Downloaded from Katja Steinkamp-Fenske, Larissa Bollinger ...jpet.aspetjournals.org/content/jpet/early/2007/05/11/jpet.107... · Chinese Medicine for the treatment of neurasthenia,

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Reciprocal regulation of eNOS and NADPH oxidase by betulinic acid in

human endothelial cells

Katja Steinkamp-Fenske, Larissa Bollinger, Hui Xu, Ying Yao,

Sven Horke, Ulrich Förstermann and Huige Li

From the

Department of Pharmacology,

Johannes Gutenberg University, Mainz, Germany

JPET Fast Forward. Published on May 11, 2007 as DOI:10.1124/jpet.107.123356

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 11, 2007 as DOI: 10.1124/jpet.107.123356

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Running title: Regulation of eNOS and NADPH oxidase by betulinic acid

Correspondence to: Huige Li, MD, PhD

Department of Pharmacology

Johannes Gutenberg University

Obere Zahlbacher Strasse 67

D-55131 Mainz

Germany

Telephone: + 49 (6131) 39-36929

Fax: + 49 (6131) 39-36611

E-mail: [email protected]

The manuscript contains

• 23 text pages;

• 8 figures;

• 0 Tables;

• 40 references;

• 224 words in Abstract;

• 486 words in Introduction and

• 1240 words in Discussion.

Abbreviations:

BH4, (6R)-5,6,7,8-tetrahydro-L-biopterin; HUVEC, human umbilical vein endothelial cells;

L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide, eNOS, endothelial NO

synthase; ZSS, Zizyphi Spinosi Semen

Section: Cardiovascular


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ABSTRACT

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is a protective

principle in the vasculature. Many cardiovascular diseases are associated with reduced NO

bioactivity and eNOS uncoupling due to oxidative stress. Compounds that reverse eNOS

uncoupling and increase eNOS expression are of therapeutic interest. Zizyphi spinosi semen

(ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the

cardiovascular system. In human umbilical vein endothelial cells (HUVEC) and HUVEC-

derived EA.hy 926 cells, an extract of ZSS increased eNOS promoter activity, eNOS mRNA

and protein expression, as well as NO production in a concentration- and time-dependent

manner. Major ZSS constituents include saponins such as jujuboside A and B and

pentacyclic triterpenes such as betulin and betulinic acid. Jujuboside A, jujuboside B, or

betulin had no significant effect on eNOS expression whereas betulinic acid increased eNOS

mRNA and protein expression in HUVEC and EA.hy 926 cells. Interestingly, betulinic acid

also attenuated the expression of NADPH oxidase subunits Nox4 and p22phox, thereby

reducing oxidative stress and improving eNOS function. Consequently, betulinic acid-treated

endothelial cells showed an increased production of bioactive NO (as indicated by a higher

efficacy in stimulating cGMP generation in RFL-6 reporter cells). Thus betulinic acid

possesses combined properties of eNOS upregulation and NADPH oxidase downregulation.

Compounds such as betulinic acid may have a therapeutic potential in cardiovascular disease.


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Introductions

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important

vasodilator and plays a protective physiological role in the vasculature (Li and Forstermann,

2000a). 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 anti-atherosclerotic 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, anti-atherosclerotic 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; Li et al., 2002c). In vivo upregulation 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 (eNOS-TG/apoE-KO) developed

larger atherosclerotic lesions than apoE-KO mice (Ozaki et al., 2002). Also in animal models

of hypertension or diabetes, eNOS has been found upregulated, however, the upregulated

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” (Forstermann 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


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many cases (Forstermann and Munzel, 2006). Suppression of oxidative stress by

downregulating 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 upregulation of eNOS

expression may be a promising therapeutic strategy for cardiovascular disease.

Zizyphi Spinosi Semen (ZSS, Suanzaoren in Chinese) is the dried seeds 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 anti-arrhythmic and blood pressure-lowering effects (Huang, 1999).

Here we report that betulinic acid from ZSS upregulates 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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Methods

Materials.

Aqueous extract of ZSS: 10g ZSS were crushed, incubated with 100 ml of water for

30 min and then boiled for another 30 min. The solution was collected using paper filter.

After adding 100 ml water, the solid phase was boiled again for 30 min, and the mixture

filtered with a paper filter. Filtrates from the both steps were pooled, mixed with an equal

volume of ethanol and placed in darkness at 4°C for 24h. The precipitate was removed by

centrifugation at 1000 g for 10 min. The supernatant was filtered using a 0.45 µm membrane.

The solution was concentrated using a rotary evaporator at 40°C to reduce the volume down

to 2 ml. This was considered a stock solution and used at different dilutions in the

experiments. A control stock solution was generated by evaporating a mixture of 100 ml

water and 100 ml ethanol down to 2 ml.

Jujuboside A (CAS number 55466-04-1) and B (CAS number 55466-05-2) were

purchased from ChromaDex (Santa Ana, CA, USA). Betulin [lup-20(29)-ene-3β,28-diol;

CAS number 473-98-3] and betulinic acid [3β-3-Hydroxy-lup-20(29)-en-28-oic acid, CAS

number 472-15-1] are from Sigma (Taufkirchen, Germany).

Cell culture.

Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase

digestion as described (Li et al., 1998). HUVEC-derived EA.hy 926 endothelial cells were

kindly provided by Dr. Cora-Jean Edgell (Chapel Hill, NC). EA.hy 926 endothelial cells

were grown under 10% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Sigma)

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


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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 compound G418. For analysis of

eNOS promoter activity, the stably transfected cells were incubated with ZSS extract for 18

hours. Then, cells lysates were prepared and luciferase activities were determined as

described (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 hours and total RNA was

isolated. The expression of eNOS mRNA was analyzed by RNase protection assay as

described previously (Li et al., 2002a; Li et al., 1998).

Real-Time RT-PCR for mRNA expression analyses.

In some experiments, eNOS mRNA expression was analyzed with quantitative Real-

Time RT-PCR using an iCyclerTM iQ System (Bio-Rad Laboratories, Munich, Germany). 500

ng of total RNA was used for Real-Time RT-PCR analysis with the QuantiTect™ Probe RT-

PCR kit (Qiagen, Hilden, Germany). Sequences of used eNOS primers were

GTGGCTGTCTGCATGGACCT (forward) and CCACGATGGTGACTTTGGCT (reverse).

The sequence of the dual-labeled TaqMan probe was

AGTGGAAATCAACGTGGCCGTGCTGC (Li et al., 2004). TaqMan Gene Expression

Assays (pre-designed probe and primer sets) were obtained from Applied Biosystems (Foster

City, CA, USA) 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 (TBP, Applied Biosystems,

assay ID Hs00427620_m1).

Western blot for eNOS protein analyses.

Confluent EA.hy 926 cells were incubated with ZSS extract for 18 hours 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


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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 hours 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 NOATM 280 NO Analyzer (Sievers, Boulder, CO). Total protein

content of the cells was determined (Bradford), 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 hours and NO

production bioassayed with RFL-6 rat lung fibroblasts as reporter cells (Li and Forstermann,

2000b). After the 18 hour-treatment, EA.hy cells and RFL-6 cells were washed twice with

Locke’s solution. Then, EA.hy 926 cells 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, 10.0

mM HEPES, pH 7.4).) containing 200 U/ml SOD, 100 µM L-arginine and 10µM betulinic

acid, and RFL-6 cells 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 10µM betulinic acid. Then, the

conditioned media containing the NO released from the EA.hy cells were transferred onto the

RFL-6 cells which contain 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).


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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). Briefly, EA.hy

926 cells were treated with 10 µM betulinic acid for 18 hours in 96-well plates and L-012-

derived chemiluminescence was measured using a Microplate Luminometer (Berthold) in

Hanks ́buffered salt solution (PAA Laboratories) containing 500 µM L-012. In additional

experiments, cells were pre-treated with 300 µM apocynin for 48h prior to 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

(ANOVA) followed by Fisher’s protected least-significant-difference test for comparison of

different means.


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Results

Increased eNOS promoter activity, eNOS expression and NO production in response to

a ZSS extract.

EA.hy 926 cells stably transfected with a 3.5 kb human eNOS promoter fragment were

treated with an aqueous extract of ZSS. This treatment resulted in a concentration-dependent

increase in eNOS promoter activity (Fig. 1A). The maximal increase was seen with a 1:100

dilution of the aqueous ZSS extract (5g raw material per ml as stock solution). Treatment

with the control solutions at any dilution did not have any effect on eNOS promoter activity.

For further experiments, control solutions at a 1:100 dilution was used as control.

Treatment of naïve EA.hy 926 cells with ZSS resulted in a concentration-dependent

increase in eNOS mRNA expression, as analyzed with RNase protection assay (Figs. 1B and

1C). Fig. 1D demonstrates the time-dependence of eNOS upregulation in response to

aqueous ZSS extract. A maximum effect was reached after 18 hours. Therefore, this time

frame was chosen for all other experiments. An 18-hour treatment of EA.hy 926 cells with

aqueous ZSS extract also increased eNOS protein (as analyzed with Western blot, Figs. 2A

and 2B).

Finally, ZSS treatment for 18 hours increased NO synthesis in EA.hy 926 cells in a

concentration-dependent manner, as determined by the NO-ozone chemiluminescence assay

(Fig. 2C).

Effects of ZSS constituents on eNOS expression in human endothelial cells.

Jujuboside A and B had no effect or on eNOS mRNA expression in human EA.hy 926

endothelial cells (Fig. 3). These compounds did not change eNOS promoter activity, either

(up to 30 µM and an 18 hour incubation, data not shown). Also betulin had no significant

effect on eNOS mRNA expression (Fig. 4A). In contrast, an 18 hour-treatment with betulinic

acid significantly increased eNOS mRNA expression in both EA.hy 926 cells (Fig. 4A) and in

HUVEC (Fig. 4B).


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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 hours 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).

Downregulation of Nox4 and p22phox expression and reduced ROS production in

response to betulinic acid.

Treatment with 10 µM betulinic acid for 18 hours resulted in marked downregulation

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-upregulating effect of betulinic

acid, because it was not prevented by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-

NAME, Fig. 7). 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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Discussion

ZSS is one of the most commonly used Chinese herbs. It is a sedative and hypnotic

drug with additional effect on the cardiovascular system (Huang, 1999).

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, lasted at least 3.5 hours and the effect declined

after 7.5 hours (Zhang et al., 2003). Moreover, treatment of hypercholesterolemic rabbits

with ZSS for three months resulted in a reduction of total cholesterol, triglyceride and LDL,

an increase in HDL, and reduced atherosclerosis (Wu et al., 1989).

Molecular mechanisms underlying these cardiovascular effects are poorly understood.

Interestingly, treatment of rats with ZSS results 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. 1B-D) and protein expression (Figs. 2A and 2B), as

well as NO production (Fig. 2C) in human endothelial cells.


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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 (Bai et al., 2003;

Huang, 1999; Wang et al., 2005). The content of jujubosides A and B in ZSS is about 400

mg/kg (Wang et al., 2005). Triterpenoids found in ZSS include betulin, 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). Also betulin had no effect on eNOS expression (Fig. 4A). Interestingly,

treatment of human endothelial cells with betulinic acid resulted in a significant upregulation

of eNOS mRNA (Fig. 4) and protein (Fig. 5A) expression. The contents of betulinic acid and

betulin in ZSS are about 7 mg/kg and 3 mg/kg, respectively (Li et al., 2005b). In the present

study, we used ZSS extract at a concentration of 5g ZSS per ml. When cells were treated with

this extract at a 1:100 dilution, the estimated final concentration of betulinic acid is in the low

micromolar range. As shown in Fig. 4B, betulinic acid increased eNOS mRNA expression

even at 1 µM. Thus, betulinic acid is likely to be one of the compounds responsible for the

eNOS upregulation produced by ZSS.

Upregulation of eNOS does not necessarily result in an increase in bioactive NO.

Under pathological conditions of oxidative stress, eNOS is often uncoupled and dysfunctional

(Forstermann 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, about 60% of vascular superoxide is derived from NADPH oxidases

(Guzik et al., 2006).

The NADPH oxidase enzyme complex consists of 2 essential membrane-bound

catalytic subunits, gp91phox/Nox2 and p22phox, which compose flavocytochrome b558, and


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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

(Griendling, 2004; Lassegue and Clempus, 2003). Both Nox2 and Nox4 are found in

endothelial cells (Griendling, 2004). Nox4 is likely 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

upregulates eNOS expression, thereby producing more eNOS protein, and (ii) it

downregulates NADPH oxidase, thereby maintaining the upregulated eNOS enzyme in a

functional state.

In the past, we have found analogous combined effects of some other compounds. The

PKC inhibitor midostaurin, for example, upregulates 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; Li et al., 2006). Also red wine and its

constituents (such as resveratrol, cinnamic acid and hydroxycinnamic acid) upregulate eNOS

while preserving eNOS functionality (Wallerath et al., 2002; Wallerath et al., 2005; Wallerath

et al., 2003). Direct antioxidant properties of these compounds as well as the demonstrated

downregulation of Nox4 by resveratrol (Li et al. unpublished data) may contribute to this

effect.

Thus, the combination of eNOS upregulation and NADPH oxidase downregulation

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


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generation in RFL-6 reporter cells; Fig. 5B), indicating that the upregulated 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. At least the latter indicates,

however, 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 (Figs. 6A and B) would favor

such an action. (iii) Betulinic acid has been shown to have pro-apoptotic 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). In HUVEC, however, 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 upregulates eNOS expression and downregulates 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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Acknowledgments

We thank Dr. Andrew Marston, Laboratoire de Pharmacognosie et Phytochimie, Ecole

de Pharmacie Genève-Lausanne, Université de Genève, Switzerland, for methodological

suggestions and helpful discussions. The manuscript contains parts of the doctoral theses of

K.S.-F. and of L.B.


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Footnotes

This work was supported by the Collaborative Research Center SFB 553 (project A1 to H.L.

and U.F.) from the DFG (Deutsche Forschungsgemeinschaft), Bonn, Germany.

Address correspondence to: Dr. Huige Li, Department of Pharmacology, Johannes Gutenberg

University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. E-mail:

[email protected]


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Legends for Figures

Fig. 1. Extract of Zizyphi Spinosi Semen (ZSS) enhances eNOS promoter activity

and eNOS mRNA expression. Panel A: Human EA.hy 926 endothelial cells were stably

transfected with a 3.5-kb human eNOS promoter fragment driving a luciferase reporter gene.

Cells were treated with the ZSS extract at the indicated dilutions (stock solution

corresponding to 5g raw ZSS per ml). Luciferase activity was analyzed as a determinant of

eNOS promoter activity. Panels B-D: Human EA.hy 926 endothelial cells were treated for 18

hours with ZSS extract and eNOS mRNA expression analyzed with RNase protection assays.

Panel B demonstrates an original gel (performed in triplicate). The gel shows the protected

bands for eNOS and for β-actin (used for normalization). Panels C and D demonstrate the

concentration- and time-dependency of ZSS’s effect on eNOS mRNA expression,

respectively. Symbols represent mean ± SEM, n = 9-15 (*p< 0.05; **p<0.01, ***p<0.001,

compared with control).

Fig. 2. ZSS extract increases eNOS protein expression and NO production. EA.hy

926 cells were treated for 18 hours with ZSS extract and eNOS protein expression analyzed

with Western blot using a monoclonal anti-eNOS antibody. Panel A shows a representative

blot of three independent experiments. β-tubulin was used for normalization. Panel B

illustrates results of densitometric analyses. Panel C: Oxidation products of NO, nitrite and

nitrate, in the cell culture supernatant were assayed by NO-ozone chemiluminescence using a

NO Analyzer. Symbols represent mean ± SEM, n = 9-18 (*p< 0.05, **p< 0.01, compared

with control).

Fig. 3. The saponins jujuboside A and B have no effect on eNOS expression.

Human EA.hy 926 cells were treated with jujuboside A or B for 18 hours (1-10 µM) and

eNOS mRNA expression was analyzed with quantitative Real-Time RT-PCR. Columns

represent mean ± SEM, n = 9-12.

Fig. 4. The triterpenoid betulinic acid increases eNOS mRNA expression. Human

EA.hy 926 cells (panel A) or HUVEC (panel B) were treated with betulin or betulinic acid (1-


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10 µM) for 18 hours and eNOS mRNA expression was analyzed with quantitative Real-Time

RT-PCR. Columns represent mean ± SEM, n = 9-15 (*p< 0.05; **p<0.01, compared with

control).

Fig. 5. Betulinic acid enhances eNOS protein expression and NO production.

EA.hy 926 cells were treated for 18 hours with betulinic acid (10 µM) and eNOS protein

expression was analyzed with Western blot using a monoclonal anti-eNOS antibody. Panel A

shows a representative blot of three independent experiments. β-tubulin was used for

normalization. Panel B: Human EA.hy 926 cells were treated with betulinic acid (10 µM) for

18 hours. Bioactive NO from EA.hy 926 cells was bioassayed using guanylyl cyclase-

containing RFL-6 reporter cells. cGMP content in RFL-6 cells was determined with

radioimmunoassay. Columns represent mean ± SEM, n = 8-18 (**p< 0.01 compared with

control).

Fig. 6. Betulinic acid downregulates the mRNA expression of Nox4 and p22phox.

Human EA.hy 926 cells (panel A) or HUVEC (panel B) were treated with 10 µM betulinic

acid (BA) for 18 hours. Co: control. mRNA expression of NADPH oxidase catalytic subunits

Nox4 and p22phox was analyzed with quantitative Real-Time RT-PCR. Columns represent

mean ± SEM, n = 9-15 (**p< 0.01).

Fig. 7. Nox4 downregulation by betulinic acid is NO-independent. Human EA.hy

926 cells were treated with betulinic acid (BA, 10 µM), the NOS inhibitor L-NAME (1 mM)

or the NO donor spermine NONOate (NONOate, 10 µM) for 18 hours. Co: control. mRNA

expression of Nox4 was analyzed with quantitative Real-Time RT-PCR. Columns represent

mean ± SEM, n = 9-12 (**p< 0.01, compared with control).

Fig. 8. Betulinic acid reduces ROS production in EA.hy 926 endothelial cells.

Human EA.hy 926 cells, with or without apocynin pre-treatment (300 µM, 48 hours), were

treated with 10 µM betulinic acid (BA) for 18 hours. Co: control. ROS production was

measured with L-012-derived chemiluminescence. Columns represent mean ± SEM, n = 9-12

(**p < 0.01).


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A

D

Figure 1

eNO

S m

RN

A(%

)

ZSS 18 hours

C

100

140

180

220

Control 1:1000 1:300 1:100

**

*

ZSS 1:100 (hours)

0 6 12 18 24

100

150

200

**

** **

100

150

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250

300

1:1001:3001:1000

eNO

S p

rom

ote

r ac

tivi

ty(%

)

ZSS***

*

Vehicle

Control

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eNOS

β-actin

Control 1:1000 1:300 1:100

ZSS 18 hoursZSS 18 hours

eNO

S m

RN

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)


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β-Tubulin

ControlZSS

1:1000

100

200

300

400

500

eNO

Sp

rote

in(%

)

Control ZSS1:100

**

Figure 2

0

100

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300

400

500

Control 1:1000 1:300 1:100

ZSS

Nit

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/Nit

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(%

)

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C


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Jujuboside A (µM) Jujuboside B (µM)

eNO

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RN

A (

%)

Figure 3

0 1 3 10 0 1 3 10


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Figure 4

HUVEC

0

50

100

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200

eNO

Sm

RN

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% o

f co

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ControlBetulinic acid (µM)

1 10

EA.hy

0

50

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eNO

Sm

RN

A (

% o

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Betulin(µM)

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cGM

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**

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Betulinic acidControl

eNOS

β-Tubulin

Figure 5This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 11, 2007 as DOI: 10.1124/jpet.107.123356

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Figure 6

HUVEC

0

25

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125

Rel

ativ

e m

RN

A(%

)

Nox4 p22phox

Co BA Co BA

** **

Nox4 p22phox

EA.hy

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25

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125R

elat

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mR

NA

(%)

Co BA Co BA

****

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Co BA L-NAME NONOate

No

x4 m

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