Statistical differences were assessed by one way-ANOVA. P < 0.05 was considered statistically significant. Data were expressed as the mean ± SEM.
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Results
Sustained incubation of H9c2 cells in hypoxia conditions increases reactive oxygen species (ROS) production and decreases antioxidant enzyme expression
Cardiovascular injury, one of the most common hypoxia complications,is linked to the elevated ROS levels, which subsequently induce cell apoptosis. Therefore, we examined the cellular ROS levels in cardiomyoblast H9c2 cells incubated in sustained hypoxia conditions.
The gp91phox is the catalytic core of this complex and p22phox is the only other membrane component of the vascular NADPH oxidases. On stimulation, p47phox becomes phosphorylated and forms a complex and translocates to the membrane, where it is associated with gp91phox and p22phox to assemble the active oxidase (Zeng, Han et al. 2010). The effects of hypoxia on gp91phox and p22phox expression and membrane translocation of p47phox and Rac-1 were examined by Western blotting in H9c2 cells with membrane and cytosolic isolation. We found that the protein levels of NADPH oxidases were increased significantly in H9c2 cells exposed to hypoxia for 0-24 h(Fig.1A). There is growing evidence indicated that AT1 and PKC induced by hypoxia have been shown to contribute to the activation of NADPH oxidase.
As show in Fig.1B, after treatement with hypoxia for different time periods, the protein levels of AT1, and phosphorylation of PKCα and δ significantly increased in H9c2 cells. Intracellular ROS levels are regulated by the balance between ROS generation and activity of antioxidant enzymes such as catalase or SOD. Thus, the involved ROS is
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able to inactivate antioxidative enzymes that additionally increase the imbalance in favor of oxidative stress. Therefore, we investigated the expression of its isoforms in H9c2 cells in response to hypoxia. Our results showed that the antioxidant enzymes SOD2 decreased in H9c2 cells treated with hypoxia (Fig.1B).
Hypoxia-induced apoptosis in cardiomyocyte
To clarify hypoxia induced cell apoptosis in cardiac myocyte, we
performed TUNEL analysis for observing cells undergoing apoptosis.After incubation with hypoxia for 24h, we observed a significant increase apoptosis bodies (Fig.2A). Next, we examined the survival protein (p-AKTser473) in different time of hypoxia condition. The results showed hypoxia downregulated the survival protein expression (Fig.2B).
Roles of MAPK family proteins in hypoxia induced H9c2 cell apoptosis
To investigate whether MAPK family protiens are involved in the
hypoxia-induced H9c2 apoptosis, we examined the expression levels of MAPK family proteins by Western blot. Our results showed that the phosphorylation of ERK, JNK, and P38 were increased after treatment with hypoxia for 0-24h (Fig.2C).Effects of HDL on hypoxa-induced NADPH oxidase complex and antioxidant enzyme protein expression
The functionality of HDL can be influenced by the abundance of various bioactive proteins and lipids, that exert anti-inflammatory, anti-oxidative,
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anti-coagulative and other atheroprotective functions(Vaisar, Mayer et al.
2010).So, we would like to know whether HDL can downregulated the effect of hypoxia in H9c2 cardiomyocyte cells.
The effects of HDL (25-100μg/ml) on gp91phox and p22phox expressions and membrane translocation of p47phox and Rac-1 were examined by Western blotting in H9c2 cells with membrane and cytosolic isolation.
We found hypoxia treatment of H9c2 cells resulted in the increase of NADPH oxidase activity. However, pretreatment of hypoxia-exposed cells with HDL (25-100μg/ml) led to a does-dependent reductions in gp91phox and Rac-1 protein expression (Fig.3A). As shown in Fig.3A, incubation of H9c2 cells with hypoxia resulted in significant phophorylation of protein kinase C and HDL attenuated this protein activation. Our result also showed that HDL (25-100μg/ml) reversed the reduced of SOD activity caused by hypoxia. In order to explore whether HDL influence ROS and superoxidase generation in H9c2 cells, we investigated the effects of HDL on generation of ROS, a potential factor related to hypoxia-induced H9c2 cells injury, by using hydroxyl radical sensitive probe 2’,7’-dichlorofluorescein acetoxymethyl ester (DCF-AM), superoxide sensitive probe dihydroethidium (DHE) and MitoSOX™ Red mitochondrial superoxide indicator. The levels of ROS and superoxide generations examined by flow cytometry have significantly decreased in H9c2 cells with pretreatment of HDL (25-100μg/ml) for 2 h before exposure to hypoxia in a dose-dependent manner (Fig.3B).
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Sustain exposure of HDL can reduce NADPH oxidase activity in neonatal cardiomyocytes treated with hypoxia.
In order to explore whether HDL influence NADPH oxidase, a major
source of ROS in H9c2 cells, we measured the effects of HDL on the formation of superoxide by using superoxide sensitive probe dihydroethidium (DHE). As shown in Fig.3C, neonatal cardiomyocytes treated with HDL (25-100μg/ml) for 2h before exposure to hypoxia for 24h, we used superoxide sensitive probe dihydroethidium (DHE) to confirm by microscopic observation. The resuls showed HDL (25-100μg/ml) enhanced superoxide generation to reduced induced by hypoxia to control similar results were obsereved by the treatments of anti-oxidant (NAC,500μM) and mitochondrial superoxide inhibitor (Rotenone,5μM).Effect of HDL on hypoxia-induced MAPK protein phosphorylations
Incubation of H9c2 cells with hypoxia resulted in significant phophorylation of p38MAPK, JNK and ERK. Therefore, we would like to examine whether HDL can inhibit MAPK protein activation. H9c2 cells were treated with HDL (25-100μg/ml) for 2h before exposure to hypoxia for 24h, it data showed HDL can downregulate the phophorylations of ERK, JNK, p38 proteins (Fig.4A).HDL attenuated the apoptoic effects of hypoxia by regulating Bcl2 family protein, and activation of casepase3
Whether HDL can influence apoptosis related proteins was investigated. Immunoblotting studies demonstrated that hypoxia
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downregulated the antiapoptotic and survival protein (BCL2, p-AKTser473), also upregulated the proapoptoic protein (Bax), whereas HDL treatment effectively repressed these hypoxia-induced proapoptoic events (Fig.4B).
Since activated caspase 3 is a key factor in the execution of mithchondrial apoptosis (Narula, Pandey et al. 1999), Whether HDL ultimately influence this factor to modulate apoptosis, we subsequently determined its pro-form and active-form by immunoblotting (Fig.4B). The data showed that active caspase 3 was significantly increased in cells that had been treated with hypoxia was suppressed in cells induced by hypoxia was suppressed by the treatment of HDL. To further confirm whether the antiapoptic effects of HDL on hypoxia-induced cell death, TUNEL and DAPI staining assays were performed and evaluated by microscopic observation. As shown in Fig.4B, cells incubated with 1%hypoxia for 24h showed typical features of apoptosis, including the formation of condensed nuclei, which were, however, not observed in the HDL-pretreated H9c2 cells. As described above, both results of cell viability assay and phenotypic observation of apoptosis under microscopy suggested that HDL is a potent inhibitor of hypoxia-induced cytotoxicity in cultured H9c2 cells. NAC (500μM) could suppress increased TUNEL-positive cell number induced by hypoxia.
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Discussion
Hypoxic condition is a general cause of cell damage, which is implicated in many pathologic conditions including stroke, myocardial infarction (MI), diabetes mellitus, multiple organ failure (Halterman and Federoff 1999; Kim, Ahn et al. 2004). Plaques formation in the heart's arteries will leading to myocardial remodeling and myocardial hypoxia (Lee, Wolf et al. 2000). It has reported that induced Angiotensin II attenuates chemical hypoxia-induced caspase-3 activation in primary cortical neuronal cultures (Chiu, Wang et al. 2010). However, in hypoxia condition, we found hypoxia downregulated the antiapoptoic proteins (BCL2 andp-AKTser473) and upregulated the proapoptoic (Bax) proteins.
Furthermore, increased caspase 3 activity in H9c2 cells with hypoxia (Fig.2B) can be decreased by treatment of HDL (Fig.4).
We want to investigate whether production of Angiotensin II lead to generate hypoxia-induced ROS.In this study, we explore the mechanisms of hypoxia-induced cell apoptosis in heart, focusing on the NADPH oxidase-generated ROS induced signaling. This study also extends the therapeutic potenial of inhibiting NADPH oxidase in hypoxia-exposed cardiac cells. The effects of ang II are mediated by two receptors, referred to as the Ang II type-1 (AT1) and type-2 (AT2) receptor subtypes.AT1 receptor is dependend on the cell and organ type, stimulation of these signal transduction pathways leads to cellular contraction, hypertrophy, proliferation, and/or apoptosis. One of the most important effects of AT1-receptor activation, particularly in the cardiovascular system, is the production and release of reactive oxygen species (ROS)(Nickenig and
89
Harrison 2002). The effect of hypoxia resulted in increased AT1 and NADPH oxidase protein expression (Fig.1).Various protein kinases have been involved in the regulation of NADPH oxidase activity (McPhail, Qualliotine-Mann et al. 1995; El Benna, Faust et al. 1996), among which the protein kinase C (PKC) family appears to play a major role (Wolfson, McPhail et al. 1985; Nauseef, Volpp et al. 1991; Combadiere, el Benna et al. 1993). As show in Fig.1B, hypoxia increased phosphorylation of PKC α and δ. Furthermore, we suggest hypoxia activated NADPH oxidase via AT1 receptor and PKCα and δ. SOD protects against superoxide-mediated cytotoxicity by rapidly dismutating O2– to H2O2.
Treatment of hypoxia decreased Cu/Zn-SOD and Mn-SOD protein expression level (Fig.1). The treatment of HDL resulted in decreased ROS generation, and subsequently attenuated hypoxia-impaired superoxide dismutase (SOD) activity and suppressed ROS-induced intracellular signaling pathways (Fig.3). Hypoxia is a prevalent cellular stress in many diseased states and stimulates MAPK signaling pathways.
(Bogoyevitch, Gillespie-Brown et al. 1996; Sugden and Clerk 1998;
Cook, Sugden et al. 1999; Nakano, Baines et al. 2000). Our results showed that hypoxia increased p-P38 and p-ERK protein expression, but not p-JNK, and HDL can decreased the expressions.
In summary, the present results indicated that HDL attenuates hypoxia-induced cardiomyocyte apoptosis and oxidative dysfunction via modulating mitochondria dependent pathway (Fig.3, 4). Therefore, reducing the downstream of superoxide-induced ROS generation and impairment of antioxidant enzymes. In addition, HDL inhibited hypoxia-induced cell death and apoptosis in cardiomyocytes (Fig.4).
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Further studies are required to confirm the effect of HDL on the inhibition of hypoxia mediated pro-atherogenic effects and the effectiveness in vivo. Our findings may provide a relevant therapeutic molecular mechanism in the improvement of cardiovascular disease. In H9c2 cells, hypoxia trigger AT1 receptor and NADPH oxidase activity.
Whether HDL protects the cells against hypoxia-induced apoptosis via blocked AT1receptor or through HDL compose such as Apo A-1, SR-B1 or other receptors will be another issue we can identify in the future.
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