Ellagic Acid from Terminalia arjuna Fruits Protects Against Chromium and Cobalt Toxicity in Primary Human Lymphocytes.

Increased accumulation of heavy metal ions such as Cr6+ and Co2+ due to release from prostheses and metallic implants has been reported. These metal ions have been shown to affect both resting and activated lymphocytes. Natural remedies towards mitigating the cytotoxic effects of metal ions are clearly warranted. Polyphenolic compounds which are part of hydrolysable tannins from natural plant sources are considered effective in cheating heavy metal ions in a biological system. We have isolated and characterized a polyphenolic compound (ellagic acid) from Terminalia arjuna fruits that has been tested for its ability to attenuate the metal ion toxicity in primary human lymphocytes in culture. Cr6+ and Co2+ (100 µM) decreased lymphocyte viability and proliferation and increased apoptosis of resting as well as CD3 and/or CD28-stimulated lymphocytes. Metal ions markedly diminished the cytokine (interleukin-2 and interferon-γ) secretion from activated lymphocytes. Pretreatment with ellagic acid at 25, 50, and 100 µM concentrations effectively improved viability and proliferative responses of both resting and activated lymphocytes, while attenuating the apoptotic index. Ellagic acid also tended to normalize the cytokine secretion from the activated lymphocytes even in the presence of metal ions, suggesting broad effects on the adaptive immune system.

Zinc ionophores isolated from Terminalia bellirica fruit rind extract protect against cardiomyocyte hypoxia/reoxygenation injury.

The study aimed to isolate and characterize zinc ionophores from Terminalia bellirica fruit using a liposome assay and test its utility in H9c2 rat cardiomyoblasts cells subjected to hypoxia/reoxygenation. Ethyl acetate extract that exhibited zinc ionophore activity was resolved to yield three polyphenols that were characterized as epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and epigallocatechin (EGC) by nuclear magnetic resonance and electrospray ionization-mass spectra. The polyphenols enhanced the uptake of zinc into the liposomes and increased FluoZin-3 fluorescence. These polyphenols in the presence of 10 µM ZnCl2 enhanced the zinc import into H9c2 cells, whose intracellular zinc levels were otherwise lowered upon hypoxia/reoxygenation. EGCG proved to be more potent than ECG, which indeed was more effective than EGC in improving cellular zinc levels and in attenuating the apoptosis of H9c2 cells after hypoxia/reoxygenation injury. The polyphenols required zinc for anti-apoptotic effect. The cardioprotective effect is indeed due to enhanced zinc uptake mediated by these polyphenols.

Andrographolide suppresses cisplatin-induced endothelial hyperpermeability through activation of PI3K/Akt and eNOS -derived nitric oxide.

Cisplatin upregulate the intercellular adhesion molecule expression on the surface of endothelium, which in turn mediates enhanced infiltration by monocytes or leukocytes, resulting in endothelial dysfunction. Here we examined changes induced by andrographolide, a diterpenoid lactone isolated from Andrographis paniculata on endothelial cell activation and hyperpermeability in cisplatin-stimulated endothelial cells. Cisplatin upregulated endothelial ICAM-1 expression, through an NF-κB dependent mechanism, that also required the enhanced translocation of Protein Kinase C-α (PKC) onto the plasma membrane, phosphorylation of transient receptor potential channel 1 (TRPC), leading to store-operated Ca2+-entry (SOCE), endothelial cell dysfunction and hyperadhesion of U937 monocytes. Pretreatment of endothelial cells with andrographolide prior to stimulation with cisplatin resulted in activation of PI3K/Akt and eNOS, production of nitric oxide (NO) and cGMP, with a consequential lowering of endothelial cell leakiness and improved transendothelial electrical resistance. Andrographolide-induced NO was essential for NF-κB inhibition, lowered ICAM-1 expression as well as prevention of SOCE and reduced the U937 binding to cisplatin-stimulated endothelial cells.

Zinc-dependent changes in oxidative and endoplasmic reticulum stress during cardiomyocyte hypoxia/reoxygenation.

Myocardial zinc dyshomeostasis is associated with caspase-3 activation, ErbB2 degradation and apoptosis during hypoxia/reoxygenation. Zinc pyrithione replenishes intracellular zinc, suppresses caspase-3, augments ErbB2 levels and improves cell survival. We hypothesize that zinc is capable of modulating redox and endoplasmic reticulum (ER) stress in the setting of cardiomyocyte hypoxia-reoxygenation. Hypoxia/reoxygenation lowered intracellular zinc, increased ER as well as oxidative stress in H9c2 cells, both of which were effectively attenuated by zinc supplementation. Silencing of gp91phox attenuated oxidative and ER stress, decreased caspase-3 activation and improved cell survival. Mimicking the oxidative insult using 50 µM H2O2 increased the caspase-3 activity that correlated with decreased ErbB2 levels, concomitant with augmented ER stress. N-acetyl cysteine (NAC) administration completely suppressed ER stress as well as caspase-3 activity. Zinc depletion using TPEN also resulted in lowered ErbB2 and increased apoptosis, along with NOX2 mRNA upregulation, increased oxidative and ER stress. Repletion with zinc suppressed NOX2 mRNA, lowered oxidative as well as ER stress and decreased cell death. These results suggest that zinc dyshomeostasis, along with oxidative stress contribute to the unfolded protein response during myocardial H/R and that zinc replenishment corrects zinc homeostasis, alleviates associated stress and improves cardiomyocyte survival.

Dietary phytate lowers K-ras mutational frequency, decreases DNA-adduct and hydroxyl radical formation in azoxymethane-induced colon cancer.

OBJECTIVES: Dietary phytate is known to protect against azoxymethane (AOM)-induced preneoplastic lesions. The present study was designed to determine whether dietary phytate affects mutation frequency in colon epithelial cells challenged with azoxymethane in vivo, through lowering the formation of O6-methyl guanosine (O6-MeG) and 8-hydroxy deoxyguanosine (8-OHdG) adducts. MATERIALS AND METHODS: We used Fisher F344 rats induced with AOM for 20 weeks and undertook 1% or 2% phytate supplementation for subsequent 16 weeks to monitor the mutation frequencies of one of the candidate genes, K-ras, along with DNA adduct load. RESULTS: Dietary phytate significantly suppressed aberrant crypt foci formation and effectively inhibited colon tumor formation in a dose-dependent manner. DNA sequencing results demonstrated that 60% of the colon tumors from AOM-treated and control diet fed animals showed GGT to GAT transition and 40% of the tumors showed GGT to GTT transversion at codon 12, along with 18% of the tumors showing GGC to CGC transversion at codon 13. Phytate supplementation at 1 and 2% lowered the frequency of GGT > GAT to 30 and 10%, respectively. Phytate supplementation also nullified the codon 13 mutations. No mutations were observed at codon 61 in any of the experimental groups. CONCLUSION: The lowered frequency of K-ras mutations correlated with decreased formation of hydroxyl radicals, O5-meG and 8-OH-dG levels in phytate-supplemented animals with lowered tumor burden.

Comparative Response of Cardiomyocyte ZIPs and ZnTs to Extracellular Zinc and TPEN.

Intracellular zinc concentrations are tightly regulated by the coordinated regulation of ZIPs and ZnTs. Very little is known about the regulation of these transporters in cardiomyocytes, in response to extracellular zinc. Adult rat cardiomyocytes express ZnTs 1, 2, 5, and 9, in addition to ZIPs 1, 2, 3, 6, 7, 9, 10, 11, 13, and 14. We have determined the intracellular free zinc levels using Zinpyr-1 fluorescence and studied response of ZIP and ZnT mRNA by real-time PCR to the changes in extracellular zinc and TPEN in adult rat ventricular myocytes. TPEN downregulated ZnT1, ZnT2, and ZIP11 mRNAs but upregulated ZnT5, ZIP2, ZIP7, ZIP10, ZIP13, and ZIP14 mRNAs. Zinc supplementation upregulated ZnT1, ZnT2 mRNA but downregulated ZnT5, ZIP1, ZIP2, ZIP3, ZIP7, ZIP9, and ZIP10 mRNA. The negative regulation of ZIPs by zinc excess can be explained in terms of zinc homeostasis as these transporters may act to protect cells from zinc over accumulation by reducing zinc influx when the extracellular concentration of zinc is high. Similarly, the ZnT expression appears to be regulated to avoid loss of zinc from the intracellular milieu, under zinc-deficient conditions.

Low Ctr1p, due to lack of Sco1p results in lowered cisplatin uptake and mediates insensitivity of rho0 yeast to cisplatin.

Copper and cisplatin share copper transporter 1 (Ctr1) for cellular import. Copper depletion increases sensitivity of wild type yeast to cisplatin, whereas mitochondrial DNA-deficient rho0 cells are resistant to cisplatin. In the current study, we sought to determine whether copper deprivation modulates sensitivity of rho0 yeast to cisplatin. Yeast cultures grown in low copper medium and exposed to bathocuproine disulfonic acid resulted in significant reduction of intracellular copper. We report here that low copper medium rendered wild type hypersensitive to cisplatin, but failed to sensitize rho0 yeast to cisplatin. Wild type yeast grown in low copper medium exhibited ~2.0 fold enhanced cytotoxicity in survival and colony-forming ability compared to copper adequate wild type cells. The effect of copper restriction on cisplatin sensitivity was associated with upregulation of copper transporter 1 mRNA as well as protein, facilitating enhanced uptake and accumulation of cisplatin. Rho0 yeast also showed increased copper transporter 1 mRNA upon copper restriction, but failed to increase corresponding protein. Loss of synthesis of cytochrome coxidase 1 protein (Sco1) in rho0 cells deregulated copper transporter 1, impaired Pt uptake and lowered cytotoxicity, despite lowered glutathione levels. Sco1Δ mutants exhibited low copper transporter 1, reduced Pt accumulation suggesting that Sco1 mediated regulation of copper transporter 1 is responsible for altered sensitivity to cisplatin. Rho0 cells demonstrated loss of Sco1, resulting in copper deficiency by lowering copper transporter 1 abundance, via mechanism involving increased turnover due to ubiquitination. These findings reveal that a Sco1-dependent mitochondrial signal regulates cellular cisplatin import and cytotoxicity.

Zinc Dyshomeostasis in Cardiomyocytes after Acute Hypoxia/Reoxygenation.

Zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. The objective of this study was to investigate the expression profile of zinc regulated transporter like- and iron-regulated transporter-like proteins (ZIPs) and zinc transporter proteins (ZnTs) in cardiomyocytes and their modulation in response to hypoxia and reoxygenation. Adult rat ventricular myocytes (ARVMs) were subjected to 6 h of hypoxia, followed by 18 h of reoxygenation. Intracellular and extracellular zinc concentrations were determined using Fluozin-3 and Newport Green fluorescence, respectively. Expression of ZnTs 1, 2, 5, and 9 along with ZIPs 1, 2, 3, 6, 7, 9, 10, 11, 13, and 14 was detectable in the cardiomyocytes by real-time reverse transcriptase polymerase chain reaction. Hypoxia elicited accumulation of intracellular free zinc, but subsequent reoxygenation resulted in striking loss of intracellular free zinc and decreased the cardiomyocyte viability. Concomitantly, extracellular zinc levels dropped rapidly during hypoxia, but increased after reoxygenation. Immunoblotting analysis revealed that hypoxia increased the expression of ZnT1, but reoxygenation significantly increased the expression of ZnTs 2 and 5. Neither hypoxia nor reoxygenation altered the levels of ZnT9. Increased intracellular zinc at the end of hypoxia is related to enhanced expression of ZIPs, whereas decreased intracellular zinc during reoxygenation appears to be due to lowered expression of all ZIPs, in addition to elevated levels of ZnTs 2 and 5. These results thus suggest that there is impaired accumulation of intracellular zinc during reoxygenation, due to overexpression of specific ZnTs and downregulation of ZIP expression.

Loss of ErbB2-PI3K/Akt signaling prevents zinc pyrithione-induced cardioprotection during ischemia/reperfusion.

OBJECTIVES: The purpose of this study was to determine if zinc homeostasis is affected during ischemia/reperfusion, if so, whether zinc pyrithione limits myocardial cell death and improves hemodynamics when administered as an adjunct to reperfusion and if ErbB receptor tyrosine kinases that are important for the long-term structural integrity of the heart are indispensable for reperfusion salvage. METHODS: Isolated perfused rat hearts were subjected to 35min of global ischemia and reperfused for 120min to determine the relative intracellular zinc levels by TSQ staining. The hearts were reperfused in the presence of incremental concentrations of zinc pyrithione for the first 10min during reperfusion. Silencing or blockade of ErbB2 using a monoclonal antibody, ErbB2 kinase inhibition and PI3kinase inhibition was used to study their critical role in zinc pyrithione-induced cardioprotection. RESULTS: We found that there was a profound decrease in intracellular zinc after ischemia/reperfusion resulting in increased apoptosis, caspase-3 activation, and infarct size. A dose-dependent reduction of infarct size with zinc pyrithione in the range of 5-20µmol/l (optimal protection was seen at 10µmol/l with infarct size of 16±2% vs. I/R vehicle, 33±2%, p<0.01). Increased TUNEL staining and caspase-3 activity observed after ischemia/reperfusion were attenuated by zinc pyrithione administration during the reperfusion. Moreover, this protection was sensitive to silencing and blockade of ErbB2, inhibition of ErbB2 kinase activity or PI3-kinase activity. Western blot analysis revealed that zinc pyrithione resulted in decreased caspase-3 activation, rapid stabilization of ErbB2/ErbB1 heterodimers, and increased activation of PI3K/Akt signaling cascade. CONCLUSIONS: Zinc pyrithione attenuates lethal perfusion-induced injury in a manner that is reliant on ErbB2/PI3K/Akt activity.

Cisplatin cytotoxicity is dependent on mitochondrial respiration in Saccharomyces cerevisiae.

OBJECTIVES: To understand the role of mitochondrial respiration in cisplatin sensitivity, we have employed wild-type and mitochondrial DNA depleted Rho0 yeast cells. MATERIALS AND METHODS: Wild type and Rho0 yeast cultured in fermentable and non-fermentable sugar containing media, were studied for their sensitivity against cisplatin by monitoring growth curves, oxygen consumption, pH changes in cytosol/mitochondrial compartments, reactive oxygen species production and respiratory control ratio. RESULTS: Wild-type yeast grown on glycerol exhibited heightened sensitivity to cisplatin than yeast grown on glucose. Cisplatin (100 µM), although significantly reduced the growth of wild- type cells, only slightly altered the growth rate of Rho0 cells. Cisplatin treatment decreased both pHcyt and pHmit to a similar extent without affecting the pH difference. Cisplatin dose-dependently increased the oxidative stress in wild-type, but not in respiration-deficient Rho0 strain. Cisplatin decreased the respiratory control ratio. CONCLUSION: These results suggest that cisplatin toxicity is influenced by the respiratory capacity of the cells and the intracellular oxidative burden. Although cisplatin per se slightly decreased the respiration of yeast cells grown in glucose, it did not disturb the mitochondrial chemiosmotic gradient.

Intracellular zinc status influences cisplatin-induced endothelial permeability through modulation of PKCα, NF-κB and ICAM-1 expression.

Platinum-based chemotherapeutic regimen induces vascular dysfunction. Action of cisplatin on endothelial cells is mediated by protein kinase C (PKC-α), which further activates nuclear factor-κB (NF-κB) and induces canonical transient receptor potential channel (TRPC1) and intercellular adhesion molecule (ICAM-1) expression. Increased ICAM-1 contributes to hyperadhesion of monocytes and endothelial dysfunction. PKC-α is also involved in phosphorylation of TRPC1, resulting in store-operated calcium entry (SOCE) and further activation of NF-κB. Although the role of altered intracellular zinc status is not known in cisplatin-induced vascular dysfunction, because of the ability of zinc to modulate PKC-α, NF-κB activity, we hypothesized that zinc can ameliorate the extent of endothelial dysfunction induced by cisplatin. Human umbilical vein endothelial cells treated with cisplatin (8.0µg/ml) showed lowered intracellular free zinc, concomitant with enhanced activation of PKC-α, NF-κΒ activation, TRPC1, SOCE and ICAM-1 levels. Zinc deficiency per se induced using membrane permeable chelator (TPEN) mimicked the cisplatin-induced PKC-α, NF-κB activation and ICAM-1 expression, but also activated Activator Protein-1 (AP-1). Zinc supplementation (2.0-10.0µM) to the endothelial cells during cisplatin treatment or TPEN-induced zinc deficiency suppressed PKC-α, NF-κB, TRPC1, SOCE activation and lowered the ICAM-1 expression. Zinc supplementation thereby effectively decreased the cisplatin-induced endothelial permeability and adherence of the activated endothelial cells to U937 monocytes.

Zinc pyrithione inhibits caspase-3 activity, promotes ErbB1-ErbB2 heterodimerization and suppresses ErbB2 downregulation in cardiomyocytes subjected to ischemia/reperfusion.

Heart tissue becomes zinc-depleted and the capacity to mobilize labile zinc is diminished, indicating zinc dyshomeostasis during ischemia/reperfusion (I/R). Apparently, zinc pyrithione restores the basal zinc levels during I/R and prevents apoptosis by activating phosphatidyl inositol-3-kinase/Akt and targeting mitochondrial permeability transition. Receptor tyrosine kinases of the ErbB family (ErbB1 to ErbB4) are cell surface proteins that can regulate cell growth, proliferation and survival. Previous studies have shown that zinc pyrithione-induced activation of PI3kinase/Akt requires ErbB2 expression. On the other hand, while I/R decreases ErbB2 levels causing cardiomyocyte dysfunction and cell death, zinc pyrithione restores ErbB2 levels and maintains cardiomyocyte function. H9c2 cells expressed all the four ErbBs, although the expression of ErbB1 and ErbB2 were higher compared to ErbB3 and ErbB4. Hypoxia/Reoxygenation (H/R) had opposing effects on the mRNA expression of ErbB1 and ErbB2. ErbB2 mRNA levels were enhanced, but corresponding ErbB2 protein levels decreased after reoxygenation. H/R induced the degradation of ErbB2 in caspase-3 dependent manner, with the formation of a 25kDa fragment. This fragment could be detected after H/R only upon treatment of the cells with a proteasomal inhibitor, ALLN, suggesting that caspase-mediated cleavage of 185kDa ErbB2 results in C-terminal cleavage and formation of 25kDa fragment, which is further degraded by proteasome. Heterodimerization and phosphorylation of ErbB2/ErbB1 which decreased upon reoxygenation, was promoted by zinc pyrithione. Zinc pyrithione effectively suppressed the caspase activation, decreased the proteolytic cleavage of ErbB2, enhanced the phosphorylation and activation of ErbB1-ErbB2 complexes and improved the cell survival after hypoxia/reoxygenation.

Silencing of PKC-α, TRPC1 or NF-κB expression attenuates cisplatin-induced ICAM-1 expression and endothelial dysfunction.

Platinum-based chemotherapy has been associated with increased long-term cardiovascular events. Also noteworthy is the accumulating awareness of early vascular toxicity occurring at the time of chemotherapy or immediately thereafter. The objective of the study was to delineate the molecular mechanisms associated with the early vascular toxicity and test the molecular silencing approach towards attenuating the endothelial dysfunction during platinum-based chemotherapy. Human umbilical vein endothelial cells (HUVECs) were treated with varying concentrations of cisplatin (1.0-10.0µg/ml) or vehicle control (0.1% dimethyl sulfoxide) for monitoring the changes in Intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression viz. a viz. altered activation of protein kinase C (PKC) isoforms, transient receptor potential channel (TRPC) 1 expression, Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), Store Operated Ca(2+) Entry (SOCE) in cisplatin-induced endothelial permeability and adherence of the activated endothelial cells to human monocyte-like U937 cells. Silencing of either PKC-α, TRPC1 or p65 subunit of NF-κB, all resulted in significant alleviation of cisplatin-induced endothelial dysfunction. At concentrations ≥8µg/ml, cisplatin induced a significant increase in the expression of ICAM-1 mRNA as well as protein. This was mediated by changes in PKC-α membrane translocation, NF-κB activation, increased expression as well as phosphorylation of TRPC1 and enhanced SOCE, leading to hyperpermeability and leakage of albumin. Increased adherence of U937 monocytes to cisplatin-activated endothelial cells was evident. Cisplatin challenge activates PKC-α, which in turn phosphorylated TRPC1 resulting in enhanced Ca(2+) entry. Increased Ca(2+) flux is required for activation of NF-κB and ICAM-1 expression. Enhanced ICAM-1 expression promotes monocyte binding to endothelial cells and increased endothelial hyperpermeability.

In vitro biological evaluation of glyburide as potential inhibitor of collagenases.

In tissues with upregulated MMP activity, MMP inhibition remains one of the key strategies. Potential inhibitors of MMPs have been tested for almost 30 years, but none have reached clinical utility due to bioavailability issues and adverse effects. This study utilized the approach of drug repurposing for exploring glyburide as a potential inhibitor against collagenases. In silico molecular docking studies were carried out to probe the interactions of glyburide with the active site Zn. Collagenase enzyme activity measurements and zymography analyses using conditioned medium from lung fibroblasts, rheumatoid synovial fibroblasts, and osteoblasts were carried out to confirm the inhibitory activity. Glyburide binds and interacts with the catalytic Zn residues of the collagenases, as evidenced by in silico molecular docking studies. Fluorescence enzyme activity measurements reveal that glyburide inhibits peptide substrate cleavage by all three collagenases in a dose-dependent manner. Collagen zymography studies validated inhibition of these collagenases by glyburide. These results identify glyburide as a potential inhibitor of collagenases and provide an insight into the mechanism of action of this small molecule. Thus, glyburide may offer additional advantages in diabetics, in controlling MMP activation and collagen degradation and could aid in the treatment of diseases with aberrant MMP activity.

Effect of dietary antioxidants on the cytostatic effect of acrylamide during copper-deficiency in Saccharomyces cerevisiae.

Acrylamide exposure increases oxidative stress and causes cytotoxicity. In order to understand the role of oxidative stress in acrylamide toxicity, we utilized Saccharomyces cerevisiae as a model organism grown in Yeast Peptone Dextrose (YPD) or Copper-Deficient Medium (CDM). Although the growth curves of yeast were comparable in these media, acrylamide treatment resulted in significant growth inhibition and colony formation only in the CDM. Copper-deficiency induced a decrease in the intracellular metallothionein levels, along with reduced Cu, Zn-SOD activity that appeared to increase the sensitivity of the yeast to the cytostatic effect of acrylamide. Increased dichlorofluorescein (DCF) fluorescence, enhanced formation of para-phenyl tertiary butyl nitrone (PBN)-hydroxyethyl adducts and a lowered reduced glutathione (GSH) content were observed under copper-deficient conditions, when challenged with acrylamide. The cytostatic effects and intracellular redox changes in response to acrylamide were ameliorated by antioxidant molecules viz. a viz. curcumin, ß-carotene, vanillin and caffeic acid, which effectively decreased the oxidative stress and improved the growth recovery.

Advanced glycation end products: role in pathology of diabetic cardiomyopathy.

Increasing evidence demonstrates that advanced glycation end products (AGEs) play a pivotal role in the development and progression of diabetic heart failure, although there are numerous other factors that mediate the disease response. AGEs are generated intra- and extracellularly as a result of chronic hyperglycemia. Then, following the interaction with receptors for advanced glycation end products (RAGEs), a series of events leading to vascular and myocardial damage are elicited and sustained, which include oxidative stress, increased inflammation, and enhanced extracellular matrix accumulation resulting in diastolic and systolic dysfunction. Whereas targeting glycemic control and treating additional risk factors, such as obesity, dyslipidemia, and hypertension, are mandatory to reduce chronic complications and prolong life expectancy in diabetic patients, drug therapy tailored to reducing the deleterious effects of the AGE-RAGE interactions is being actively investigated and showing signs of promise in treating diabetic cardiomyopathy and associated heart failure. This review shall discuss the formation of AGEs in diabetic heart tissue, potential targets of glycation in the myocardium, and underlying mechanisms that lead to diabetic cardiomyopathy and heart failure along with the use of AGE inhibitors and breakers in mitigating myocardial injury.

Attenuation of non-enzymatic thermal glycation of bovine serum albumin (BSA) using ß-carotene.

Although heat treatment of proteins is believed to promote and accelerate glycation, the accompanying structural changes in proteins because of heat denaturation and/or glycation are not completely understood. In addition, there is an increasing interest for inhibitors of thermal glycation in food processing. ß-Carotene is a naturally occurring carotenoid found in many vegetables, fruits and herbs, with known antioxidant activity. However, it has not been identified if ß-carotene possesses anti-glycation activity. We have tested the anti-glycation capacity of ß-carotene in the bovine serum albumin (BSA)/glucose system that was heated to 55 and 65 °C for 3 days and studied the level of glycation, conformational alterations in BSA, and changes in hydrophobicity, due to thermal treatment and/or glycation. ß-Carotene exhibited significant inhibitory effects on the formation of advanced glycation end products (AGEs) and also prevented the secondary structural changes in BSA due to thermal glycation. Our results represent the anti-glycative properties of ß-carotene in food systems where such thermal conditions prevail.

Short-term, long-term and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary dysplasia (BPD) remains a main complication of extreme prematurity and currently lacks efficient treatment. Rat bone marrow-derived mesenchymal stem cells (MSC) prevent lung injury in an oxygen-induced model of BPD. Human cord is an advantageous source of stem cells that is especially appealing for the treatment of neonatal diseases. The therapeutic benefit after established lung injury and long-term safety of cord-derived stem cells is unknown. METHODS: Human cord-derived perivascular cells (PCs) or cord blood-derived MSCs were delivered prophylactically or after established alveolar injury into the airways of newborn rats exposed to hyperoxia, a well-established BPD model. RESULTS: Rat pups exposed to hyperoxia showed the characteristic arrest in alveolar growth with air space enlargement and loss of lung capillaries. PCs and MSCs partially prevented and rescued lung function and structure. Despite therapeutic benefit, cell engraftment was low, suggesting that PCs and MSCs act via a paracrine effect. Accordingly, cell free-derived conditioned media from PCs and MSCs also exerted therapeutic benefit when used either prophylactically or therapeutically. Finally, long-term (6 months) assessment of stem cell or conditioned media therapy showed no adverse lung effects of either strategy, with persistent improvement in exercise capacity and lung structure. CONCLUSIONS: Human umbilical cord-derived PCs and MSCs exert short- and long-term therapeutic benefit without adverse lung effects in this experimental model and offer new therapeutic options for lung diseases characterised by alveolar damage.

Tissue protection and endothelial cell signaling by 20-HETE analogs in intact ex vivo lung slices.

The capacity to follow cell type-specific signaling in intact lung remains limited. 20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous fatty acid that mediates signaling for a number of key physiologic endpoints in the pulmonary vasculature, including cell survival and altered vascular tone. We used confocal microscopy to identify enhanced reactive oxygen species (ROS) production in endothelial cell (EC)s in intact lung evoked by two stable analogs of 20-HETE, 20-5,14-HEDE (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid) and 20-5,14-HEDGE (N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine). These analogs generated increased ROS in cultured pulmonary artery endothelial cells as well. 20-HETE analog treatment decreased apoptosis of pulmonary tissue exposed to hypoxia-reoxygenation (HR) ex vivo. Enhanced ROS production and apoptosis were confirmed by biochemical assays. Our studies identify physiologically critical, graded ROS from ECs in live lung tissue ex vivo treated with 20-HETE analogs and protection from HR-induced apoptosis. These methodologies create exciting possibilities for studying signaling by stable 20-HETE analogs and other factors in pulmonary endothelial and other lung cell types in their native milieu.

Loss of angiotensin-converting enzyme-2 exacerbates diabetic cardiovascular complications and leads to systolic and vascular dysfunction: a critical role of the angiotensin II/AT1 receptor axis.

RATIONALE: Diabetic cardiovascular complications are reaching epidemic proportions. Angiotensin-converting enzyme-2 (ACE2) is a negative regulator of the renin-angiotensin system. We hypothesize that loss of ACE2 exacerbates cardiovascular complications induced by diabetes. OBJECTIVE: To define the role of ACE2 in diabetic cardiovascular complications. METHODS AND RESULTS: We used the well-validated Akita mice, a model of human diabetes, and generated double-mutant mice using the ACE2 knockout (KO) mice (Akita/ACE2(-/y)). Diabetic state was associated with increased ACE2 in Akita mice, whereas additional loss of ACE2 in these mice leads to increased plasma and tissue angiotensin II levels, resulting in systolic dysfunction on a background of impaired diastolic function. Downregulation of SERCA2 and lipotoxicity were equivalent in Akita and Akita/ACE2KO hearts and are likely mediators of the diastolic dysfunction. However, greater activation of protein kinase C and loss of Akt and endothelial nitric oxide synthase phosphorylation occurred in the Akita/ACE2KO hearts. Systolic dysfunction in Akita/ACE2KO mice was linked to enhanced activation of NADPH oxidase and metalloproteinases, resulting in greater oxidative stress and degradation of the extracellular matrix. Impaired flow-mediated dilation in vivo correlated with increased vascular oxidative stress in Akita/ACE2KO mice. Treatment with the AT1 receptor blocker, irbesartan rescued the systolic dysfunction, normalized altered signaling pathways, flow-mediated dilation, and the increased oxidative stress in the cardiovascular system. CONCLUSIONS: Loss of ACE2 disrupts the balance of the renin-angiotensin system in a diabetic state and leads to an angiotensin II/AT1 receptor-dependent systolic dysfunction and impaired vascular function. Our study demonstrates that ACE2 serves as a protective mechanism against diabetes-induced cardiovascular complications.