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Inhibition of experimental abdominal aortic aneurysm in a ratmodel by way of tanshinone IIA

Tao Shang, MD,a,1 Zhao Liu, MD, PhD,a,1 Min Zhou, MD, PhD,a

Christopher K. Zarins, MD,b Chengpei Xu, MD, PhD,b and Chang-jian Liu, MDa,*aDepartment of Vascular Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School, Nanjing, ChinabDivision of Vascular Surgery, Stanford University, Stanford, CA, USA

a r t i c l e i n f o

Article history:

Received 16 January 2012

Received in revised form

26 April 2012

Accepted 27 April 2012

Available online xxx

Keywords:

Abdominal aortic aneurysm

Tanshinone IIA

Metalloproteinase

Inflammation

Vascular smooth muscle cell

Inducible nitric oxide synthase

* Corresponding author. Department of VasMedical School, No. 321 Zhongshan Road, N

E-mail address: dr_cjliu@hotmail.com (C.1 These authors equally contributed to thi

0022-4804/$ e see front matter ª 2012 Elsevdoi:10.1016/j.jss.2012.04.068

a b s t r a c t

Background: The purpose of the present study was to investigate whether tanshinone

IIA (Tan IIA), one of the major lipophilic components of Salvia miltiorrhiza Bunge, could

inhibit the development of elastase-induced experimental abdominal aortic aneurysms

(AAAs).

Methods: Male Sprague-Dawley rats (n ¼ 12/group) were randomly distributed into three

groups: Tan IIA, control, and sham. The rats from the Tan IIA and control groups under-

went intra-aortic elastase perfusion to induce AAAs, and the rats in the sham group were

perfused with saline. Only the Tan IIA group received Tan IIA (2 mg/rat/d). The maximum

luminal diameter of the abdominal aorta was measured before and 5, 12, 18, and 24 d after

perfusion. The systolic blood pressure was measured twice using the tail cuff technique

before administration and death. Aortic tissue samples were harvested at 24 d and

evaluated using reverse transcriptase-polymerase chain reaction, Western blot, immuno-

histochemistry, and Miller’s elastin-Van Gieson staining.

Results: The rats in the control group had significantly increased aortic sizes compared with

the sham group after 24 days (P < 0.05), and the Tan IIA group had a significant reduction in

aortic size (Tan IIA versus control, P < 0.05) without affecting blood pressure (P > 0.05). The

overexpression of matrix metalloproteinase-2, metalloproteinase-9, monocyte chemo-

tactic protein-1, and inducible nitric oxide synthase and the depletion of elastic fibers and

vascular smooth muscle cells induced by elastase perfusion were significantly decreased

by Tan IIA treatment (P < 0.05).

Conclusions: Tan IIA inhibited the development of elastase-induced experimental AAAs by

suppressing proteolysis, inflammation, and oxidative stress and preserving vascular

smooth muscle cells. It could be a new pharmacologic therapy for AAAs.

ª 2012 Elsevier Inc. All rights reserved.

cular Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing Universityanjing 210008, China. Tel.: þ86 025 8330 4616 11801; fax: þ86 025 8330 8417.-j. Liu).s study.ier Inc. All rights reserved.

j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 2 ) 1e92

1. Introduction containing 81.6 U of elastase was performed for 10 min using

Abdominal aortic aneurysm (AAA) can cause death from

aneurysm rupture, which has a mortality rate of up to 90% [1].

This disease has been 1 of the 15 leading causes of death in the

United States [2]. Two randomized controlled trials have

shown that early surgery offered no survival advantage for

small AAAs (diameter less than 5.5 cm) in patients diagnosed

with ultrasound surveillance [3,4]. Therefore, surgical repair is

the only recommended treatment for patients with large

AAAs (diameter 5.5 cm or larger). However, from aneurysm

screening programs performed in the United States about

10 years ago, an estimate of 360,000 reported aneurysms were

small AAAs (greater than 90%) [5e7]. Those patients could not

receive effective therapy, except for “watchful waiting” in

clinics. Also, they were more likely to acquire psychological

problems and a reduced quality of life [8]. For the slow

development of small AAAs, it would take a long period before

they reach the threshold size for surgical intervention [9].

Thus, an effective pharmacologic therapy might be a good

option to limit or stop the growth of small AAAs.

Tanshinone IIA (Tan IIA) is one of the major lipophilic

components extracted from the root of Salvia miltiorrhiza

Bunge, which has been used to treat various types of ischemic

disease as a traditional Chinese medicine for many years [10].

Recent studies have shown that Tan IIA might have the

potential to suppress AAA formation and affect AAA devel-

opment [10e15]. Therefore, we studied the effect of Tan IIA on

experimental AAAs and investigated the possible molecular

mechanisms.

2. Methods

2.1. Experimental groups and AAA model

All experiments in our study were conducted in compliance

with the Guide for the Care and Use of Laboratory Animals

approved by the Ethical Committee of Researches of Nanjing

University and were approved by the Committee on the Use

and Care of Animals of Medical School of Nanjing University.

Six-week-old male rats (SLAC Laboratory Animal, Shanghai,

China) weighing 180 to 220 g were used in our study. All rats

(n ¼ 12/group) were randomly distributed into three groups:

Tan IIA, control, and sham. The rats were housed with a 12-h

light/dark cycle and standard diet and water. They were

anesthetized and underwent laparotomy mainly according to

previous methods, with some modifications [16,17]. In brief,

the abdominal aorta was isolated from the level of the left

renal vein to the bifurcation. A tiny incision was made on the

aorta bifurcation. Next, a polyethylene-10 tube (Smiths

Medical International, Ashford, UK) was introduced through

the incision into the abdominal aorta. The aorta was clamped

above the tip level of the polyethylene tube and ligated with

a 4-0 silk suture (Ethicon, Johnson & Johnson, Somerville, NJ)

near the aortic bifurcation, followed by perfusion with

27.2 U/mL type I porcine pancreatic elastase (Sigma-Aldrich,

St. Louis, MO) in the Tan IIA and control groups and isotonic

saline in the sham group. Aortic perfusion with 10 mL saline

a microinfusion pump at 2 atm. After perfusion, the clamp

and ligatures were removed, and the polyethylene tube was

withdrawn. The incision was sutured with an 8-0 poly-

propylene suture (Prolene, Ethicon, Johnson & Johnson).

Aortic segments were harvested for additional study 24 d after

perfusion. The systolic blood pressure of the rats was

measured using the tail cuff technique before administration

and death.

2.2. Drug administration

From the findings of previous reports and experiments, 2 mg

Tan IIA with 0.4 mL saline (Shanghai No.1 Biochemical &

Pharmaceutical, Shanghai, China) was given to each rat daily

by intraperitoneal injection in the Tan IIA group (n ¼ 12) [20].

Tan IIA injection was started 1 d before elastase perfusion

until the rats were killed. The same volume of saline was

given to the sham (n ¼ 12) and control (n ¼ 12) groups for the

same period by intraperitoneal injection.

2.3. Measurement of aortic size using ultrasonography

An ultrasound system (SonoSite, Bothell, WA) containing

a linear transducer (25 MHz) was used to demonstrate the

dilation of the rat aortas. We measured the maximum inner

luminal diameter of aortas. The aortic size was measured

before laparotomy and 5, 12, 18, and 24 d after. Two experi-

enced operators who were unaware of the protocols

performed the quantitative analysis of the ultrasound data.

2.4. Histologic studies

All rats were killed 24 d after the operation. The excised aorta

was fixed in 10% neutral-buffered formalin and processed

using routine paraffin embedding. Aortic tissue cross-sections

(5 mm) were stained with hematoxylin and eosin and Miller’s

elastin-Van Gieson according to standard procedures. The

percentage of the surface area occupied by the elastin-Van

Giesonestained elastic fibers was quantified using the

morphometry system MacScope, version 2.2 (Mitani, Tokyo,

Japan).

2.5. Immunohistochemical staining

Mouse monoclonal antibodies for matrix metalloproteinase

(MMP)-9 (ab58803, Abcam, Cambridge, MA) and MMP-2

(ab3158, Abcam) were used to analyze the local expression of

matrix-degrading proteinases in the aorta walls 24 d after

operation. Also, mouse monoclonal antibody for a-smooth

muscle cell (SMC) actin (ab7817, Abcam) was used to analyze

the depletion of medial SMCs. Immunohistochemical staining

was performed using an immunoperoxidase avidin-biotin

complex system. After blocking the activity of endogenous

peroxidase, the sections were incubated in the primary anti-

bodies (1:100) overnight at 4�C. Next, according to the manu-

facturer’s specifications (Vectastain Elite ABC Kit, Vector

Laboratories, Burlingame, CA), we incubated the sectionswith

biotinylated anti-mouse IgG antibody for 30 min (Vector

Table 1 e Effect of tanshinone IIA (2 mg/rat/d) on systolicblood pressure.

Group (n) Beforeadministration

(mm Hg)

24 d Afterperfusion(mm Hg)

P value

Sham (12) 113 � 3 112 � 3 0.304

Control (12) 113 � 4 115 � 3 0.287

Tan IIA (12) 112 � 4 113 � 4 0.226

P value 0.201 0.275

Tan IIA ¼ tanshinone IIA.

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Laboratories) and then with avidin-biotinylated horseradish

peroxidase complex in phosphate-buffered saline for

10 min. Immune complexes were visualized using 0.05%

3,3’-diaminobenzidine (Vector Laboratories). The slides were

counterstained using hematoxylin.

2.6. Quantitative real-time reverse transcriptase-polymerase chain reaction

The expression of MMP-2 and MMP-9 mRNA in the aortic

tissues was determined using quantitative real-time reverse

transcriptase-polymerase chain reaction (PCR). TotalmRNAwas

extracted from the aorta using TRIzol reagent (Invitrogen, Carls-

bad, CA), and cDNA produced by reverse transcription using

oligo-(dT) primer 5.0 and Moloney Murine Leukemia Virus

Reverse Transcriptase (Fermentas, Vilnius, Lithuania). The PCRs

were performed in quadruplicate with SYBR Green PCR Core

Reagents (Toyobo, Osaka, Japan), and the fluorescence signals

were analyzedusing theDA7600 SequenceDetectionSystem (Da

An Gene, Guangzhou, China). The results for each sample were

normalized to the concentration of b-actin mRNA. PCR amplifi-

cation was performed under the following conditions: denatur-

ation for 15 s at 95�C, annealing for 30 s at 60�C, and extension for

30sat72�C.PrimersofMMP-2,MMP-9,andb-actinweredesigned

according to published rat sequences, and the amplified frag-

ments were 135 bp, 228 bp, and 136 bp, respectively. The primer

sequences were as follows: MMP-2 primer (GenBank reference

no. NM031054, 135 bp), sense primer: 50-CTGATAACCTGGATGCAGTCGT-30; antisense primer: 50-CCAGCCAGTCCGATTTGA-30;MMP-9 primer (GenBank reference no. NM031055, 228 bp); sense

primer: 50-TTCAAGGACGGTCGGTATT-30; antisense primer: 50-CTCTGAGCCTAGCCCCAACTTA-30; and b-actin primer (GenBank

referenceno.NM031144, 136bp), senseprimer: 50-GCAGAAGGAGATTACTGCCCT-30; antisense primer: 50-GCTGATCCACATCTGCTGGAA-30.

2.7. Western blot analysis

Aortic tissues were obtained and homogenized 24 d after the

operation, and the total proteins were extracted from the

frozen aorta tissue. The samples (70 mg) were electrophoresed

in sodium dodecyl sulfate-polyacrylamide gel at 80 V, trans-

ferred onto polyvinylidene difluoride membranes at 300 mA,

and incubated for 1 h in Tris-buffered saline, 5% nonfat milk,

and 0.2% Tween-20 at room temperature. The membranes

were then incubated for 24 h at 4�C with different antibodies,

including mouse monoclonal antibodies for MMP-9 (1:2000

dilution; Abcam), MMP-2 (1:2000 dilution; Abcam), and

inducible nitric oxide synthase (iNOS) (1:4000 dilution;

Abcam), rabbit polyclonal antibody for monocyte chemo-

attractant protein-1 (MCP-1) (1:5000 dilution; Abcam).They

were washed in Tris-buffered saline and 0.1% Tween-20 and

then incubated for 2 h at room temperature with sheep anti-

mouse IgG secondary antibody for MMP-2, MMP-9, and iNOS

(1:5000; Amersham Biosciences, Uppsala, Sweden), and goat

anti-rabbit IgG forMCP-1 (1:5000; AmershamBiosciences). The

membranes were visualized using an ECL plus chemilumi-

nescent kit (Amersham Biosciences), according to the manu-

facturer’s instructions, and exposed to X-ray film (Kodak,

Rochester, NY). b-Actin levels were used to standardize

protein loading. To quantify and compare the levels of

proteins, the density of each band was measured using

densitometry (Shimazu, Kyoto, Japan).

2.8. Statistical analysis

All values are presented as the mean � standard error of the

mean. Statistical analysis was performed using SPSS, version

16.0 (SPSS, Chicago, IL). Multiple comparisons were analyzed

using one-way analysis of variance followed by Bonferroni’s

post hoc test. The threshold for significance was P < 0.05.

3. Results

3.1. Blood pressure

At 24 d after intra-aortic perfusion, the systolic blood pressure

in all three groups was not significantly different (Table 1).

This result indicated that Tan IIA (2 mg/rat/d) did not affect

the blood pressure (P > 0.05).

3.2. Development of elastase-induced AAAs

AAA was induced by elastase perfusion. Next, the aortic size

was measured using ultrasonography before perfusion and 5,

12, 18, and 24 d afterward (Fig. 1A1, A2). Compared with the

sham group, we found that the aortic size of the control group

was significantly increased (P < 0.05; Fig. 1A3), which sug-

gested that the AAA model was successfully established.

However, the aortic size of the Tan II A group was obviously

smaller than that of the control group (P < 0.05; Fig. 1A3).

Therefore, Tan IIA inhibited the progression of the AAAs.

3.3. Histologic changes in aortic walls

The aortas of the rats were harvested after 24 d. On hema-

toxylin and eosin staining, it was found that the control group

had a thinner aortic wall and more mural thrombus than

other groups (Fig. 2A1, A2). Elastin-Van Gieson staining

showed observable degeneration of elastic lamellae in the

control group (control versus sham, P < 0.05; Fig. 2B2). In

contrast, no remarkable degeneration of the medial elastic

lamellae was seen in the Tan IIA or sham groups (Tan IIA

versus control, P < 0.05; Fig. 2B2).

Fig. 1 e Effects of Tan IIA on the aortic size in AAAs. (A1) Aortic dilation 24 d after operation detected using ultrasonography.

(A2) Abdominal aorta velocity 24 d after operation detected using ultrasonography. (A3) Development of aortic size after

operation as assessed by ultrasonography. In sham group, aorta was perfusedwith saline and treatedwith saline. In control

group,aortawasperfusedwithelastaseand treatedwithsaline. InTan IIAgroup,aortawasperfusedwithelastaseand treated

with Tan IIA (2 mg/rat/d). Pre[ before elastase perfusion; n[ 12 per group. *P< 0.05 versus sham; **P< 0.05 versus control.

j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 2 ) 1e94

3.4. Expression of MMP-2 and MMP-9

Activated MMP-2 and MMP-9 in the aortic walls were

analyzed using immunohistochemical staining (Fig. 3). The

strongest staining of the activated MMP-2 or MMP-9 subunits

was detected in the control group (Fig. 3A1, A2). The staining

was weaker in the Tan IIA group than in control group but

stronger than in the sham group. Western blotting showed

that both MMP-2 and MMP-9 within the aortic segment were

overexpressed in the control group (control versus sham,

P < 0.05; Fig. 3B2). However, the expression of MMP-2 and

MMP-9 was significantly downregulated in the Tan IIA group

(Tan IIA versus control, P < 0.05; Fig. 3B2). Compared with the

sham group, the expression of MMP-9 in the Tan IIA group

was not significantly different (P ¼ 0.165; Fig. 3B2); however,

the expression of MMP-2 was still significantly greater

(P < 0.05; Fig. 3B2). The mRNA expression analysis by quan-

titative real-time reverse transcriptase-PCR had a similar

result (control versus sham, P < 0.05; Tan IIA versus control,

P < 0.05; Fig. 3C).

Fig. 2 e Effects of Tan IIA on preservation of arterial wall. Histologic sections of rat aorta stained with hematoxylin and eosin

staining (A1, 403; A2, 2003). (B1) Histologic sections of rat aorta stained with Miller’s elastin-Van Gieson staining (1003).

Elastin stained dark purple. (B2) Percentage of area positive for elastic fibers (n [ 12 per group, *P < 0.05 versus sham;

**P < 0.05 versus control).

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3.5. Depletion of vascular SMCs

Immunohistochemical staining revealed a marked depletion

of medial vascular SMCs (VSMCs) in the control group but

preservation of VSMCs in the Tan IIA group (Fig. 4).

3.6. Production of MCP-1 and iNOS

The expression of MCP-1and iNOS in the aortic walls was

increased in the control group after 24 d (control versus

sham, P < 0.05; Fig. 5A2). However, they were obviously

lower in Tan IIA group (Tan IIA versus control, P < 0.05;

Fig. 5A2).

4. Discussion

Tan IIA is one of the major lipophilic components from Salvia

miltiorrhiza Bunge,which has been used in Asia for hundreds of

years. Because of the effects of anti-atherosclerosis and

vasodilation, it has been widely used in the treatment of

peripheral artery disease and the prevention of myocardial

infarction. Recent experimental studies have demonstrated

that Tan IIAhas someother vascular protective effects, suchas

inhibiting the expression of MMPs [10,12], anti-inflammation

[11,18,19], attenuating oxidative stress [14,15,19], and pre-

venting the loss of VSMCs [12,13]. Thus, we supposed that Tan

IIA could suppress the development of AAAs.

Fig. 3 e Effects of Tan IIA on expression of MMP-2 andMMP-9. (A1) Immunohistochemical staining for MMP-2 (403) in aortic

tissue; (A2) Immunohistochemical staining forMMP-9 (403) in aortic tissue; (B1)Western blot results ofMMP-2 andMMP-9 in

aneurysmwall. (B2) Quantitative analysis of MMP-2 andMMP-9 expression by densitometry. (C) mRNA expression of MMP-2

and MMP-9 measured using real-time reverse transcriptase-PCR. All data displayed as mean ± standard error of mean for

transcript levels for MMP-2 and MMP-9 as normalized to b-actin (n [ 12). *P < 0.05 versus sham; **P < 0.05 versus control.

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Fig. 4 e Effects of Tan IIA on the preservation of VSMCs. Immunohistochemical staining for a-SMC actin in aorta tissues

(4003).

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The elastase-induced AAA in our experiments is a stan-

dard small animal aneurysm model. Compared with other

AAA-induced methods, it achieves pathologic characteristics

similar to those of human AAAs [20]. The animal surgery was

performed according to a previously published protocol, with

some modifications [21]. Previous studies have indicated that

AAA formation correlated with the pump pressure and elas-

tase concentration [21]. We increased the perfusion pressure

to 2 atm with an elastase concentration of 8.16 U/mL to

shorten the period of anesthesia and allow the operator to

suture the incision in the bifurcation of the abdominal aorta in

time. These modifications resulted in successful AAAmodels.

AAA is a complex multifactorial disease with an unknown

etiology. It occurs after the development of an inflammatory

response, destruction of elastin and collagen, the loss of

VSMCs, and other histologic changes [21e26]. Among these

characteristics, chronic inflammation is prominent and could

leadmacrophages and lymphocytes to infiltrate into the outer

aortic wall [27]. The recruitedmacrophages could secret MMPs

and pro-inflammatory cytokines, contributing to the devel-

opment of AAAs [28]. Increased expression of MMPs (espe-

cially MMP-2 and MMP-9) could promote elastin degradation,

which is considered to cause the direct enlargement of AAA

[24,29]. In addition, some of the pro-inflammatory cytokines

such asMCP-1 could induce themigration ofmonocytes to the

aneurysm walls [18]. These infiltrated monocytes would

further secret MMPs and pro-inflammatory cytokines and

Fig. 5 e (A1) Western blot results of MCP-1 and iNOS expression

by densitometry (n [ 12 per group, *P < 0.05 versus sham, **P

continue to deteriorate the pathologic process [19,30]. Our

results showed that MMP-2, MMP-9, and MCP-1 were highly

expressed in the aortic walls of AAA rats. In accordance with

previous studies of atherosclerotic and inflammatory animal

models, our studies showed that, along with inhibiting the

overexpression of MMP-2, MMP-9, and MCP-1, Tan IIA could

attenuate the inflammatory response and downregulate the

expression of MMP-2 and MMP-9 in an AAA model.

Apoptosis of VSMCs is another major pathologic feature of

AAA. It impairs the reparation for connective tissues and

reduces the capacity for anti-pressure. The loss of VSMCs is

generally caused by two mechanisms, one associated with

inflammation. Previous research has shown that Fas ligand

and inflammatory cytokines (e.g., interleukin-6) from infil-

trated inflammatory cells play a crucial role in the depletion of

medial VSMCs [21,31]. The other mechanism is related to the

activation of nuclear factor-kB (NF-kB) caused by oxidative

stress [32]. In our study, the loss of VSMCs in the control group

was observed by immunohistochemical staining. Some

investigators have reported that Tan IIAwas able to inhibit the

migration of human aortic SMCs and the proliferation of

VSMCs [12,13]. Our study has demonstrated that Tan IIA

treatment can preserve the medial level of VSMCs in the rat

aortic wall. Therefore, it might inhibit the development of

AAAs by preventing the loss of VSMCs.

Oxidative stress involved in inflammatory response is

associated with aortic aneurysm formation in human tissues

. (A2) Quantitative analysis of MCP-1 and iNOS expression

< 0.05 versus control).

j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 2 ) 1e98

and animal models [19,33]. Previous studies have suggested

thatnitric oxidesynthesized fromiNOScandamage thearterial

wall by producing reactive oxidative stress. Thus, the expres-

sion of iNOS could promote oxidative vascular injury in AAAs

[34e36]. According toour study, theelastase-inducedAAAshad

overexpressed iNOS, and the Tan IIA-treated rats did not share

this phenomenon. Thus, it can be inferred that Tan IIA might

inhibit the development of AAAs by downregulating the

expression of iNOS and attenuating oxidative stress.

5. Conclusions

Wefound that Tan IIA could significantly inhibit the expansion

of elastase-induced experimental AAAs. The possible mecha-

nisms include downregulating the expression ofMMP-2,MMP-

9, and MCP-1, preventing the loss of VSMCs, suppressing the

infiltration of monocytes, and decreasing oxidative stress by

inhibiting iNOS synthesis. Our findings have shown that it

could be a new potential drug for AAA therapy.

Acknowledgment

The authors thank Dr. Wei Wang and Zhu Cheng-yan for

technical assistance with the duplex ultrasound examination

of the rat aorta. This work was supported by grants from

Nature Science Foundation of Jiangsu Province, China (grant

BK2009035).

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