Baicalein, a flavonoid derived from Scutellaria baicalensis Georgi, possesses cardioprotection against oxidant injury by scavenging reactive oxygen species (ROS). Few studies investigate whether baicalein protection is mediated by attenuating mitochondrial ROS and modulating the prosurvival and proapoptotic signaling. Primary cultured chick cardiomyocytes were used to study the role of baicalein in mitochondrial superoxide [Formula: see text] generation and signaling of Akt and JNK. Cells were exposed to H 2 O 2 for 2 h and baicalein was given 2 h prior to and during 2 h of H 2 O 2 exposure. Cell viability was assessed by propidium iodide and DNA fragmentation. H 2 O 2 (500 ?M) significantly induced 45.3 ± 6.2% of cell death compared to the control (p < 0.001) and resulted in DNA laddering. Baicalein (10, 25 or 50 ?M) dose-dependently reduced the cell death to 38.7 ± 5.6% (p = 0.226); 31.2 ± 3.9% (p < 0.01); 30.3 ± 5.3% (p < 0.01), respectively. It also attenuated DNA laddering. Further, baicalein decreased intracellular ROS and mitochondrial [Formula: see text] generation that was confirmed by superoxide dismutase PEG-SOD and mitochondria electron transport chain complex III inhibitor stigmatellin. In addition, baicalein increased Akt phosphorylation and decreased JNK phosphorylation in H 2 O 2-exposed cells. Moreover, baicalein augmented mitochondrial phosphorylation of Akt Thr308 and GSK3? Ser9, and prevented mitochondrial cytochrome c release assessed by cellular fractionation. Our results suggest that baicalein cardioprotection may involve an attenuation of mitochondrial [Formula: see text] and an increase in mitochondrial phosphorylation of Akt and GSK3? while decreasing JNK activation.
Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-?PTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-?p110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-?p110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-?PTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury.
Previous studies suggest baicalein, in addition to its antioxidant effects, protects against hypoxia/reoxygenation injury via its pro-oxidant properties. We hypothesize that a brief period of baicalein treatment prior to ischemia/reperfusion (I/R) may trigger preconditioning protection via a mitochondrial pro-oxidant mechanism. Using an established chick cardiomyocyte model of I/R, cells were preconditioned with baicalein (10 ?M) for 10 min followed by 10-min wash prior to I/R. Intracellular oxidants were measured using 2, 7-dichlorofluorescin diacetate (DCFH/DA). Cell viability was assessed by propidium iodide and apoptosis determined by DNA fragmentation. Baicalein induced a transient but significant increase of DCF fluorescence within the 10-min preconditioning period, and led to significant reduction of cell death (38.9 ± 1.8% vs. 58.7 ± 1.2% in I/R control, n = 6, p < 0.001) and DNA fragmentation after I/R. Cotreatment with N-acetylcysteine (500 ?M), mitochondrial complex III electron transport chain inhibitor myxothiazol (1 ?M), mitochondrial KATP channel blocker 5-hydroxydecanoate-Na (5-HD, 500 ?M) or anion channel inhibitor 4, 4-diisothiocyanato-stilbene-2, 2-disulfonic acid (DIDS, 200 ?M) resulted in significant abrogation of oxidant increase during induction as well as the protection conferred by baicalein preconditioning. These results suggest that baicalein preconditioning exhibits significant anti-apoptotic protection against cardiomyocyte I/R injury by mitochondrial oxidant signaling, which was in part mediated by mitochondrial KATP channel and anion channel opening.
The cardiotoxicity of doxorubicin limits its clinical use in the treatment of a variety of malignancies. Previous studies suggest that doxorubicin-associated cardiotoxicity is mediated by reactive oxygen species (ROS)-induced apoptosis. We therefore investigated if baicalein, a natural antioxidant component of Scutellaria baicalensis, could attenuate ROS generation and cell death induced by doxorubicin. Using an established chick cardiomyocyte model, doxorubicin (10 µM) increased cell death in a concentration- and time-dependent manner. ROS generation was increased in a dose-response fashion and associated with loss of mitochondrial membrane potential. Doxorubicin also augmented DNA fragmentation and increased the phosphorylation of ROS-sensitive pro-apoptotic kinase c-Jun N-terminal kinase (JNK). Adjunct treatment of baicalein (25 µM) and doxorubicin for 24 h significantly reduced both ROS generation (587 ± 89 a.u. vs. 932 a.u. ± 121 a.u., P < 0.01) and cell death (30.6 ± 5.1% vs. 46.8 ± 8.3%, P < 0.01). The dissipated mitochondrial potential and increased DNA fragmentation were also ameliorated. Along with the reduction of ROS and apoptosis, baicalein attenuated phosphorylation of JNK induced by doxorubicin (1.7 ± 0.3 vs. 3.0 ± 0.4-fold, P < 0.05). Co-treatment of cardiomyocytes with doxorubicin and JNK inhibitor SP600125 (10 µM; 24 h) reduced JNK phosphorylation and enhanced cell survival, suggesting that the baicalein protection against doxorubicin cardiotoxicity was mediated by JNK activation. Importantly, concurrent baicalein treatment did not interfere with the anti-proliferative effects of doxorubicin in human breast cancer MCF-7 cells. In conclusion, baicalein adjunct treatment confers anti-apoptotic protection against doxorubicin-induced cardiotoxicity without compromising its anti-cancer efficacy.
Doxorubicin (Dox) is one of the most widely used and successful chemotherapeutic antitumor drugs. Its clinical application is highly limited due to its cumulative dose-related cardiotoxicity. Proposed mechanisms include the generation of reactive oxygen species (ROS)-mediated oxidative stress. Therefore, reducing oxidative stress should be protective against Dox-induced cardiotoxicity. To determine whether antioxidant, grape seed proanthocyanidin extract (GSPE) attenuates Dox-induced ROS generation and protects cardiomyocytes from Dox-induced oxidant injury, cultured primary cardiomyocytes were treated with doxorubicin (Dox, 10 microM) alone or GSPE (50 microg/ml) with Dox (10 microM) for 24 hours. Dox increased intracellular ROS production as measured by 6-carboxy-2,7-dichlorodihydrofluorescein diacetate, induced significant cell death as assessed by propidium iodide, and declined the redox ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) and disrupted mitochondrial membrane potential as determined by 5,5,6,6-tetrachloro-1,1,3,3-tetraethlbenzimidazole-carbocyanide iodine (JC-1). Analysis of agarose gel electrophoresis revealed Dox-induced nuclear DNA damage with the ladder like fragmentation. GSPE treatment suppressed those alterations. Electron Spin Resonance (ESR) spectroscopy data also showed that GSPE strongly scavenged hydroxyl radical, superoxide and DPPH radicals. Together, these findings indicate that GSPE in combination with Dox has protective effect against Dox-induced toxicity in cardiomyocytes, which may be in part attributed to its antioxidative activity. Importantly, flow cytometric analysis demonstrated that co-treatment of Dox and GSPE did not decrease the proliferation-inhibitory effect of Dox in MCF-7 human breast carcinoma cells. Thus, GSPE may be a promising adjuvant to prevent cardiotoxicity without interfering with antineoplastic activity during chemotherapeutic treatment with Dox.
Therapeutic hypothermia (TH) is a promising cardioprotective treatment for cardiac arrest and acute myocardial infarction, but its cytoprotective mechanisms remain unknown. In this study, we developed a murine cardiomyocyte model of ischemia-reperfusion injury to better determine the mechanisms of TH cardioprotection. We hypothesized that TH manipulates Akt, a survival kinase that mediates mitochondrial protection by modulating reactive oxygen species (ROS) and nitric oxide (NO) generation. Cardiomyocytes, isolated from 1- to 2-day-old C57BL6/J mice, were exposed to 90 min simulated ischemia and 3 h reperfusion. For TH, cells were cooled to 32 degrees C during the last 20 min of ischemia and the first hour of reperfusion. Cell viability was evaluated by propidium iodide and lactate dehydrogenase release. ROS production was measured by 6-carboxy-2,7-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential (DeltaPsim) by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazoly-carbocyanine iodide (JC-1). Phospho (p)-Akt (Thr308), p-Akt (Ser473), and phosphorylated heat shock protein 27 (p-HSP27) (Ser82) were analyzed by Western blot analysis. TH attenuated reperfusion ROS generation, increased NO, maintained DeltaPsim, and decreased cell death [19.3 + or - 3.3% (n = 11) vs. 44.7 + or - 2.7% (n = 10), P < 0.001]. TH also increased p-Akt during ischemia before reperfusion. TH protection and attenuation of ROS were blocked by the inhibition of Akt and NO synthase but not by a cGMP inhibitor. HSP27, a regulator of Akt, also exhibited increased phosphorylation (Ser82) during ischemia with TH. We conclude that TH cardioprotection is mediated by enhanced Akt/HSP27 phosphorylation and enhanced NO generation, resulting in the attenuation of ROS generation and the maintenance of DeltaPsim following ischemia-reperfusion.
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 microg/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 Nomega-nitro-L-arginine methyl ester (L-NAME, 200 microM) 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 microM) 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.
A simple, polishable and renewable DNA biosensor was fabricated based on a zirconia modified carbon paste electrode. Zirconia was mixed with graphite powder and paraffin wax to produce the paste for the electrode, and response-optimized at 56% graphite powder, 19% ZrO(2) and 25% paraffin wax. An oligonucleotide probe with a terminal 5-phosphate group was attached to the surface of the electrode via the strong affinity of zirconia for phosphate groups. DNA immobilization and hybridization were characterized by cyclic voltammetry and differential pulse voltammetry, using methylene blue as indicator. Examination of changes in response with complementary or non-complementary DNA sequences showed that the developed biosensor had a high selectivity and sensitivity towards hybridization detection (< or =2x10(-10) M complementary DNA detectable). The surface of the biosensor can be renewed quickly and reproducibly (signal RSD+/-4.6% for five successive renewals) by a simple polishing step.
Reactive oxygen species (ROS) can induce oxidative injury via iron interactions (i.e. Fenton chemistry and hydroxyl radical formation). Our prior work suggested that American ginseng berry extract and ginsenoside Re were highly cardioprotective against oxidant stress. To extend this study, we evaluated the protective effect of protopanaxadiol-type ginsenoside Rb1 (gRb1) on H(2)O(2)-induced oxidative injury in cardiomyocytes and explored the ROS-mediated intracellular signaling mechanism. Cultured embryonic chick cardiomyocytes (4-5 day) were used. Cell death was assessed by propidium iodide and lactate dehydrogenase release. Pretreatment with gRb1 (0.01, 0.1, or 1 ?M) for 2 h and concurrent treatment with H(2)O(2) (0.5 mM) for 2 h resulted in a dose-dependent reduction of cell death, 36.6 ± 2.9% (n = 12, p < 0.05), 30.5 ± 5.1% (n = 12, p < 0.05) and 28.6 ± 3.1% (n = 12, p < 0.01) respectively, compared to H(2)O(2)-exposed cells (48.2 ± 3.3%, n = 12). This cardioprotective effect of gRb1 was associated with attenuated intracellular ROS generation as measured by 6-carboxy-2, 7-dichlorodihydrofluorescein diacetate, preserved the mitochondrial membrane potential as determined using JC-1. In the ESR study, gRb1 exhibited the scavenging DPPH and hydroxyl radical activities. Furthermore, our data showed the increased JNK phosphorylation (p-JNK) in H(2)O(2)-exposed cells was suppressed by the pretreatment with gRb 1 (1 ?M) (p < 0.01). Co-treatment of gRb1 with a specific inhibitor of JNK SP600125 (10 ?M) further reduced the p-JNK and enhanced the cell survival after H(2)O(2) exposure. Collectively, our results suggest that gRb1 conferred cardioprotection that was mediated via attenuating ROS and suppressing ROS-induced JNK activation.
Microparticles possess therapeutic potential regarding angiogenesis. We have demonstrated the contribution of apoptotic human CEM T lymphocyte-derived microparticles (LMPs) as inhibitors of angiogenic responses in animal models of inflammation and tumor growth. In the present study, we characterized the antivascular endothelial growth factor (VEGF) effects of LMPs on pathological angiogenesis in an animal model of oxygen-induced retinopathy and explored the role of receptor-mediated endocytosis in the effects of LMPs on human retinal endothelial cells (HRECs). LMPs dramatically inhibited cell growth of HRECs, suppressed VEGF-induced cell migration in vitro experiments, and attenuated VEGF-induced retinal vascular leakage in vivo. Intravitreal injections of fluorescently labeled LMPs revealed accumulation of LMPs in retinal tissue, with more than 60% reductions of the vascular density in retinas of rats with oxygen-induced neovascularization. LMP uptake experiments demonstrated that the interaction between LMPs and HRECs is dependent on temperature. In addition, endocytosis is partially dependent on extracellular calcium. RNAi-mediated knockdown of low-density lipoprotein receptor (LDLR) reduced the uptake of LMPs and attenuated the inhibitory effects of LMPs on VEGF-A protein expression and HRECs cell growth. Intravitreal injection of lentivirus-mediated RNA interference reduced LDLR protein expression in retina by 53% and significantly blocked the antiangiogenic effects of LMPs on pathological vascularization. In summary, the potent antiangiogenic LMPs lead to a significant reduction of pathological retinal angiogenesis through modulation of VEGF signaling, whereas LDLR-mediated endocytosis plays a partial, but pivotal, role in the uptake of LMPs in HRECs.
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