The role of Nrf2 and PPARgamma in the improvement of oxidative stress in hypertension and cardiovascular diseases.

Reactive oxygen species are an important element of redox regulation in cells and tissues. During physiological processes, molecules undergo chemical changes caused by reduction and oxidation reactions. Free radicals are involved in interactions with other molecules, leading to oxidative stress. Oxidative stress works two ways depending on the levels of oxidizing agents and products. Excessive action of oxidizing agents damages biomolecules, while a moderate physiological level of oxidative stress (oxidative eustress) is necessary to control life processes through redox signaling required for normal cellular operation. High levels of reactive oxygen species (ROS) mediate pathological changes. Oxidative stress helps to regulate cellular phenotypes in physiological and pathological conditions. Nrf2 (nuclear factor erythroid 2-related factor 2, NFE2L2) transcription factor functions as a target nuclear receptor against oxidative stress and is a key factor in redox regulation in hypertension and cardiovascular disease. Nrf2 mediates transcriptional regulation of a variety of target genes. The Keap1-Nrf2-ARE system regulates many detoxification and antioxidant enzymes in cells after the exposure to reactive oxygen species and electrophiles. Activation of Nrf2/ARE signaling is differentially regulated during acute and chronic stress. Keap1 normally maintains Nrf2 in the cytosol and stimulates its degradation through ubiquitination. During acute oxidative stress, oxidized molecules modify the interaction of Nrf2 and Keap1, when Nrf2 is released from the cytoplasm into the nucleus where it binds to the antioxidant response element (ARE). This triggers the expression of antioxidant and detoxification genes. The consequence of long-term chronic oxidative stress is activation of glycogen synthase kinase 3beta (GSK-3beta) inhibiting Nrf2 activity and function. PPARgamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor playing an important role in the management of cardiovascular diseases, hypertension and metabolic syndrome. PPARgamma targeting of genes with peroxisome proliferator response element (PPRE) has led to the identification of several genes involved in lipid metabolism or oxidative stress. PPARgamma stimulation is triggered by endogenous and exogenous ligands - agonists and it is involved in the activation of several cellular signaling pathways involved in oxidative stress response, such as the PI3K/Akt/NOS pathway. Nrf2 and PPARgamma are linked together with their several activators and Nrf2/ARE and PPARgamma/PPRE pathways can control several types of diseases.

Chronic NOS Inhibition Affects Oxidative State and Antioxidant Response Differently in the Kidneys of Young Normotensive and Hypertensive Rats.

Deficiency of nitric oxide (NO) and oxidative stress can be a cause, a consequence, or, more often, a potentiating factor for hypertension and hypertensive renal disease. Both NO and superoxide anions are radical molecules that interact with each other, leading to oxidative damage of such organs as the kidney. In the present study, we investigated the effect of chronic-specific (neuronal NOS inhibition) and nonspecific NOS inhibition on the oxidative state and antioxidant response and associated oxidative damage of the kidney of young normotensive and hypertensive rats. Young male normotensive Wistar rats (WRs, age 4 weeks) and spontaneously hypertensive rats (SHRs, age 4 weeks) were divided into three groups for each strain by the type of administered compounds. The first group was treated with 7-nitroindazole (WR+7-NI; SHR+7-NI), the second group was treated with N(G)-nitro-L-arginine-methyl ester (WR+L-NAME; SHR+L-NAME), and the control group was treated with pure drinking water (WR; SHR) continuously for up to 6 weeks. Systolic blood pressure increased in WR+L-NAME after the first week of administration and increased slightly in SHR+L-NAME in the third week of treatment. 7-NI had no effect on blood pressure. While total NOS activity was not affected by chronic NOS inhibition in any of the WR groups, it was attenuated in SHR+7-NI and SHR+L-NAME. Nitration of proteins (3-nitrotyrosine expression) was significantly reduced in WR+7NI but not in WR+L-NAME and increased in SHR+7-NI and SHR+L-NAME. Immunoblotting analysis of SOD isoforms showed decreased SOD2 and SOD3 expressions in both WR+7-NI and WR+L-NAME followed by increased SOD activity in WR+L-NAME. Conversely, increased expression of SOD2 and SOD3 was observed in SHR+L-NAME and SHR+7-NI, respectively. SOD1 expression and total activity of SOD did not change in the SHR groups. Our results show that the antioxidant defense system plays an important role in maintaining the oxidative state during NO deficiency. While the functioning antioxidant system seeks to balance the oxidation state in the renal cortex of normotensive WRs, the impaired antioxidant activity leads to the development of oxidative damage of proteins in the kidney induced by peroxynitrite in SHRs.

The peroxisome proliferator-activated receptor gamma agonist pioglitazone improves nitric oxide availability, renin-angiotensin system and aberrant redox regulation in the kidney of pre-hypertensive rats.

The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear receptor. It plays an important role in kidney physiology, where it might contribute to arterial blood pressure regulation and hypertension development by modulation of several signaling pathways. In our study we focused on the effect of PPARγ agonist pioglitazone on changes in the nitric oxide synthase (NOS) expression and activity, the renin-angiotensin system (RAS) cascade, and redox homeostasis signaling pathways in the renal cortex of young pre hypertensive rat models. Young (5-weeks old) spontaneously hypertensive (SHR) and borderline hypertensive (BHR) rats were treated by pioglitazone (PIO, 10 mg/kg/day) during 10 days. Blood pressure (BP) was determined by plethysmography method. Changes in lipid profile were detected in plasma with standard kits using biochemical analyser. Gene expression has been detected by qRT-PCR and protein level was determined using Western blot analysis. Superoxide dismutase (SOD) and catalase (CAT) activities were determined spectrophotometrically and the total enzyme activity of NOS was measured using a radioactive assay based on conversion of [3H] L-arginine to [3H] L- citrulline. Administration of pioglitazone decreased BP in BHR and slowed down the development of BP increase in young SHR animals. For NOS, activation by PPARγ correlated with increase in gene and protein expression of NOS isoforms and in total enzyme activity both in BHR and SHR. In the AT1R/Nox pathway, the treatment did not significantly influence mRNA expression of the p22phox subunit of NADPH oxidase (Nox) and AT1R, but up-regulated the 'pro-vasodilatatory' Mas and AT2R receptors in both BHR and SHR groups. Pioglitazone treatment affected redox regulation. Increase in gene expression of nuclear factor E2-related factor 2 (Nrf2) and SOD isoforms correlated with SOD and CAT enzyme activities. The group treatment-to-control ratios, BHR Pioglitazone to BHR control and SHR Pioglitazone to SHR control for gene expression increased by 10% to 230%. The largest effect of PPARγ has been observed in SOD1, SOD3 and the Mas receptor gene treatment-to-control ratios. The most prominent differences between BHR and SHR were observed in SOD1 and Mas receptor expressions, with large effects of opposite sign in BHR versus SHR. Our data indicate that an increase of NO release activates signaling in the renal cortex of pre-hypertensive rats after pioglitazone treatment. Improvement of NO availability, AT2R, Mas receptors and aberrant redox regulation is thought to be the major correlated mechanisms mediating the BP decrease affected by the PPARγ agonist treatment. We also observed that the most sensitive tissue responses to PPARγ-dependent activation of Nrf2 have been primarily found in the kidney of young hypertensive animals.

The role of PPARgamma in cardiovascular diseases.

The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear superfamily of ligand-activated transcription factors. PPARgamma acts as a nutrient sensor that regulates several homeostatic functions. Its disruption can lead to vascular pathologies, disorders of fatty acid/lipid metabolism and insulin resistance. PPARgamma can modulate several signaling pathways connected with blood pressure regulation. Firstly, it affects the insulin signaling pathway and endothelial dysfunction by modulation of expression and/or phosphorylation of signaling molecules through the PI3K/Akt/eNOS or MAPK/ET-1 pathways. Secondly, it can modulate gene expression of the renin- angiotensin system - cascade proteins, which potentially slow down the progression of atherosclerosis and hypertension. Thirdly, it can modulate oxidative stress response either directly through PPAR or indirectly through Nrf2 activation. In this context, activation and functioning of PPARgamma is very important in the regulation of several disorders such as diabetes mellitus, hypertension and/or metabolic syndrome.

Nrf2 as a key player of redox regulation in cardiovascular diseases.

The oxidative stress plays an important role in the development of cardiovascular diseases (CVD). In CVD progression an aberrant redox regulation was observed. In this regulation levels of reactive oxygen species (ROS) play an important role in cellular signaling, where Nrf2 is the key regulator of redox homeostasis. Keap1-Nrf2-ARE system regulates a great set of detoxificant and antioxidant enzymes in cells after ROS and electrophiles exposure. In this review we focus on radical-generating systems in cardiovascular system as well as on Nrf2 as a target against oxidative stress and a key player of redox regulation in cardiovascular diseases. We also summarize the current knowledge about the role of Nrf2 in pathophysiology of several CVD (hypertension, cardiac hypertrophy, cardiomyopathies) as well as in cardioprotection against myocardial ischemia/ reperfusion injury.

Ageing related down-regulation of myocardial connexin-43 and up-regulation of MMP-2 may predict propensity to atrial fibrillation in experimental animals.

Mechanisms underlying atrial fibrillation (AF), the most common cardiac arrhythmia, particularly in aged population, are not fully elucidated. We have previously shown an increased propensity of old guinea pigs (GPs) heart to inducible AF when comparing to young animals. This study aimed to verify our hypothesis that susceptibility of aged heart to AF may be attributed to abnormalities in myocardial connexin-43 (Cx43) and extracellular matrix that affect cardiac electrical properties. Experiments were conducted on male and female 4-week-old and 24-week-old GPs. Atrial tissue was processed for analysis of Cx43 topology using immunohistochemistry, expression of Cx43 protein using immunobloting, and expression of mRNA of Cx43 and extracellular matrix metalloproteinase-2 (MMP-2) using real time PCR. Immunohistochemistry revealed uniform Cx43 distribution predominantly on lateral sides of the cardiomyocytes of young male and female GP atria. In contrast, non-uniform distribution, mislocalization and reduced immunolabeling of Cx43 were detected in atria of old GPs. In parallel, the atrial tissue levels of Cx43 mRNA were significantly decreased, while mRNA expression of MMP-2 was significantly increased in old versus young GPs. The changes were more pronounced in old GPs males comparing to females. Findings indicate that age-related down-regulation of atrial Cx43 and up-regulation of MMP-2 as well as disordered Cx43 distribution can facilitate development of AF in old guinea pig hearts.

Novel 3,6-bis(imidazolidine)acridines as effective photosensitizers for photodynamic therapy.

The photoeffect of new proflavine derivatives with DNA-binding and antitumour activities, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was studied to evaluate them as potential photosensitizers for photodynamic antitumor therapy. EPR measurements showed that superoxide radical anion and singlet oxygen were produced upon irradiation of AcrDIMs with UV-A light (>300nm) in the presence of molecular oxygen. This indicates that AcrDIMs may act as photosensitizers. The most active pentyl-AcrDIM and hexyl-AcrDIM displayed photocytotoxic effect toward the mouse lymphocytic leukemia cell line L1210 and human ovarian cancer cells A2780. Antitumor activity of pentyl-AcrDIM increased as high as about 12 times (72h incubation) after irradiation of A2780 cells (365nm, 1.05J/cm(2)). The photocytotoxicity seems to be associated with oxidative stress. Concerning the cell cycle, flow cytometry showed an arrest in the S-phase already 4h after irradiation. In a comet assay, no genotoxicity of AcrDIMs was found. Typical morphologic changes and formation of DNA-ladders indicated induction of apoptotic cell death, though no activation of caspase-3 was observed. Investigation of intracellular localization of pentyl-AcrDIM confirmed its partial accumulation in mitochondria and lysosomes. After irradiation of the A2780 cells, colocalization of pentyl-AcrDIM with monodansylcadaverine, a lysosomal dye, was proven, suggesting that lysosomes in the irradiated cells may be involved in the cell death.

The role of oxidative stress in acetylcholine-induced relaxation of endothelium-denuded arteries.

Nitric oxide (NO) is produced in the endothelium in response to vasorelaxants, such as acetylcholine, and acts on vascular smooth muscle cells to induce vasorelaxation. Previously, we found that the smooth muscle of endothelium-denuded arteries expresses functional NO synthase. We hypothesized that the destruction of arterial anatomical integrity induced by denuding arteries of their endothelial layers causes the vessels to become insensitive to vasodilators as a consequence of oxidative stress. In this study, we examined whether the acetylcholine-induced vasorelaxation observed in deendothelialized arteries is mediated by NO and/or affected by oxidative stress. For functional relaxation studies, the isolated thoracic aorta and pulmonary artery of male Wistar rats were used. Vessel superoxide production was assessed in preserved and endothelium-denuded arteries by the lucigenin chemiluminescence method. In all arteries with intact endothelia, acetylcholine evoked vasorelaxation; this effect was inhibited in endothelium-denuded rings. Pretreatment of denuded rings with the free-radical scavenger tempol improved acetylcholine-induced relaxation. This effect was inhibited by the coadministration of 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), an inhibitor of guanylate cyclase, or N(G)-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase. The chemiluminescent assay revealed that endothelial denudation of both vessel types increased the production of superoxide radicals which has been decreased after tempol administration. Our results show that non-endothelial NO could represent an additional source of physiologically active NO and that the insensitivity of endothelium-denuded vessels to vasodilators could be a consequence of oxidative stress. These findings question the concept that endothelial cells play an obligatory role in vasorelaxation.

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy.

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.

Hypoxia increases cell death in multidrug-resistant leukemia cells. Differences in viability and ultrastructure between sensitive and multidrug-resistant L1210 mouse leukemic cells under hypoxia.

The comparative study of sensitive and multidrug-resistant L1210 cells under 24 hours of hypoxia (2% O2 and 5% CO2 at 37 degrees C) was done to see if differences in energetic metabolism between both cell lines are paralleled by differences in cellular morphology. During the dye exclusion assay the viability of sensitive cells was about 70 to 90%, whereas only 30 to 50% of resistant cells were viable. Electron microscopic study of sensitive and resistant L1210 cells under hypoxia has shown cells of different ultrastructural appearance in both cell lines. Cells with necrotic changes (swollen mitochondria, lysed cells) prevailed in resistant cells. The highest incidence of cells with normal or slightly dense mitochondria was found among the sensitive L1210 cells. Additionally, cells with pyknotic nuclei, shrunken cytoplasm and dense mitochondria, reminiscent of apoptosis, could be found sporadically, especially in the sensitive L1210 cell line. These results are in agreement with flow cytometry measurements: in resistant cells the number of necrotic cells was on the average 2.3 times higher than in sensitive cells. Ultrastructural differences and differences in the numbers of necrotic cells as measured by flow cytometry between sensitive and resistant L1210 cells under hypoxia are consistent with differences in energetic metabolism between these cell lines, as described in earlier studies, and document an increased cell death in the resistant L1210 cell line.

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents.

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.

Main targets of tetraaza macrocyclic copper complex on L1210 murine leukemia cells.

Several metal complex agents have already been introduced into clinical tumor therapy and others are subject of antitumor studies. In this study we focused on the tetraaza macrocyclic copper complex (Cu(TAAB)Cl(2)). We studied the influence of the substance on cell growth, cell cycle, membrane integrity, necrosis, apotosis and glutathione level on the leukemic cell line L1210 in 1-day (22 h) and 3-day (72 h) experiments. The metal complex shows a dose-dependent antiproliferative effect, without affecting cell cycle phases. The present results confirm that copper complex can damage plasmatic membranes and trigger apoptosis, and that after treatment of leukemic cells with the copper complex, glutathione levels were increased.

Antitumor activity of benzamide riboside and its combination with cisplatin and staurosporine.

Benzamide riboside (BR), a new synthetic nucleoside analogue, has demonstrated a potent cytotoxic activity in murine leukemia in vitro. The purpose of the present investigation was to examine the antitumor activity of BR in mice bearing leukemia L1210. The results revealed that BR possesses a potent antitumor activity in vivo. It increases life-span of mice with leukemia. Synergistic cytotoxicity of BR with select DNA damaging agents, cisplatin (cis-Pt) and staurosporine (STP) was examined in MTT chemosensitivity assay, FACS analyses and apoptotic DNA fragmentation on L1210 cells in culture. A simultaneous treatment of leukemia L1210 cells with the combination of BR and STP resulted in synergistic cytotoxicity that correlated with increased apoptotic activity in those cells. On the other hand, treatment of L1210 cells with combination of BR and cis-Pt resulted in antagonistic cytotoxic effect. Finally, to elucidate the synergistic effect of BR and STP in inducing apoptosis, the attention was directed to the activation of cell death processes through various cell cycle signals. This is the first report describing in vivo antitumor activity of BR and its utilization in combination chemotherapy.

Combined effect of lipoic acid and doxorubicin in murine leukemia.

Our experiments indicate that administration of a toxic drug with high rate of free-radical formation (doxorubicin, DOX) combined with an antioxidant (alpha-lipoic acid, LA) may lead to a decrease in drug-toxicity. However, the effects of antioxidant may be concentration-dependent and it is therefore crucial to choose its appropriate dosage. LA at a low concentration (1 micromol/l) acts as a growth factor and at a higher concentration (100 micromol/l) acts as an antiproliferation agent. Both concentrations of LA in combination with DOX were examined in cytotoxic and antitumor effects in L1210 mouse leukemia cells employing a MTT chemosensitivity assay. In most concentration combinations, DOX and LA effect were antagonistic and synergistic action was only found at the higher concentration of both agents (DOX 2.5 micromol/l and LA 100 micromol/l). Use of LA in doxorubicin therapy lead to an increase (though marginally significant) in survival of animals. Combined single-dose administration of DOX (5 mg/kg) and LA (16 mg/kg) lead to super-additive effect of the combination on survival of leukemic mice.

Potential carcinogenicity of some transition metal ions.

Potential carcinogenicity of some transition metal ions was tested using a direct-current polarography method. The measurements were based on the reduction of tested compounds in an anhydrous solution using alpha-lipoic acid as the detection compound. The potential carcinogenicity was expressed in terms of the parameter tg alpha, which is known to directly correlate with the carcinogenicity of tested compounds. For the metal ions tested, tg alpha was found to decrease in the following sequence: Fe(III) > Pb(II) > V(IV) > Fe(II) > Mn(II) > Cu(II). Zero values of tg alpha were found for Cd(II) and Mn(III).

Application of NMR spectroscopy in biochemical studies of tumor cells sensitive and resistant to anticancer drugs.

Drug resistance is a prominent problem of cancer therapy. Differences in quantity and quality of many metabolites in normal and malignant cells and their changes after treatment by anticancer drugs can be detected by nuclear magnetic resonance (NMR) both in vivo and in vitro. The results of in vivo and in vitro 1H, 13C, 19F and 31P NMR spectroscopy and their correlation with the degree of resistance to anticancer drugs are discussed. Monitoring of treatment and development of drug resistance by this non-invasive method could be useful not only in cancer research related to drug resistance but also in clinical medical oncology.

Macrocyclic Cu(II)tetraanhydroaminobenzaldehyde complex--antiproliferative activity in vitro and in vivo.

The macrocyclic Cu(II)-tetraanhydroaminobenzaldehyde complex Cu(TAAB)C12 induces various cytotoxic effects in the dependence on a cell line, concentration and time of exposition. The highest complex concentration of 914 nmol/l induces degeneration of certain part of the HeLa cells population after 24 hours of cultivation. The concentration of 183 nmol/l causes a delayed cytotoxic effect on HeLa and HepG2 cells. After 24 hours of culturing 15.4-28.4% of cell population proliferated but after 48 and 72 hours 2.0-42.3% of the cell population degenerated. The cytotoxic effect on V79 cells is directly dependent on the actual concentration and time of the complex influence. The cytotoxic concentrations of Cu(TAAB)Cl2 induce an integrity damage of cytoplasmatic membrane and two phase unbalanced growth. Cu(TAAB)Cl2 possesses an antileukemic activity which at the dose of 10 mg/kg of body weight is not accompanied by side toxic effects on mice.

[alpha-Lipoic acid--a natural disulfide cofactor and antioxidant with anticarcinogenic effects]. / Kyselina alpha-lipoová--prírodný disulfidový kofaktor a antioxidant s antikarcinogénnymi úcinkami.

The present survey summarizes the data about the structure, function and methods of investigation of the natural substance alpha-lipoic acid. This compound is an important growth factor of many microorganisms and at the same time a disulfide cofactor of dehydrogenases in oxidative phosphorylation. It is a physiological constituent of biological membranes, an efficient antioxidant and a scavenger of free radicals. Lipoic acid possesses anticarcinogenic and preventive effects which protect the calls from damage.

Calmodulin interaction with mesocaine-modified lipid bilayer.

Calmodulin (CaM) interactions with bilayer lipid membranes (BLM) were studied by measuring modulus of elasticity in direction perpendicular to the membrane plane (E perpendicular) and intramembrane potential delta psi. Upon interaction of CaM with egg phosphatidylcholine and asolectin BLM the parameter E perpendicular grew slightly (not more than by 10% as compared to the respective vale for nonmodified BLM), suggesting a weak effect on the ordering of the hydrophobic moiety of the lipid bilayer. In the presence of mesocaine (Mes), a calmodulin inhibitor, CaM affected the incorporation of Mes into the membrane. It can be concluded that CaM affects the ordering of the polar (superficial) membrane region.