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The main treatment for myocardial infarction is early reperfusion of ischemic tissue. Ischemia and reperfusion (IR) produces reactive oxygen species that oxidize membrane phospholipids. The production of oxidized lipids and their role on cell death in cardiac IR injury is unknown. Using in vitro model of IR, our goal was to identify oxidized phosphatidylcholines (OxPC) from cardiomyocytes, to determine their bioactivity on cardiomyocyte viability and mitochondrial permeability, and using an OxPC specific EO6 antibody inhibit OxPC activity on cardiomyocytes. Rat cardiomyocytes were exposed to IR and lipid extracts underwent lipidomic analysis with HPLC-MS/MS to quantitate 82 novel OxPC species. Cell viability and mitochondrial permeability were determined in vehicle control, non-oxidized control PC, and fragmented OxPC molecules. EO6 antibody was applied and cell viability was assessed. Cardiomyocytes under IR demonstrated increased relevant OxPCs particularly fragmented species. OxPC treatment resulted in loss of cardiomyocyte viability, increased mitochondrial permeability when compared to control. EO6 antibody blocked the loss of cardiomyocyte viability. We have shown for the first time that OxPCs are generated cardiomyocytes during IR and they have detrimental effects on cardiomyocyte viability. Additionally the EO6 antibody inhibits the bioactivity of the OxPCs on cardiomyocytes and could be part of a future treatment regimen.

Cardiovascular disease is one of the main causes of mortality and one of the significant burdens to society. Major cardiovascular diseases such as acute myocardial infarction (heart attack), heart failure and cardiac arrhythmia often result in the development of ischemia/reperfusion (I/R) injury. Untreated I/R injury is known to cause cardiac contractile dysfunction. It is established that matrix metalloproteinase-2 (MMP-2) is activated and degrades contractile proteins during I/R, and many other factors including metabolic enzymes, kinases and structural proteins are affected by I/R. However, the molecular mechanisms responsible for these changes are unclear. Since MMP-2 is known to its broad spectrum of action, I hypothesize that, in addition to contractile proteins, proteins related to regulation of energy metabolism are MMP-2 targets during I/R, and protein kinase such as myosin light chain kinase (MLCK) is also involved in this process. The use of proteomics in studying heart injury triggered by I/R will reveal new potential targets for pharmacological protection of heart from I/R induced contractile dysfunction. In addition, selective inhibition of MMP-2 using MMP-2 siRNA protects the heart from I/R injury. In this study, we investigated the protein modulation during I/R using proteomic approach. In order to study the effect of protein kinases (MLCK) and MMP-2, their selective inhibitors were used to inhibit those factors and evaluate the changes in energy metabolic proteins during I/R. Proteomic analysis revealed that six proteins are involved in energy metabolism: ATP synthase β subunit, cytochrome b-c1 complex subunit 1, 24-kDa mitochondrial NADH dehydrogenase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, cytochrome c oxidase subunit, and succinyl-CoA ligase subunit, resulting in decreased levels in I/R hearts. The data suggests that energy metabolic proteins, especially the metabolic enzymes involved in the electron transport chain in the mitochondria may contribute to I/R injury. In addition, our data provides evidence that the right and left ventricles of the heart respond differently to I/R injury, in terms of the regulation of contractile proteins and energy metabolic enzymes. Studies using MLCK inhibitor, ML-7, and MMP-2 inhibitor, MMP-2 siRNA to investigate the effect of myosin light chain kinase (MLCK) and MMP-2 in energy metabolic proteins have shown that succinyl-CoA ligase and ATP synthase are affected by MLCK and MMP-2 respectively. These results demonstrate that the effect of inhibition of the MLCK and MMP-2 involves optimization of energy metabolism in I/R injury, likely resulting in increased energy production. Hence, the observed proteins increase in cardiac recovery after I/R. Also, inhibition of MLCK and MMP-2 by ML-7 and MMP-2 respectively shows cardio protective effect during I/R. In summary, this study provides a novel pathogenesis in the development of I/R-induced cardiac contractile dysfunction. Moreover, we suggest a new therapeutic approach whereby using MMP-2 siRNA can be a promising gene therapy in the development of new preventive or treatment strategies against I/R injury.

Stroke is a devastating and debilitating condition resulting from a blockage or hemorrhage in the vasculature of the brain. Despite extensive research, the etiology and pathophysiology of the disease at the level of the cell membrane are poorly understood, and effective treatment has been elusive. Though much research has shown marked increases in lipid metabolism following stroke, the impact of these changes have often been overlooked given the technical challenges associated with identifying regionally specific changes in degenerating tissue. The advent of lipidomics – a systems biology approach to the large-scale profiling of individual lipid species in tissues – has renewed interest in understanding the role of membrane lipids and their metabolites in the cell and in ischemic injury. In this thesis, I have used an unbiased LC-ESI-MS-based lipidomic approach to profile the small molecular weight glycerophosphocholine second messenger lipidome in anterior and posterior regions of cortex and striatum in the forebrain of wild-type and platelet activating factor receptor (PAFR) null-mutant mice before and after middle cerebral artery occlusion (MCAO). From these profiles, I have outlined the potential use of lipid second messenger distribution as topographic landmarks to identify functional subdomains within neural tissue. Further, I have demonstrated that ischemia does not simply disrupt lipid second messenger metabolism globally but produces regionally specific changes in discrete species and that these changes are altered by the loss of lipid regulatory effectors (i.e., PAFR null mutation). Based on the lipid species identified in this profile of healthy and ischemic tissue, I proposed that tight regulation of PC(O-22:6/2:0) homeostasis by PAFR-expressing microglia is ii required for proper dopaminergic signaling in prefrontal cortex. Finally, I have outlined a model whereby increased PAF synthesis following ischemia contributes the inflammatory response by promoting blood-brain barrier permeability, microglial activation and immune cell infiltration in a PAFR-dependent manner.

Ischaemic stroke is a complex disorder with many different aetiologies, but previous studies of stroke often did not differentiate aetiological subtypes of ischaemic stroke. However, different stroke subtypes may have different risk factors, and to target preventive treatments more effectively, we need to understand these associations. I studied the association of established vascular risk factors with different aetiological stroke subtypes in population-based cohorts of stroke patients. I studied Diffusion Weighted Magnetic Resonance Imaging (DWI) in patients with subacute minor stroke and TIA to determine whether DWI may be a useful addition to the management of such patients, and whether it may be a useful tool in future epidemiological studies of stroke. To determine whether carotid anatomy may be a risk factor for large vessel atheroma I studied angiographical data from the European Carotid Surgery Trial. My main findings are that the prevalence of risk factors differs between stroke subtypes. It also differs between hospitalised and non-hospitalised patients, highlighting that risk factor studies should be performed in population-based cohorts. Analysis of family history data suggests that future genetic studies may best be targeted at non-cardioembolic stroke and at younger patients, and that genetic studies of hypertension may help to unravel some of the genetic factors contributing to stroke risk. DWI is sensitive in subacute minor stroke, and inter- and intra-observer reproducibility are high. DWI frequently adds useful information and may influence patient management. More widespread use of DWI in patients with subacute stroke and TIA should be considered, and DWI may also be a useful tool in future epidemiological studies of stroke. Carotid anatomy varies considerably between individuals, is very asymmetrical within individuals, and it differs between men and women. These findings may partly explain differences in plaque development between individuals, asymmetrical plaque formation within individuals, and sex differences in the distribution of carotid plaque and in the prevalence of carotid atheroma in the general population. Carotid anatomy may be a risk factor for local plaque development. Although not amenable to treatment, knowing which anatomical configuration is associated with atheroma formation could help to identify high-risk individuals in whom other risk factors should be treated aggressively.

This thesis is based on a worldwide overview (meta-analysis) of prospective observational studies of blood pressure and cholesterol, involving a centralised collection of data on over one million individuals from 59 studies, which I have co- ordinated since its inception. Analytically, the aim has been to develop and to use appropriate statistical techniques to assess the age- and sex-specific associations of usual blood pressure and of usual cholesterol with cause-specific mortality. Since the data set is uniquely large, and because appropriate methods of analysis (with full account taken of the time-dependent nature of the regression dilution bias) have been developed and used, these associations have been established more reliably. An integral part of the methodological element of the thesis has been to investigate the systematic underestimation of associations between risk factor and disease that are obtained when only a single baseline measurement is used to assess levels of such risk factors (the regression dilution bias). The extent of this bias has been investigated in each study that had repeat measurements of risk factors during follow-up. One particularly novel aspect has been the emphasis on, and methods developed to account for, the regression dilution bias in several studies simultaneously and in an appropriately time-dependent way. This thesis illustrates the extent to which random error and inappropriate statistical analysis lead to misleading conclusions concerning the importance of blood pressure and blood cholesterol, particularly in premature death. Only by studying adequate numbers of deaths (136,000 deaths among 1 million adults during 13 million person- years of follow-up) and by using appropriate statistical techniques - taking proper account of (a) the regression dilution bias; (b) the full range of blood pressure and cholesterol; (c) the opposing effects of HDL.and the remaining non-HDL cholesterol; and (d) age at death - did it become possible to provide reliable results on the true relationships between blood pressure, cholesterol fractions and vascular and other causes of death. These analyses have demonstrated reliably that, as causes of IHD death in early middle age, blood pressure and blood lipids are three to five times more important than suggested by inappropriate analyses, with no clinically relevant inverse associations with cancer or other non-vascular mortality (except, surprisingly, COPD).

No description available.

Šio darbo tikslas ištirti išemijos/reperfuzijos poveikį žiurkės inkstų mitochondrijų funkcijoms. Pagrindiniai darbo uždaviniai: 1) įvertinti išemijos (20, 40, 60 minučių) in vitro poveikį inkstų mitochondrijų kvėpavimo funkcijoms; 2) įvertinti išemijos (60 minučių) ir reperfuzijos (30 minučių) in vivo poveikį inkstų mitochondrijų kvėpavimo funkcijoms; 3) ištirti išemijos poveikį inkstų mitochondrijų kvėpavimo grandinės I komplekso aktyvumui. Išemijos poveikio žiurkės inkstų mitochondrijų funkcijų tyrimui buvo pasirinkti du eksperimentiniai modeliai, t.y. in vitro ir in vivo modelis. Mitochondrijas išskyrėme diferencinio centrifugavimo būdu, baltymą nustatėme biureto metodu. Išskirtų mitochondrijų kvėpavimo greitį vertinome poliarografiniu metodu. I komplekso aktyvumą nustatėme spektrofotometriniu būdu. Rezultatai parodė, kad mitochondrijų pažaida išryškėja labai anksti - jau po 20 minučių, t.y. slopinamas maksimalus (ADP stimuliuotas) mitochondrijų kvėpavimo greitis tiek oksiduojant glutamatą/malatą, tiek sukcinatą. Ilginant išemijos trukmę, mitochondrijų pažaida didėjo. Išorinės mitochondrijų membranos pažaida taip pat didėjo ilginant išemijos trukmę. Tiek išemija (60 minučių), tiek reperfuzija (30 minučių) slopina mitochondrijų kvėpavimo greitį trečioje metabolinėje būsenoje. 60 minučių išemija/30 minučių reperfuzija nepadidino mitochondrijų vidinės membranos pralaidumo. Rezultatai rodo, jog išemijos poveikyje (60 minučių) mitochondrijos pasižeidžia labiau nei... [toliau žr. visą tekstą] / The aim of this work is to explore the effect of ischemia/reperfusion on rat kidney mitochondrial function. The main objectives of this work are to: 1) evaluate the effect of ischemia (20, 40, 60 minutes) in vitro on kidney mitochondrial function; 2) evaluate the effect of ischemia (60 minutes) and reperfusion (30 minutes) in vivo on kidney mitochondrial function; 2) explore the effect of ischemia/reperfusion on kidney mitochondrial respiratory chain complex I activity. We chose two experimental models of ischemia for investigation of its effects on rat kidney mitochondrial functions, i.e. in vitro and in vivo models. The mitochondria were isolated by a differential centrifugation method, while the protein was determined using biuret method. Mitochondrial respiration rate was measured polarographically. Complex I activity was evaluated spectrophotometrically. The results showed that mitochondrial damage become apparent very early - after 20 minutes, i.e. the maximal (ADP- stimulated) mitochondrial respiration rate decreased with both, glutamate/malate and succinate. It is obvious that by increasing the duration of ischemia, the mitochondrial damage increased. The damage of mitochondrial outer membrane increased by increasing the duration of ischemia. We found that both, ischemia (60 minutes) and reperfusion (30 minutes) decreased the mitochondrial State 3 respiration rate. 60 minutes ischemia/30 minutes reperfusion had no effect on mitochondrial inner membrane permeability... [to full text]

Pastaruoju metu daug mokslinių tyrimų atliekama su flavonoidų klasei priskiriamais antocianinais – augaliniais pigmentais, kurie aptinkami uogose, vaisiuose ir daržovėse ir suteikia jiems mėlyną, raudoną ir violetinę spalvas. Literatūroje aprašytas antocianinų poveikis, apsaugantis nuo širdies ir kraujagyslių ligų, siejamas su antocianinų antioksidaciniu veikimu. Mes iškėlėme hipotezę, kad antocianinai gali apsaugoti širdį redukuodami citozolyje esantį citochromą c. Šio darbo tikslas yra ištirti antocianinų poveikį žiurkės širdies mitochondrijų oksidacinio fosforilinimo sistemos efektyvumui bei nustatyti, kokius už ląstelės žūtį atsakingus viduląstelinius signalinius kelius reguliuoja šie junginiai. Iškelti uždaviniai: 1. Ištirti tiesioginį penkių dažniausiai gamtoje sutinkamų antocianinų poveikį žiurkės širdies mitochondrijų kvėpavimui skirtingose metabolinėse būsenose. 2. Nustatyti įvairių antocianinų gebėjimą redukuoti citochromą c in vitro. 3. Nustatyti, ar antocianinai – citochromo c reduktoriai – apsaugo nuo išemijos sukeltų oksidacinio fosforilinimo sistemos pažeidimų ir kaspazių aktyvacijos bei nuo išemijos/reperfuzijos sukeltos kardiomiocitų apoptozės ir nekrozės. 4. Ištirti, ar stipriu citochromą c redukuojančiu aktyvumu pasižymintys antocianinai gali atstatyti mitochondrijų oksidacinio fosforilinimo sistemos efektyvumą po išemijos. 5. Patikrinti, ar tirtųjų antocianinų poveikis širdies mitochondrijų funkcijoms bei apsauginis nuo išemijos ir išemijos/reperfuzijos. / A lot of research is carried out with anthocyanins, which are widely prevalent among colored berries, fruits, and vegetables and give them blue, red, violet, and purple colors. It is known that consumption of dietary plants and products rich in anthocyanins can reduce the risk of cardiovascular diseases. This protective effect is related to the antioxidant properties of anthocyanins. We have raised the hypothesis that anthocyanins could protect cardiomyocytes from ischemia/reperfusion-induced cell death reducing cytosolic cytochrome c. The aim of this work was to investigate the effect of anthocyanins on heart mitochondrial oxidative phosphorylation and determine what intracellular signaling pathways responsible for cell death are regulated by these compounds. The tasks of the study were: 1. To investigate the direct effect of anthocyanins on rat heart mitochondrial respiration rates; 2. To determine the ability of anthocyanins to reduce cytochrome c in vitro; 3. To determine whether anthocyanins – cytochrome c-reducing compounds – protect heart mitochondria from ischemia-induced mitochondrial dysfunction and ischemia/reperfusion-induced cardiomyocyte apoptosis and necrosis; 4. To examine whether anthocyanins with high cytochrome-c reducing activity can restore the efficiency of mitochondrial oxidative phosphorylation after ischemia; 5. To verify whether the effect of the investigated anthocyanins on cardiac mitochondrial function and their protective effect on... [to full text]

Spontaneous and evoked potentials (EPs) were recorded with cross-cortical microelectrode arrays following partial occlusion of the MCA and ACA in urethane-anaesthetised rats. The control group received no occlusion, while the treatment group was injected with anti-stroke peptide Tat-NR2B9c 5min before ischemia. Spontaneous EEG power significantly decreased in the stroke-only group when compared to controls (p<0.001). A greater loss of EEG power was observed on anterior electrodes closer to the occluded area versus posterior contacts in stroke-only rats (p<0.05). The Tat-NR2B9c+stroke group lost significantly less power when compared to stroke-only animals (p<0.05). EP amplitude in the stroke-only group was significantly reduced following ischemia when compared to control and Tat-NR2B9c+stroke animals (p<0.001). Epileptiform discharges were observed in 8/10 untreated stroke rats and 3/5 stroke rats treated with Tat-NR2B9c. The characteristic features of spontaneous and evoked potentials validate this rat focal stroke model for in vivo testing of pharmacological agents.