to post-treatment day 6) than that revealed for gait improvement. The shorter effect of EVs coincided with both full normalization of TH expression and Nissl bodies count in the nigrostriatal structures, while slight but significant increase in the 6-OHDA-decreased Nissl count persisted in the substantia nigra even on the post-treatment day 20, supposedly due to the continuation of protein synthesis in the living cells. The obtained data indicate the usefulness of further studies to find the optimal administration regimen which could be translated into clinical trials on PD patients, as well as to clarify other-apart from dopaminergic-neuromodulatory pathways involved in the EVs mechanism of action.
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http://dx.doi.org/10.1007/s10571-020-00865-8DOI Listing
April 2021

Astroglial atrophy in Alzheimer's disease.

Pflugers Arch 2019 10 13;471(10):1247-1261. Epub 2019 Sep 13.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya street 16/10, Moscow, 117997, Russia.

Astrocytes, a class of morphologically and functionally diverse primary homeostatic neuroglia, are key keepers of neural tissue homeostasis and fundamental contributors to brain defence in pathological contexts. Failure of astroglial support and defence facilitate the evolution of neurological diseases, which often results in aberrant synaptic transmission, neurodegeneration and death of neurones. In Alzheimer's disease (AD), astrocytes undergo complex and multifaceted metamorphoses ranging from atrophy with loss of function to reactive astrogliosis with hypertrophy. Astroglial asthenia underlies reduced homeostatic support and neuroprotection that may account for impaired synaptic transmission and neuronal demise. Reactive astrogliosis which mainly develops in astrocytes associated with senile plaque is prominent at the early to moderate stages of AD manifested by mild cognitive impairment; downregulation of astrogliosis (reflecting astroglial paralysis) is associated with late stages of the disease characterised by severe dementia. Cell-specific therapies aimed at boosting astroglial supportive and defensive capabilities and preventing astroglial paralysis may offer new directions in preventing, arresting, or even curing AD-linked neurodegeneration.
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http://dx.doi.org/10.1007/s00424-019-02310-2DOI Listing
October 2019

Extracellular Vesicles Suppress Basal and Lipopolysaccharide-Induced NFκB Activity in Human Periodontal Ligament Stem Cells.

Stem Cells Dev 2019 08 24;28(15):1037-1049. Epub 2019 May 24.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.

Periodontitis is an infectious disease characterized by chronic inflammation and progressive destruction of periodontal tissues. Chronic inflammatory environment may affect immunomodulatory function of periodontal ligament stem cells (PDLSCs) and promote shift toward proinflammatory phenotype contributing to propagation of periodontitis. Therefore, suppression of inflammatory response in PDLSCs represents a novel therapeutic approach. Extracellular vesicles (EVs) have been shown to display anti-inflammatory and immunosuppressive actions in different tissues and could represent a potent therapeutic tools against chronic inflammation during periodontitis. In the present study, we investigated the effects of EVs on the basal and lipopolysaccharide (LPS)-induced activity of NFκB signaling pathway in PDLSCs. We also examined the impact of EVs on the osteogenic differentiation and expression of osteogenesis-related genes. EVs were purified by differential ultracentrifugation from PDLSCs grown on gelatin-coated alginate microcarriers in a bioreactor. NFκB reporter assays demonstrated that EVs permanently suppressed basal and LPS-induced activity of NFκB in PDLSCs. Combined treatment with EVs and anti-TLR4 antibody (Ab) resulted in attenuation of the inhibitory effect on the NFκB activity, suggesting a possible interference through a competition for TLR4 signaling pathway. EVs also increased phosphorylation of Akt and its downstream target GSK3β (Ser 9) indicating that PI3K/Akt signaling pathway may act as suppressor of NFκB activity. LPS stimulated osteogenic mineralization of PDLSCs. Unexpectedly, anti-TLR4 blocking Ab per se significantly decreased osteogenic mineralization of PDLSCs. EVs did not affect osteogenic mineralization, but partially suppressed inhibitory effect of anti-TLR4 blocking Ab. Gene expression studies revealed significant effects of EVs on osteogenesis-related genes and possible interference with TLR4 signaling in PDLSCs. In conclusion, our study demonstrates that EVs suppress basal and LPS-induced activity of NFκB signaling pathway in PDLSCs and could potentially be used for targeting of chronic inflammation during periodontitis.
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http://dx.doi.org/10.1089/scd.2019.0021DOI Listing
August 2019

Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats.

Stem Cells Transl Med 2019 05 1;8(5):490-499. Epub 2019 Feb 1.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting millions of people worldwide. At present, there is no effective cure for PD; treatments are symptomatic and do not halt progression of neurodegeneration. Extracellular vesicles (EVs) can cross the blood-brain barrier and represent promising alternative to the classical treatment strategies. In the present study, we examined therapeutic effects of intranasal administration of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on unilateral 6-hydroxydopamine (6-OHDA) medial forebrain bundle (MFB) rat model of PD. CatWalk gait tests revealed that EVs effectively suppressed 6-OHDA-induced gait impairments. All tested gait parameters (stand, stride length, step cycle, and duty cycle) were significantly improved in EV-treated animals when compared with 6-OHDA-lesion group rats. Furthermore, EVs slowed down numbers of 6-OHDA-induced contralateral rotations in apomorphine test. Improvements in motor function correlated with normalization of tyrosine hydroxylase expression in the striatum and substantia nigra. In conclusion, we demonstrated, for the first time, the therapeutic efficacy of intranasal administration of EVs derived from SHEDs in a rat model of PD induced by 6-OHDA intra-MFB lesion. Our findings could be potentially exploited for the development of new treatment strategies against PD.
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http://dx.doi.org/10.1002/sctm.18-0162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477008PMC
May 2019

Extracellular vesicles can act as a potent immunomodulators of human microglial cells.

J Tissue Eng Regen Med 2019 02 28;13(2):309-318. Epub 2019 Jan 28.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.

Functional impairments of microglia have been recently associated with several neurological conditions. Therefore, modulation of anti-inflammatory and phagocytic properties of microglial cells could represent a novel therapeutic approach. In the present study, we investigated the effects of extracellular vesicles (EVs) derived from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs) on the inflammatory response and functional properties of immortalized human microglial cells. NFκB reporter assays demonstrated that EVs suppressed LPS-induced activation of NFκB signalling pathway in human microglial cells. The effect was similar to that obtained with anti-TLR4 blocking antibody. We also show that EVs differentially affected phagocytic activity of unpolarized (M0) and polarized (M1 and M2) microglial cells. EVs induced significant upregulation of phagocytic activity in M0 cells (by 39%), slight decrease in M1 cells, and moderate increase (by 21%) in M2 cells. The Seahorse XF Glycolysis Stress Test revealed that EVs induced an immediate and sustained increase of glycolytic activity in M0, M1, and M2 cells. Interestingly, EVs acted in an inverse dose-dependent manner. These findings indicate that EVs can induce glycolytic reprogramming of unpolarized and polarized human microglial cells. In conclusion, our pilot study demonstrates that EVs derived from SHEDs can act as a potent immunomodulators of human microglial cells. These findings could be potentially exploited for the development of new therapeutic strategies targeting neuroinflammatory microglia.
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http://dx.doi.org/10.1002/term.2810DOI Listing
February 2019

Advances in stem cell therapy for amyotrophic lateral sclerosis.

Expert Opin Biol Ther 2018 08;18(8):865-881

b Department of Biotechnology and Biosciences , University Milano Bicocca , Milano , Italy.

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a progressive, incurable neurodegenerative disease that targets motoneurons. Cell-based therapies have generated widespread interest as a potential therapeutic approach but no conclusive results have yet been reported either from pre-clinical or clinical studies.

Areas Covered: This is an integrated review of pre-clinical and clinical studies focused on the development of cell-based therapies for ALS. We analyze the biology of stem cell treatments and results obtained from pre-clinical models of ALS and examine the methods and the results obtained to date from clinical trials. We discuss scientific, clinical, and ethical issues and propose some directions for future studies.

Expert Opinion: While data from individual studies are encouraging, stem-cell-based therapies do not yet represent a satisfactory, reliable clinical option. The field will critically benefit from the introduction of well-designed, randomized and reproducible, powered clinical trials. Comparative studies addressing key issues such as the nature, properties, and number of donor cells, the delivery mode and the selection of proper patient populations that may benefit the most from cell-based therapies are now of the essence. Multidisciplinary networks of experts should be established to empower effective translation of research into the clinic.
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http://dx.doi.org/10.1080/14712598.2018.1503248DOI Listing
August 2018

Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements.

Arch Oral Biol 2018 Feb 27;86:108-115. Epub 2017 Nov 27.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, LT-08406, Vilnius, Lithuania.

Objective: To compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann's Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs).

Design: HGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test. In order to determine the primary mechanism of the cell death induced by extracts from different luting cements, the real-time monitoring of caspase-3/-7 activity and membrane integrity of cells was employed.

Results: The extracts from the RMGIC and ZPC decreased the metabolic activity and numbers of viable cells. Unexpectedly, the extracts from the RC evoked only small effects on the metabolic activity of HGFs with a decreasing number of viable cells in a dose-and time-dependent manner. The live cell imaging revealed that the apoptosis was the primary mechanism of a cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death through a necrotic and caspase-independent pathway.

Conclusions: The apoptosis was the primary mechanism of the cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death via a necrotic pathway. We suggest that metabolic assays commonly used to assess the cytotoxicity of luting cements should be validated by alternative methods.
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http://dx.doi.org/10.1016/j.archoralbio.2017.11.011DOI Listing
February 2018

Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells.

J Craniomaxillofac Surg 2017 Jun 29;45(6):845-854. Epub 2017 Mar 29.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine (Head: Prof. Algirdas Venalis, M.D., Ph.D.), LT-08406, Vilnius, Lithuania.

Regeneration of periodontal tissue represents a major challenge to modern tissue engineering, since cell-based therapies require large amounts of periodontal ligament stromal cells (PLSC), which can be obtained only by in vitro expansion. Ideally, the period of the in vitro expansion should be optimized for the generation of large enough numbers of pre-specified progenitor cells ready to contribute to the restoration of periodontal tissues. In the present study, we used a commercially available, three-dimensional culturing platform and alginate microcarrier cell culture system for the propagation of human PLSCs, which were derived using the explant outgrowth method. Induction of osteogenic differentiation resulted in rapid and robust mineralization of the extracellular matrix in PLSCs grown on microcarriers, but not in PLSCs grown under standard culture conditions. Gene expression studies revealed upregulation of osteogenesis-related genes, BMP2, ALP, RUNX2, MSX2, cementum protein 23, bone sialoprotein, osteopontin and periostin, in undifferentiated and differentiating microcarrier cultures of PLSCs. In addition, the microcarrier culture enhanced the expression of β-catenin, intermediate filament protein vimentin and focal adhesion proteins vinculin and paxillin. Our study shows that microcarrier culture allows rapid generation of large numbers of PLSCs pre-specified towards an osteogenic-like phenotype. This method may be useful for the development of new tissue engineering protocols for the reconstruction of periodontal tissues.
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http://dx.doi.org/10.1016/j.jcms.2017.03.009DOI Listing
June 2017

Neuroprotective properties of extracellular vesicles derived from mesenchymal stem cells.

Neural Regen Res 2016 Jun;11(6):904-5

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.

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http://dx.doi.org/10.4103/1673-5374.184480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962579PMC
June 2016

Exosomes as a potential novel therapeutic tools against neurodegenerative diseases.

Pharmacol Res 2016 11 6;113(Pt B):816-822. Epub 2016 Feb 6.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Santariškių 5, LT-08406 Vilnius, Lithuania. Electronic address:

Exosomes are extracellular vesicles that can transfer biological information over long distances affecting normal and pathological processes throughout organism. It is known that very often composition and therapeutic properties of exosomes depends on cell type and its physiological state. Thus, depending on tissue of origin and physiological context exosomes may act as promoters, or suppressors of pathological processes in CNS. From the therapeutic perspective, the most promising cellular sources of exosomes are mesenchymal stem cells, dendritic cells and inducible pluripotent stem cells. In this review, we will summarize the current state of knowledge on the molecular mechanisms underlying neuroprotective actions of exosomes derived from these cells. New therapies for the neurodegenerative disorders are often halted by the inability of drugs to cross blood-brain barrier. In this respect exosomes have a critical advantage, because they can cross blood-brain barrier. Despite the great importance, surprisingly little is known about mechanistic details of this process. Therefore we will discuss some recent findings that may explain mechanisms of exosomal entry into the brain.
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http://dx.doi.org/10.1016/j.phrs.2016.02.002DOI Listing
November 2016

Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

Cytotherapy 2015 Jul 13;17(7):932-9. Epub 2015 May 13.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania. Electronic address:

Background Aims: Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA).

Methods: ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 μmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent.

Results: Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions.

Conclusions: Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease.
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http://dx.doi.org/10.1016/j.jcyt.2014.07.013DOI Listing
July 2015

Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

Inflammation 2015 Oct;38(5):1933-41

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Žygimantų 9, LT-01102, Vilnius, Lithuania.

The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.
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http://dx.doi.org/10.1007/s10753-015-0173-6DOI Listing
October 2015

A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth.

J Mol Neurosci 2013 Jun 26. Epub 2013 Jun 26.

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Žygimantų 9, 01102, Vilnius, Lithuania.

Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF β-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.
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http://dx.doi.org/10.1007/s12031-013-0046-0DOI Listing
June 2013

Upregulation of iHsp70 by mild heat shock protects rabbit myogenic stem cells: involvement of JNK signalling and c-Jun.

Cell Biol Int 2012 ;36(12):1089-96

Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Zygimantu, Lithuania.

iHsp70 [inducible Hsp70 (heat-shock protein 70)] family members (iHsp70, Hsp72 and Hsp70) are highly conserved proteins that act as molecular chaperones and promote cell survival during various forms of stress. Our data indicate that cultured adult rabbit myoblasts do not express iHsp70 under normal growth conditions, although increased expression was detectable 0.5-72 h following a 42°C heat shock for 15-60 min. The intracellular iHsp70 level reached a maximum 8 h after onset of the heat shock, which correlated with its increased accumulation in nuclei. Inhibition of iHsp70 expression by quercetin showed that sustained activation of JNK (c-Jun N-terminal kinase) 2 and suppression of c-Jun phosphorylation were responsible for myoblast death after heat shock. The data also demonstrate that activation of transcription factor c-Jun depends mostly on JNK1, whereas JNK2 had higher affinity and was translocated to nuclei together with c-Jun. We have also shown that the JNK signalling pathway is an upstream effect of iHsp70 expression. These findings provide further in-depth understanding of the implication of the pro-survival signalling kinases JNK1 and JNK2 and their target, c-Jun, in expression of iHsp70 and regulation of myogenic stem cell survival and death mechanisms after heat shock. Mild heat shock before transplantation might be a way of improving myogenic stem cell survival.
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http://dx.doi.org/10.1042/CBI20120143DOI Listing
April 2013

Effects of different sera on adipose tissue-derived mesenchymal stromal cells.

J Tissue Eng Regen Med 2011 Oct 29;5(9):733-46. Epub 2010 Dec 29.

Department of Experimental and Clinical Medicine, State Research Institute Centre for Innovative Medicine, LT-01102 Vilnius, Lithuania.

Current cell therapy protocols require considerable numbers of mesenchymal stromal cells (MSCs), which can be obtained only by in vitro expansion. The most important issue is a choice of optimal growth supplements for cell culture. Ideally, these should be of known composition, free of animal components and allow production of large homogenic populations of MSCs in a considerably short period of time. Since this standard has not been achieved to date, we aimed to assess the molecular responses of MSCs to different growth supplements commonly in use. MSCs were isolated from breast or abdominal adipose tissue and plated into DMEM supplemented with one of four different sera: fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS). MSCs cultivated with different serum supplements demonstrated distinct morphologies, high adipogenic and osteogenic differentiation potential and expressed characteristic antigens. Using real-time PCR, we found a large increase in PPARγ and Msx2 gene expression in both lines of proliferating MSCs cultivated with AS. We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.
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http://dx.doi.org/10.1002/term.374DOI Listing
October 2011

Effects of major human antiprotease alpha-1-antitrypsin on the motility and proliferation of stromal cells from human exfoliated deciduous teeth.

Regen Med 2010 Jul;5(4):633-43

State Institute of Science "Centre of Innovative Medicine", Vilnius University, Zygimantu 9, Vilnius LT-01102, Lithuania.

Aim: Intrinsic tissue regeneration mechanisms are still not fully understood. The destruction/reconstruction processes are usually in fine balance; however, this can be easily destroyed, for example in the environment of chronic inflammation. One of the major proteins present at the inflammatory sites is the multifunctional protein alpha-1-antitrypsin (AAT). In this study, potential therapeutic effects of this major human antiprotease on progenitor cells are assessed.

Materials & Methods: Stromal cells from human exfoliated deciduous teeth (SHEDs) were used, which are similar to the mesenchymal stromal cells isolated from other tissues. SHEDs were cultivated in the presence of subphysiological, physiological and inflammatory concentrations of AAT, and their proliferation and motility traits were assayed. Some intracellular signaling pathways, AAT internalization by SHEDs and their matrix metalloprotease profile were studied in parallel.

Results: Physiologic and inflammatory concentrations of AAT significantly increased the cell proliferation rate, induced phosphorylation of several key protein kinases and increased the amount of secreted active gelatinases. Moreover, cells exposed to physiologic and inflammatory levels of AAT were able to invade and migrate more efficiently. Subphysiologic AAT levels did not change cell behavior significantly.

Conclusion: AAT at physiologic and inflammatory concentrations positively modulates the proliferation and motility of SHEDs in vitro. These results suggest the importance of AAT in the maintenance and regulation of tissue progenitor cells in vivo.
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http://dx.doi.org/10.2217/rme.10.18DOI Listing
July 2010

PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells.

Mol Cell Biochem 2007 Aug 8;302(1-2):9-18. Epub 2007 Feb 8.

Department of Experimental Research, Institute of Experimental and Clinical Medicine, Zygimantu 9, 01102, Vilnius, Lithuania.

Despite the understanding of the importance of phosphoinositide 3-kinase (PI 3-K) signaling pathway in the regulation of cellular proliferation, little is known about its role during phorbol 12-myristate 13-acetate (PMA)-induced differentiation in human leukemia cells. Here, we report a novel finding that PI 3-K inhibition by LY294002 significantly increases p21WAF1/Cip1 expression in PMA-stimulated human leukemia cells NB4 and THP1. LY294002 potentiated expression of p21WAF1/Cip1 via a p53-independent mechanism and did not affect mitogen activated protein kinase (MAPK) activation. Electrophoretic mobility shift (EMSA) experiments revealed that blocking of PI 3-K was associated with increased binding of transcription factor Sp1 to the PMA-responsive sites on the p21WAF1/Cip1 promoter. Pretreatment with rapamycin, an inhibitor of mTOR kinase, decreased the expression of p21WAF1/Cip1 protein in PMA-stimulated NB4 cells. The level of PMA-induced p21WAF1/Cip1 protein expression was lower in NB4 cells overexpressing wild type protein kinase C zeta (PKC zeta) compared to those transfected with empty vector or with kinase inactive PKC zeta. Sp1 binding to the p21WAF1/Cip1 promoter was completely lost in a wild type PKC zeta overexpressing and PMA-stimulated NB4 cells. We demonstrate that PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells, and that this effect is partly mediated by PKC zeta.
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http://dx.doi.org/10.1007/s11010-007-9419-4DOI Listing
August 2007

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway.

Microbes Infect 2005 Dec 1;7(15):1512-8. Epub 2005 Jul 1.

Division of Medical Microbiology, Department of Molecular and Clinical Medicine, Linköping University, SE-58185, Sweden.

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.
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http://dx.doi.org/10.1016/j.micinf.2005.05.012DOI Listing
December 2005

Sp1 and NF-kappaB transcription factor activity in the regulation of the p21 and FasL promoters during promyelocytic leukemia cell monocytic differentiation and its associated apoptosis.

Ann N Y Acad Sci 2004 Dec;1030:569-77

Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius, Lithuania.

Treatment of human acute promyelocytic leukemia cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest and differentiation toward monocytes, which subsequently die by apoptosis. However, the relationship between terminal differentiation and apoptosis remains unclear. Here we have studied Sp1 and nuclear factor kappaB (NF-kappaB) transcription factor activity in controlling promoters of cell cycle-regulating (p21/WAF1/CIP1) and cell death (FasL) genes during monocytic differentiation and apoptosis of the human acute promyelocytic leukemia cell lines NB4 and HL-60. Using the electrophoretic mobility shift assay, we observed that PMA treatment of NB4 cells caused an early response in Sp1 binding to the p21 and FasL promoters at 8 h. The firmly adherent cell phenotype, characteristic of differentiated cells, retained Sp1-binding activity to either promoter, but it was often lost completely in detached, apoptotic cells. The association of Sp1 with the p21 promoter during monocytic differentiation correlated with the levels of expressed p21 in the cytoplasmic fraction, as detected by immunoblotting. In HL-60 cells, very weak or no Sp1 binding to either promoter was observed. Low NF-kappaB affinity for its consensus sites and to the FasL promoter was characteristic of apoptotic cells. The results of this study suggest a positive role of Sp1 and NF-kappaB, as regulators of p21 and FasL genes, in leukemic cell survival and monocytic differentiation and a negative role in apoptotic cell death.
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http://dx.doi.org/10.1196/annals.1329.066DOI Listing
December 2004