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HIF-dependent signaling pathway
Journal of Ethnopharmacology 133 (2011) 517–523
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Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway Jun Qing Liang a,b,1 , Kun Wu a,1 , Zhen Hua Jia b , Chang Liu a , Jin Ding a , Shan Na Huang a , Pei Pei Yin a , Xiang Chun Wu b , Cong Wei b , Yi Ling Wu b,∗∗ , Hong Yang Wang a,c,∗ a The International Cooperation Laboratory on Signal Transduction of Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China b Department of Pharmacology, Hebei Yiling Pharmaceutical Research Institute, 238 Tianshan Street, Shijiazhuang, Hebei 050035, China c State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute and Cancer Institute of Shanghai Jiao Tong University, No. 25 Lane 2200 Xietu Road, Shanghai 200032, China
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Article history: Received 13 July 2010 Received in revised form 21 September 2010 Accepted 13 October 2010 Available online 20 October 2010 Keywords: Tongxinluo Hypoxia-inducible factor Signal transduction eNOS
a b s t r a c t Aim of the study: To investigate the molecular mechanisms whereby the Chinese medicinal compound Tongxinluo improves vascular endothelial function through studying the induction of endothelial nitric oxide synthase (eNOS) and its upstream signaling pathway. Materials and methods: Hyperhomocysteinemia was induced in Wistar rats by a methionine-rich diet followed by Tongxinluo treatment. The aorta ring was isolated for measuring vascular dilation of aorta and eNOS expression. Human umbilical vein endothelial cells (HUVECs) were transfected with AP-1, NFB, HRE or eNOS reporter plasmid followed by Tongxinluo exposure. Expression of the reporter genes was measured by luciferase assay. The level of eNOS was studied by western blot and the nitric oxide content was measured using the nitrate reductase method. HUVECs were also transiently transfected with the dominant negative mutant of HIF-1, PI-3K or Akt to explore the role of HIF and PI-3K/Akt pathway in eNOS induction by Tongxinluo. Results: Tongxinluo could significantly up-regulate the expression of eNOS in the aortic tissue and improve the endothelium-dependent vasodilation of the aorta ring. Additionally, Tongxinluo at various doses could significantly enhance the expression of HRE and eNOS reporter gene as well as up-regulate the protein level of eNOS. Meanwhile, Tongxinluo caused a dose-dependent increase in the NO content in the supernatant of HUVECs. Suppression of HIF-1 activation by DN-HIF or inhibition of PI-3K/Akt pathway by P85 or DN-Akt both attenuated HRE reporter gene activation and eNOS induction by Tongxinluo. Conclusion: Tongxinluo, a compound Chinese traditional medicine, up-regulates the expression of eNOS via the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the endothelium-dependent vasodilation. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Tongxinluo is a Chinese herbal compound that has been widely used in the treatment of cardiovascular and cerebrovascular diseases in China (Chen et al., 2009; Zhang et al., 2009). Up to now, about 6 million patients with cardiovascular and cerebrovascular diseases have been treated with Tongxinluo. It has been demonstrated that this herbal compound is cardioprotective against coronary heart disease and unstable angina pectoris (Tang et al.,
∗ Corresponding author. Tel.: +86 021 81875361; fax: +86 021 65566851. ∗∗ Corresponding author. Tel.: +86 0311 85901718; fax: +86 0311 85901088. E-mail addresses: [email protected] (Y.L. Wu), [email protected] (H.Y. Wang). 1 These authors have contributed equally to this work. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.10.031
2006). It not only alleviates the symptoms of angina pectoris, but also decreases the frequency and duration of anginal episodes. In addition, Tongxinluo has been verified to significantly reduce the doses of nitroglycerin for angina pectoris and result in improvements in ECG patterns and hemorheology variables in patients with angina pectoris (Xu et al., 2000; Ji, 2001). Tongxinluo also alleviates the symptoms of patients with cardiac failure caused by the onset of acute coronary events via improving myocardial blood supply and enhancing myocardial contractility. Furthermore, it has been found that Tongxinluo promotes the recovery of myocardial functions and the revascularization of the ventricular myocardium and improves global systolic function after myocardial infarction (Yang et al., 2006). These aforementioned therapeutic effects of Tongxinluo are closely associated with the improvement of cardiovascular functions. However, the molecular mechanisms whereby Tongxinluo improves vascular endothelial function remain unelucidated.
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Hypoxia is an obligatory component in the ischemic heart or brain. In recent years, hypoxia inducible factor-1 (HIF-1) has been shown to play an important role in the protection of the compromised myocardium during myocardial infarction. HIF-1 consists of two subunits, HIF-1␣ and HIF-1. HIF-1 is constitutively expressed while HIF-1␣ is regulated by oxygen tension and determines the activity of HIF-1. HIF-1 is activated under hypoxic conditions and is the principal transcription factor involved in the regulation of transcriptional responses to hypoxia. HIF-1␣ can bind to the hypoxia response element (HRE) of its target genes such as vascular endothelial growth factor (VEGF), erythropoietin (Epo), and glucose transporter-1 (Glut-1) and thereby modulate their transcription, thus inducing a cascade of compensatory response against hypoxia and promoting the growth and metabolism of cells (Goldberg et al., 1988; Semenza, 1999; Farmer et al., 2003; Resar et al., 2005; Kaur et al., 2007). We hypothesized that Tongxinluo might exert its cardiovascular protective function by modulating the activation of HIF-1. In this study, we investigated the effects of Tongxinluo on the expression of endothelial nitric oxide synthase (eNOS) and endothelium-dependent vasodilation in a vascular endothelial dysfunction model by inducing hyperhomocysteinemia in rats using a methionine-rich diet. We further investigated the effects of Tongxinluo on the eNOS expression and HIF activation in human umbilical vein endothelial cells (HUVECs) and its effect on the upstream PI-3K/Akt signaling pathway. 2. Materials and methods
dissolving 4.11 g of Tongxinluo ultra fine powder in 100 mL 0.5% sodium carboxymethylcellulose. Besides the same treatment as the hyperhomocysteinemia group, the positive control group was administrated with 10 mL/kg body weight of 4 mg/mL folic acid (FA) (Tianjin Lisheng Pharmaceutical Co., Tianjin, China) in 0.5% sodium carboxymethylcellulose by gavage. 2.3. Isolation of the aorta rings in rats The thoracic aorta was isolated as previously described (Resta and Walker, 1996). Briefly, a midline cut was made along the sternum, and with the removal of the lungs and heart, the thoracic aorta was exposed, isolated and then placed in cold Krebs’ solution containing 95% O2 and 5% CO2 . The isolated blood vessel was cut into two 3–4 mm wide vascular rings with the careful preservation of the vascular endothelium. The vascular rings were vertically hung in an organ bath containing 10 mL Krebs’ solution and connected with a BL-420E+ biological function experimental system (Chengdu Technology and Market, Chengdu, China). The vascular rings were subjected to precontraction with 1 M phenylephrine (PE) (Sigma–Aldrich Co., St. Louis, MO, USA) or 60 mM KCl, followed by dilation with acetylcholine (ACH) (Sigma–Aldrich Co., St. Louis, MO, USA) at progressive final concentrations of 0.01 M, 0.1 M, 1 M, 10 M and the tension curve was observed and recorded. The results were presented as the percentage of ACH dilation value relative to the maximal PE tension value. The change in tension rate of the vascular ring was calculated as follows: Tension rate (%) =
PE tension peak value − ACH dilation peak value × 100% PE tension peak value − value of basic tension
2.1. Preparation of Tongxinluo ultra fine powder solution 2.4. Immunohistochemistry Tongxinluo ultra fine powder (Lot Number: 071201, Shijiazhuang Yiling Pharmaceutical Co., Shijiazhuang, China) was dissolved in serum-free Dulbecco’s Modified Eagle’s Medium (DMEM). The solution was sonicated for 1 h followed by centrifuge at 3500 rpm for 10 min. The supernatant was filtrated using 0.22 m micro-pore filter. Meanwhile, the precipitate was heated and dried at 60 ◦ C in order to calculate the practical volume of Tongxinluo ultra fine powder dissolved. The solution was adjusted to a final concentration of 2 mg/mL and preserved at −20 ◦ C for subsequent use. Before each experiment, the solution was diluted in serum-free DMEM to a working concentration (containing 0.1% fetal bovine serum). 2.2. Animals A total of 180 healthy male Wistar rats, weighing 220–250 g, were bred in the New Drug Evaluation Center, Hebei Institute of Integrated Chinese Traditional Medicine and Western Medicine, Hebei, China. This study was approved by the Animal Care and Use Committee of Shijiazhuang, China. After they were bred for 5 days for adaptation, the animals were randomly divided into four groups: the negative control group, the hyperhomocysteinemia group, the Tongxinluo group, and the positive control group. The negative control group was fed normally with free access to drinking water and was administered with 10 mL/kg body weight of 0.5% sodium carboxymethylcellulose by gavage. Hyperhomocysteinemia was induced in rat as previously described (Joseph et al., 2002; Walker et al., 2004). The hyperhomocysteinemia group was administrated with 10 mL/kg body weight of 3% l-methionine (Sigma–Aldrich Co., St. Louis, MO, USA) in 0.5% sodium carboxymethylcellulose by gavage for the time period indicated. In addition to the same treatment as the hyperhomocysteinemia group, the animals in the Tongxinluo group were administered by gavage, at 9–10 o’clock everyday with 10 mL/kg body weight of Tongxinluo suspension, which was prepared by
Tissue sections were prepared of the thoracic aortas after fixation in 4% paraformaldehyde, dehydration and embedding in paraffin. The expression of eNOS in the aorta tissue was examined using the SP immunohistochemistry kit according to the manufacturer’s instructions (Zhongshan Co., Beijing, China). Densitometric analysis of immunocytochemical staining of eNOS was carried out and eNOS staining intensity was expressed in optical density (OD) units. 2.5. DNA transfections Human eNOS luciferase reporter plasmid was kindly provided by Dr. Philip A. Marsden at University of Toronto, Toronto, ON, Canada. AP-1 luciferase reporter plasmid, NF-B luciferase reporter gene vector and HRE reporter plasmid were used for transient transfections as previously described (Ding et al., 2006a). The plasmid vector DN-HIF encoded a dominant negative mutant of HIF-1␣, lacking both the basic DNA binding domain and the C-terminal transactivation domain as previously described (Jiang et al., 1996). The plasmid vector DN-Akt expressed a full length Akt protein mutated at Thr308 and Ser473 , rendering it inactivated. The plasmid vector P85 expressed the dominant-negative mutant form of PI-3K (Ding et al., 2006b). For DNA transfections, HUVECs were inoculated in a 6-well plate at a density of 2 × 105 cells/well and incubated at 37 ◦ C for 24 h. These cells were transfected with vectors carrying eNOS, AP-1 or NF-B reporter, where indicated, using Lipofectamine according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Stable cells expressing the appropriate genes were selected using geneticin (G418, Gibco-BRL) at 300 g/mL and were identified by determining luciferase activities. Additionally, HUVECs stably expressing eNOS-luc were grown to 80% to 90% confluence in a 10-cm dish and transfected with 5 g of plasmid vectors for DN-HIF, P85 or DN-Akt, or GFP control vectors using Lipofectamine as instructed by the manufacturer. The cells were typically used 24–48 h after transfection.
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Fig. 1. Tongxinluo promotes eNOS expression and improves aorta dilation in rats. Hyperhomocysteinemia rats and Tongxinluo treatment were administrated for four weeks as described in Materials and Methods. Their aorta rings were isolated and subjected to immunochemical staining using anti-eNOS antibody (A) ensitometric analysis of immunocytochemical staining of eNOS (B) (p < 0.01). The change in tension rate of the isolated aorta in response to 1 M phenylephrine (PE) and 1 M acetylcholine (ACH) was measured (C and D). Rats in control group, hyperhomocysteinemia group, Tongxinluo group and folic acid group were administrated for two weeks before their aorta rings were isolated and subjected to KCl precontracted vasodilation (E) or PE precontracted vasodilation (F) by ACH at doses indicated. HCY, hyperhomocysteinemia group; TXL, Tongxinluo group; FA, folic acid group. *p < 0.05 compared with controls; # p < 0.05 compared with HCY.
2.6. Luciferase reporter gene assay
2.9. Statistical analysis
HUVECs were inoculated in a 96-well plate at a density of 8 × 103 cells/well. When they reached 80% to 90% confluent, the cells were treated with Tongxinluo at the doses and time points indicated. Both the treated cells and untreated control cells were lysed in a 50-l lysis buffer at 4 ◦ C for 20 min. The luciferase activities were measured using a luciferase reporter gene detection kit (Promega) in a luminometer as instructed by the manufacturer. The induced expression of eNOS or the enhanced activity of AP-1, NFB or HIF-1 was represented as the ratio between the luciferase activity in Tongxinluo-treated cells and that in untreated cells.
The data were expressed as mean ± S.D. and analyzed using SPSS software (SPSS Inc., Chicago, IL, USA). Each data point represents the average of three independent experiments; bars, ±S.D. One-way ANOVA was used in the analysis of the data between the groups. The data with significant difference were compared using Newman–Kuels q-test. p < 0.05 was considered statistically significant.
2.7. Immunoblotting studies Cellular lysates of HUVECs were prepared as previously described. The protein samples (30 g each) were resolved by SDSPAGE. Immunoblotting procedures were performed as previously depicted (Wen et al., 2010). Anti-GAPDH, anti--actin, anti-eNOS antibodies (from Santa Cruze Biotechnology, Santa Cruz, CA) and anti-phosphorylated JNK, anti-total JNK antibodies (from Cell Signaling Technology, Danvers, MA) were used. 2.8. Determination of nitric oxide content The nitric oxide (NO) content was examined using the nitrate reductase method by using NO detection kit as instructed by the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
3. Results 3.1. Tongxinluo promotes eNOS expression and improves aorta dilation in rats We induced hyperhomocysteinemia in rats using a methioninerich diet and examined the effect of Tongxinluo on the expression of eNOS in the aorta. We found that eNOS was positively expressed in the normal aortic tissue (Fig. 1A). The intensity of eNOS expression, however, was significantly attenuated in rats with hyperhomocysteinemia. Tongxinluo treatment, on the other hand, significantly enhanced the intensity of eNOS expression compared with that of both the control rats and the untreated hyperhomocysteinemia rats (p < 0.05) (Fig. 1A and B). We also studied the effect of Tongxinluo on the vasodilation of the aorta in rats with induced hyperhomocysteinemia. We showed that, in the rats with hyperhomocysteinemia, the change in tension rate of the isolated aorta ring decreased significantly compared with that in the control
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Fig. 2. Tongxinluo enhances the expression of eNOS and the production of NO. HUVECs stably expressing eNOS-luc were treated with Tongxinluo for 24 h at doses indicated (A) or for 12–48 h at 100 g/mL (B). The eNOS transcription level was assessed by measuring the luciferase activities. The induction of eNOS was expressed as fold over that of medium control. HUVEC cells were treated with Tongxinluo for 24 h at doses indicated (C and D). Cellular lysates were prepared from these cells for immunoblotting analysis of eNOS and GADPH (C) and their supernatant were collected for nitric oxide (NO) detection (D). The supernatant of HUVECs treated with Tongxinluo (50 g/mL) and/or l-NMMA (400 M) for 24 h were collected and subjected to nitric oxide detection (E).
group (p < 0.05) (Fig. 1C and D). Tongxinluo administration, however, significantly increased the change in tension rate of isolated aorta ring compared with that of the untreated rats with induced hyperhomocysteinemia. In order to further investigate the effect of Tongxinluo on the relationship between increasing ACH levels and the vasodilation effect of ACH on aorta rings, the isolated aorta rings precontracted with KCl (Fig. 1E) or PE (Fig. 1F) were exposed to progressive concentrations of ACH. We discovered that ACH induced a similar pattern of change in tension rate in aorta rings precontracted by KCl or PE. In consistence with the results of single-dose ACH induced vasodilation, the change in tension rate of aorta rings was much lower in the hyperhomocysteinemia group compared with the control group. On the other hand, the treatment of Tongxinluo and folic acid increased the change in tension rate of aorta rings compared with that of hyperhomocysteinemia rats. These results indicated that ACH dilated aorta rings precontracted by KCl or PE in a dose-dependent manner.
3.3. HIF-1 is activated in HUVECs exposed to Tongxinluo As shown in Fig. 3A, Tongxinluo treatment did not influence the phosphorylation of JNK and Erks, and exhibited marginal effect on p38 phosphorylation, indicating MAPKs were not involved for Tongxinluo-induced eNOS expression. Moreover, phosphorylation of STAT3 was not increased but decreased in HUVEC cells exposed to Tongxinluo, suggesting STAT3 is not required in Tongxinluoinduced eNOS expression (Fig. 3B). We also studied the effects of Tongxinluo on the activation of AP-1 and NF-B in HUVECs. We observed that EGF could markedly induce the activation of AP-1 in HUVECs while Tongxinluo at 100 g/mL exerted virtually no effect in these cells (Fig. 3C). Similarly, TNF-␣ induced a noticeable increase in the activity of NF-B in HUVECs, while Tongxinluo at 100 g/mL did not exert any effect in these cells (Fig. 3D). On the other hand, Tongxinluo treatment induced a dose- and time-dependent increase in the activities of HRE (Fig. 3E and F), suggesting that HIF-1 transcriptional activity was up-regulated by Tongxinluo in HUVECs.
3.2. Tongxinluo enhances the expression of eNOS and the production of NO
3.4. HIF-1 is required in Tongxinluo-induced eNOS expression
We further examined the effects of Tongxinluo on the expression of eNOS in HUVECs stably expressing eNOS-luc. We observed that Tongxinluo induced the expression of eNOS in a dose and timedependent manner (Fig. 2A and B). Additionally, immunoblotting studies revealed that, the expression of eNOS markedly increased upon Tongxinluo treatment as compared with that of the control (Fig. 2C). Furthermore, we observed that Tongxinluo induced a dose-dependent significant increase in the level of NO, which was abolished by the NOS inhibitor l-NMMA (p < 0.01) (Fig. 2D and E).
We further examined whether the up-regulation of eNOS by Tongxinluo in HUVECs was mediated by its effect on HIF-1. We transfected HUVEC eNOS-luc cells with the DN-HIF expression vectors and GFP control vectors and exposed these cells to Tongxinluo for various time points at the doses indicated. We observed that Tongxinluo induced a remarkable time- and dose-dependent increase in the expression of eNOS in the HUVEC eNOS-luc cells transfected with the GFP expression vector (Fig. 4A and B). This apparent increase in eNOS expression, however, was significantly attenuated in the HUVEC eNOS-luc cells transfected with the DN-
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Fig. 3. HIF-1 is activated in HUVECs exposed to Tongxinluo. (A and B) HUVECs treated with Tongxinluo for 24 h were subjected to immunoblotting assay. EGF and TNF-␣ treated HUVECs were used as positive control, respectively. HUVECs were transfected with vectors expressing NF-B or AP-1 reporter and then treated with TNF-␣, EGF or Tongxinluo. The transcription level of AP-1 (C) and the transcriptional activities of NF-B (D) were measured by luciferase assay. HUVECs were also transfected with the expression vector for HRE reporter and were treated with Tongxinluo for 48 h at the doses indicated (E) or for 12–48 h at 100 g/mL (F). The induction of HRE was examined by luciferase assays relative to that of the medium control.
HIF expression vector, which was markedly lower than that of the HUVEC eNOS-luc cells transfected with the GFP control vectors at each time point and dose examined (p < 0.01). In addition, compared with that in the GFP control group treated with Tongxinluo, the expression of eNOS in the HUVEC eNOS-luc cells transfected
with DN-HIF and treated with Tongxinluo was markedly decreased (Fig. 4C). 3.5. Tongxinluo up-regulates eNOS expression via PI-3K/Akt-dependent pathway We also investigated whether Tongxinluo had any effect on the PI-3K/Akt pathway, which functions upstream of HIF-1. Tongxinluo induced a marked dose-dependent rise in the activities of HRE in HUVEC cells, and transfection of these cells with DN-Akt or P85 both significantly attenuated the activation of HRE (p < 0.01) (Fig. 5A and B). Similarly, transfection of these cells with DN-Akt significantly attenuated the rise of eNOS expression induced by Tongxinluo as well (p < 0.01) (Fig. 5C and D). 4. Discussion
Fig. 4. HIF-1 is required in Tongxinluo-induced eNOS expression. HUVEC eNOS-luc cells were transfected with the expression vector of DN-HIF or GFP and then treated with 100 g/mL of Tongxinluo for 12–48 h (A) or with 50–200 g/mL of Tongxinluo for 24 h (B) before luciferase assays. Cellular lysates from HUVEC eNOS-luc cells transfected with the DN-HIF expression vector or GFP control vectors were used for immunoblotting analysis with anti-eNOS or -actin antibody (C).
Tongxinluo is a traditional Chinese medicine which is extracted, concentrated, freeze-dried and standardized from a mixture of 12 medicinal constituents as previously described (Chen et al., 2009) and has been widely used in the treatment of cardiovascular and cerebrovascular diseases over recent years. Numerous basic and clinical studies have indicated that Tongxinluo can significantly improve the vascular endothelial function (Zhang et al., 2009), although the underlying molecular mechanism is unclear. Hyperhomocysteinemia was considered as an independent risk factor for cardiovascular and cerebrovascular disease (Maron and Loscalzo, 2009), and folic acid was reported to counteract the level of plasma homocysteine in rats with hyperhomocysteinemia (Gilani et al., 2001). In this study, we investigated the effects of Tongxinluo on the vasodilatory function of the aorta in rats
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Fig. 5. Tongxinluo up-regulates eNOS expression via PI-3K/Akt/-dependent pathway. HUVEC cells were co-transfected with the HRE reporter plasmid together with GFP control plasmid or the expression vectors for DN-Akt (A) or P85 (B) and then treated with various dosages of Tongxinluo for 48 h. Luciferase activity was then measured. HUVEC eNOS-luc cells were transfected with the GFP control plasmid or the expression vectors for DN-Akt (C and D) and then treated with Tongxinluo as indicated followed by luciferase assay.
the intracellular and extracellular route (Liang and Lv, 2002). However, the effect of HIF-1␣, which has a transcriptional activity on the expression of eNOS (Kobayashi et al., 2003; Martinive et al., 2009), has not been reported. Therefore, in this study, we investigated the effects of Tongxinluo on such transcription factors as NF-B, AP-1 and HIF-1␣ and STAT3. We found that Tongxinluo did not elicit the activation of AP-1, NF-B, STAT3, but could significantly induce the expression of the HRE reporter gene, suggesting that Tongxinluo could improve the vascular endothelial function in a HIF-dependent manner. In addition, we found that HIF downregulation by a dominant-negative form of HIF attenuated the rise of eNOS induced by Tongxinluo treatment, suggesting that the upregulation of eNOS by Tongxinluo is at least partly dependent upon HIF. It has been reported that the activation of HIF is related to the PI3K/Akt signaling pathway (Sakamoto et al., 2009). Chen et al. (2005) found that the inhibition of the activity of PI-3K by Ly294001, a PI-3K inhibitor, in the renal vascular endothelium inhibited the expression of HIF. Many previous studies also confirmed that the activation of Akt could up-regulate intracellular eNOS expression (Dimmeler et al., 1999; Fulton et al., 1999; Gao et al., 2000; Kureishi et al., 2000; Zeng et al., 2000; Chavakis et al., 2001). Activated PI3K phosphorylates downstream PKB/Akt and activates eNOS, thus leading to the production of endothelium-derived NO (Scotland et al., 2002). It has not been reported whether Tongxinluo can up-regulate eNOS expression by activating PI-3K/Akt pathway to induce HIF expression. Therefore, in this study, we used P85 and DN-Akt to inhibit the PI-3K/Akt signaling pathway and found that the activation of the HRE reporter gene and the up-regulation of eNOS by Tongxinluo was markedly inhibited, indicating that the induction by Tongxinluo of HIF and eNOS expression partly depends on the activation of the PI-3K/Akt signaling pathway. 5. Conclusion
with vascular endothelial dysfunctions caused by hyperhomocysteinemia and found that the herbal compound could not only improve the endothelium-dependent vasodilation of the compromised artery, but also increase the aortic response of TXL-treated rats to phenylephrine, indicating that Tongxinluo may protect vascular smooth muscle cells against homocysteine-induced oxidative stress. Strikingly, the expression of eNOS was up-regulated in the arterial tissue in rats treated with Tongxinluo compared with that in hyperhomocysteinemia group. Our in vitro experiments also confirmed that Tongxinluo could up-regulate eNOS expression in vascular endothelium via the PI-3K/Akt/HIF-dependent signaling pathway, thus leading to the up-regulation of NO levels. Under normal condition, eNOS is a major NO source in the cardiovascular system and plays an important role in dilating blood vessel, adjusting blood pressure, inhibiting platelet aggregation and suppressing smooth muscle cell proliferation (Kavdia and Popel, 2004). In this study, we found that Tongxinluo could enhance the expression of eNOS, elevate NO levels and consequently improve the vascular endothelial function. It is reported that enhanced eNOS activity by carbon monoxide (CO) inhalation may result from the activation of MAPKs (Fujimoto et al., 2004). Whereas, our study revealed JNK, ERK and p38 are not required in eNOS induction by Tongxinluo in HUVECs. Previous studies have suggested that NF-B, a transcription factor, could be activated and induce the expression of eNOS during cerebral ischemia-reperfusion, which serves to antagonize the condition (Min et al., 2007). A previous study also confirmed that in human neutrophils, transcription factor AP1 could modulate not only the expression of plasma C-reactive protein, nitrotyrosine and IL-8, but also the expression of eNOS (Khreiss et al., 2005). In addition, Liang et al. studied the expression of eNOS and HIF-1 in hypoxia-preconditioned mice and found that HIF-1␣ and eNOS could protect the cerebral function from both
The traditional Chinese medicinal compound Tongxinluo can up-regulate eNOS expression and enhance the release of NO through activating the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the vascular endothelial function. This finding not only helps us to understand the pharmacological mechanism of Tongxinluo, but also provides a theoretical basis for Tongxinluo in treating cardiovascular and cerebrovascular diseases. It is worth mentioning that, in this study, it is revealed that Tongxinluo has an inductive effect on HIF-1, suggesting that the drug may improve the cell’s adaptability to hypoxia and increases the capacity of cells against hypoxic injury, which is worthwhile to be further studied. Acknowledgements This work was supported by grants from Ministry of Science and Technology Key Program 2005CB523301, and 2006DFB32460 and 91-10-02, Shanghai International Collaboration grant 08400701000 and Shanghai Pujiang Program. References Chavakis, E., Dernbach, E., Hermann, C., Mondorf, U.F., Zeiher, A.M., Dimmeler, S., 2001. Oxidized LDL inhibits vascular endothelial growth factor-induced endothelial cell migration by an inhibitory effect on the Akt/endothelial nitric oxide synthase pathway. Circulation 103 (16), 2102–2107. Chen, T.H., Wang, J.F., Chan, P., Lee, H.M., 2005. Angiotensin II stimulates hypoxia-1␣ inducible factor 1alpha accumulation in glomerular mesangial cells. Annals of the New York Academy of Sciences 1042, 286–293. Chen, W.Q., Zhong, L., Zhang, L., Ji, X.P., Zhao, Y.X., Zhang, C., Jiang, H., Wu, Y.L., Zhang, Y., 2009. Chinese medicine tongxinluo significantly lowers serum lipid levels and stabilizes vulnerable plaques in rabbit model. Journal of Ethnopharmacology 124 (1), 103–110.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway Jun Qing Liang a,b,1 , Kun Wu a,1 , Zhen Hua Jia b , Chang Liu a , Jin Ding a , Shan Na Huang a , Pei Pei Yin a , Xiang Chun Wu b , Cong Wei b , Yi Ling Wu b,∗∗ , Hong Yang Wang a,c,∗ a The International Cooperation Laboratory on Signal Transduction of Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China b Department of Pharmacology, Hebei Yiling Pharmaceutical Research Institute, 238 Tianshan Street, Shijiazhuang, Hebei 050035, China c State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute and Cancer Institute of Shanghai Jiao Tong University, No. 25 Lane 2200 Xietu Road, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 13 July 2010 Received in revised form 21 September 2010 Accepted 13 October 2010 Available online 20 October 2010 Keywords: Tongxinluo Hypoxia-inducible factor Signal transduction eNOS
a b s t r a c t Aim of the study: To investigate the molecular mechanisms whereby the Chinese medicinal compound Tongxinluo improves vascular endothelial function through studying the induction of endothelial nitric oxide synthase (eNOS) and its upstream signaling pathway. Materials and methods: Hyperhomocysteinemia was induced in Wistar rats by a methionine-rich diet followed by Tongxinluo treatment. The aorta ring was isolated for measuring vascular dilation of aorta and eNOS expression. Human umbilical vein endothelial cells (HUVECs) were transfected with AP-1, NFB, HRE or eNOS reporter plasmid followed by Tongxinluo exposure. Expression of the reporter genes was measured by luciferase assay. The level of eNOS was studied by western blot and the nitric oxide content was measured using the nitrate reductase method. HUVECs were also transiently transfected with the dominant negative mutant of HIF-1, PI-3K or Akt to explore the role of HIF and PI-3K/Akt pathway in eNOS induction by Tongxinluo. Results: Tongxinluo could significantly up-regulate the expression of eNOS in the aortic tissue and improve the endothelium-dependent vasodilation of the aorta ring. Additionally, Tongxinluo at various doses could significantly enhance the expression of HRE and eNOS reporter gene as well as up-regulate the protein level of eNOS. Meanwhile, Tongxinluo caused a dose-dependent increase in the NO content in the supernatant of HUVECs. Suppression of HIF-1 activation by DN-HIF or inhibition of PI-3K/Akt pathway by P85 or DN-Akt both attenuated HRE reporter gene activation and eNOS induction by Tongxinluo. Conclusion: Tongxinluo, a compound Chinese traditional medicine, up-regulates the expression of eNOS via the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the endothelium-dependent vasodilation. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Tongxinluo is a Chinese herbal compound that has been widely used in the treatment of cardiovascular and cerebrovascular diseases in China (Chen et al., 2009; Zhang et al., 2009). Up to now, about 6 million patients with cardiovascular and cerebrovascular diseases have been treated with Tongxinluo. It has been demonstrated that this herbal compound is cardioprotective against coronary heart disease and unstable angina pectoris (Tang et al.,
∗ Corresponding author. Tel.: +86 021 81875361; fax: +86 021 65566851. ∗∗ Corresponding author. Tel.: +86 0311 85901718; fax: +86 0311 85901088. E-mail addresses: [email protected] (Y.L. Wu), [email protected] (H.Y. Wang). 1 These authors have contributed equally to this work. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.10.031
2006). It not only alleviates the symptoms of angina pectoris, but also decreases the frequency and duration of anginal episodes. In addition, Tongxinluo has been verified to significantly reduce the doses of nitroglycerin for angina pectoris and result in improvements in ECG patterns and hemorheology variables in patients with angina pectoris (Xu et al., 2000; Ji, 2001). Tongxinluo also alleviates the symptoms of patients with cardiac failure caused by the onset of acute coronary events via improving myocardial blood supply and enhancing myocardial contractility. Furthermore, it has been found that Tongxinluo promotes the recovery of myocardial functions and the revascularization of the ventricular myocardium and improves global systolic function after myocardial infarction (Yang et al., 2006). These aforementioned therapeutic effects of Tongxinluo are closely associated with the improvement of cardiovascular functions. However, the molecular mechanisms whereby Tongxinluo improves vascular endothelial function remain unelucidated.
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Hypoxia is an obligatory component in the ischemic heart or brain. In recent years, hypoxia inducible factor-1 (HIF-1) has been shown to play an important role in the protection of the compromised myocardium during myocardial infarction. HIF-1 consists of two subunits, HIF-1␣ and HIF-1. HIF-1 is constitutively expressed while HIF-1␣ is regulated by oxygen tension and determines the activity of HIF-1. HIF-1 is activated under hypoxic conditions and is the principal transcription factor involved in the regulation of transcriptional responses to hypoxia. HIF-1␣ can bind to the hypoxia response element (HRE) of its target genes such as vascular endothelial growth factor (VEGF), erythropoietin (Epo), and glucose transporter-1 (Glut-1) and thereby modulate their transcription, thus inducing a cascade of compensatory response against hypoxia and promoting the growth and metabolism of cells (Goldberg et al., 1988; Semenza, 1999; Farmer et al., 2003; Resar et al., 2005; Kaur et al., 2007). We hypothesized that Tongxinluo might exert its cardiovascular protective function by modulating the activation of HIF-1. In this study, we investigated the effects of Tongxinluo on the expression of endothelial nitric oxide synthase (eNOS) and endothelium-dependent vasodilation in a vascular endothelial dysfunction model by inducing hyperhomocysteinemia in rats using a methionine-rich diet. We further investigated the effects of Tongxinluo on the eNOS expression and HIF activation in human umbilical vein endothelial cells (HUVECs) and its effect on the upstream PI-3K/Akt signaling pathway. 2. Materials and methods
dissolving 4.11 g of Tongxinluo ultra fine powder in 100 mL 0.5% sodium carboxymethylcellulose. Besides the same treatment as the hyperhomocysteinemia group, the positive control group was administrated with 10 mL/kg body weight of 4 mg/mL folic acid (FA) (Tianjin Lisheng Pharmaceutical Co., Tianjin, China) in 0.5% sodium carboxymethylcellulose by gavage. 2.3. Isolation of the aorta rings in rats The thoracic aorta was isolated as previously described (Resta and Walker, 1996). Briefly, a midline cut was made along the sternum, and with the removal of the lungs and heart, the thoracic aorta was exposed, isolated and then placed in cold Krebs’ solution containing 95% O2 and 5% CO2 . The isolated blood vessel was cut into two 3–4 mm wide vascular rings with the careful preservation of the vascular endothelium. The vascular rings were vertically hung in an organ bath containing 10 mL Krebs’ solution and connected with a BL-420E+ biological function experimental system (Chengdu Technology and Market, Chengdu, China). The vascular rings were subjected to precontraction with 1 M phenylephrine (PE) (Sigma–Aldrich Co., St. Louis, MO, USA) or 60 mM KCl, followed by dilation with acetylcholine (ACH) (Sigma–Aldrich Co., St. Louis, MO, USA) at progressive final concentrations of 0.01 M, 0.1 M, 1 M, 10 M and the tension curve was observed and recorded. The results were presented as the percentage of ACH dilation value relative to the maximal PE tension value. The change in tension rate of the vascular ring was calculated as follows: Tension rate (%) =
PE tension peak value − ACH dilation peak value × 100% PE tension peak value − value of basic tension
2.1. Preparation of Tongxinluo ultra fine powder solution 2.4. Immunohistochemistry Tongxinluo ultra fine powder (Lot Number: 071201, Shijiazhuang Yiling Pharmaceutical Co., Shijiazhuang, China) was dissolved in serum-free Dulbecco’s Modified Eagle’s Medium (DMEM). The solution was sonicated for 1 h followed by centrifuge at 3500 rpm for 10 min. The supernatant was filtrated using 0.22 m micro-pore filter. Meanwhile, the precipitate was heated and dried at 60 ◦ C in order to calculate the practical volume of Tongxinluo ultra fine powder dissolved. The solution was adjusted to a final concentration of 2 mg/mL and preserved at −20 ◦ C for subsequent use. Before each experiment, the solution was diluted in serum-free DMEM to a working concentration (containing 0.1% fetal bovine serum). 2.2. Animals A total of 180 healthy male Wistar rats, weighing 220–250 g, were bred in the New Drug Evaluation Center, Hebei Institute of Integrated Chinese Traditional Medicine and Western Medicine, Hebei, China. This study was approved by the Animal Care and Use Committee of Shijiazhuang, China. After they were bred for 5 days for adaptation, the animals were randomly divided into four groups: the negative control group, the hyperhomocysteinemia group, the Tongxinluo group, and the positive control group. The negative control group was fed normally with free access to drinking water and was administered with 10 mL/kg body weight of 0.5% sodium carboxymethylcellulose by gavage. Hyperhomocysteinemia was induced in rat as previously described (Joseph et al., 2002; Walker et al., 2004). The hyperhomocysteinemia group was administrated with 10 mL/kg body weight of 3% l-methionine (Sigma–Aldrich Co., St. Louis, MO, USA) in 0.5% sodium carboxymethylcellulose by gavage for the time period indicated. In addition to the same treatment as the hyperhomocysteinemia group, the animals in the Tongxinluo group were administered by gavage, at 9–10 o’clock everyday with 10 mL/kg body weight of Tongxinluo suspension, which was prepared by
Tissue sections were prepared of the thoracic aortas after fixation in 4% paraformaldehyde, dehydration and embedding in paraffin. The expression of eNOS in the aorta tissue was examined using the SP immunohistochemistry kit according to the manufacturer’s instructions (Zhongshan Co., Beijing, China). Densitometric analysis of immunocytochemical staining of eNOS was carried out and eNOS staining intensity was expressed in optical density (OD) units. 2.5. DNA transfections Human eNOS luciferase reporter plasmid was kindly provided by Dr. Philip A. Marsden at University of Toronto, Toronto, ON, Canada. AP-1 luciferase reporter plasmid, NF-B luciferase reporter gene vector and HRE reporter plasmid were used for transient transfections as previously described (Ding et al., 2006a). The plasmid vector DN-HIF encoded a dominant negative mutant of HIF-1␣, lacking both the basic DNA binding domain and the C-terminal transactivation domain as previously described (Jiang et al., 1996). The plasmid vector DN-Akt expressed a full length Akt protein mutated at Thr308 and Ser473 , rendering it inactivated. The plasmid vector P85 expressed the dominant-negative mutant form of PI-3K (Ding et al., 2006b). For DNA transfections, HUVECs were inoculated in a 6-well plate at a density of 2 × 105 cells/well and incubated at 37 ◦ C for 24 h. These cells were transfected with vectors carrying eNOS, AP-1 or NF-B reporter, where indicated, using Lipofectamine according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Stable cells expressing the appropriate genes were selected using geneticin (G418, Gibco-BRL) at 300 g/mL and were identified by determining luciferase activities. Additionally, HUVECs stably expressing eNOS-luc were grown to 80% to 90% confluence in a 10-cm dish and transfected with 5 g of plasmid vectors for DN-HIF, P85 or DN-Akt, or GFP control vectors using Lipofectamine as instructed by the manufacturer. The cells were typically used 24–48 h after transfection.
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Fig. 1. Tongxinluo promotes eNOS expression and improves aorta dilation in rats. Hyperhomocysteinemia rats and Tongxinluo treatment were administrated for four weeks as described in Materials and Methods. Their aorta rings were isolated and subjected to immunochemical staining using anti-eNOS antibody (A) ensitometric analysis of immunocytochemical staining of eNOS (B) (p < 0.01). The change in tension rate of the isolated aorta in response to 1 M phenylephrine (PE) and 1 M acetylcholine (ACH) was measured (C and D). Rats in control group, hyperhomocysteinemia group, Tongxinluo group and folic acid group were administrated for two weeks before their aorta rings were isolated and subjected to KCl precontracted vasodilation (E) or PE precontracted vasodilation (F) by ACH at doses indicated. HCY, hyperhomocysteinemia group; TXL, Tongxinluo group; FA, folic acid group. *p < 0.05 compared with controls; # p < 0.05 compared with HCY.
2.6. Luciferase reporter gene assay
2.9. Statistical analysis
HUVECs were inoculated in a 96-well plate at a density of 8 × 103 cells/well. When they reached 80% to 90% confluent, the cells were treated with Tongxinluo at the doses and time points indicated. Both the treated cells and untreated control cells were lysed in a 50-l lysis buffer at 4 ◦ C for 20 min. The luciferase activities were measured using a luciferase reporter gene detection kit (Promega) in a luminometer as instructed by the manufacturer. The induced expression of eNOS or the enhanced activity of AP-1, NFB or HIF-1 was represented as the ratio between the luciferase activity in Tongxinluo-treated cells and that in untreated cells.
The data were expressed as mean ± S.D. and analyzed using SPSS software (SPSS Inc., Chicago, IL, USA). Each data point represents the average of three independent experiments; bars, ±S.D. One-way ANOVA was used in the analysis of the data between the groups. The data with significant difference were compared using Newman–Kuels q-test. p < 0.05 was considered statistically significant.
2.7. Immunoblotting studies Cellular lysates of HUVECs were prepared as previously described. The protein samples (30 g each) were resolved by SDSPAGE. Immunoblotting procedures were performed as previously depicted (Wen et al., 2010). Anti-GAPDH, anti--actin, anti-eNOS antibodies (from Santa Cruze Biotechnology, Santa Cruz, CA) and anti-phosphorylated JNK, anti-total JNK antibodies (from Cell Signaling Technology, Danvers, MA) were used. 2.8. Determination of nitric oxide content The nitric oxide (NO) content was examined using the nitrate reductase method by using NO detection kit as instructed by the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
3. Results 3.1. Tongxinluo promotes eNOS expression and improves aorta dilation in rats We induced hyperhomocysteinemia in rats using a methioninerich diet and examined the effect of Tongxinluo on the expression of eNOS in the aorta. We found that eNOS was positively expressed in the normal aortic tissue (Fig. 1A). The intensity of eNOS expression, however, was significantly attenuated in rats with hyperhomocysteinemia. Tongxinluo treatment, on the other hand, significantly enhanced the intensity of eNOS expression compared with that of both the control rats and the untreated hyperhomocysteinemia rats (p < 0.05) (Fig. 1A and B). We also studied the effect of Tongxinluo on the vasodilation of the aorta in rats with induced hyperhomocysteinemia. We showed that, in the rats with hyperhomocysteinemia, the change in tension rate of the isolated aorta ring decreased significantly compared with that in the control
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Fig. 2. Tongxinluo enhances the expression of eNOS and the production of NO. HUVECs stably expressing eNOS-luc were treated with Tongxinluo for 24 h at doses indicated (A) or for 12–48 h at 100 g/mL (B). The eNOS transcription level was assessed by measuring the luciferase activities. The induction of eNOS was expressed as fold over that of medium control. HUVEC cells were treated with Tongxinluo for 24 h at doses indicated (C and D). Cellular lysates were prepared from these cells for immunoblotting analysis of eNOS and GADPH (C) and their supernatant were collected for nitric oxide (NO) detection (D). The supernatant of HUVECs treated with Tongxinluo (50 g/mL) and/or l-NMMA (400 M) for 24 h were collected and subjected to nitric oxide detection (E).
group (p < 0.05) (Fig. 1C and D). Tongxinluo administration, however, significantly increased the change in tension rate of isolated aorta ring compared with that of the untreated rats with induced hyperhomocysteinemia. In order to further investigate the effect of Tongxinluo on the relationship between increasing ACH levels and the vasodilation effect of ACH on aorta rings, the isolated aorta rings precontracted with KCl (Fig. 1E) or PE (Fig. 1F) were exposed to progressive concentrations of ACH. We discovered that ACH induced a similar pattern of change in tension rate in aorta rings precontracted by KCl or PE. In consistence with the results of single-dose ACH induced vasodilation, the change in tension rate of aorta rings was much lower in the hyperhomocysteinemia group compared with the control group. On the other hand, the treatment of Tongxinluo and folic acid increased the change in tension rate of aorta rings compared with that of hyperhomocysteinemia rats. These results indicated that ACH dilated aorta rings precontracted by KCl or PE in a dose-dependent manner.
3.3. HIF-1 is activated in HUVECs exposed to Tongxinluo As shown in Fig. 3A, Tongxinluo treatment did not influence the phosphorylation of JNK and Erks, and exhibited marginal effect on p38 phosphorylation, indicating MAPKs were not involved for Tongxinluo-induced eNOS expression. Moreover, phosphorylation of STAT3 was not increased but decreased in HUVEC cells exposed to Tongxinluo, suggesting STAT3 is not required in Tongxinluoinduced eNOS expression (Fig. 3B). We also studied the effects of Tongxinluo on the activation of AP-1 and NF-B in HUVECs. We observed that EGF could markedly induce the activation of AP-1 in HUVECs while Tongxinluo at 100 g/mL exerted virtually no effect in these cells (Fig. 3C). Similarly, TNF-␣ induced a noticeable increase in the activity of NF-B in HUVECs, while Tongxinluo at 100 g/mL did not exert any effect in these cells (Fig. 3D). On the other hand, Tongxinluo treatment induced a dose- and time-dependent increase in the activities of HRE (Fig. 3E and F), suggesting that HIF-1 transcriptional activity was up-regulated by Tongxinluo in HUVECs.
3.2. Tongxinluo enhances the expression of eNOS and the production of NO
3.4. HIF-1 is required in Tongxinluo-induced eNOS expression
We further examined the effects of Tongxinluo on the expression of eNOS in HUVECs stably expressing eNOS-luc. We observed that Tongxinluo induced the expression of eNOS in a dose and timedependent manner (Fig. 2A and B). Additionally, immunoblotting studies revealed that, the expression of eNOS markedly increased upon Tongxinluo treatment as compared with that of the control (Fig. 2C). Furthermore, we observed that Tongxinluo induced a dose-dependent significant increase in the level of NO, which was abolished by the NOS inhibitor l-NMMA (p < 0.01) (Fig. 2D and E).
We further examined whether the up-regulation of eNOS by Tongxinluo in HUVECs was mediated by its effect on HIF-1. We transfected HUVEC eNOS-luc cells with the DN-HIF expression vectors and GFP control vectors and exposed these cells to Tongxinluo for various time points at the doses indicated. We observed that Tongxinluo induced a remarkable time- and dose-dependent increase in the expression of eNOS in the HUVEC eNOS-luc cells transfected with the GFP expression vector (Fig. 4A and B). This apparent increase in eNOS expression, however, was significantly attenuated in the HUVEC eNOS-luc cells transfected with the DN-
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Fig. 3. HIF-1 is activated in HUVECs exposed to Tongxinluo. (A and B) HUVECs treated with Tongxinluo for 24 h were subjected to immunoblotting assay. EGF and TNF-␣ treated HUVECs were used as positive control, respectively. HUVECs were transfected with vectors expressing NF-B or AP-1 reporter and then treated with TNF-␣, EGF or Tongxinluo. The transcription level of AP-1 (C) and the transcriptional activities of NF-B (D) were measured by luciferase assay. HUVECs were also transfected with the expression vector for HRE reporter and were treated with Tongxinluo for 48 h at the doses indicated (E) or for 12–48 h at 100 g/mL (F). The induction of HRE was examined by luciferase assays relative to that of the medium control.
HIF expression vector, which was markedly lower than that of the HUVEC eNOS-luc cells transfected with the GFP control vectors at each time point and dose examined (p < 0.01). In addition, compared with that in the GFP control group treated with Tongxinluo, the expression of eNOS in the HUVEC eNOS-luc cells transfected
with DN-HIF and treated with Tongxinluo was markedly decreased (Fig. 4C). 3.5. Tongxinluo up-regulates eNOS expression via PI-3K/Akt-dependent pathway We also investigated whether Tongxinluo had any effect on the PI-3K/Akt pathway, which functions upstream of HIF-1. Tongxinluo induced a marked dose-dependent rise in the activities of HRE in HUVEC cells, and transfection of these cells with DN-Akt or P85 both significantly attenuated the activation of HRE (p < 0.01) (Fig. 5A and B). Similarly, transfection of these cells with DN-Akt significantly attenuated the rise of eNOS expression induced by Tongxinluo as well (p < 0.01) (Fig. 5C and D). 4. Discussion
Fig. 4. HIF-1 is required in Tongxinluo-induced eNOS expression. HUVEC eNOS-luc cells were transfected with the expression vector of DN-HIF or GFP and then treated with 100 g/mL of Tongxinluo for 12–48 h (A) or with 50–200 g/mL of Tongxinluo for 24 h (B) before luciferase assays. Cellular lysates from HUVEC eNOS-luc cells transfected with the DN-HIF expression vector or GFP control vectors were used for immunoblotting analysis with anti-eNOS or -actin antibody (C).
Tongxinluo is a traditional Chinese medicine which is extracted, concentrated, freeze-dried and standardized from a mixture of 12 medicinal constituents as previously described (Chen et al., 2009) and has been widely used in the treatment of cardiovascular and cerebrovascular diseases over recent years. Numerous basic and clinical studies have indicated that Tongxinluo can significantly improve the vascular endothelial function (Zhang et al., 2009), although the underlying molecular mechanism is unclear. Hyperhomocysteinemia was considered as an independent risk factor for cardiovascular and cerebrovascular disease (Maron and Loscalzo, 2009), and folic acid was reported to counteract the level of plasma homocysteine in rats with hyperhomocysteinemia (Gilani et al., 2001). In this study, we investigated the effects of Tongxinluo on the vasodilatory function of the aorta in rats
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Fig. 5. Tongxinluo up-regulates eNOS expression via PI-3K/Akt/-dependent pathway. HUVEC cells were co-transfected with the HRE reporter plasmid together with GFP control plasmid or the expression vectors for DN-Akt (A) or P85 (B) and then treated with various dosages of Tongxinluo for 48 h. Luciferase activity was then measured. HUVEC eNOS-luc cells were transfected with the GFP control plasmid or the expression vectors for DN-Akt (C and D) and then treated with Tongxinluo as indicated followed by luciferase assay.
the intracellular and extracellular route (Liang and Lv, 2002). However, the effect of HIF-1␣, which has a transcriptional activity on the expression of eNOS (Kobayashi et al., 2003; Martinive et al., 2009), has not been reported. Therefore, in this study, we investigated the effects of Tongxinluo on such transcription factors as NF-B, AP-1 and HIF-1␣ and STAT3. We found that Tongxinluo did not elicit the activation of AP-1, NF-B, STAT3, but could significantly induce the expression of the HRE reporter gene, suggesting that Tongxinluo could improve the vascular endothelial function in a HIF-dependent manner. In addition, we found that HIF downregulation by a dominant-negative form of HIF attenuated the rise of eNOS induced by Tongxinluo treatment, suggesting that the upregulation of eNOS by Tongxinluo is at least partly dependent upon HIF. It has been reported that the activation of HIF is related to the PI3K/Akt signaling pathway (Sakamoto et al., 2009). Chen et al. (2005) found that the inhibition of the activity of PI-3K by Ly294001, a PI-3K inhibitor, in the renal vascular endothelium inhibited the expression of HIF. Many previous studies also confirmed that the activation of Akt could up-regulate intracellular eNOS expression (Dimmeler et al., 1999; Fulton et al., 1999; Gao et al., 2000; Kureishi et al., 2000; Zeng et al., 2000; Chavakis et al., 2001). Activated PI3K phosphorylates downstream PKB/Akt and activates eNOS, thus leading to the production of endothelium-derived NO (Scotland et al., 2002). It has not been reported whether Tongxinluo can up-regulate eNOS expression by activating PI-3K/Akt pathway to induce HIF expression. Therefore, in this study, we used P85 and DN-Akt to inhibit the PI-3K/Akt signaling pathway and found that the activation of the HRE reporter gene and the up-regulation of eNOS by Tongxinluo was markedly inhibited, indicating that the induction by Tongxinluo of HIF and eNOS expression partly depends on the activation of the PI-3K/Akt signaling pathway. 5. Conclusion
with vascular endothelial dysfunctions caused by hyperhomocysteinemia and found that the herbal compound could not only improve the endothelium-dependent vasodilation of the compromised artery, but also increase the aortic response of TXL-treated rats to phenylephrine, indicating that Tongxinluo may protect vascular smooth muscle cells against homocysteine-induced oxidative stress. Strikingly, the expression of eNOS was up-regulated in the arterial tissue in rats treated with Tongxinluo compared with that in hyperhomocysteinemia group. Our in vitro experiments also confirmed that Tongxinluo could up-regulate eNOS expression in vascular endothelium via the PI-3K/Akt/HIF-dependent signaling pathway, thus leading to the up-regulation of NO levels. Under normal condition, eNOS is a major NO source in the cardiovascular system and plays an important role in dilating blood vessel, adjusting blood pressure, inhibiting platelet aggregation and suppressing smooth muscle cell proliferation (Kavdia and Popel, 2004). In this study, we found that Tongxinluo could enhance the expression of eNOS, elevate NO levels and consequently improve the vascular endothelial function. It is reported that enhanced eNOS activity by carbon monoxide (CO) inhalation may result from the activation of MAPKs (Fujimoto et al., 2004). Whereas, our study revealed JNK, ERK and p38 are not required in eNOS induction by Tongxinluo in HUVECs. Previous studies have suggested that NF-B, a transcription factor, could be activated and induce the expression of eNOS during cerebral ischemia-reperfusion, which serves to antagonize the condition (Min et al., 2007). A previous study also confirmed that in human neutrophils, transcription factor AP1 could modulate not only the expression of plasma C-reactive protein, nitrotyrosine and IL-8, but also the expression of eNOS (Khreiss et al., 2005). In addition, Liang et al. studied the expression of eNOS and HIF-1 in hypoxia-preconditioned mice and found that HIF-1␣ and eNOS could protect the cerebral function from both
The traditional Chinese medicinal compound Tongxinluo can up-regulate eNOS expression and enhance the release of NO through activating the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the vascular endothelial function. This finding not only helps us to understand the pharmacological mechanism of Tongxinluo, but also provides a theoretical basis for Tongxinluo in treating cardiovascular and cerebrovascular diseases. It is worth mentioning that, in this study, it is revealed that Tongxinluo has an inductive effect on HIF-1, suggesting that the drug may improve the cell’s adaptability to hypoxia and increases the capacity of cells against hypoxic injury, which is worthwhile to be further studied. Acknowledgements This work was supported by grants from Ministry of Science and Technology Key Program 2005CB523301, and 2006DFB32460 and 91-10-02, Shanghai International Collaboration grant 08400701000 and Shanghai Pujiang Program. References Chavakis, E., Dernbach, E., Hermann, C., Mondorf, U.F., Zeiher, A.M., Dimmeler, S., 2001. Oxidized LDL inhibits vascular endothelial growth factor-induced endothelial cell migration by an inhibitory effect on the Akt/endothelial nitric oxide synthase pathway. Circulation 103 (16), 2102–2107. Chen, T.H., Wang, J.F., Chan, P., Lee, H.M., 2005. Angiotensin II stimulates hypoxia-1␣ inducible factor 1alpha accumulation in glomerular mesangial cells. Annals of the New York Academy of Sciences 1042, 286–293. Chen, W.Q., Zhong, L., Zhang, L., Ji, X.P., Zhao, Y.X., Zhang, C., Jiang, H., Wu, Y.L., Zhang, Y., 2009. Chinese medicine tongxinluo significantly lowers serum lipid levels and stabilizes vulnerable plaques in rabbit model. Journal of Ethnopharmacology 124 (1), 103–110.
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