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CARDIOVASCULAR
From the Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany
Received March 24, 2007; accepted May 10, 2007.
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Abstract |
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). Blockade of NO synthesis with pharmacological NOS inhibitors causes significant peripheral vasoconstriction and elevation of blood pressure. Mice with a disrupted eNOS gene are hypertensive and lack endothelium-dependent, NO-mediated vasodilation (Huang et al., 1995). Besides its role in controlling blood pressure, NO protects the blood vessels from thrombosis by inhibiting platelet aggregation and adhesion. In addition, endothelial NO possesses multiple antiatherosclerotic properties (Li and Forstermann, 2000a). In agreement with this concept, pharmacological inhibition of eNOS causes accelerated atherosclerosis in rabbits (Cayatte et al., 1994) and in mice (Kauser et al., 2000), and deficiency in eNOS accelerates the development of atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice (Kuhlencordt et al., 2001).
Due to the antithrombotic, antiatherosclerotic, and antihypertensive properties of endothelial NO, the eNOS enzyme could be an interesting target for the prevention or therapy of cardiovascular disease. NO synthesis can be modulated by eNOS activity and/or eNOS gene expression (Li et al., 2002b,2002c). In vivo up-regulation of eNOS gene expression had been considered to be a reasonable strategy to prevent cardiovascular disease.
However, results from such experiments were disappointing. For example, eNOS-overexpressing transgenic mice on an apoE-KO background developed larger atherosclerotic lesions than apoE-KO mice (Ozaki et al., 2002). In addition, in animal models of hypertension or diabetes, eNOS has been found to be up-regulated; however, the up-regulated enzyme turned out to be dysfunctional (Hink et al., 2001; Mollnau et al., 2002). A dysfunctional eNOS produces reactive oxygen species (ROS) at the expense of NO (Xia et al., 1998). This has been referred to as eNOS "uncoupling" (Förstermann and Munzel, 2006). The main reason for eNOS uncoupling is a deficiency of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) (Channon, 2004). Oxidation of BH4 due to NADPH oxidase-mediated vascular oxidative stress may represent a major cause of BH4 deficiency in many cases (Förstermann and Munzel, 2006). Suppression of oxidative stress by down-regulating the expression or activity of vascular NADPH oxidase has been shown to restore eNOS functionality (Hink et al., 2001; Mollnau et al., 2002). Therefore, a correction of eNOS uncoupling combined with up-regulation of eNOS expression may be a promising therapeutic strategy for cardiovascular disease.
Zizyphi Spinosi semen (ZSS, Suanzaoren in Chinese) is the dried seed of Zizyphus jujuba Mill. var. spinosa (Bunge). ZSS has been widely used since antiquity in Traditional Chinese Medicine for the treatment of neurasthenia, anxiety, and insomnia. However, modern pharmacological studies revealed that this herb also has potent actions on the cardiovascular system, such as antiarrhythmic and blood pressure-lowering effects (Huang, 1999).
Here we report that betulinic acid from ZSS up-regulates eNOS and, at the same time, reduces the expression of NADPH oxidase in human endothelial cells. This results in enhanced bioactive NO. Therefore, compounds like betulinic acid may have therapeutic potential for cardiovascular diseases.
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Materials and Methods |
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Jujuboside A (Chemical Abstracts Service (CAS) number 55466-04-1) and B (CAS number 55466-05-2) were purchased from ChromaDex (Santa Ana, CA). Betulin [lup-20(29)-ene-3
,28-diol; CAS number 473-98-3] and betulinic acid [3-3-hydroxy-lup-20(29)-en-28-oic acid; CAS number 472-15-1] are from Sigma-Aldrich (Taufkirchen, Germany).
Cell Culture. Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion as described previously (Li et al., 1998). HUVEC-derived EA.hy 926 endothelial cells were kindly provided by Dr. Cora-Jean Edgell (Pathology Department, University of North Carolina, Chapel Hill, NC). EA.hy 926 endothelial cells were grown under 10% CO2 in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1x HAT (hypoxanthine, aminopterin, and thymidine) (Invitrogen, Karlsruhe, Germany) (Li and Forstermann, 2000b).
Analysis of eNOS Promoter Activity in Stably Transfected EA.hy 926 Cells. A stable EA.hy 926 cell line was generated by transfection of EA.hy 926 cells with pGL3-eNOS-Hu-3500-neo, which contains a neomycin-resistance gene and a 3.5-kb promoter fragment of human eNOS driving the luciferase reporter gene (Li et al., 1998). These EA.hy 926 cells were cultured in medium containing 1 mg/ml G418 compound. For analysis of eNOS promoter activity, the stably transfected cells were incubated with ZSS extract for 18 h. Cell lysates then were prepared, and luciferase activities were determined as described previously (Li et al., 1998). The luciferase activity, normalized for protein concentration of cell lysates, was used as a determinant of eNOS promoter activity.
RNase Protection Assay for eNOS mRNA Analyses. EA.hy 926 cells were incubated with ZSS extract for 18 h, and total RNA was isolated. The expression of eNOS mRNA was analyzed by RNase protection assay as described previously (Li et al., 1998, 2002a).
Real-Time RT-PCR for mRNA Expression Analyses. In some experiments, eNOS mRNA expression was analyzed with quantitative real-time RT-PCR using an iCycler iQ System (Bio-Rad Laboratories, Munich, Germany). Total RNA (500 ng) was used for real-time RT-PCR analysis with the QuantiTect Probe RT-PCR kit (QIAGEN, Hilden, Germany). Sequences of used eNOS primers were 5'-GTGGCTGTCTGCATGGACCT-3' (forward) and 5'-CCACGATGGTGACTTTGGCT-3' (reverse). The sequence of the dual-labeled TaqMan probe was 5'-AGTGGAAATCAACGTGGCCGTGCTGC-3' (Li et al., 2004). TaqMan Gene Expression Assays (predesigned probe and primer sets) were obtained from Applied Biosystems (Foster City, CA) for analyzing the mRNA expression of Nox4 and p22phox (assay ID Hs00276431_m1 and Hs00609145_m1, respectively). mRNA expression levels of target genes were normalized to TATA box-binding protein mRNA (Applied Biosystems; assay ID Hs00427620_m1).
Western Blot for eNOS Protein Analyses. Confluent EA.hy 926 cells were incubated with ZSS extract for 18 h, and total protein was isolated. Western blotting was performed using 50 µg of protein and a monoclonal anti-eNOS antibody (BD Biosciences PharMingen, San Diego, CA), as described previously (Li and Forstermann, 2000b). Immunocomplexes were developed using an enhanced horseradish peroxidase/luminol chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's instructions.
Determination of NO Synthesis Using an NO Analyzer. EA.hy 926 cells were treated with ZSS extract for 18 h, and the oxidation products of NO, nitrite and nitrate, were assayed as a measure of NO synthesis in the supernatants. After reduction of nitrate with nitrate reductase, total nitrite was determined by NO-ozone chemiluminescence using a NOA 280 NO Analyzer (Sievers, Boulder, CO). Total protein content of the cells was determined (Bradford method), and nitrite levels were normalized for protein (Li et al., 2002a).
Reporter Cell Assay for Determination of NO Production. EA.hy 926 cells were treated with 10 µM betulinic acid for 18 h, and NO production was bioassayed with RFL-6 rat lung fibroblasts as reporter cells (Li and Forstermann, 2000b). After the 18 h-treatment, EA.hy cells and RFL-6 cells were washed twice with Locke's solution. EA.hy 926 cells then were incubated with Locke's solution (154.0 mM NaCl, 5.6 mM KCl, 2.0 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5.6 mM glucose, and 10.0 mM HEPES, pH 7.4).) containing 200 U/ml SOD, 100 µM L-arginine, and 10 µM betulinic acid, and RFL-6 cells were incubated with Locke's solution containing 600 µM IBMX for 30 min. After the preincubation, EA.hy cells were incubated for 3 min at 37°C in Locke's solution containing 200 U/ml SOD, 300 µM IBMX, 100 µM L-arginine, and 10 µM betulinic acid. The conditioned media containing the NO released from the EA.hy cells then were transferred onto the RFL-6 cells, which contained guanylyl cyclase, and another incubation of 3 min at 37°C was performed. The reaction was stopped by aspiration of the solution, adding 1 ml of ice-cold 50 mM sodium acetate, pH 4.0, and rapidly freezing the cells with liquid nitrogen. The cGMP content of the RFL-6 samples was determined by radioimmunoassay as described. The basal cGMP content of the RFL-6 cells (receiving no conditioned media) was subtracted from all samples (Li and Forstermann, 2000b).
Measurement of ROS Production by L-012 Chemiluminescence. Production of ROS was determined using the luminol derivative L-012 [8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4(2H,3H)dione] (Li et al., 2006). In brief, EA.hy 926 cells were treated with 10 µM betulinic acid for 18 h in 96-well plates, and L-012-derived chemiluminescence was measured using a Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany) in Hanks' buffered salt solution (PAA Laboratories GmbH, Coelbe, Germany) containing 500 µM L-012. In additional experiments, cells were pretreated with 300 µM apocynin for 48 h before betulinic acid treatment. The photon counts were normalized for protein content of the cell lysates.
Statistics. Statistical differences between mean values were determined by analysis of variance followed by Fisher's protected least-significant-difference test for comparison of different means.
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Treatment of naive EA.hy 926 cells with ZSS resulted in a concentration-dependent increase in eNOS mRNA expression, as analyzed with RNase protection assay (Fig. 1, B and C). Figure 1D demonstrates the time dependence of eNOS up-regulation in response to aqueous ZSS extract. A maximal effect was reached after 18 h. Therefore, this time frame was chosen for all other experiments. An 18-h treatment of EA.hy 926 cells with aqueous ZSS extract also increased eNOS protein (as analyzed with Western blot, Fig. 2, A and B). Finally, ZSS treatment for 18 h increased NO synthesis in EA.hy 926 cells in a concentration-dependent manner, as determined by the NO-ozone chemiluminescence assay (Fig. 2C).
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Increased eNOS Protein Expression and NO Production in Response to Betulinic Acid. Treatment of EA.hy 926 cells with 10 µM betulinic acid for 18 h resulted in a significant induction of eNOS protein, as determined by Western blot (Fig. 5A). The conditioned media from these cells showed higher potency in stimulating cGMP generation in RFL-6 reporter cells, indicating an increased NO production by EA.hy 926 cells (Fig. 5B).
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Down-Regulation of Nox4 and p22phox Expression and Reduced ROS Production in Response to Betulinic Acid. Treatment with 10 µM betulinic acid for 18 h resulted in marked down-regulation of Nox4 and p22phox mRNA expression in EA.hy 926 cells (Fig. 6A) as well as in HUVEC (Fig. 6B). This effect seems to be independent of the eNOS-up-regulating effect of betulinic acid, because it was not prevented by the NOS inhibitor NG-nitro-L-arginine methyl ester (Fig. 7, L-NAME). In addition, treatment of EA.hy 926 cells with the NO donor spermine NONOate did not reduce Nox4 expression (Fig. 7). The reduced expression of NADPH oxidase subunits by betulinic acid was associated with a reduction of ROS production (Fig. 8). The effect of betulinic acid on ROS production was likely to be NADPH oxidase-dependent, because it was absent in the presence of apocynin, a NADPH oxidase inhibitor (Fig. 8).
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).
ZSS protects cardiomyocytes from ischemic injury. Damage of cultured neonatal rat myocardial cells by deprivation of oxygen and glucose was markedly reduced by ZSS total saponins (Chen et al., 1990). Anoxia reoxygenation of cultured neonatal rat myocardial cells results in increased intracellular malondialdehyde and lipid peroxides, increased intercellular calcium concentration, and decreased SOD activity. All of these parameters were reversed by ZSS total saponins (Chen et al., 1990; Wan et al., 1997). In an animal model of endotoxin fever, the reduced SOD protein levels were prevented by treatment with ZSS (Peng et al., 1995).
ZSS has also blood pressure-lowering effects. Intravenous injection of an aqueous solution of ZSS extract markedly decreased blood pressure in anesthetized rats, dogs, and cats without any significant effect on the coronary blood flow, heart rates, or myocardial contractility (Gu et al., 1987). Oral treatment of spontaneously hypertensive rats with ZSS jujubosides resulted in reduction of blood pressure (Zhang et al., 2003). Blood pressure reduction was observed as early as 30 min and lasted at least 3.5 h, and the effect declined after 7.5 h (Zhang et al., 2003). Moreover, treatment of hypercholesterolemic rabbits with ZSS for 3 months resulted in a reduction of total cholesterol, triglyceride, LDL, and atherosclerosis and an increase in HDL (Wu et al., 1989).
Molecular mechanisms underlying these cardiovascular effects are poorly understood. Interestingly, treatment of rats with ZSS resulted in increased plasma levels of NO by unknown mechanisms (Wang and Xie, 2004). Expression or activity of NOS isoforms was not analyzed in that study.
The present study is the first that systematically investigates the effect of ZSS and its constituents on the gene expression of eNOS. We found that ZSS increases eNOS promoter activity (Fig. 1A), eNOS mRNA (Fig. 1, B–D), and protein expression (Fig. 2, A and B), as well as NO production (Fig. 2C) in human endothelial cells.
The active constituents of ZSS include saponins, triterpenoids, flavonoids, alkaloids, and fatty acids (Huang, 1999; Li et al., 2005b; Zhao et al., 2006). The most important ZSS saponins are triterpenoid oligoglycosides, such as jujubosides A and B (Huang, 1999; Bai et al., 2003; Wang et al., 2005). The content of jujubosides A and B in ZSS is approximately 400 mg/kg (Wang et al., 2005). Triterpenoids found in ZSS include betulin and betulinic acid (Bai et al., 2003; Li et al., 2005b).
Jujuboside A and B showed no effect on eNOS promoter activity or eNOS mRNA expression (Fig. 3). Furthermore, betulin had no effect on eNOS expression (Fig. 4A). Interestingly, treatment of human endothelial cells with betulinic acid resulted in a significant up-regulation of eNOS mRNA (Fig. 4) and protein (Fig. 5A) expression. The contents of betulinic acid and betulin in ZSS are approximately 7 and 3 mg/kg, respectively (Li et al., 2005b). In the present study, we used ZSS extract at a concentration of 5 g/ml. When cells were treated with this extract at a 1:100 dilution, the estimated final concentration of betulinic acid was in the low micromolar range. As shown in Fig. 4B, betulinic acid increased eNOS mRNA expression even at 1 µM. Thus, betulinic acid is probably one of the compounds responsible for the eNOS up-regulation produced by ZSS.
Up-regulation of eNOS does not necessarily result in an increase in bioactive NO. Under pathological conditions of oxidative stress, eNOS is often uncoupled and dysfunctional (Förstermann and Munzel, 2006). In the vasculature, ROS can be produced by several enzyme systems, including enzymes of the respiratory chain, cytochrome P450 monoxygenases, uncoupled eNOS, xanthine oxidase, and NADPH oxidases. Although all of these enzymes contribute to the oxidative burden, evidence is accumulating that an initial generation of ROS by NADPH oxidases triggers the release of ROS by the other enzymes (Griendling, 2004). Moreover, NADPH oxidase-mediated oxidative stress is involved in the development and progression of atherosclerosis (Lassegue and Clempus, 2003). In diseased human coronary arteries, approximately 60% of vascular superoxide is derived from NADPH oxidases (Guzik et al., 2006).
The NADPH oxidase enzyme complex consists of two essential membrane-bound catalytic subunits, gp91phox/Nox2 and p22phox, which compose flavocytochrome b558, and several cytosolic regulatory components (Lassegue and Clempus, 2003). In the vasculature, the catalytic subunit gp91phox/Nox2 is a member of a family of homologous proteins termed Nox. In addition to gp91phox/Nox2, Nox1 and Nox4 are also expressed in blood vessels (Lassegue and Clempus, 2003; Griendling, 2004). Both Nox2 and Nox4 are found in endothelial cells (Griendling, 2004). Nox4 is probably the major Nox isoform and the predominant ROS source in endothelial cells (Ago et al., 2004; Hu et al., 2005).
Interestingly, betulinic acid significantly reduced the expression of NADPH oxidase catalytic subunits Nox4 and p22phox (Fig. 6), which was associated with a reduction in ROS production (Fig. 8).
Therefore, betulinic acid possesses a dual protective action on the vasculature. i) It up-regulates eNOS expression, thereby producing more eNOS protein, and ii) it down-regulates NADPH oxidase, thereby maintaining the up-regulated eNOS enzyme in a functional state.
In the past, we have found analogous combined effects of some other compounds. For example, the PKC inhibitor midostaurin up-regulates eNOS expression by a PKC-independent mechanism (Li and Forstermann, 2000b) and reduces vascular Nox1 expression by PKC inhibition (Li et al., 2006). By reducing NADPH oxidase-mediated oxidative stress, midostaurin reverses eNOS uncoupling in spontaneously hypertensive rats and in atherosclerosis-prone apoE-KO mice, which was associated with NO-mediated vasodilation and blood pressure reduction (Li et al., 2005a, 2006). In addition, red wine and its constituents (such as resveratrol, cinnamic acid, and hydroxycinnamic acid) upregulate eNOS while preserving eNOS functionality (Wallerath et al., 2002, 2003, 2005). Direct antioxidant properties of these compounds, as well as the demonstrated down-regulation of Nox4 by resveratrol (H. Li, H. Xu, G. Spanier, and U. Forstermann, unpublished data), may contribute to this effect.
Thus, the combination of eNOS up-regulation and NADPH oxidase down-regulation may result in enhanced levels of bioactive NO and thus vascular protection. Indeed, treatment of endothelial cells with betulinic acid increased bioactive NO (that stimulated cGMP generation in RFL-6 reporter cells; Fig. 5B), indicating that the up-regulated eNOS by betulinic acid is maintained under a functional state.
The current study has certain limitations. i) The measurements of NO were indirect as nitrite/nitrate or stimulation of soluble guanylyl cyclase; however, at least the latter indicated that bioactive NO was increased by betulinic acid. ii) The main effects are shown on healthy cells that are not stressed. Further experiments are needed to find out whether betulinic acid can reverse eNOS uncoupling under conditions of oxidative stress. The suppression of NADPH oxidase expression by betulinic acid (Fig. 6, A and B) would favor such an action. (iii) Betulinic acid has been shown to have proapoptotic effects in cancer cells (Rzeski et al., 2006). We have confirmed this effect in the alveolar epithelial carcinoma cell line A549/8 (data not shown). However, in HUVEC, betulinic acid did not induce apoptosis. At concentrations of 1 and 10 µM, betulinic acid even reduced caspase 3/7 activity in HUVEC (data not shown).
In conclusion, the present study demonstrates that betulinic acid from the Chinese herb ZSS up-regulates eNOS expression and down-regulates NADPH oxidase expression in human endothelial cells. This results in an increased amount of functional eNOS protein, enhanced production of bioactive NO, and thus vasoprotection (even in disease conditions with increased oxidative stress).
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Acknowledgements |
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Footnotes |
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This article contains parts of the doctoral theses of K.S.-F. and of L.B.: Steinkamp-Fenske K (2007) Identifikation protektiver Naturstoffe aus kardiovaskulär wirksamen chinesischen Heilpflanzen. Doctoral dissertation, Johannes Gutenberg University, Mainz, Germany; and Bollinger L (2005) Regulation der gen-expression der endothelialen NO-synthase durch extrakte chinesischer Heilpflanzen. Doctoral dissertation, Johannes Gutenberg University, Mainz, Germany.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NO, nitric oxide; eNOS, endothelial NO synthase; BH4,(6R)-5,6,7,8-tetrahydro-L-biopterin; HUVEC, human umbilical vein endothelial cells; ZSS, Zizyphi Spinosi semen; apoE-KO, apolipoprotein E-knockout; ROS, reactive oxygen species; kb, kilobase; RT, reverse transcription; PCR, polymerase chain reaction; SOD, superoxide dismutase; IBMX, 3-isobutyl-1-methylxanthine; L-012, 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4(2H,3H)dione; PKC, protein kinase C; CAS, Chemical Abstracts Service.
Address correspondence to: Dr. Huige Li, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. E-mail: huigeli{at}uni-mainz.de
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