Sarada D. Tetali

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana

BACKGROUND AND AIMS The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. METHODS Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. KEY RESULTS In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. CONCLUSIONS These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III.

Gum resin of Boswellia serrata inhibited human monocytic (THP-1) cell activation and platelet aggregation.

ETHNOPHARMACOLOGICAL RELEVANCE Stem bark gum resin extract of Boswellia serrata is traditionally used in India for its hemostatic, antiinflammatory and cardiovascular health effects and it is named as Śallakī in Ayurvedic medicine. AIM OF THE STUDY This study was conducted to evaluate the antioxidative and antithrombotic properties of stem bark gum resin extracts of Boswellia serrata (BS). MATERIALS AND METHODS The inhibitory activity of the BSWE and BSAE on FeCl(3) induced lipid peroxidation (in vitro) in rat liver and heart homogenates was measured spectrophotometrically. Their effect on H(2)O(2) induced reactive oxygen species (ROS) generation in human monocytic (THP-1) cells was investigated by tracking intensity of a cell permeable fluorescent dye, H(2)DCFDA and subjecting the cell samples to confocal microscopy. Further, the effect of BSAE and BSWE on ADP-induced platelet aggregation was assessed using a multimode detection plate reader, plasma coagulation times using an automated blood coagulation analyzer and on human blood clotting factors Xa and XIa using chromogenic substrate. Phytomarker analysis of the water (BSWE) and hydroalcoholic (BSAE) extracts of BS-gum resin was done through HPLC using a standard compound AKβBA. RESULTS BSAE and BSWE inhibited, to varied extents, the lipid peroxidation in liver (80%) and heart (50%) tissue homogenates of male Wistar rats. Further, BSAE (30 μg dwt/mL) and BSWE (300 μg dwt/mL) attenuated ≥ 60% of H(2)O(2) mediated ROS generation in THP-1 cells. In case of standard compounds, ascorbate (20 μg dwt/mL) and butylated hydroxytoluene (BHT) (10 μg dwt/mL) completely scavenged ROS in the cells. BSAE and BSWE at 3 mg dwt/mL completely inhibited ADP induced platelet aggregation and activities were comparable to 20 μg/mL of heparin. The extracts also showed very high activity in prolonging coagulation time periods. Both types of extracts extended prothrombin time (PT) from ∼13 to >60s and activated partial thromboplastin time (APTT) from ∼32s to >90s. BSAE inhibited clotting factors Xa and XIa remarkably at 6 μg of dwt where as BSWE did not show much effect on FXa and showed 30% inhibition on FXIa at 120 μg. 10 μg of heparin was required to inhibit about 30% activity of the above factors. HPLC analyses suggested that BSAE and BSWE had AKβBA of 9% (w/w) and 7.8% (w/w) respectively. CONCLUSION Present study demonstrated antioxidant and antithrombotic anticoagulant activities of water and hydroalcoholic extracts of Boswellia serratas gum resin. We suggest that BS-gum resin as a good source for lead/therapeutic compounds possessing antioxidant, antiplatelet and anticoagulant activities.

VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL∼LDL<VLDL<VLDL+lipoprotein lipase. Moreover, the conformation of apoE4 depended on the lipoprotein fraction: VLDL-associated apoE4 had a more linear conformation than apoE4 associated with LDL or HDL. Further, by changing VLDL fluidity, VLDL lipolysis products significantly altered apoE4 into a more expanded conformation. Our studies indicate that after every meal, VLDL fluidity is increased causing apoE4 associated with VLDL to assume a more expanded conformation, potentially enhancing the pathogenicity of apoE4 in vascular tissue.

Stem-bark of Terminalia arjuna attenuates human monocytic (THP-1) and aortic endothelial cell activation.

ETHNOPHARMACOLOGICAL RELEVANCE Terminalia arjuna - stem bark extract is traditionally used as cardiotonic in Ayurvedic medicine. AIM OF THE STUDY The present study was aimed to evaluate the molecular basis for cardioprotective potential of Terminalia arjuna (TA) stem bark, using cell cultures of human monocytic (THP-1) and human aortic endothelial cells (HAECs). MATERIALS AND METHODS Inhibitory effect of alcoholic (TAAE) and aqueous (TAWE) extracts of TA-stem bark was assessed on human 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lipoprotein lipase (LpL) and lipid peroxidation in rat (wistar) liver and heart homogenates. The patterns of H2O2 induced reactive oxygen species (ROS) generation were observed by confocal microscopy. The activities of antioxidant enzymes and reducing power of the cells were measured in a microplate reader. Gene transcripts of proinflammatory markers in THP-1 and HAECs were assayed by real time PCR and levels of inflammatory protein markers by ELISA or flow cytometry. Phytochemical analyses of TAAE and TAWE were done using liquid chromatography, coupled to mass spectrometry (LC-MS). RESULTS TAAE and TAWE inhibited the lipid peroxidation and HMG-CoA reductase but had no effect on LpL. Both the extracts attenuated H2O2 mediated ROS generation in THP-1 cells by promoting catalase (CAT), glutathione peroxidase (GPx) activities, and by sustaining cellular reducing power. TAAE was highly effective in attenuating proinflammatory gene transcripts in THP-1 cells and HAECs, whereas the response to TAWE depended on the type of transcript and cell type. Both extracts decreased the levels of typical inflammatory marker proteins, viz. LPS induced tumor necrosis factor (TNF)-α secreted by THP-1 cells and TNF-α induced cell surface adhesion molecules on HAECs, namely vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Phytochemical analyses indicated the richness in phenolic compounds and terpenes of TAAE and TAWE, while revealing variability in their metabolite profile. CONCLUSION Our study scientifically validates the antioxidative and antiinflammatory properties of Terminalia arjuna stem bark. The marked effects on cultured human monocytic and aortic endothelial cells (HAEC) provide the biochemical and molecular basis for therapeutic potential of TA-stem bark against cardiovascular diseases (CVD).

Ocimum sanctum leaf extracts attenuate human monocytic (THP-1) cell activation

ETHNOPHARMACOLOGICAL RELEVANCE Ocimum sanctum (OS), commonly known as Holy basil/Tulsi, has been traditionally used to treat cardiovascular diseases (CVD) and manage general cardiac health. The present study is designed to evaluate the antiinflammatory effect of O. sanctum and its phenolic compound and eugenol (EUG) in human monocytic (THP-1) cells and validate its traditional use for treating cardiovascular diseases. MATERIALS AND METHODS The phytochemical analysis of alcoholic and water extracts of OS-dry leaves (OSAE and OSWE) was done using LC-QTOF-MS. A phenolic compound, EUG was quantified in both OSAE and OSWE by an LC-MS technique using a mass hunter work station software quantitative analysis system. The effect of both OSAE, OSWE, pure compound EUG and positive control imatinib (IMT) was investigated in THP-1 cells by studying the following markers: lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNF-α) secretion by ELISA, gene expression of inflammatory markers (TNF-α, IL-6, MIP-1α and MCP-1) by real time PCR and translocation of nuclear factor kappa B (NF-κB) by confocol microscopy. Furthermore, the effect of the extracts, EUG and IMT, was studied on phorbol-12-myristate-13-acetate (PMA) induced monocyte to macrophage differentiation and gene expression of CD14, TLR2 and TLR4. RESULTS The LC-MS analysis of OSAE and OSWE revealed the presence of several bioactive compounds including eugenol. Quantitative analysis revealed that OSAE and OSWE had EUG of 12 ng/mgdwt and 19 ng/mgdwt respectively. OSAE, OSWE (1 mg dwt/mL) pure compound EUG (60 µg/mL) and positive control IMT (20 µg/mL) showed marked inhibition on LPS induced TNF-α secretion by THP-1 cells. At the selected concentration, the plant extracts, EUG and IMT inhibited gene expression of cytokines and chemokines (IL-6, TNF-α, MIP-1α, MCP-1) and translocation of NF-κB-p65 to the nuclei. In addition, they showed significant inhibition on PMA induced monocyte to macrophage differentiation and the gene expression of CD14, TLR2 and TLR4 markers. CONCLUSION The result of the present study validated traditional use of Ocimum sanctum for treating cardiovascular disease for the first time by testing antiinflammatory activity of Ocimum sanctum in acute inflammatory model, LPS induced THP-1 cells. The plant extracts showed significant antiinflammatory activity, however, further to be evaluated using chronic inflammatory animal models like diabetic or apolipoprotein E-deficient mice to make it evidence based medicine. The phenolic compound eugenol (60 µg/mL) showed significant antiinflammatory activity. However the amount of eugenol present in 1mg of OSAE and OSWE (12 ng/mg and 19 ng/mg dwt respectively) used for cell based assays was very low. It suggests that several other metabolites along with eugenol are responsible for the efficacy of the extracts.

Testosterone-derived estradiol production by male endothelium is robust and dependent on p450 aromatase via estrogen receptor alpha

Vascular endothelium expresses both the estrogen receptors (ERs) α and β, and ERα mediates development of early atherosclerosis in male mice. This process is thought to be testosterone-dependent. We hypothesized that male murine aortic endothelium produces robust levels of estradiol by aromatase conversion of testosterone, and that regulation of this process is mediated by the presence of ERs, primarily ERα. Aortic endothelium was isolated from ERα knockout (ERα -/-) and wild-type (ERα +/+) male mice and treated with testosterone or the 5α reduction product dihydrotestosterone (DHT), with or without the P450 aromatase inhibitor anastrazole, or a non-specific estrogen receptor antagonist. Aromatase gene expression and estradiol production were assayed. Treatment with testosterone, but not DHT, caused increased aromatase expression and estradiol production in ERα +/+ endothelium that was attenuated by disruption of ERα in the ERα -/- group. Anastrazole inhibition of aromatase reduced testosterone-induced aromatase expression and estradiol levels in both ERα -/- and ERα +/+ endothelium. Antagonism of both ERs decreased testosterone-induced aromatase expression in both wild-type and knockout groups. The effects of the receptor antagonist on estradiol production differed between the two groups, however, with a reduction in estradiol release from the ERα +/+ cells and complete abolition of estradiol release from the ERα -/- cells. Thus, estradiol production in vascular endothelium from male mice is robust, depends on the aromatic conversion of testosterone and requires functional ERα to achieve maximal levels of estradiol generation. Local vascular production of aromatase-mediated estradiol in response to circulating testosterone may affect ERα-dependent mechanisms to increase susceptibility to early atheroma formation in male mice. This pathway may have important therapeutic relevance for reducing the risk of atherosclerotic cardiovascular disease in human males.

Adsorption capacity of poly(ether imide) microparticles to uremic toxins

Uremia is a phenomenon caused by retention of uremic toxins in the plasma due to functional impairment of kidneys in the elimination of urinary waste products. Uremia is presently treated by dialysis techniques like hemofiltration, dialysis or hemodiafiltration. However, these techniques in use are more favorable towards removing hydrophilic than hydrophobic uremic toxins. Hydrophobic uremic toxins, such as hydroxy hipuric acid (OH-HPA), phenylacetic acid (PAA), indoxyl sulfate (IDS) and p-cresylsulfate (pCRS), contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease. Therefore, objective of the present study is to test adsorption capacity of highly porous microparticles prepared from poly(ether imide) (PEI) as an alternative technique for the removal of uremic toxins. Two types of nanoporous, spherically shaped microparticles were prepared from PEI by a spraying/coagulation process.PEI particles were packed into a preparative HPLC column to which a mixture of the four types of uremic toxins was injected and eluted with ethanol. Eluted toxins were quantified by analytical HPLC. PEI particles were able to adsorb all four toxins, with the highest affinity for PAA and pCR. IDS and OH-HPA showed a partially non-reversible binding. In summary, PEI particles are interesting candidates to be explored for future application in CKD.

Postprandial apoE Isoform and Conformational Changes Associated with VLDL Lipolysis Products Modulate Monocyte Inflammation

Objective Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation. Methods and Results We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4. Conclusion Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

Genetic engineering of AtAOX1a in Saccharomyces cerevisiae prevents oxidative damage and maintains redox homeostasis

This study aimed to validate the physiological importance of Arabidopsis thaliana alternative oxidase 1a (AtAOX1a) in alleviating oxidative stress using Saccharomyces cerevisiae as a model organism. The AOX1a transformant (pYES2AtAOX1a) showed cyanide resistant and salicylhydroxamic acid (SHAM)‐sensitive respiration, indicating functional expression of AtAOX1a in S. cerevisiae. After exposure to oxidative stress, pYES2AtAOX1a showed better survival and a decrease in reactive oxygen species (ROS) when compared to S. cerevisiae with empty vector (pYES2). Furthermore, pYES2AtAOX1a sustained growth by regulating GPX2 and/or TSA2, and cellular NAD+/NADH ratio. Thus, the expression of AtAOX1a in S. cerevisiae enhances its respiratory tolerance which, in turn, maintains cellular redox homeostasis and protects from oxidative damage.

Influence of nanoporous poly(ether imide) particle extracts on human aortic endothelial cells (HAECs)

Accumulated uremic toxins like indoxyl sulphate, hippuric acid and p-cresyl sulphates in renal failure patients stimulate proinflammatory effects, and consequently kidney and cardiovascular diseases. Low clearance rate of these uremic toxins from the blood of uremic patients by conventional techniques like hemodialysis is due to their strong covalent albumin binding (greater than 95%) and hydrophobic nature, which led to alternatives like usage of hydrophobic adsorbers in removing these toxins from the plasma of kidney patients. Polymers like polyethylene, polyurethane, polymethylmethacrylate, cellophane and polytetrafluoroethylene were already in use as substitutes for metal devices as dialysis membranes. Among new synthetic polymers, one such ideal adsorber material are highly porous microparticles of poly(ether imide) (PEI) with diameters in the range from 50-180μm and a porosity around 88±2% prepared by a spraying and coagulation process.It is essential to make sure that these synthetic polymers should not evoke any inflammatory or apoptotic response during dialysis. Therefore in our study we evaluated in vitro effect of PEI microparticle extracts in human aortic endothelial cells (HEACs) concerning toxicity, inflammation and apoptosis. No cell toxicity was observed when HAECs were treated with PEI extracts and inflammatory/apoptotic markers were not upregulated in presence of PEI extracts. Our results ensure biocompatibility of PEI particles and further hemocompatibility of particles will be tested.