Yimin Qin

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt‐NOS signaling

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R‐induced cell death [18.0 ± 1.8% (GSPE, 50 µg/ml) vs. 42.3 ± 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 200 µM) significantly reduced GSPE‐induced NO release and its associated cardioprotection [32.7 ± 2.7% (GSPE + L‐NAME) vs. 18.0 ± 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt‐eNOS pathway, we utilized the Akt inhibitor API‐2. API‐2 (10 µM) abrogated GSPE‐induced protection [44.3% ± 2.2% (GSPE + API‐2) vs. 27.0% ± 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE‐treated cells and attenuated GSPE‐induced NO increases. Simultaneously blocking NOS activation (L‐NAME) and Akt (API‐2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt‐eNOS signaling. J. Cell. Biochem. 107: 697–705, 2009.

Synergistic effect of Scutellaria baicalensis and grape seed proanthocyanidins on scavenging reactive oxygen species in vitro.

Scutellaria baicalensis (SbE) is a commonly used Chinese herb medicine and grape seed proanthocyanidins is a popular herbal supplement in the United States. Both herbs have been shown to possess potent antioxidant effects. Using an in vitro model to produce the reactive oxygen species (ROS) generation (H2O2/FeSO4 for hydroxyl radicals, xanthine/xanthine oxidase for suproxide), we observed that Scutellaria baicalensis and grape seed proanthocyanidins acted synergistically to scavenge ROS. Our data suggest that a combination of these two herbs can potentially enhance their antioxidant efficacy, allowing lower dosages of each drug to be used. This has the advantage of avoiding possible side effects that may arise when higher doses of a single herb are used in an attempt to achieve a maximum degree of antioxidant activity.

Cytotoxicity induced by grape seed proanthocyanidins: role of nitric oxide.

Grape seed proanthocyanidin extract (GPSE) at high doses has been shown to exhibit cytotoxicity that is associated with increased apoptotic cell death. Nitric oxide (NO), being a regulator of apoptosis, can be increased in production by the administration of GSPE. In a chick cardiomyocyte study, we demonstrated that high-dose (500 μg/ml) GSPE produces a significantly high level of NO that contributes to increased apoptotic cell death detected by propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. It is also associated with the depletion of intracellular glutathione (GSH), probably due to increased consumption by NO with the formation of S-nitrosoglutathione. Co-treatment with L-NAME, a NO synthase inhibitor, results in reduction of NO and apoptotic cell death. The decline in reduced GSH/oxidized GSH (GSSG) ratio is also reversed. N-Acetylcysteine, a thiol compound that reacts directly with NO, can reduce the increased NO generation and reverse the decreased GSH/GSSG ratio, thereby attenuating the cytotoxicity induced by high-dose GSPE. Taken together, these results suggest that endogenous NO synthase (NOS) activation and excessive NO production play a key role in the pathogenesis of high-dose GSPE-induced cytotoxicity.

Fas Resistance of Leukemic Eosinophils Is Due to Activation of NF-κB by Fas Ligation

TNF family receptors can lead to the activation of NF-κB and this can be a prosurvival signal in some cells. Although activation of NF-κB by ligation of Fas (CD95/Apo-1), a member of the TNFR family, has been observed in a few studies, Fas-mediated NF-κB activation has not previously been shown to protect cells from apoptosis. We examined the Fas-induced NF-κB activation and its antiapoptotic effects in a leukemic eosinophil cell line, AML14.3D10, an AML14 subline resistant to Fas-mediated apoptosis. EMSA and supershift assays showed that agonist anti-Fas (CH11) induced nuclear translocation of NF-κB heterodimer p65(RelA)/p50 in these cells in both a time- and dose-dependent fashion. The influence of NF-κB on the induction of apoptosis was studied using pharmacological proteasome inhibitors and an inhibitor of IκBα phosphorylation to block IκBα dissociation and degradation. These inhibitors at least partially inhibited NF-κB activation and augmented CH11-induced cell death. Stable transfection and overexpression of IκBα in 3D10 cells inhibited CH11-induced NF-κB activation and completely abrogated Fas resistance. Increases in caspase-8 and caspase-3 cleavage induced by CH11 and in consequent apoptotic killing were observed in these cells. Furthermore, while Fas-stimulation of resistant control 3D10 cells led to increases in the antiapoptotic proteins cellular inhibitor of apoptosis protein-1 and X-linked inhibitor of apoptosis protein, Fas-induced apoptosis in IκBα-overexpressing cells led to the down-modulation of both of these proteins, as well as that of the Bcl-2 family protein, Bcl-xL. These data suggest that the resistance of these leukemic eosinophils to Fas-mediated killing is due to induced NF-κB activation.

Grape seed proanthocyanidins induce pro-oxidant toxicity in cardiomyocytes.

Grape seed proanthocyanidin extract (GSPE), a polyphenolic compound with antioxidant properties, may protect against cardiac ischemia and reperfusion injury. However, its potential toxicity at higher doses is unknown. The authors tested the effects of GSPE on reactive oxygen species (ROS) generation, cell survival, lactate dehydrogenase (LDH) release, and caspase-3 activity using chick cardiomyocytes incubated with GSPE at 5, 10, 50, 100, or 500 μg/mL in medium for 8 h. Exposure to increasing concentrations of GSPE (100 or 500 μg/mL) resulted in an increase in ROS generation and cell death as measured by propidium iodide uptake and LDH release. Caspase-3 activity was significantly increased fourfold in cells exposed to GSPE 500 μg/mL compared to controls; this was abolished by the selective caspase-3 inhibitor AC-Asp-Gln-Thr-Asp-H(50 μM), which aslso significantly reduced the cell death resulting from GSPE (500 μg/mL). The antioxidant N-acetylcysteine (NAC, 100 μM) reduced cell death induced by GSPE (500 μg/mL) but failed to attenuate caspase-3 activation. Collectively, the authors conclude that higher doses of GSPE could cause apoptotic cell injury via effector caspase-3 activation and subsequent induction of ROS generation. Consumers may take higher doses of dietary supplements in the belief that natural herbs have no major side effects. This study demonstrates that dosages of GSPE should be optimized to avoid potential harmful pro-oxidant effects.

Regulation of adhesion of AML14.3D10 cells by surface clustering of β2-integrin caused by ERK-independent activation of cPLA2

We examined the role of cell surface clustering of β2‐integrin caused by protein kinase C (PKC)‐activated‐cPLA2 in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12‐myristate 13‐acetate (PMA) caused time‐ and concentration‐dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti‐CD11b or anti‐CD18 monoclonal antibodies (mAb) directed against β2‐integrin. Inhibition of PKC with Ro‐31‐8220 or rottlerin blocked PMA‐induced cell adhesion in a concentration‐dependent fashion. Inhibition of cytosolic phospholipase A2 (cPLA2) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA‐induced cell adhesion. PMA caused time‐dependent p42/44 mitogen‐activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal‐regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA‐induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c‐Jun N‐terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA2 activity, which was inhibited by Ro‐31‐8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA2 inhibition. PMA stimulation also caused up‐regulation of CD11b on the AML cell surface. However, this up‐regulation was not affected by cPLA2‐ or PKC‐inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through β2‐integrin by PKC activation of cPLA2. This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering – rather than conformational change or up‐regulation of CD11b/CD18 – causes PMA stimulated adhesion of AML cells.

Blockade of caspase-2 Activity inhibits ischemia/ Reperfusion-induced Mitochondrial Reactive Oxygen Burst and cell Death in cardiomyocytes

We previously showed that initiator caspases-2 and -8 are prominently activated in ischemia/reperfusion (I/R)-induced injury in cardiomyocytes, but while blockade of caspase-2 activity enhanced cell survival, blockade of caspase-8 activity did not protect cardiomyocytes. Because apoptotic death in these cells is characterized by a burst of reactive oxygen species (ROS) at reperfusion and their survival by inhibition of this burst, we examined the effects of blocking caspase-2 and caspase-8 activities on ROS production. Caspase-2 inhibition blocked the reperfusion-induced ROS burst, while inhibition of caspase-8 did not. We also examined effects of caspase inhibition on mitochondrial membrane potential (∆ψ m) and mitochondrial function and found that blocking caspase-2, but not caspase-8, allowed recovery of ∆ψ m and mitochondrial functionality. Furthermore, knockdown of caspase-2 by small-interfering (si)RNA confirmed caspase-2 participation in cytochrome c release, which correlates with loss of ∆ψm and cell death in these cardiomyocytes.