Isheemia reperfusiooni kahjustuse terapeutilised mehhanismid

Väitekirja elektrooniline versioon ei sisalda publikatsiooneHüpoksilis-isheemiline kahjustus avaldub, kui peaaju või südame verevarustus on häiritud, mistõttu tekib hapniku- ja toitainevaegus. Ilma nendeta surevad rakud kiiresti. Antud töö aitabki paremini mõista, millised tegurid võiksid kaitsta kude hapnikupuuduse eest, mis kaasneb erinevate südameveresoonkonna haigustega. Tegu on molekulaarsel tasemel alusuuringutega rakkudel ja hiirtel, mille käigus uurisime kolme potentsiaalset terapeutilist mehhanismi isheemia reperfusiooni kahjustuse ravis - hüpotermia, vesiniksulfiid ja lümfangiogenees. Uued ravimeetodid aitavad laiendada arusaamist hüpoksilis-isheemilisest kahjustusest ja parandada patsientide taastumisvõimalusi. Kliinilises praktikas on kehatemperatuuri langetamine kasutusel eelkõige sekundaarse ajukahjustuse vähendamiseks, samas näitavad mõningad uuringud, et hüpotermia on võimeline vähendama ka südamelihase kahjustust. Seni on hüpotermiat vaadeldud kui tegurit, mis vähendab metabolismi ja hapniku tarbimist. Meie tulemused näitavad, et lisaks sellele võimaldab hüpotermia suurendada vastupanu hapnikupuudusest tulenevale stressile läbi Nrf2 ja HIF1 signaaliradade. Vesiniksulfiidi peeti pikka aega mürgiseks gaasiks, kuid sellel põhinevad ravimimeetodid võivad saada oluliseks mitmete südame-veresoonkonna haiguste puhul. Südamekahjustus vähendab rakus mitokondrite hulka, mis varustab aga rakku eluks vajaliku energiaga. Meie tulemused näitavad, et vesiniksulfiid reguleerib rakus mitokondrite hulka läbi AMPK-PGC1α signaaliraja. Südamekahjustuse puhul aitab eksogeenne vesiniksulfiid (SG-1002) taastada vesiniksulfiidi tasemed. Selle tulemusena suureneb südame mitokondrite hulk ja paraneb südame funktsioon. Lümfangiogeneesile ehk lümfisoonte tekkele on isheemia reperfusiooni kahjustuse ravis oluliselt vähem tähepanu pööratud kui angiogeneesile ehk veresoonte tekkele, kuid isheemia reperfusiooni kahjustuse tulemusena muutub südame lümfisüsteem. Meie tulemused näitavad, et endogeense lümfangiogeneesi inhibeerimine südamekahjustuse olukorras tekitab põletikuvastust, südame talitushäiret ning ei toimunud iseloomulikku lümfisoonte tiheduse suurenemist. Selle leevendamiseks manustati hiirtele lokaalselt VEGFC-d, mis vähendas põletikku ja parandas südame talitushäiret. Seega on lümfangiogenees üks oluline uus terapeutiline lähenemine isheemia reperfusiooni kahjustuse ravis.Ischemic-hypoxic injury is caused by diminished or absent blood flow and result of oxygen deprivation (e.g. stroke, heart attack). It encompasses a complex constellation of pathophysiological and molecular injuries, if left untreated can result in death. In this dissertation we studied therapeutic mechanisms that might be protective against ischemia reperfusion injury in order to enhance both our knowledge of ischemia reperfusion injury pathophysiology and mechanisms of protection. Clinical hypothermia is considered as one of the most effective intervention for a range of hypoxic ischemic pathologies of the central nervous system, however, the mechanisms that underlie its protective effects are uncertain. Currently, it is widely accepted that therapeutic effects of hypothermia are due to reduced metabolism and oxygen consumption. Our study demonstrates for the first time that mild hypothermia activates major transcription factors Nrf2 and HIF1A, which orchestrate adaptive responses to hypoxic stress. Hydrogen sulfide (H2S) is now recognized as a novel gaseous signaling molecule along with nitric oxide and carbon monoxide, although it was considered to be a toxic gas for hundreds of years. We show here that not only does H2S regulate mitochondrial biogenesis via AMPK-PGC1α signaling pathway but also that correcting H2S levels with SG-1002 (H2S-releasing prodrug) could protect against heart failure. The lymphatic system has been suggested to play an important role in cardiovascular disease. Our results show that inhibition of cardiac lymphangiogenesis exaggerates ischemia reperfusion injury even more. Furthermore, stimulation of cardiac lymphangiogenesis with VEGF-C improves cardiac function following ischemia reperfusion injury. This dissertation provides clues to additional opportunities that could be translated into more efficient therapeutic approaches towards ischemia reperfusion injury.https://www.ester.ee/record=b5249899~S

Promoter-Specific Expression and Genomic Structure of IgLON Family Genes in Mouse

IgLON family is composed of five genes: Lsamp, Ntm, Opcml, Negr1, and Iglon5; encoding for five highly homologous neural adhesion proteins that regulate neurite outgrowth and synapse formation. In the current study we performed in silico analysis revealing that Ntm and Opcml display similar genomic structure as previously reported for Lsamp, characterized by two alternative promotors 1a and 1b. Negr1 and Iglon5 transcripts have uniform 5' region, suggesting single promoter. Iglon5, the recently characterized family member, shares high level of conservation and structural qualities characteristic to IgLON family such as N-terminal signal peptide, three Ig domains, and GPI anchor binding site. By using custom 5'-isoform-specific TaqMan gene-expression assay, we demonstrated heterogeneous expression of IgLON transcripts in different areas of mouse brain and several-fold lower expression in selected tissues outside central nervous system. As an example, the expression of IgLON transcripts in urogenital and reproductive system is in line with repeated reports of urogenital tumors accompanied by mutations in IgLON genes. Considering the high levels of intra-family homology shared by IgLONs, we investigated potential compensatory effects at the level of IgLON isoforms in the brains of mice deficient of one or two family members. We found that the lack of IgLONs is not compensated by a systematic quantitative increase of the other family members. On the contrary, the expression of Ntm 1a transcript and NEGR1 protein was significantly reduced in the frontal cortex of Lsamp-deficient mice suggesting that the expression patterns within IgLON family are balanced coherently. The actions of individual IgLONs, however, can be antagonistic as demonstrated by differential expression of Syp in deletion mutants of IgLONs. In conclusion, we show that the genomic twin-promoter structure has impact on both anatomical distribution and intra-family interactions of IgLON family members. Remarkable variety in the activity levels of 1a and 1b promoters both in the brain and in other tissues, suggests complex functional regulation of IgLONs by alternative signal peptides driven by 1a and 1b promoters.Peer reviewe

Mathematical reconstruction of the metabolic network in an in-vitro multiple myeloma model

It is increasingly apparent that cancer cells, in addition to remodelling their metabolism to survive and proliferate, adapt and manipulate the metabolism of other cells. This property may be a telling sign that pre-clinical tumour metabolism studies exclusively utilising in-vitro mono-culture models could prove to be limited for uncovering novel metabolic targets able to translate into clinical therapies. Although this is increasingly recognised, and work towards addressing the issue is becoming routinary much remains poorly understood. For instance, knowledge regarding the biochemical mechanisms through which cancer cells manipulate non-cancerous cell metabolism, and the subsequent impact on their survival and proliferation remains limited. Additionally, the variations in these processes across different cancer types and progression stages, and their implications for therapy, also remain largely unexplored. This study employs an interdisciplinary approach that leverages the predictive power of mathematical modelling to enrich experimental findings. We develop a functional multicellular in-silico model that facilitates the qualitative and quantitative analysis of the metabolic network spawned by an in-vitro co-culture model of bone marrow mesenchymal stem- and myeloma cell lines. To procure this model, we devised a bespoke human genome constraint-based reconstruction workflow that combines aspects from the legacy mCADRE & Metabotools algorithms, the novel redHuman algorithm, along with 13C-metabolic flux analysis. Our workflow transforms the latest human metabolic network matrix (Recon3D) into two cell-specific models coupled with a metabolic network spanning a shared growth medium. When cross-validating our in-silico model against the in-vitro model, we found that the in-silico model successfully reproduces vital metabolic behaviours of its in-vitro counterpart; results include cell growth predictions, respiration rates, as well as support for observations which suggest cross-shuttling of redox-active metabolites between cells

Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation

C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv) mice display different coping strategies in stressful conditions. Our aim was to evaluate biomarkers related to different adaptation strategies in the brain of male 129Sv and Bl6 mice. We focused on signaling pathways related to the dopamine (DA) system, N-methyl-D-aspartate (NMDA) receptor and epidermal growth factor (EGF) family, shown as the key players in behavioral adaptation. Mice from Bl6 and 129Sv lines were divided into either home cage controls (HCC group) or exposed to repeated motility testing and treated with saline for 11 days (RMT group). Distinct stress responses were reflected in severe body weight loss in 129Sv and the increased exploratory behavior in Bl6 mice. Besides that, amphetamine caused significantly stronger motor stimulation in Bl6. Together with the results from gene expression (particularly Maob), this study supports higher baseline activity of DA system in Bl6. Interestingly, the adaptation is reflected with opposite changes of DA markers in dorsal and ventral striatum. In forebrain, stress increased the gene expressions of Egf-Erbb1 and Nrg1/Nrg2-Erbb4 pathways more clearly in 129Sv, whereas the corresponding proteins were significantly elevated in Bl6. We suggest that not only inhibited activity of the DA system, but also reduced activity of EGF family and NMDA receptor signaling underlies higher susceptibility to stress in 129Sv. Altogether, this study underlines the better suitability of 129Sv for modelling neuropsychiatric disorders than Bl6

Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon

Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin

Chronic Alcohol Use Induces Molecular Genetic Changes in the Dorsomedial Thalamus of People with Alcohol-Related Disorders

The Mediodorsal (MD) thalamus that represents a fundamental subcortical relay has been underrepresented in the studies focusing on the molecular changes in the brains of subjects with alcohol use disorder (AUD). In the current study, MD thalamic regions from AUD subjects and controls were analyzed with Affymetrix Clariom S human microarray. Long-term alcohol use induced a significant (FDR ≤ 0.05) upregulation of 2802 transcripts and downregulation of 1893 genes in the MD thalamus of AUD subjects. A significant upregulation of GRIN1 (glutamate receptor NMDA type 1) and FTO (alpha-ketoglutarate dependent dioxygenase) was confirmed in western blot analysis. Immunohistochemical staining revealed similar heterogenous distribution of GRIN1 in the thalamic nuclei of both AUD and control subjects. The most prevalent functional categories of upregulated genes were related to glutamatergic and GABAergic neurotransmission, cellular metabolism, and neurodevelopment. The prevalent gene cluster among down-regulated genes was immune system mediators. Forty-two differentially expressed genes, including FTO, ADH1B, DRD2, CADM2, TCF4, GCKR, DPP6, MAPT and CHRH1, have been shown to have strong associations (FDR p < 10−8) with AUD or/and alcohol use phenotypes in recent GWA studies. Despite a small number of subjects, we were able to detect robust molecular changes in the mediodorsal thalamus caused by alcohol emphasizing the importance of deeper brain structures such as diencephalon, in the development of AUD-related dysregulation of neurocircuitry

Hypothermia Alleviates Reductive Stress, a Root Cause of Ischemia Reperfusion Injury

Ischemia reperfusion injury is common in transplantation. Previous studies have shown that cooling can protect against hypoxic injury. To date, the protective effects of hypothermia have been largely associated with metabolic suppression. Since kidney transplantation is one of the most common organ transplant surgeries, we used human-derived renal proximal tubular cells (HKC8 cell line) as a model of normal renal cells. We performed a temperature titration curve from 37 &deg;C to 22 &deg;C and evaluated cellular respiration and molecular mechanisms that can counteract the build-up of reducing equivalents in hypoxic conditions. We show that the protective effects of hypothermia are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component), with the highest overlap between activating and suppressing mechanisms emerging in the window of mild hypothermia (32 &deg;C). Hypothermia decreased hypoxia-induced rise in the extracellular lactate:pyruvate ratio, increased ATP/ADP ratio and mitochondrial content, normalized lipid content, and improved the recovery of respiration after anoxia. Importantly, it was observed that in contrast to mild hypothermia, moderate and deep hypothermia interfere with HIF1 (hypoxia inducible factor 1)-dependent HRE (hypoxia response element) induction in hypoxia. This work also demonstrates that hypothermia alleviates reductive stress, a conceptually novel and largely overlooked phenomenon at the root of ischemia reperfusion injury

Altered Expression Profile of IgLON Family of Neural Cell Adhesion Molecules in the Dorsolateral Prefrontal Cortex of Schizophrenic Patients

Neural adhesion proteins are crucial in the development and maintenance of functional neural connectivity. Growing evidence suggests that the IgLON family of neural adhesion molecules LSAMP, NTM, NEGR1, and OPCML are important candidates in forming the susceptibility to schizophrenia (SCZ). IgLON proteins have been shown to be involved in neurite outgrowth, synaptic plasticity and neuronal connectivity, all of which have been shown to be altered in the brains of patients with the diagnosis of schizophrenia. Here we optimized custom 5′-isoform-specific TaqMan gene-expression analysis for the transcripts of human IgLON genes to study the expression of IgLONs in the dorsolateral prefrontal cortex (DLPFC) of schizophrenic patients (n = 36) and control subjects (n = 36). Uniform 5′-region and a single promoter was confirmed for the human NEGR1 gene by in silico analysis. IgLON5, a recently described family member, was also included in the study. We detected significantly elevated levels of the NEGR1 transcript (1.33-fold increase) and the NTM 1b isoform transcript (1.47-fold increase) in the DLPFC of schizophrenia patients compared to healthy controls. Consequent protein analysis performed in male subjects confirmed the increase in NEGR1 protein content both in patients with the paranoid subtype and in patients with other subtypes. In-group analysis of patients revealed that lower expression of certain IgLON transcripts, mostly LSAMP 1a and 1b, could be related with concurrent depressive endophenotype in schizophrenic patients. Additionally, our study cohort provides further evidence that cannabis use may be a relevant risk factor associated with suicidal behaviors in psychotic patients. In conclusion, we provide clinical evidence of increased expression levels of particular IgLON family members in the DLPFC of schizophrenic patients. We propose that alterations in the expression profile of IgLON neural adhesion molecules are associated with brain circuit disorganization in neuropsychiatric disorders, such as schizophrenia. In the light of previously published data, we suggest that increased level of NEGR1 in the frontal cortex may serve as molecular marker for a wider spectrum of psychiatric conditions