Terve ja kahjustatud maksa regeneratsioonis ning Dupuytrenʼi kontraktuuri progressioonis osalevate rakuliste ja molekulaarsete mehhanismide uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Maks on imetaja suurim siseorgan, mis omab unikaalset taastumisvõimet, kuid pidev krooniline maksakahjustus põhjustab tema regeneratsioonivõime vähenemist, fibroosse armkoe tekkimist ja maksa funktsioonide kadumist. Sageli on lõppstaadiumis oleva kroonilise maksahaiguse ainsaks ravivõimaluseks maksasiirdamine. Leidmaks maksasiirdamisele alternatiivseid ravimeetodeid, on oluline uurida maksa regeneratsiooni ja haiguslike protsessidega seotud molekulaarseid ja rakulisi mehhanisme. Käesolevas töös uurisime, kuidas maksa aeglaselt jagunevad rakud panustavad koe taastamisse, millised rakuvälise maatriksi muudatused leiavad aset vastusena maksakahjustusele ja kuidas need võiksid mõjutada rakkude käitumist. Samuti uurisime, kas signaalirajad, mis on olulised maksafibroosis, omavad rolli ka teist tüüpi fibrootilises haiguses ja lõpuks viisime läbi suuremahulise kemikaalide skriinimise, leidmaks AKT1-PDPK1 interaktsiooni inhibiitorit, mis vähendaks kasvajate-ja fibroosiseoselise AKT1 aktivatsiooni. Töö tulemusena leidsime, et maksa aeglaselt jagunevad rakud on unipotentsed sapijuharakud, mis aktiveeruvad ainult kroonilise kahjustuse korral. Samuti selgus, et maksa rakuvälise maatriksi muutused sõltuvad kahjustuse tüübist ning erinevad maatriksi komponendid omavad hepatotsüütide ja sapijuharakkude proliferatsioonile erinevat mõju. Uurides fibrootilisi protsesse Dupuytren´i kontraktuuri koes, avastasime, et haige koe erinevad komponendid sünteesivad erinevaid fibroosi ja proliferatsiooni soodustavaid molekule, moodustades haiguse arenguks sobiliku keskonna. Töö viimases osas kirjeldasime AKT1-PDPK1 interaktsiooni inhibiitori, väikesemolekulaarse ühendi NSC156529, identifitseerimise protsessi. NSC156529 vähendas AKT valgu ja tema sihtmärkvalkude aktiivsust ning pidurdas rakkude kasvu in vitro ja tuumori kasvu in vivo. Seega on NSC156529 sobiv alusmolekul uudsete ravimite väljatöötamiseks selliste haiguste nagu kasvajad ning fibroos vastu, mille puhul AKT signaaliraja aktiivsus on kõrgenenud.The liver is the largest mammalian visceral organ with a remarkable regenerative capacity. However, in case of persistent chronic liver injury, this regenerative potential declines, the parenchyma becomes fibrotic, and finally, the liver is not able to exert its biological functions anymore. Since liver transplantation is often the only treatment option for end-stage chronic liver diseases, development of alternative therapies has become increasingly important. In order to generate new treatments, it is necessary to understand the cellular and molecular mechanisms that control liver regeneration and disease progression. The first objective of this thesis was to determine the role of slowly-cycling liver cells in liver regeneration. Secondly, we studied the changes in extracellular matrix (ECM) that occur in response to different liver injuries, and the effect of these alterations on the regenerative properties of liver cells. We also studied, how the signaling pathways that are associated with liver fibrosis, are involved in another type of fibrotic disease–Dupuytren’s contracture (DC). And finally we conducted a large-scale chemical screening to find inhibitors for AKT1-PDPK1interaction that would suppress AKT pathway, which is aberrantly activated in many cancers and fibrotic diseases. We discovered that slowly-cycling liver cells are unipotent biliary cells which are activated and take part in liver regeneration only in response to chronic liver injury. The changes in ECM appear to be dependent on the type of injury, and the hepatocytes and cholangiocytes have different proliferative activity on different matrix components. When studying fibrotic processes in DC we found that different compartments in DC tissue synthesize distinct fibrosis-and proliferation-inducing molecules, which synergize in forming a supportive microinvironment for disease progression. In the last part of this work we described the identification process of NSC156529–the novel inhibitor of AKT1-PDPK1 interaction. NSC156529 is a small molecule compound that inhibited AKT pathway and restricted cell growth in vitro and tumor growth in vivo. We proposed that NSC156529 is a new strong candidate for the development of novel anti-tumor therapeutics

Assessing the effects of bovine embryo-derived extracellular vesicles on the development of individually cultured bovine embryos

In vitro embryo production requires an enriched microenvironment with various vital cell-secreted factors. In vitro cultured single bovine embryos have demonstrated lower blastocyst rate compared to grouped cultured embryos. We assumed that extracellular vesicles (EVs) within an embryo culture system may affect normal in vitro development. This study aimed to assess the supplementation effects of bovine embryo-derived EVs on the development of individually cultured bovine embryos. Bovine oocytes were in vitro maturated (IVM) for 24 h and then in vitro fertilized (IVF). In preliminary experiments, we established that group cultured embryos in EV depleted Bovine Serum Albumin (BSA) media successfully completed their development; while single cultured embryos were only able to reach the morula stage and then degenerated. Hence, we tested EVs supplementation effects in droplets of EV depleted BSA media covered by mineral oil. EVs used for supplementation were produced from single embryos cultured for 8 days in droplets of BSA culture media under mineral oil. Conditioned medium was collected on day 5. EVs were purified, using Izon columns, from embryos which reached the blastocyst stage and embryos which cleaved on day 2 then degenerated. Non-EV supplemented single embryos cultured in BSA media were considered as control. Purified EVs were characterized by nanoparticle tracking analysis and transmission electron microscope (TEM). A total of 8.8 ×106 particles/ml, which we assumed to be the approximate amount of EVs that a single embryo may release during in vitro culture, was supplemented to each droplet on day 4 post-fertilization. Cleavage rates were 70 and 80% for the supplemented groups and 86% for the control. Morula rates were 40%, 47%, and 47% respectively. No blastocyst was observed within the supplemented groups while the control group counted 33% of blastocysts. Our study suggests that BSA EVs support single cultured embryos to complete their development and that a single embryo needs a significant amount of EVs to reach the blastocyst stage. More researches are needed to understand the role of culture media EVs in supporting single embryo development

Elocalcitol, a fluorinated vitamin D derivative, prevents high-fat diet-induced obesity via SCAP downregulation and miR-146a-associated mechanisms

BackgroundObesity is an emerging health problem worldwide as it is associated with increased risk of cardiovascular, metabolic, mental disorders, and cancer. Therapeutic weight management remains one of the options for the treatment of excess weight and associated comorbidities. In this study, the therapeutic potential of elocalcitol, a fluorinated derivative of vitamin D, was studied on the model of high-fat diet (HFD)-induced obesity in mice.ResultsIt was demonstrated that co-administration of elocalcitol in the doses 15 ug/kg (i.p.) twice a week for 16 weeks prevented body weight gain by approximately 15%. The significant retardation in the body weight gain was observed already on the second week of elocalcitol treatment. Administration of elocalcitol also reduced visceral and epididymal fat accumulation by 55% and 35%, respectively, metabolic syndrome development, and lipid droplets accumulation in the liver of mice exposed to HFD. In contrast, the administration of cholecalciferol (vitamin D)—a precursor to calcitriol, the biologically active form of vitamin D, did not affect significantly the signs of obesity and metabolic syndrome, suggesting that the anti-obese effects of elocalcitol are not related to the canonical vitamin D receptor (VDR). Further studies have demonstrated that the preventive effect of elocalcitol is associated with the decreased levels of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and upregulation insulin-inducing gene-1 (Insig1) mRNA expression suggesting that the anti-obese effect of elocalcitol is mediated via inhibition of SREBP-mediated lipogenesis. We also demonstrated that elocalcitol prevents an increase in the expression of proinflammatory cytokines such as interleukin-1 beta (Il1b), tumor necrosis factor-alpha (Tnf), and interleukin-18 (Il18), and this effect was associated with upregulation of microRNA-146a (miR-146a). Deletion of the miR-146a gene reduced the anti-obese effects of elocalcitol and prevented its actions on the SCAP levels. The data indicate that elocalcitol’s reduction of SCAP is at least partly mediated by miR-146a modulation.ConclusionThe study demonstrates that elocalcitol prevents HFD-induced obesity and metabolic syndrome in mice, likely by inhibiting SREBP-mediated lipogenesis and upregulating miR-146a. These findings provide valuable insights into the anti-obesity mechanisms of fluorinated D-vitamin analogs and suggest potential therapeutic strategies for obesity prevention

Laminin-rich blood vessels display activated growth factor signaling and act as the proliferation centers in Dupuytren’s contracture

INTRODUCTION: Dupuytren’s contracture (DC) is a chronic fibroproliferative disease of the hand, which is characterized by uncontrolled proliferation of atypical myofibroblasts at the cellular level. We hypothesized that specific areas of the DC tissue are sustaining the cell proliferation and studied the potential molecular determinants that might contribute to the formation of such niches. METHODS: We studied the expression pattern of cell proliferation marker Ki67, phosphorylated AKT (Ak mouse strain thymoma) kinase, DC-associated growth factors (connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2)) and extracellular matrix components (laminins, fibronectin, collagen IV) in DC tissue and normal palmar fascia using immunofluorescence microscopy and quantitative real-time polymerase chain reaction (qPCR). RESULTS: We found that proliferative cells in the DC nodules were concentrated in the immediate vicinity of small blood vessels and localized predominantly in the myofibroblast layer. Correspondingly, the DC-associated blood vessels contained increased levels of phosphorylated AKT, a hallmark of activated growth factor signaling. When studying the expression of potential activators of AKT signaling we found that the expression of bFGF was confined to the endothelium of the small blood vessels, IGF-2 was present uniformly in the DC tissue and CTGF was expressed in the DC-associated sweat gland acini. In addition, the blood vessels in DC nodules contained increased amounts of laminins 511 and 521, which have been previously shown to promote the proliferation and stem cell properties of different cell types. CONCLUSIONS: Based on our findings, we propose that in the DC-associated small blood vessels the presence of growth factors in combination with favorable extracellular matrix composition provide a supportive environment for sustained proliferation of myofibroblasts and thus the blood vessels play an important role in DC pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13075-015-0661-y) contains supplementary material, which is available to authorized users

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p Peer reviewe

Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk

Background Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. Methods We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. Results We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. Conclusion Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.Peer reviewe

A Novel Inhibitor of AKT1–PDPK1 Interaction Efficiently Suppresses the Activity of AKT Pathway and Restricts Tumor Growth <i>In Vivo</i>

Abstract The serine/threonine kinase AKT/PKB has a critical role in the regulation of cell proliferation. Because AKT signaling is deregulated in numerous human malignancies, it has become an attractive anticancer drug target. A number of small molecule AKT kinase inhibitors have been developed; however, severe side effects have prevented their use in clinical trials. To find inhibitors of AKT1 signaling with principally novel mechanism of action, we carried out a live cell-based screen for small molecule inhibitors of physical interaction between AKT1 and its primary activator PDPK1. The screen revealed one molecule—NSC156529, which downregulated AKT1 signaling, efficiently decreased the proliferation of human cancer cells in vitro, and substantially inhibited the growth of prostate tumor xenografts in vivo. Interestingly, the treated tumor xenografts exhibited higher expression level of normal prostate differentiation markers but did not show augmented cell death, suggesting that the identified compound primarily enhances the differentiation of malignant cells toward normal prostate epithelium and thus poses as an attractive lead compound for developing novel antitumor agents with less cytotoxic side effects. Mol Cancer Ther; 14(11); 2486–96. ©2015 AACR.</jats:p

Supplementary Tables 1 through 5, Supplementary Materials and Methods, and Supplementary Figure Legends from A Novel Inhibitor of AKT1–PDPK1 Interaction Efficiently Suppresses the Activity of AKT Pathway and Restricts Tumor Growth <i>In Vivo</i>

Table S1. Antibodies used for Western Blotting. Table S2. Primers used for RT-QPCR. Table S3. Antibodies used for immunofluorescence analysis. Table S4. Vectors used for screening experiments. Table S5. 36 chemicals from 2000 selected for further investigation. Supplementary Methods. Supplementary figure legends.</p

Supplementary Figure S3 from A Novel Inhibitor of AKT1–PDPK1 Interaction Efficiently Suppresses the Activity of AKT Pathway and Restricts Tumor Growth <i>In Vivo</i>

NSC156529 interacted with PDPK1 and AKT1</p

Supplementary Figure S1 from A Novel Inhibitor of AKT1–PDPK1 Interaction Efficiently Suppresses the Activity of AKT Pathway and Restricts Tumor Growth <i>In Vivo</i>

Four selected chemicals did not alter the morphology of H1299 cell culture</p