Salidroside protects cardiomyocyte against hypoxia-induced death: A HIF-1α-activated and VEGF-mediated pathway

European Journal of Pharmacology 607 (2009) 6–14

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

Salidroside protects cardiomyocyte against hypoxia-induced death: A HIF-1α-activated and VEGF-mediated pathway Jinping Zhang a, Anheng Liu b, Rongrong Hou a, Juan Zhang a, Xin Jia a, Weifeng Jiang a, Jianzong Chen a,⁎ a Laboratory of Senile Encephalopathy with the Integrated Traditional Chinese Medicine and Western Medicine, Research Center of Traditional Chinese Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China b Department of Cardiology, 307 Hospital of PLA, Academy of Military Medical Sciences, Beijing 100071, China

a r t i c l e

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Article history: Received 24 October 2007 Received in revised form 11 January 2009 Accepted 27 January 2009 Available online 5 February 2009 Keywords: Salidroside Hypoxia Cardiomyocyte Necrosis Apoptosis HIF-1α VEGF

a b s t r a c t Cardiomyocyte death (necrosis and apoptosis) plays a critical role in the progress of heart diseases. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, has shown cardioprotective effects in vivo. However, whether salidroside has a protective effect against cardiomyocyte death is poorly understood. The present study was aimed to investigate the cardioprotective role of salidroside and the underlying mechanisms in hypoxia-induced cardiomyocyte death. Cardiomyocytes pretreated with or without salidroside for 24 h were exposed to hypoxic condition for 6 h and then cell viability, necrosis, apoptosis, the expressions of HIF-1α and VEGF were investigated. Pretreatment with salidroside markedly attenuated hypoxia-induced cell viability loss, cell necrosis and apoptosis in a dose-dependent manner. Mechanistically, pretreatment with salidroside up-regulated the HIF-1α protein expression and induced its translocation. Moreover, the level of VEGF, a downstream target of HIF, was significantly increased in parallel with the level of HIF-1α following pretreatment with salidroside. However, 2-methoxyestradiol (2-ME2), a HIF-1α inhibitor, attenuated the protection of salidroside and blocked the increase of HIF-1α and VEGF. These data indicated that salidroside has protective effect against hypoxia-induced cardiomyocytes necrosis and apoptosis by increasing HIF-1α expression and subsequently up-regulating VEGF levels. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction There are two general mechanisms of cell death: necrosis and apoptosis (Arends and Wyllie, 1991; Iliodromitis et al., 2007), which show characteristic but morphologically and biologically distinct features (Iliodromitis et al., 2007). Countless studies have shown the association and/or causation between the apoptotic loss of myocytes and the progression of heart diseases, including heart failure, in humans and in animal models (Haunstetter and Izumo, 1998; Foo et al., 2005). While considerably less studied, necrosis may also be a critical mechanism underlying myocyte loss in heart failure or with aging (Kajstura et al., 1996; Goldspink et al., 2003; Nakayama et al., 2007). A variety of stimuli that are known to participate in the pathogenesis of heart failure have been shown to induce cardiomyocyte necrosis and apoptosis, such as hypoxia (Mehrhof et al., 2001; Danial and Korsmeyer, 2004), ischemia and reperfusion (Gao et al., 2002), and oxidative stress (von Harsdorf et al., 1999; Danial and Korsmeyer, 2004). Therefore, therapeutic strategies aimed at preventing or delaying cardiomyocytes death induced by these stimuli might be a reasonable choice for the ⁎ Corresponding author. Tel.: +86 29 84775517; fax: +86 29 82555145. E-mail address: [email protected] (J. Chen).

treatment of related heart diseases. Many chemicals such as UCF-101, insulin, erythropoietin have been proved to be cardioprotective (Abdallah and Schafer, 2006; Ma et al., 2006; Bhuiyan and Fukunaga, 2007; Fu and Arcasoy, 2007), but they usually have some adverse effects. Recently, interests have been focused on searching for natural substances with cardioprotective potential which can treat certain types of heart disease through antinecrotic, antiapoptotic, or a combination of both therapeutic agents for slowing or halting the progression of cellular attrition in heart failure. Salidroside (p-hydroxyphenethyl-β-D-glucoside, structure shown in Fig. 1A, C14H20O7: 300.30), which is extracted from Rhodiola rosea L and has long been used as an adaptogen in traditional Tibetan medicine, has been reported to possess various pharmacological properties including resisting anoxia, anti-aging, anti-cancer, antiinflammation, antioxidative, hepatoprotective and cardioprotective effects (Diaz Lanza et al., 2001; Kelly, 2001; Nan et al., 2003; Kucinskaite et al., 2004; Kanupriya et al., 2005; Zhang et al., 2007). As reported by Maslova et al. (1994), salidroside can protect the cardiovascular system from stress-induced catecholamine release and alleviate adrenaline-induced arrhythmias in rats. The antiarrhythmic effect of salidroside is supposed to be secondary to its ability to induce opioid peptide biosynthesis, and to be related to the stimulation of peripheral kappa-opioid receptors (Lishmanov et al., 1993; Maslova

0014-2999/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.01.046

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Fig. 1. Protective effect of salidroside on hypoxia-induced cytotoxicity in cardiomyocytes. A: Chemical structure of salidroside; B: time-dependent toxic effect of hypoxia on cardiomyocytes viability. Cardiomyocytes were exposed to hypoxia for 0–8 h. Viable cells were identified with MTT assay. C: Effect of salidroside on hypoxia-induced cardiomyocytes death. Cardiomyocytes were pretreated to various concentrations of salidroside (10, 50, 100 µg/ml) or 10− 7 M insulin for 24 h and then were exposed to hypoxia for 6 h and cell viability was determined with MTT assay. D: Cell viability with pretreatment by 1–10 µM 2-ME2 for 24 h in a concentration-dependent manner. All data were shown as mean ± S.E.M. of three experiments and each included triplicate sets. n = 6. Sal: salidroside; Ins: insulin; 2-ME2: 2-methoxyestradiol. ##P b 0.01 vs Control; ⁎P b 0.05, ⁎⁎P b 0.01 vs hypoxia.

et al., 1994; Lishmanov et al., 1997; Maimeskulova et al., 1997). Nevertheless, the mechanism underlying its cardioprotective effects remains unclear, especially at the cellular level. The clarification of the effects of salidroside on cardiomyocytes necrosis and apoptosis induced by hypoxia may provide a new insight into the mechanism of cardioprotection. It is well known that hypoxia can induce cardiomyocytes necrosis and apoptosis; thus, it is important to find ways to protect cardiomyocytes from hypoxia-induced cell death apart from the regeneration of injured heart. Control of the hypoxic response in mammalian cells occurs through a number of mechanisms, primary transcriptional and post transcriptional mechanisms. A growing body of evidence has shown that the transcription factor hypoxia inducible factor-1 (HIF-1) is one of the major regulators in hypoxic response. HIF-1 consists of a constitutively expressed subunit HIF-1β and an oxygen-regulated subunit HIF-1α (Wenger, 2002; Pugh and Ratcliffe, 2003). HIF-1α protein is ubiquitously expressed whereas its homologues HIF-2α and HIF-3α have more restricted expression patterns. Under lower oxygen tension, hydroxylation is inhibited because of substrate (O2) deprivation, and HIF-1α accumulates, dimerizes with HIF-1β, and mediates profound changes in hypoxia-inducible genes (HIGs) expression (Lee et al., 2004; Chi et al., 2006). To our knowledge, more than 60 target genes which are activated by HIF-1 have been identified (Ke and Costa, 2006), including vascular endothelial growth factor (VEGF) and other cytoprotective proteins. There is now increasing evidence that VEGF, an endothelial cell-specific angiogenic factor, induces expression of Bcl-2 which eventually functions to enhance cell survival in the anoxic and oxygen-deficient environment (Nor et al., 1999) and activates the myocardial PI-3K pathway to decrease myocardial infarct size (Zhou et al., 2005).

This study was to investigate the protective effect of salidroside against cardiomyocytes death induced by hypoxia, and furthermore to explore the possible mechanisms involved. We hypothesized that salidroside attenuates hypoxia-induced cardiomyocytes necrosis and apoptosis by up-regulating cardioprotective protein, HIF-1α in parallel with its target gene VEGF, under lower oxygen tension environment. 2. Materials and methods 2.1. Materials All chemical reagents were purchased from Sigma (A.R) unless indicated otherwise. Salidroside (purity N 99%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. Rabbit polyclonal antibody to HIF-1α (NB100-479) was obtained from Novus Biologicals, Rabbit anti-VEGF antibody was from Beijing Biosynthesis Biotechnology Co., LTD. 2-methoxyestradiol (2-ME2) and proteinase K were purchased from Merck. ECL reagent and bicinchoninic acid (BCA) protein Assay Kit were from Pierce (Rockford, IL, USA). The kits for determination of lactate dehydrogenase (LDH) and creatine phosphokinase (CK) contents were obtained from Jiancheng Bioengineering Institute (Nanjing,China). The purity of all chemical reagents was of at least analytical grade. 2.2. Primary neonatal rat cardiomyocyte cultures Primary cultures of neonatal rat cardiomyocytes were prepared as previously described with minor modifications (Takahashi et al., 2005; Liu et al., 2008). The hearts from 1- to 2-day-old Sprague–

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Dawley rats were isolated and minced with scissors into 1–2 mm fragments, and were then enzymatically digested 6–7 times for 5–7 min each in HEPES containing 0.1% collagenase (Sigma, type I). The liberated cells from each digestion were collected in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO) and centrifuged, and then were re-suspended in DMEM with 10% FBS, penicillin (100 U/ml) and streptomycin (100 U/ml). The cell suspension was filtered through a 100-µm diameter filter and the cells were plated onto a 10-cm Petri dish for 1 h cultivation (37 °C in a 5% CO2 incubator) for purification. After that, cells were plated onto 96- and 6-well plates at a density of 1.2× 105/well and 8.0 ×105/well, respectively and were maintained in a humidified atmosphere with 5% CO2 at 37 °C. 5-Bromo-29-deoxyuridine (BrdU; 100 mM) was added during the first 48 h to inhibit proliferation of non-myocytes. We routinely obtained contractile, myocytes-enriched cells. The cells were allowed to grow in culture medium for 72 h before being used. 2.3. In vitro hypoxia model

cation of apoptotic myocytes. Briefly, following exposure, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After several washes with PBS, the cells were exposed to Hoechst 33258 (5 µg/ml in PBS) and incubated for 10 min at room temperature. After a final rinse with PBS, the cells were examined under a fluorescence microscope with an appropriate filter. The percentage of apoptotic cells displaying chromatin condensation and nuclear fragmentation was determined. 2.7. Analysis of DNA fragmentation Cardiomyocyte apoptosis was analyzed both qualitatively and semiquantitatively by detection of DNA fragmentation (DNA ladder) as described previously (Xu et al., 2007). In brief, following hypoxia, cardiomyocytes were resuspended in PBS and homogenized in TNE buffer (0.5% SDS, 10 mM EDTA, 100 mM NaCl, 10 mM Tris pH 7.4) containing 20 µg/ml RNase A and 200 µg/ml proteinase K. The cell lysate was incubated at 50 °C for 2 h and isopropanol was added. DNA was precipitated by means of centrifugation and washed with 70% ethanol. DNA (8 µg) was then analyzed by using 2% agarose gel electrophoresis and visualized under a UV (302 nm) transilluminator.

To mimic the hypoxia injury in vitro, cells were incubated in a hypoxia solution, which contained 0.9 mM NaH2PO4, 6.0 mM NaHCO3, 1.0 mM CaCl2, 1.2 mM MgSO4, 40 mM Natrium lacticum, 20 mM HEPES, 98.5 mM NaCl, 10.0 mM KCl (pH adjusted to 6.8 ) and was bubbled with N2 for 30 min before application. The pO2 of the hypoxia solution was adjusted to reach a level of ≤4.0 kPa. Hypoxic condition was produced by placing the plates of cultured cardiomyocytes in a hypoxic incubator (Kendro, Germany) and oxygen was adjusted to 1.0% and CO2 to 5.0%. Prior to hypoxia, cells were pretreated with various concentrations (10, 50, 100 µg/ml) of salidroside for 24 h. To confirm the role of HIF-1α in the protection of salidroside on cardiomyocyte, cells were pretreated with different concentrations (1, 5, 10 µM) of 2-ME2, a HIF-1α inhibitor, for 24 h in some experiments. Normal culture (10% FBS regular medium under 20% oxygen and 5% CO2) served as a control.

To quantify the level of necrosis and apoptosis, flow cytometry (FCM) was determined to detect cellular events that have undergone cell death. At the end of hypoxia, cells were washed and resuspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells grown adherent were gently and mechanically detached and resuspended. Subsequently cells were incubated for 10 min in the dark with Annexin V-FITC (1 µg/ml, Roche, Germany) at 4 °C. To discriminate necrosis, cells were incubated with PI solution (5 µg/ml, Roche, Germany) and analyzed with a FACScan flow cytometer (BD. FACS Calibur, USA).

2.4. Cell viability assays

2.9. Western blot analysis

Cell viability was assessed by the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and experiments were performed in three independent assays. Briefly, cells seeded on 96-well culture plates at 1.2× 105/well were incubated with the test compounds for indicated time period. Following exposure to hypoxia, 20 µl of the MTT solution (5 mg/ml) was added into each well and made the final concentration 0.5 mg/ml, and then the plates were incubated for an additional 2 h. After the medium was removed, DMSO (150 µl) was added into each well before reading the microplates at 490 nm. Cell viability was expressed as a percentage of the control culture value.

After being washed with cold PBS, cells were homogenized in lysis buffer containing proteinase inhibitors. After protein concentration quantitation with BCA protein assay, denatured protein (50 µg) was then analyzed using 6% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polywinylidene difluoride (PVDF) membranes (Pierce). After being blocked with 5% defatted milk for 1 h at 37 °C, membranes were immunoblotted overnight at 4 °C with polyclonal antibodies including HIF-1α (1:1000), VEGF (1:400) and β-actin (1:1000) followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ECL-plus reagent and the results were analyzed by LabImage version 2.7.1 (Kapelan GmbH, Halle, Germany).

2.5. LDH and CK activity assay It has been shown that LDH and CK are two reliable markers of myocyte necrosis that correlates well with cell death (Nakano et al., 1998; Wang et al., 2006). Therefore, cardiomyocytes necrosis or viability was further confirmed by measurement of LDH and CK activity. Briefly, LDH and CK levels in the cultured supernatant were measured in duplicate following hypoxia with a colorimetric assay that measures the conversion of pyruvic acid to lactic acid by LDH and triphosphare and creatine to phosphagen by CK, respectively. The activities of LDH and CK were analyzed with microplates at 440 nm and 660 nm, respectively.

2.8. Analysis of flow cytometry

2.10. Immunofluorescence for HIF-1α For HIF-1α staining, cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.2% TritonX-100 in PBS for 5 min. After being blocked for 1 h in the blocking buffer containing 10% goat serum, the cells were incubated overnight at 4 °C with rabbit polyclonal anti-HIF-1α (1:200). After washing thoroughly, the FITC-conjugated secondary antibody (1:100) was applied at room temperature for 25 min. Fluorescent images were processed using a fluorescence microscope.

2.6. Nuclear staining for assessment of apoptosis 2.11. Statistical analysis Hoechst 33258 staining, which allows clear distinction between apoptotic and normal cells on the basis of nuclear morphology (chromatin condensation and fragmentation), was used for quantifi-

The results were expressed as mean± S.E.M. of at least 3 independent experiments, unless stated otherwise. Statistical significance was

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Fig. 2. Effect of salidroside on hypoxia-induced morphological alterations in cardiomyocytes (× 200). A: Control cells; B: hypoxia cells; C: hypoxia + 100 µg/ml salidroside cells; D: hypoxia + 10− 7 M insulin cells.

determined using ANOVA followed by Bonferroni correction for post hoc t test. A value of P b 0.05 was considered significant.

increased after exposure to hypoxia for 6 h. However, marked reductions in LDH and CK activity in the culture supernatant were observed in myocytes pretreated with 10–100 µg/ml salidroside in a

3. Results 3.1. Salidroside attenuated hypoxia-induced cell viability loss Cell viability was shown in Fig. 1B using the MTT assay. Hypoxia for 1 h to 8 h induced significant decreases in cell survival in a timedependent manner. Exposed to hypoxia for 6 h, there were only 47.7 ± 10.1% viable cells as compared to the control cells. Therefore, hypoxia for 6 h was used as a standard apoptosis induction in the subsequent experiments. As illustrated in Fig. 1C, salidroside (10, 50, 100 µg/ml) prevented cells from hypoxia-induced damage in a dose-dependent manner, restoring cell survival to 56.3 ± 15.2%, 64.5 ± 9.4% and 66.6 ± 12.8%, respectively. Insulin (10− 7 M), which has been confirmed to protect cardiomyocytes against apoptosis induced by several stimuli, increased the cell viability to 77.1 ± 11.5%, serving as a positive control. However, the protection of salidroside in cell survival was abolished by pretreating cells with 2-ME2 (1–10 µM), a HIF-1α inhibitor, in a concentration-dependent manner (Fig. 1D). Moreover, the protective effect of salidroside could also be confirmed by the morphological observation (Fig. 2). With hypoxia injury, cardiomyocyte contracted, tending to get round in shape and its pseudopods decreased, while marked morphological changes were greatly decreased with salidroside or insulin pretreatment. It should be noted that salidroside at each concentration applied alone did not cause any apparent cytotoxicity (data not shown). 3.2. Salidroside protected cardiomyocytes against hypoxia-induced necrosis and apoptosis To examine how salidroside influence hypoxia-induced cardiomyocyte necrosis which is characterized by cell membrane disruption and cell content release, the releases of LDH and CK in the medium were measured as indexes of cell necrosis. As shown in Fig. 3, LDH and CK activity in the culture supernatant significantly

Fig. 3. Effect of salidroside on lactate dehydrogenase (LDH) and creatine phosphokinase (CK) activity in the culture supernatant of cardiomyocytes subjected to hypoxia. All data were expressed as mean ± S.E.M. n = 6; ##P b 0.01 vs Control; ⁎⁎P b 0.01 vs hypoxia. Sal: salidroside; Ins: insulin.

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dose-dependent manner. Similarly, significant decreases were observed in the insulin-pretreated cells as compared to the hypoxic cells. Nuclei morphological change was observed by Hoechst 33258 staining, which illustrated that the control cells exhibited uniformly dispersed chromatin, normal organelle and intact cell membrane. Exposed to hypoxia for 6 h, myocytes featured typical characteristics of apoptosis, including the condensation of chromatin, the shrinkage of nuclei and the appearance of a few apoptotic bodies and about 43.0% underwent apoptosis. However, in 100 µg/ml salidroside or 10− 7 M insulin pretreated cells, the morphological changes were significantly attenuated and the number of cells with nuclear condensation and fragmentation was markedly decreased. However, the protection by salidroside was blocked by pretreating cells with 2-ME2 (5 µM) (Fig. 4). To further qualitatively and semiquantitatively assess cardiomyocyte apoptosis, internucleosomal fragmentation of DNA was analyzed.

Fig. 5A indicated that after hypoxia for 6 h, characteristic “DNA laddering” was elicited, which was significantly attenuated with 100 µg/ml salidroside or 10− 7 M insulin pretreatment. However, the prevention of salidroside was markedly attenuated by 2-ME2 (5 µM) pretreatment (Fig. 5B). Cardiomyocytes were stained with both PI and FITC-labeled Annexin V (AV-FITC) to further quantitatively assess the percentage of necrotic and apoptotic cells with FCM. Apoptotic myocytes were defined as AnnV positive and PI negative while necrotic ones were defined as AnnV and PI positive. In the control group, most cells were viable (93.7%) (Fig. 6A). Hypoxia increased the percentage of both necrotic and apoptotic cells (13.7% and 41.7%, respectively) (Fig. 6B). While with the pretreatment by salidroside, the necrotic and apoptotic cells were decreased in a dose-dependent manner (Fig. 6C–F). Furthermore, interestingly, the apoptotic cells were affected more significantly than the necrotic ones, which may be because of the secondary necrosis.

Fig. 4. Effect of salidroside on morphological features of hypoxia-induced cardiomyocytes (× 200). A: Normoxia control; B: hypoxia alone; C: hypoxia + 100 µg/ml salidroside group; D: hypoxia + 100 µg/ml salidroside + 5 µM 2-ME2 group (co-pretreatment of cells with 2-ME2 for 24 h). E: Percentage of nuclei staining positive in cardiomyocytes from normoxia control group or pretreated groups. The apoptotic index was determined by the number of positively stained apoptotic myocytes/the total number of myocytes counted × 100%. Arrowheads in the pictures indicate the nuclei of apoptotic cells. All data were expressed as mean ± S.E.M. n = 5; ##P b 0.01 vs Control; ⁎⁎P b 0.01 vs hypoxia; ††P b 0.01 vs hypoxia + 100 µg/ml salidroside. Sal: salidroside; Ins: insulin.

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to protect cardiomyocytes against apoptosis induced by hypoxia. As illustrated in Fig. 9, compared with the control cells, a 1.9-fold increase in VEGF protein expression was found in hypoxia cells. Furthermore, cells pretreated with salidroside elicited a 5.9-fold induction of the VEGF protein. Also the level of VEGF protein was decreased by pretreatment with 5 µM 2-ME2. 4. Discussion

Fig. 5. Effect of salidroside on DNA fragmentation. A: Lane 1: DNA marker; Lane 2: normoxia control; Lane 3: hypoxia alone; Lane 4: hypoxia + 100 µg/ml salidroside; Lane 5: hypoxia + 10− 7 M insulin. B: Lane 1: DNA marker; Lane 2: normoxia control; Lane 3: hypoxia alone; Lane 4: hypoxia + 100 µg/ml salidroside; Lane 5: hypoxia + 100 µg/ml salidroside + 5 µM 2-ME2.

3.3. Salidroside up-regulated expression of HIF-1α It is generally known that HIF-1α pathway is activated when cells are exposed to lower oxygen tension, which render the cells adaptive response to hypoxia. But it was doubted whether HIF-1α pathway was also associated with the process of salidroside protection against hypoxia-induced cardiomyocytes necrosis and apoptosis. HIF-1α was mainly localized in the cytosol under normal conditions (Fig. 7A). Hypoxia induced the translocation of HIF-1α into the nuclei or to perinucleus areas (Fig. 7B). Interestingly, pretreating cardiomyocytes with 100 µg/ml salidroside led to significant up-regulation and translocation of HIF-1α protein (Fig. 7C). However, the up-regulation and translocation of HIF-1α protein were completely abolished by pretreating cells with 5 µM 2-ME2 (Fig. 7D). To confirm these results from immunofluorescence detection, western blot analysis was adopted to determine the changes in the expression of HIF-1α protein. Compared with the control cells, densitometric quantitation of the immunoblots revealed a 3.6- to 10.1-fold increase in the levels of HIF-1α protein of the hypoxia-treated cells relative to 100 µg/ml salidroside pretreated cells. While after pretreating by 5 µM 2-ME2, the expression of HIF-1α protein was significantly reduced and the densitometric quantitation attenuated to 5.8-fold compared with the control cells (Fig. 8). 3.4. Salidroside up-regulated VEGF expression Accumulated data have suggested that HIF-1 is activated in the hypoxia conditions and can participate in the hypoxic protective effect on apoptosis. Indeed, some target genes of HIF-1 have already been shown to protect cells against apoptosis such as VEGF. It was wondered whether VEGF was also activated via the HIF-1 pathway

It is well known that cardiomyocyte loss as a result of necrosis and apoptosis plays an important role in various heart diseases which may ultimately lead to heart failure (Kumar et al., 1999, 2001; Foo et al., 2005; Nakayama et al., 2007); Given that hypoxia stands as a crucial initiator to cause the loss of cardiomyocytes (Kakinuma et al., 2005), blocking the necrosis and apoptosis process induced by hypoxia may help to slow down or even prevent the occurrence and progression of heart failure. Thus it is of vital importance to find a critical molecular involved in the mechanisms of protecting cardiomyocyte against hypoxia-induced necrosis and apoptosis. Salidroside, a major active ingredient occurring naturally in R. rosea L., has been reported to have cardioprotective effects in vivo. Although opioid peptide biosynthesis and the stimulation of petipheal kappa-opioid receptors were supposed to be a possible molecular mechanism, the mechanism for the beneficial effect of salidroside remains to be clarified, especially at the cellular level. In the present study, we have focused on demonstrating the possible molecular mechanisms underlying the protective effect of salidroside to protect cardiomyocytes against hypoxia-induced necrosis and apoptosis. The construction of hypoxia model in vitro was performed as described previously (Koyama et al., 1991; Wang et al., 2005). Our present study confirmed that hypoxia for 6 h significantly decreases the cell viability and increases cardiomyocyte necrosis and apoptosis as evidenced by elevated LDH and CK activity in the culture medium, condensed and fragmented chromatin in the nuclei and characteristic “DNA laddering”. However, pretreatment with different concentrations (10, 50, 100 µg/ml) of salidroside greatly decreased the cell viability loss in a dose-dependent way, reduced LDH and CK activity, mitigated morphological changes and nuclear condensation, reduced the percentage of apoptotic cells and attenuated the characteristic DNA laddering. These findings strongly suggested that salidroside may exert a protective effect against the hypoxia-induced cytotoxicity and inhibited cardiomyocyte necrosis and apoptosis in response to hypoxia. On the basis of the obtained results that salidroside protected cardiomyocytes against necrosis and apoptosis induced by hypoxia, further investigation was performed with focuses on the possible mechanisms involved in the protective effect of salidroside. It is well known that oxygen homeostasis represents a fundamental physiological process that requires the coordinated regulation of an extensive array of genes, and deprivation of oxygen (hypoxia) is a serious extracellular and intracellular stress that compromises cellular survival. In most cell types, hypoxia inducible factor-1 (HIF-1), a transcription complex, controls gene expression in response to reduce oxygen tension (Semenza, 1998, 1999; Piret et al., 2002). As noted above, HIF-1 activity is regulated by the cellular levels of the HIF-1α subunit, which then heterodimerizes with HIF-1β to form the active transcription factor (Jiang et al., 1996). Much of the programmatic response directed by HIF-1 is involved in protecting cells against hypoxic, ischemic stress or injury. The induction of erythropoietin, VEGF and other pro-angiogenic factors are examples of this protective program. It is well established that the activation of HIF-1 and the upregulation of its target genes function to promote cell survival and restore tissue homeostasis under hypoxic conditions (Semenza, 2004; Semenza et al., 2006). For example, HIF regulates the expression of iNOS and indirectly (via VEGF) influences the level and activity of eNOS, thus modulates intracellular NO availability (Giordano, 2005).

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Fig. 6. Effect of salidroside on hypoxia-induced cardiomyocytes necrosis and apoptosis was determined with flow cytometry. A: Normoxia control; B: hypoxia alone; C: hypoxia + 10 µg/ml salidroside group; D: hypoxia + 50 µg/ml salidroside group; E: hypoxia + 100 µg/ml salidroside group; F: hypoxia + 10− 7 M insulin group.

HIF also has marked effects on the expression of genes involved in cardiac metabolism, including the glycolytic enzymes, a critical glucose transporter, and lactate dehydrogenase (Huang et al., 2004). Stabilization of HIF-1 and induction of the adaptive hypoxia response can particularly participate in myocardial remodeling in children with congenital cardiac defects and chronic hypoxemia (Qing et al., 2007).

Fig. 7. Effect of salidroside on the amount of HIF-1α protein. HIF-1α protein was revealed by immunofluorescence using a rabbit polyclonal antibody to HIF-1α (green) (×200). A: HIF-1α was scattered in cytosol of normoxia cardiomyocytes; B: HIF-1α was highly concentrated in peri-nucleus and in nuclei after cardiomyocytes were exposoed to hypoxia for 6 h; C: Compared with hypoxia alone, pretreatment of cardiomyocytes with 100 µg/ml salidroside significantly increased the expression and translocation of HIF-1α. D: Pretreatment of cells with 2-ME2 (5 µM) for 24 h significantly abolished the translocation of HIF-1α. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Besides, HIF-1α also contributed to the protective effect of bone marrow stem cells on cardiomyocytes with subsequent up-regulation of VEGF (Dai et al., 2007). In this study, we hypothesized that the mechanism by which salidroside inhibited hypoxic stress-induced cardiomyocyte necrosis and apoptosis lies in the up-regulation of HIF-1α protein. In agreement with the previous studies (Dai et al., 2007), HIF-1α protein was significantly increased in low oxygen tension environment and further translocated to the nucleus and peri-nuclear areas as shown in Fig. 7. Compared to cells exposed to hypoxia, the expression levels of HIF-1α protein in cells pretreated with salidroside were further elevated. These results from immunofluorescence detection were further confirmed by Western blot analysis (Fig. 8). In addition, 2-ME2, a special HIF-1α inhibitor, has been well documented to downregulate HIF-1 expression. Mabjeesh et al. (2003) reported that this effect of 2-ME2 is due to inhibition of

Fig. 8. Effect of salidroside on the expression of HIF-1α protein. Total cell extracts were analyzed with Western blotting using a rabbit polyclonal antibody to HIF-1α. β-actin levels were performed to assess the total amount of proteins loaded on the gel. Data obtained from quantitative densitometry were presented as mean ± S.E. of 5 independent experiments. ##P b 0.01 vs Control; ⁎⁎P b 0.01 vs hypoxia; ††P b 0.01 vs hypoxia +100 µg/ml salidroside. Sal: salidroside.

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in the level of VEGF protein compared with the hypoxia-treated cells and 2-ME2 markedly blocked the increase in VEGF level, which were consistent with the changes in HIF-1α. These results further confirmed that salidroside protects cardiomyocytes from hypoxiainduced necrosis and apoptosis via a HIF-1-dependent and VEGFmediated pathway. In summary, the data obtained from this study provide direct evidence that salidroside can protect cardiomyocytes against hypoxiainduced necrosis and apoptosis. The protective effect of salidroside is closely associated with the activation of the transcription factor HIF-1α along with its target gene VEGF. Further study on the detailed mechanisms is now in progress. Acknowledgment This study was supported by a grant from the Chinese Army Foundation of New Drug (NO: 2006192). Fig. 9. Effect of salidroside on the expression of VEGF protein. Total cell extracts were analyzed with Western blotting using a rabbit anti-VEGF antibody. β-actin expression was performed to make sure the equal amount of proteins loaded on the gel. Data obtained from quantitative densitometry were presented as mean ± S.E. of 5 independent experiments. ##P b 0.01 vs Control; ⁎⁎P b 0.01 vs hypoxia; ††P b 0.01 vs hypoxia +100 µg/ml salidroside. Sal: salidroside.

HIF-1α protein synthesis and dependent on the disruption of interphase microtubules by 2-ME2 in prostate cancer PC-3 cells and breast cancer MDA-MB-231 cells. Hagen et al. (2004) demonstrated that 2-ME2 reduced HIF-1α protein accumulation following hypoxia, but not that resulting from inhibition of proteasomal protein degradation or inhibition of prolyl hydroxylase activity in HEK293 cells. It has also been reported in human breast and prostate cancer that 2-ME2 downregulates HIF-1α at post-transcriptional level and inhibits HIF-1α-induced transcriptional activation of VEGF expression (Kimbro and Simons, 2006). The data from our present study demonstrated that 2-ME2 blocked salidroside elicited cardioprotection in vitro in a concentration-dependent manner by downregulating the HIF-1α protein levels and blocking its translocation. It is well known that vascular endothelial growth factor (VEGF), a downstream gene of HIF-1, is a specific mitogen for vascular endothelial cells that plays a crucial role in vascular development, angiogenesis, collateral growth and enhancement of collateral perfusion under diverse circumstances, such as embryonic development, wound healing, tumor growth, inflammatory diseases and ischemia (Ferrara, 2004). It has been shown to exert neuroprotective actions directly through the inhibition of apoptosis and the stimulation of neurogenesis (Gora-Kupilas and Josko, 2005). In myocardial ischemia VEGF is highly up-regulated in cardiomyocytes and contributes to angiogenesis, collateral formation and ventricular remodeling (Tomanek et al., 2004). Tang et al. have reported that implantation of mesenchymal stem cells significantly elevated VEGF expression, which led to increase vascular density and regional blood flow in the infarct area and more importantly protect cells against apoptosis (Tang et al., 2004). Furthermore, the ACE inhibitor ramipril is cardio- and vasoprotective in SHR-SP by preventing hypertrophy and cellular necrosis associated with significantly decreased mRNA levels for proteins of the extracellular matrix, but increased VEGF gene expression (Zimmermann et al., 1999). However, the mechanism of VEGF in the prevention of necrosis or apoptosis has not been clear yet. It was proposed that VEGF may bind to its receptors and trigger the phosphatidylinositol 3-kinase(PI3K)/Akt signal transduction system thus resulting in the inhibition of apoptosis by activating antiapoptotic proteins through the transcription factor NFKB and by suppressing proapoptotic signaling by Bad, caspase-9, caspase-3 and other effectors (Gora-Kupilas and Josko, 2005). In the present study, we observed that hypoxia promoted the expression of VEGF protein and furthermore, salidroside treatment produced additional increase

References Abdallah, Y., Schafer, C., 2006. Insulin: an overall cardiovascular protector? Cardiovasc. Res. 69, 4–6. Arends, M.J., Wyllie, A.H., 1991. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223–254. Bhuiyan, M.S., Fukunaga, K., 2007. Inhibition of HtrA2/Omi ameliorates heart dysfunction following ischemia/reperfusion injury in rat heart in vivo. Eur. J. Pharmacol. 557, 168–177. Chi, J.T., Wang, Z., Nuyten, D.S., Rodriguez, E.H., Schaner, M.E., Salim, A., Wang, Y., Kristensen, G.B., Helland, A., Borresen-Dale, A.L., Giaccia, A., Longaker, M.T., Hastie, T., Yang, G.P., van de Vijver, M.J., Brown, P.O., 2006. Gene expression programs in response to hypoxia: cell type specificity and prognostic significance in human cancers. PLoS. Med. 3, e47. Dai, Y., Xu, M., Wang, Y., Pasha, Z., Li, T., Ashraf, M., 2007. HIF-1alpha induced-VEGF overexpression in bone marrow stem cells protects cardiomyocytes against ischemia. J. Mol. Cell Cardiol. 42, 1036–1044. Danial, N.N., Korsmeyer, S.J., 2004. Cell death: critical control points. Cell 116, 205–219. Diaz Lanza, A.M., Abad Martinez, M.J., Fernandez Matellano, L., Recuero Carretero, C., Villaescusa Castillo, L., Silvan Sen, A.M., Bermejo Benito, P., 2001. Lignan and phenylpropanoid glycosides from Phillyrea latifolia and their in vitro antiinflammatory activity. Planta. Med. 67, 219–223. Ferrara, N., 2004. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, 581–611. Foo, R.S., Mani, K., Kitsis, R.N., 2005. Death begets failure in the heart. J. Clin. Invest. 115, 565–571. Fu, P., Arcasoy, M.O., 2007. Erythropoietin protects cardiac myocytes against anthracyclineinduced apoptosis. Biochem. Biophys. Res. Commun. 354, 372–378. Gao, F., Gao, E., Yue, T.L., Ohlstein, E.H., Lopez, B.L., Christopher, T.A., Ma, X.L., 2002. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia– reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 105, 1497–1502. Giordano, F.J., 2005. Oxygen, oxidative stress, hypoxia, and heart failure. J. Clin. Invest. 115, 500–508. Goldspink, D.F., Burniston, J.G., Tan, L.B., 2003. Cardiomyocyte death and the ageing and failing heart. Exp. Physiol. 88, 447–458. Gora-Kupilas, K., Josko, J., 2005. The neuroprotective function of vascular endothelial growth factor (VEGF). Folia Neuropathol. 43, 31–39. Hagen, T., D'Amico, G., Quintero, M., Palacios-Callender, M., Hollis, V., Lam, F., Moncada, S., 2004. Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol. Biochem. Biophys. Res. Commun. 322, 923–929. Haunstetter, A., Izumo, S., 1998. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ. Res. 82, 1111–1129. Huang, Y., Hickey, R.P., Yeh, J.L., Liu, D., Dadak, A., Young, L.H., Johnson, R.S., Giordano, F.J., 2004. Cardiac myocyte-specific HIF-1alpha deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J. 18, 1138–1140. Iliodromitis, E.K., Lazou, A., Kremastinos, D.T., 2007. Ischemic preconditioning: protection against myocardial necrosis and apoptosis. Vasc. Health Risk Manag. 3, 629–637. Jiang, B.H., Rue, E., Wang, G.L., Roe, R., Semenza, G.L., 1996. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem. 271, 17771–17778. Kajstura, J., Cheng, W., Sarangarajan, R., Li, P., Li, B., Nitahara, J.A., Chapnick, S., Reiss, K., Olivetti, G., Anversa, P., 1996. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am. J. Physiol. 271, H1215–1228. Kakinuma, Y., Ando, M., Kuwabara, M., Katare, R.G., Okudela, K., Kobayashi, M., Sato, T., 2005. Acetylcholine from vagal stimulation protects cardiomyocytes against ischemia and hypoxia involving additive non-hypoxic induction of HIF-1alpha. FEBS Lett. 579, 2111–2118. Kanupriya, Prasad, D., Sai Ram, M., Kumar, R., Sawhney, R.C., Sharma, S.K., Ilavazhagan, G., Kumar, D., Banerjee, P.K., 2005. Cytoprotective and antioxidant activity of Rhodiola

14

J. Zhang et al. / European Journal of Pharmacology 607 (2009) 6–14

imbricata against tert-butyl hydroperoxide induced oxidative injury in U-937 human macrophages. Mol. Cell. Biochem. 275, 1–6. Ke, Q., Costa, M., 2006. Hypoxia-inducible factor-1 (HIF-1). Mol. Pharmacol. 70, 1469–1480. Kelly, G.S., 2001. Rhodiola rosea: a possible plant adaptogen. Altern. Med. Rev. 6, 293–302. Kimbro, K.S., Simons, J.W., 2006. Hypoxia-inducible factor-1 in human breast and prostate cancer. Endocr. Relat. Cancer 13, 739–749. Koyama, T., Temma, K., Akera, T., 1991. Reperfusion-induced contracture develops with a decreasing [Ca2+]i in single heart cells. Am. J. Physiol. 261, H1115–1122. Kucinskaite, A., Briedis, V., Savickas, A., 2004. Experimental analysis of therapeutic properties of Rhodiola rosea L. and its possible application in medicine. Medicina (Kaunas) 40, 614–619. Kumar, D., Kirshenbaum, L., Li, T., Danelisen, I., Singal, P., 1999. Apoptosis in isolated adult cardiomyocytes exposed to adriamycin. Ann. N. Y. Acad. Sci. 874, 156–168. Kumar, D., Kirshenbaum, L.A., Li, T., Danelisen, I., Singal, P.K., 2001. Apoptosis in adriamycin cardiomyopathy and its modulation by probucol. Antioxid. Redox. Signal. 3, 135–145. Lee, J.W., Bae, S.H., Jeong, J.W., Kim, S.H., Kim, K.W., 2004. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp. Mol. Med. 36, 1–12. Lishmanov, IuB., Maslova, L.V., Maslov, L.N., Dan'shina, E.N., 1993. The anti-arrhythmia effect of Rhodiola rosea and its possible mechanism. Biull. Eksp. Biol. Med. 116, 175–176. Lishmanov, IuB., Naumova, A.V., Afanas'ev, S.A., Maslov, L.N., 1997. Contribution of the opioid system to realization of inotropic effects of Rhodiola rosea extracts in ischemic and reperfusion heart damage in vitro. Eksp. Klin. Farmakol. 60, 34–36. Liu, A.H., Cao, Y.N., Liu, H.T., Zhang, W.W., Liu, Y., Shi, T.W., Jia, G.L., Wang, X.M., 2008. DIDS attenuates staurosporine-induced cardiomyocyte apoptosis by PI3K/Akt signaling pathway: activation of eNOS/NO and inhibition of Bax translocation. Cell. Physiol. Biochem. 22, 177–186. Ma, H., Zhang, H.F., Yu, L., Zhang, Q.J., Li, J., Huo, J.H., Li, X., Guo, W.Y., Wang, H.C., Gao, F., 2006. Vasculoprotective effect of insulin in the ischemic/reperfused canine heart: role of Akt-stimulated NO production. Cardiovasc. Res. 69, 57–65. Mabjeesh, N.J., Escuin, D., LaVallee, T.M., Pribluda, V.S., Swartz, G.M., Johnson, M.S., Willard, M.T., Zhong, H., Simons, J.W., Giannakakou, P., 2003. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 3, 363–375. Maimeskulova, L.A., Maslov, L.N., Lishmanov, IuB., Krasnov, E.A., 1997. The participation of the mu-, delta- and kappa-opioid receptors in the realization of the antiarrhythmia effect of Rhodiola rosea. Eksp. Klin. Farmakol. 60, 38–39. Maslova, L.V., Kondrat'ev, BIu., Maslov, L.N., Lishmanov, IuB., 1994. The cardioprotective and antiadrenergic activity of an extract of Rhodiola rosea in stress. Eksp. Klin. Farmakol. 57, 61–63. Mehrhof, F.B., Muller, F.U., Bergmann, M.W., Li, P., Wang, Y., Schmitz, W., Dietz, R., von Harsdorf, R., 2001. In cardiomyocyte hypoxia, insulin-like growth factor-I-induced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinase-dependent and mitogen-activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein. Circulation 104, 2088–2094. Nakano, M., Knowlton, A.A., Dibbs, Z., Mann, D.L., 1998. Tumor necrosis factor-alpha confers resistance to hypoxic injury in the adult mammalian cardiac myocyte. Circulation 97, 1392–1400. Nakayama, H., Chen, X., Baines, C.P., Klevitsky, R., Zhang, X., Zhang, H., Jaleel, N., Chua, B.H., Hewett, T.E., Robbins, J., Houser, S.R., Molkentin, J.D., 2007. Ca2+- and mitochondrialdependent cardiomyocyte necrosis as a primary mediator of heart failure. J. Clin. Invest. 117, 2431–2444.

Nan, J.X., Jiang, Y.Z., Park, E.J., Ko, G., Kim, Y.C., Sohn, D.H., 2003. Protective effect of Rhodiola sachalinensis extract on carbon tetrachloride-induced liver injury in rats. J. Ethnopharmacol. 84, 143–148. Nor, J.E., Christensen, J., Mooney, D.J., Polverini, P.J., 1999. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am. J. Pathol. 154, 375–384. Piret, J.P., Mottet, D., Raes, M., Michiels, C., 2002. Is HIF-1alpha a pro- or an antiapoptotic protein? Biochem. Pharmacol. 64, 889–892. Pugh, C.W., Ratcliffe, P.J., 2003. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9, 677–684. Qing, M., Gorlach, A., Schumacher, K., Woltje, M., Vazquez-Jimenez, J.F., Hess, J., Seghaye, M.C., 2007. The hypoxia-inducible factor HIF-1 promotes intramyocardial expression of VEGF in infants with congenital cardiac defects. Basic Res. Cardiol. 102, 224–232. Semenza, G.L., 1998. Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr. Opin. Genet. Dev. 8, 588–594. Semenza, G.L., 1999. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell Dev. Biol. 15, 551–578. Semenza, G.L., 2004. O2-regulated gene expression: transcriptional control of cardiorespiratory physiology by HIF-1. J. Appl. Physiol. 96, 1173–1177 discussion 1170–1172. Semenza, G.L., Shimoda, L.A., Prabhakar, N.R., 2006. Regulation of gene expression by HIF-1. Novartis Found Symp. 272, 2–8 discussion 8–14, 33–36. Takahashi, N., Wang, X., Tanabe, S., Uramoto, H., Jishage, K., Uchida, S., Sasaki, S., Okada, Y., 2005. ClC-3-independent sensitivity of apoptosis to Cl− channel blockers in mouse cardiomyocytes. Cell Physiol. Biochem. 15, 263–270. Tang, Y.L., Zhao, Q., Zhang, Y.C., Cheng, L., Liu, M., Shi, J., Yang, Y.Z., Pan, C., Ge, J., Phillips, M.I., 2004. Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium. Regul. Pept. 117, 3–10. Tomanek, R.J., Zheng, W., Yue, X., 2004. Growth factor activation in myocardial vascularization: therapeutic implications. Mol. Cell Biochem. 264, 3–11. von Harsdorf, R., Li, P.F., Dietz, R., 1999. Signaling pathways in reactive oxygen speciesinduced cardiomyocyte apoptosis. Circulation 99, 2934–2941. Wang, X., Takahashi, N., Uramoto, H., Okada, Y., 2005. Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia–reperfusion in mouse cardiomyocytes. Cell Physiol. Biochem. 16, 147–154. Wang, H.C., Zhang, H.F., Guo, W.Y., Su, H., Zhang, K.R., Li, Q.X., Yan, W., Ma, X.L., Lopez, B.L., Christopher, T.A., Gao, F., 2006. Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation. Apoptosis 11, 1453–1460. Wenger, R.H., 2002. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J. 16, 1151–1162. Xu, M.F., Rhota, U., Dai, Y., Wang, Y.G., Zeeshan, P., Muhammad, A., 2007. In vitro and in vivo effects of bone marrow stem cells on cardiac structure and function. J. Mol. Cell Cardiol. 42, 441–448. Zhang, L., Yu, H., Sun, Y., Lin, X., Chen, B., Tan, C., Cao, G., Wang, Z., 2007. Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells. Eur. J. Pharmacol. 564, 18–25. Zhou, L., Ma, W., Yang, Z., Zhang, F., Lu, L., Ding, Z., Ding, B., Ha, T., Gao, X., Li, C., 2005. VEGF165 and angiopoietin-1 decreased myocardium infarct size through phosphatidylinositol-3 kinase and Bcl-2 pathways. Gene Ther. 12, 196–202. Zimmermann, R., Kastens, J., Linz, W., Wiemer, G., Scholkens, B.A., Schaper, J., 1999. Effect of long-term ACE inhibition on myocardial tissue in hypertensive strokeprone rats. J. Mol. Cell. Cardiol. 31, 1447–1456.