CONTENTS sample-01-01-20091015034517... · 2011. 9. 7. · the Tecnic Carbostent (P=0.038). Primary...

Chinese Medicine, 2009, 1, 1-41 Published Online September 2009 in SciRes (www.SciRP.org/journal/cm)

Copyright © 2009 SciRes CM

CONTENTS

Midterm Outcomes of Prospective, Randomized, Single-Center Study of the Janus

Tacrolimus-Eluting Stent for Treatment of Native Coronary Artery Lesions…………………1

Heat Shock Protein 70 Gene Transfection Protects Rat Myocardium Cell Against

Anoxia-Reoxygeneration Injury…………………………………………………………………………………7

Surgical Outcome and Clinical Follow-up in Patients with Symptomatic Myocardial

Bridging……………………………………………………………………………………………………………15

T3/T4 Thoracic Sympathictomy and Compensatory Sweating in Treatment of Palmar

Hyperhidrosis………………………………………………………………………………………………………19

Therapeutic Strategy for Traumatic Instability of Subaxial Cervical Spine………………………23

Original Article Inhibitory Effect of Ginsenoside Rg3 on Ovarian Cancer Metastasis………30

Association of CALCA Genetic Polymorphism with Essential Hypertension……………………36

Chinese Medicine (CM)

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Midterm Outcomes of Prospective, Randomized, Single-Center Study of the Janus Tacrolimus-Eluting Stent for Treatment of Native Coronary Artery Lesions

ABSTRACT

From February 20, 2006 to August 26, 2006, a total of 200 patients were enrolled and randomly assigned to receive either Janus stent (n=100) or bare metal stent (Tecnic Carbostent, n=100). All patients were administered with clopi-dogrel for 4 months and aspirin for life long after stenting. Baseline clinical and angiographic characteristics were comparable between the two groups. AMI was present in 37% of patients with Janus and 36% with Tecnic Carbostent. At an average of 246-day follow-up, major adverse cardiac events (MACE) was 6% with the Janus stent and 15% with the Tecnic Carbostent (P=0.038). Primary events included 1 cardiac death, 1 myocardial infarction (MI) due to subacute stent thrombosis and 13 target lesion revascularizations (TLR) due to restenosis in patients with Tecnic Car-bostent and 6 TLR due to restenosis in patients with Janus stent. Although all patients had discontinued clopidogrel for an average of 126 days, there was no additional thrombotic event in the two groups. Keywords: drug-eluting stent, acute myocardial infarction, angioplasty, transluminal, percutaneous coronary

1. Introduction

Drug-eluting stent (DES) has been reported to dramati-cally reduce the incidence of restenosis and target lesion revascularization (TLR), and is widely used in clinical practice in recent years. However, controversies remain with regard to the long-term efficacy and safety for the first generation DES1. Pathological findings had indi-cated that polymer based DES delayed vessel healing, which might lead to late severe adverse events such as in-stent thrombosis. The permanent existence of non- degradable polymer coatings, which promote local vessel inflammation, is considered as one of the leading causes reducing vessel healing. The Janus tacrolimus-eluting stent (SORIN, Italy), a novel DES without polymer coating, is deemed capable to eliminate the adverse ef-fects of non-degradable polymer coatings of DES. Pre-liminary clinical outcomes from Jupiter I and II studies demonstrated that Janus stent was as safe as bare metal stent (BMS) and tended to reduce restenosis rate. How-ever, the study population in Jupiter I and II comprised of only low to mid-risk patients and lesions. We con-ducted a prospective, randomized, single-center study aiming at evaluating the safety and efficacy of Janus stent for treating coronary artery disease in “real world”

clinical practice of percutaneous coronary intervention (PCI) for the first time.

2. Methods

2.1. Study Population

Between February 20, 2006 and August 26, 2006, a total of 200 patients with symptomatic or documented myo-cardial ischemia, including acute myocardial infarction (AMI), were enrolled in this prospective, randomized study. Patients were considered eligible for enrollment if they were: fit for coronary stent implantation; to be treated exclusively with one kind of stent, no more than 3 stents for one target vessel (or total length of stents ≤85 mm), and providing written informed consent. The major exclusion criteria were in-stent restenosis lesion, graft lesion, not eligible for DES implantation, such as intol-erant of anti-platelet treatment or planned to undergo surgery, and administration of IIb/IIIa antagonist.

2.2. Study Protocols

Patients were randomly assigned to receive Janus stent

Y. L. HAN ET AL. 2

(n=100, Janus group) or Tecnic Carbostent (SORIN, Italy, n=100. Tecnic group) according to the computer generated randomization list. A 300-600 mg loading dose clopidogrel (Plavix, Sanofi aventis) were given for all patients at admission. Coronary angiography and stenting were performed according to the standard tech-niques2. After stent implantation, all patients received dual antiplatelet therapy: aspirin 300 mg per day for the initial one month continued with 100 mg per day for life-long and clopidogrel 75 mg per day for 4 months. In patients with AMI, the antiplatelet regimen was mostly the same except clopidogrel 150 mg per day for the ini-tial one week. Clinical follow-up was performed at 1-, 6-, 12- and 24-month and follow-up angiography was per-formed at 9 months after the index procedure.

2.3. End Points and Definitions

The primary end point was the 12-month rate of major adverse cardiac events (MACE), defined as the compos-ite of cardiac death, nonfatal myocardial infarction (MI) or TLR. The secondary end points were 9-month rate of angiographic in-stent restenosis and MACE at 24-month. MI was defined either as the development of pathologi-cal Q waves in at least 2 contiguous leads with or with-out elevated cardiac enzymes or, in the absence of pathological Q waves, as an elevation in creatinine kinase levels to greater than twice the upper limit of normal in the presence of an elevated creatinine kinase-MB level. TLR was defined as repeat revascularization for ische-mia owing to stenosis ≥50% of the lumen diameter any-

where within the stent or within the 5-mm borders proximal or distal to the stent. Restenosis was defined as the diameter stenosis of ≥50% of the target lesion.

2.4. Statistical Analysis

Categorical discrete variables were compared by the x2

test or the Fisher exact test when appropriate. Continu-ous variables were presented as mean±standard deviation (SD) and were compared with the use of the Student’s t test. A P value <0.05 was considered statistically sig-nificant. Data were analyzed using SPSS 10.0.

3. Results

3.1. Baseline Clinical Characteristics

The baseline clinical characteristics of the two groups were well matched (Table 1). Onset of AMI within 24 hours occurred in 37 patients in Janus group (including 4 non-ST segment elevation MI) and in 36 in Tecnic group (including 3 non-ST segment elevation MI). The propor-tions of acute coronary syndromes (ACS) were 78% in Janus group and 75% in Tecnic group, respectively.

3.2. Baseline Angiographic Characteristics

The target lesions (113 vs 122) and target vessels (107 vs 113) were similar between the two groups. Lesion char-acteristics of the two groups were well matched (Table 2). Multivessel disease was present in 7% of patients in Janus group and 13% of patients in Tecnic group (All were dual vessel disease, P>0.05).

Table 1. Baseline clinical characteristics

Janus (n=100) Tecnic(n=100) P value

Age (years） 57.94±11.52 58.79±11.26 NS

Male (%) 75 78 NS

Smoking (%) 55 62 NS

Hypertension (%) 58 57 NS

Diabetes (%) 27 17 NS

Family history of CAD (%) 12 14 NS

AMI within 24 hours (%) 37 36 NS

Killip class III−IV 4 2 NS

Unstable angina (%) 41 39 NS

LMWH before PCI (%) 11 14 NS

LMWH post PCI (%) 47 46 NS

Hypertriglyceridemia (%) 35 40 NS

Hypercholesteremia (%) 31 41 NS

NYHA class III−IV (%) 19 20 NS

CAD: coronary artery disease. AMI: acute myocardial infarction. LMWH: low molecular weight heparin. PCI: percu-taneous coronary intervention. NYHA: New York Heart Association. NS: not significant.


Y. L. HAN ET AL. 3

Table 2. Baseline angiographic characteristics


Reference vessel diameter (mm) 3.08±0.36 3.07±0.37 NS

Lesion length (mm) 27.0±10.30 27.1±11.12 NS

Diameter stenosis (%) 89.9±11.89 89.3±13.10 NS

Location of target lesions (n (%))

LM 3(2.7) 4(3.3) NS

LAD 69(61.1) 61(50.0) NS

LCX 17(15.0) 25(20.5) NS

RCA 24(21.2) 32(26.2) NS

ACC/AHA lesion type (n (%))

A/B1 lesions 6(5.3) 7(5.7) NS

B2/C lesions 107(94.7) 115(94.3) NS

Bifurcation lesions (n (%)) 45(39.8) 36(29.5) NS

CTO lesions (n (%)) 11(9.7) 10(8.2) NS

Thrombotic lesions (n (%)) 25(22.1) 17(13.9) NS

Ostial lesions (n (%)) 5(4.4) 3(2.4) NS

Small vessel lesions (≤2.75 mm) (n (%)) 12(10.6) 14(11.5) NS

Long lesions (≥20 mm) (n (%)) 81(71.7) 71(58.2) NS

99%−100% diameter stenosis (n (%)) 48(42.5) 48(39.3) NS

LM: left main. LAD: left anterior descending coronary artery. LCX: left circumflex coronary artery. RCA: right coro-nary artery. CTO: chronic total occlusion. ACC/AHA: American College of Cardiology/American Heart Association. NS: not significant.

Table 3. Stent implantation and procedural results


Mean stent diameter (mm) 3.16±0.38 3.17±0.42 NS

Mean stent length (mm) 30.0±11.56 30.5±14.94 NS

Stents per lesion 1.07±0.28 1.11±0.31 NS

Stents per patient 1.22±0.82 1.35±1.08 NS

Maximum deployment pressure (kPa) 1530.0±375.9 1509.7±314.1 NS

2.5 mm diameter stent (n (%)) 11(9.0) 12(8.9) NS

2.75 mm diameter stent (n (%)) 4(3.3) 11(8.1) NS

3.0 mm - 4.0 mm diameter stent (n (%)) 107(87.7) 112(83.0) NS

Stent overlapping (n (%)) 17(13.9) 21(15.6) NS

Post-dilatation (n (%)) 78(63.9) 81(60.0) NS

Direct stenting (n (%)) 25(20.5) 22(16.3) NS

Kissing balloon (n (%)) 13(10.7) 10(7.4) NS

Distal protective device (n (%)) 0(0) 1(1) NS

Procedural success (n (%)) 100(100) 100(100) NS

NS: not significant.


Y. L. HAN ET AL.


4

3.3. Procedural Results

There were totally 235 target lesions in 220 target vessel of 200 observed patients underwent coronary stenting. The procedure and device-deployment success rates achieved 100% in the two groups. Procedural results were similar for the two groups (Table 3). The maximum total stent length in one vessel was 81 mm in patients implanted with the Janus stent and 71 mm in patients implanted with Tecnic carbostent (overlapped by 3 stents). Stents with small diameter (2.5-2.75 mm) ac-counted for 12.3% in Janus group and 17.0% in Tecnic group (P>0.05).

3.4. Overall Clinical Outcomes

Up to January 30, 2007, all patients were clinically fol-lowed up for an average of (246±48) days (ranged 150 to 340 days). All the patients discontinued clopidogrel at the end of the fourth month after PCI according to study protocol. The mean interval from the discontinuation of clopidogrel was (126±46) days (ranged 34 to 220 days). Primary events occurred in 15 patients in Tecnic group, including 1 cardiac death due to cardiac rupture secon-dary to anterior AMI, 1 AMI caused by subacute in-stent thrombosis, 12 repeat PCI and 1 CABG due to in-stent restenosis, so the overall and stent-related MACE rates were 15% and 14%, respectively. In Janus group, there were 6 primary events of repeat PCI due to in-stent restenosis, so the overall and stent-related MACE rates were all 6%. The overall MACE rate was significantly lower in Janus group as compared with Tecnic group (6% vs 15%, P=0.038). For the TLR and stent-related MACE, there was an obvious tendency of lower inci-dence associated with the Janus stent but not statistically significant (6% vs 14%, P=0.059).

3.5. Clinical Outcomes of AMI Subgroup

For the subgroup of AMI patients who underwent emer-gent PCI within 24 hours of symptom onset, the mean clinical follow-up was (250±46) days (ranged 154 to 323 days) in Janus group and (243±52) days (ranged 150 to 340 days) in Tecnic group, respectively. There was no death or any thrombotic event in 37 patients in Janus group except 3 repeat PCI due to in-stent restenosis. In 36 patients in Tecnic group, there were 4 primary end points, including 1 cardiac death, 1 MI caused by subacute in-stent thrombosis and 2 repeat PCI due to in- stent restenosis. The incidence of death or MI was simi-lar between the two groups (0 vs 2%, P=0.24). Although all patients had discontinued clopidogrel, there was no additional death or thrombotic event in the two groups.

4. Discussion

Controversies about DES regarding its late clinical out-

comes were emerged and spread after the initial years of inspiritment. Because of the growing concern that de-layed endothelialization after implantation of a DES may cause late stent thrombosis, prolonged dual antiplatelet therapy with clopidogrel and aspirin is currently recom-mended after DES implantation. Unfortunately, even with 6 months or longer period of dual antiplatelet ther-apy, the incidence of late thrombosis or deadly cardiac events after DES implantation were still higher than those after BMS implantation3. Therefore, it is important to develop a new generation of DES which might de-crease the restenosis not at the expense of safety.

The Janus tacrolimus-eluting stent is one of such new generation DES. As the platform of Janus stent, the Tec-nic Carbostent is coated with Carbofilm to increase the biocompatibility and hemocompatibility4,5.

Phantom IV

study demonstrated the efficacy and safety of Tecnic Carbostent. In that study, there was no death or MI at 6-month clinical follow-up after Tecnic Carbostent im-plantation, and the angiographic restenosis rate was 14%6. Of the 100 patients received the Tecnic Carbostent in the present study, the stent related thrombotic events rate was only 1% at an average of 8-month follow-up, which confirms that the Tecnic Carbostent is a safe BMS platform. Having kept the structural features of the Tec-nic Carbostent, the Janus stent is coated with tacrolimus, an immunosuppressant, which was demonstrated to be efficient in inhibiting neointima hyperplasia of porcine coronary artery7,8. Differed from the first generation DES, i.e. Cypher and TAXUS, Janus stent has some unique features. First, drugs are loaded in the embedded reser-voirs on the outer stent surface, which enables drugs re-leasing directly to vessel wall without being washed away in the bloodstream. Second, there is no polymer coating in Janus stent, which may decrease the potential risks of late in-stent thrombosis caused by unabsorbable polymer coatings of traditional DES9.

Jupiter I study, the first-in-man registry of Janus stent, enrolled 58 patients. Of whom, 19% were ACS (includ-ing 6.9% of AMI). The target lesions included only 27% complex lesions of type B2/C with (11.5±5.9) mm of lesion length and (70.3±14.9)% of stenosis diameter. The randomized, controlled Jupiter II study presented at 2005 TCT showed that the total rate of MACE in the 157 pa-tients of Janus group was 7.6%, in which 6.4% of MACE was related to the stent, suggesting Janus stent having better clinical effects and safety. However, ACS ac-counted for only 27.6% of patients in Janus group in Jupiter II. Furthermore, ST segment elevation MI (STEMI) within 7 days and non-ST segment elevation MI (NSTEMI) within 3 days were all excluded in Jupiter II. It also excluded those with complicated lesions such as ostial lesions, left main diseases, chronic total occlu-sions (CTO), bifurcations, lesion in small vessels (refer-ence diameter ≤ 2.75 mm), long lesions (lesion length

Y. L. HAN ET AL. 5

>20 mm) and thrombotic lesions. Complex lesions of Type B2/C took only 34.9%. Because of the relatively simple lesion type, 75.9% of patients underwent suc-cessful direct stenting. Compared with Jupiter I and II studies, the population in the present study included 78% of ACS patients. The target lesions included many com-plicated lesions of high risk, such as left main or ostial lesions, thrombotic lesions, CTO, bifurcations, long le-sions, small vessel lesions with 2.5 mm in diameter. Some of the patients were highly endangered by some risk factors of coronary heart disease, for instance, dia-betes, smoking and hypertension, which generally re-flected the “real world” of clinical PCI daily practice. Despite of the higher risk for clinic and restenosis in subjects of present study, the incidence of MACE was 6% without occurrence of death or thrombosis in Janus group during the mean 8-month follow-up. Besides, as compared with the Tecnic group, incidence of MACE was dramatically reduced (P=0.038). The rate of stent related MACE had an obvious lower tendency in Janus group but no statistical significance (P=0.059). The above result was similar to that of Jupiter I and II, and further confirms the safety and clinical efficacy of Janus stent. Different from the present study, Jupiter II did not achieve statistical differences in the total rate of MACE, which might mainly due to the differences in study population. The enrolled patients in Jupiter II were mostly at lower risk for thrombosis. It has been reported that the effect of DES in reducing restenosis is not supe-rior to that of BMS for lesions with length <15 mm and vessel diameter >2.8 mm10. Moreover, Tecnic stent itself has certain antithrombotic function which may reduce the thrombosis related events in the control group in Jupiter II, in which late lumen lost was only 0.64 mm, and the incidence of in-stent restenosis was 14.8% in patients implanted with Tecnic stent. This was probably the reason for Jupiter II to come to the conclusion that the total incidences of MACE and stent related MACE in Janus group were not significantly lower than those in Tecnic group.

Up to now, the safety of DES implantation in primary PCI is uncertain. The randomized Typhoon trial showed that sirolimus eluting stent for treatment of STEMI was efficient in reducing MACE and TLR compared with BMS, but not beneficial for death, MI or in-stent throm-bosis11. In the present study, more than a third of study population were those with STEMI or NSTEMI under-going emergent PCI within 24 hours of symptom onset, which were excluded in Jupiter I and II study. For 37 AMI patients (including 33 STEMI) in Janus group, no acute, subacute or late thrombosis occurred at an average 8-month follow-up, suggesting Janus stent is safe in treating AMI.

It was reported that incomplete endothelialization

even appeared 1-2 years after implantation of Cypher or TAXUS in some cases12. The reports of very late in-stent thrombosis (>1 year) after DES implantation were also not rare13. Long-term dual antiplatelet therapy may re-duce the risk of thrombosis, but simultaneously accom-panies the increased adverse effects, such as hemorrhage, and the financial burden. Therefore, it is always confused to decide how long patients should take dual antiplatelet therapy after DES implantation. In Jupiter II study, about 60% patients took clopidogrel for over 6 months. Al-though patients in present study took clopidogrel for 4 months and then aspirin alone, no thrombosis was found after discontinuation of clopidogrel for 126 days on av-erage, preliminarily proving the feasibility and safety of this antiplatelet regimen, which provides an opportunity of receiving DES for patients who are intolerable of long-term dual antiplatelet therapy. The relatively shorter dual antiplatelet regimen may also associate with a de-crease of the hemorrhage risk, adverse effects and medi-cal expenses.

The main limitation of our study lies in that the mean follow-up period is only 8 months, which does not reach the designed observation time and only reflects the mid-term clinical result. Because of the limited observa-tion time, angiographic follow-up rate is low as well (19%).

In conclusion, for the first time we investigated the ef-ficacy and safety of the Janus stent, a novel DES without polymer coating, in “real world” PCI practice in this randomized single-center study. At our mean 8-month follow-up, Janus stent is efficient in reducing MACE compared with BMS, and is safe and efficient for treat-ing AMI patients. Discontinuation of clopidogrel at 4-month after Janus implantation is safe.

References

[1] Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, et al. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 2003; 349: 1315-1323.

[2] Han YL, Wang G, Jing QM, Wang SL, Wang ZL, Wang DM, et al. Percutaneous coronary intervention in acute coronary syndrome: single center experience from 4670 patients. Natl Med J China (Chin) 2005; 85: 1040-1044.

[3] Pfisterer M, Brunner-La Rocca HP, Buser PT, Ricken-bacher P, Hunziker P, Mueller C, et al. Late clinical events after clopidogrel discontinuation may limit the benefit of drug-eluting stents. J Am Coll Cardiol 2006; 48: 2584-2591.

[4] Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Ha-yashi E, Perin M, et al. A randomized comparison of a


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sirolimus-eluting stent with standard stent for coronary revascularization. N Engl J Med 2002; 346: 1773-1780.

[5] Sousa JE, Costa MA, Abizaid AC, Rensing BJ, Abizaid AS, Tanajura LF, et al. Sustained suppression of neoin-timal proliferation by sirolimus-eluting stents: one-year angiographic and intravascular ultrasound follow-up. Circulation 2001; 104: 2007-2011.

[6] Danzi GB, Capuano C, Sesana M, Baglini R, Bartorelli AL, Trabattoni D, et al. Six-month clinical and an-giographic outcomes of the tecnic carbostent

TM coronary

system: the phantom IV study. J In vasasive Cardiol 2004; 11: 641-644.

[7] Kollum M, Farb A, Schreiber R, Terfera K, Arab A, Geist A, et al. Particle debris from a nanoporous stent coating obscures potential antiproliferative effects of tac-rolimus-eluting stents in a porcine model of restenosis. Catheter Cardiovasc Interv 2005; 64: 85-90.

[8] Scheller B, Grandt A, Wnendt S, Lorenz G, Bohm M, Nickenig G. Comparative study of tacrolimus and pacli-taxel stent coating in the porcine coronary model. Z Kar-diol 2005; 94: 445-452.

[9] Bartorelli A, Trabattoni D, Fabbiocchi F, Montorsi P, Martini SD, Calligaris G, et al. Synergy of passive coat-ing and targeted drug delivery: the tacrolimus-eluting Janus Carbostent. J Interven Cardiol 2003; 16: 499-505.

[10] Pache J, Dibra A, Mehili J, Dirschinger J, Schomig A, Kastrati A. Drug-eluting stents compared with thin-strut bare stents for the reduction of restenosis: a prospective, randomized trial. Eur Heart J 2005; 26: 1262-1268.

[11] Spaulding C, Henry P, Teiger E, Beatt K, Bramucci E, Carre D, et al. Sirolimus-eluting versus uncoated stents in acute myocardial infarction. N Engl J Med 2006; 355: 1093-1104.

[12] Kotani J, Awata M, Nanto S, Uematsu M, Oshima F, Minamiquchi H, et al. Incomplete neointimal coverage of sirolimus-eluting stents: angioscopic findings. J Am Coll Cardiol 2006; 47: 2108-2111.

[13] Takahashi S, Kaneda H, Tanaka S, Miyashita Y, Shiono T, Taketani Y, et al. Late angiographic stent thrombosis after sirolimus-eluting stent implantation. Circ J 2007; 71: 226-228.



Heat Shock Protein 70 Gene Transfection Protects Rat Myocardium Cell Against Anoxia-Reoxygeneration Injury

ABSTRACT

The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups (n=8): control group, A/R group, HS+A/R group and pCDNA HSP70 +A/R group. A liposome-coated HSP70 pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP70 mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell viability was assayed by monotetra-zolium (MTT) and the lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity of cells during incuba-tion and the changes in cells ultrastructure were examined. NF-κB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry. Keywords: gene transfection, HSP70 gene, NF-κB, cardiac myocyte, anoxia-reoxygeneration injury

1. Introduction

Heat shock proteins (HSPs) are a family of inducible and constitutively expressed self-preservation proteins which maintain cell homeostasis under environmental stress. In a protocol involving prolonged cardioplegic arrest and reperfusion, Amrani et al1 has shown improved recovery of both ventricular and coronary endothelial function of rat hearts after heat stress. Heat shock protein 70 (HSP70) is one of the members which is strongly induced in the myocardium under various forms of stress. It appears that the induction of HSP70 confers a protective effect on cardiac function against exposure to an ischemia-reperfusion injury. Currie et al2 has shown that a rise in levels of a particular HSP70, induced by heat stress, is associ-ated with protection against ischemia-reperfusion injury. However, the protective effects may be contaminated by other factors resulting from stress, such as expression of catalase, superoxide dismutase (SOD) or other members of the HSP family3. To study the role of individual HSP70 separated from the many complex pathways, the tech-nique of gene transfection was adopted in this study with cultured primary cardiomyocytes.

2. Methods

2.1. Preparation of Neonatal Rat Cardiac Myocyte Cultures

Ventricular myocytes were isolated from the hearts of neonatal rats (Sprague-Dawley) which were less than 3 days old and were cultured prepared according to Simp-son and Savion4 with the following modifications: neo-natal rat pups were killed by swift decapitation within the first 3 days after birth. The heart was immediately removed under aseptic conditions and placed in ice-cold sterile Hank’s balanced salt solution (HBSS; no Ca++ or Mg++). Enzymatic and mechanical dissociation of car-diomyocytes were then performed using the Neonatal Cardiomyocyte Isolation System. The hearts were minced on ice and digested overnight at 4°C with puri-fied trypsin (10 µg/ml) in HBSS. In the following morn-ing, the digested tissue was transferred to a 50-ml conical tube and purified soybean trypsin inhibitor (40 µg/ml) was added to terminate trypsinization. The tissue was oxygenated and warmed to 37°C. Purified collagenase (10 U/ml) was added and digestion proceeded for 45 minutes at 37°C with intermittent gentle swirling. Mild titration was then used to mechanically dissociate the

J. C. LIU ET AL. 8

digested tissue and single-cell suspensions were obtained by filtering this digested material through 70 µm sterile mesh filters. The cells were collected by low-speed cen-trifugation. The supernatant was discarded and the cell pellet resuspended in DMEM (GIBCO, USA) culture medium containing 10% defined iron-supplemented bo-vine calf serum, 2 mmol/L glutamine, and gentamicin sulfate (50 µg/ml). The cells were then “preplated” to remove fibroblasts and the remaining cells counted and seeded onto multi-well culture plates at a density of 105 cells/well. The media was changed every other day be-ginning the day after seeding. Bromodeoxyuridine (0.1 mmol/L) was added to the culture media to further minimize contamination from fibroblasts. At the third day the myocytes were grouped at random.

2.2. Anoxia-reoxygeneration Model

Cells were plated in 14-mm-diameter glass bottom mi-crowell dishes and ischemia was introduced by a buffer exchange to ischemia-mimetic solution (NaH2PO4 0.9 mmol/L, NaHCO3 6.0 mmol/L, CaCl2 1.8 mmol/L, MgSO4 1.2 mmol/L, HEPES 20 mmol/L, NaCl 98.5 mmol/L, KCl 10.0 mmol/L, pH 6.8, 37°C) and placing of the dishes in hypoxic pouches equilibrated with 95% N2-5% CO2. After 3 hours of ischemia, reoxygeneration was initiated by a buffer exchange to normoxic solution (NaCl 129.5 mmol/L, KCl 5.0 mmol/L, NaH2PO4 0.9 mmol/L, NaHCO3 20 mmol/L, CaCl2 1.8 mmol/L, MgSO4 1.2 mmol/L, glucose 55 mmol/L, HEPES 20 mmol/L, pH 7.4, 37°C) and incubation at 95% room air-5% CO2

5,6. Controls incubated in normoxic solution were run in parallel for each condition for periods of time that corresponded with those of the experimental groups. Under control conditions, cell viability was not compromised.

2.3. Heat Shock Model

In heat shock group, the cells were subjected to hyper-

thermia of 42°C for 1 hour in a water bath. Forty-eight hours after treatment, cells were used for the following experiments.

2.4. Construction of Expression Vector and Lipofection

Full-length human HSP70 DNA (donated by Prof. WANG Shan-ming, Chicago University, USA) was cloned at the EcoRI/BamHI site of pCDNA, which has a cytomegalovirus promoter. pCDNA HSP70 was mixed with liposomes according to the protocol provided by the manufacturer. Briefly, for each transfection, 2 µl of li-posomes (1 mg/ml) were diluted with 100 µl serum-free Opti-MEM and kept at room temperature for 35 minutes. They were then mixed with 100 µl serum-free Opti-MEM containing 2 µg plasmid DNA and the mix-ture (0.2 ml DNA-liposome complex) was left at room temperature for 10 minutes before being diluted with 0.8 ml serum-free Opti-MEM. The diluted DNA–liposome complex was added to the cultures. Prior to transfection cultures were rinsed twice with a serum-free medium. After incubation at 37°C for 12 hours the transfection medium was replaced with a fresh culture medium con-taining 10% fetal bovine serum (FBS)7.

2.5. Experiment Protocols and Grouping

The cultures were grouped at random, including control group, anoxia-reoxygeneration group (A/R group), heat shock (HS)+ A/R group and pCDNA HSP70 + A/R group. The protocols of each group were showed in Figure 1.

2.6. MTT Assay for Cell Viability

Cardiomyocytes were seeded in 96-well plates at a den-sity of 105 cells/well, 20 µl monotetrazolium (MTT) were added to each well under sterile conditions (with a final concentration of 0.5 mg/ml) and the plates were incubated for 4 hours at 37°C. Untransformed MTT was removed by aspiration and formazan crystals were

Figure 1. Experimental protocols and grouping


J. C. LIU ET AL. 9

dissolved in dimethyl sulfoxide (150 µl/well). Formazan was quantified spectroscopically at 490 nm using an automated enzyme immunoassay analyzer8.

2.7. Biochemistry Detection

After the experiment, 200 µl of the culture supernatant were assayed and the activity of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) were detected by a biochemical autoanalyser (Beckman, USA).

2.8. Reverse Transcripta e Polymerase Chain sReaction (RT-PCR)9

Cultures harvested for total RNA immediately after treatment were washed in phosphate buffered saline solution (PBS) and RNA from five wells pooled after Trizol har-vesting (Promega, China). Total RNA, 1000 ng was re-verse transcribed for polymerase chain reaction (PCR) using avian myeloblastosis virus (AMV) reverse tran-scriptase (Promega). Primer sequences for HSP70 were 5-AAC-GTG-CTG-CGG-ATC-ATC-AA-3(sense), 5-CT G-GAT-GGA-CGT-GTA-GAA-GT-3 (antisense) (347 bp)10 for β-actin 5-TCA-TCA-CCA-TTG-GCA-ATC- AG-3 (sense), 5-GTC-TTG-GCG-TAC-AGG-3 (antisense) (154 bp); PCR was performed at 55°C (annealing tem-perature) for 30 cycles. Products were analyzed on ethid- ium bromide-stained 1.5% agarose gels. β-Actin was used as RNA input control.

2.9. Western Blotting Analysis

After incubation in serum-free DMEM cells were washed with ice-cold PBS and homogenized in buffer con-taining 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluo-ride, 2 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml pepstatin, centrifuged at 12 000 r/min for 10 minutes at 4°C and the supernatants were collected. The protein concentration in the supernatant was determined by us-ing a protein assay reagent (Bio-Rad Laboratories, Her-cules, USA). The same amounts (10 µg each lane) of proteins from cell homogenates were electrophoresed on 8% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes by electro blotting. The membranes were blocked for 1 hour at room tem-perature with 5% nonfat dried milk and 0.1% bovine serum albumin in tris-buffered saline (TBS) containing 0.1% (vol/vol) Tween 20 (TBS-T) and incubated for 1 hour with sheep anti-rat antibody diluted in TBS-T con-taining 5% FBS. After washing with TBS-T the blots were developed by enhanced chemiluminescence and exposed to X-ray film11.

2.10. Transmission Electron Microscope (TEM) for Ultramicrostructure12

Cells were washed twice with PBS at 4°C and immedi-ately fixed in 2.5% glutaraldehyde solution and then kept in the refrigerator at 4°C for two hours. Samples were later post-fixed in 1% osmium tetroxide, dehydrated in ascending concentration series of ethyl alcohol and em-bedded in Spur’s resin. Ultrathin sections were prepared using diamond knives, stained with uranyl acetate and lead acetate and then examined at 80 kV under the trans-mission electron microscope (Hitachi, H-600 Japan).

2.11. Detection of NFκB by Flow Cytometric Assay13

After reoxygeneration cardiac myocytes were washed with PBS containing 10% endotoxin-free FCS. Nuclei were prepared by incubating the cells with 200 µl Pipes- Triton buffer (10 mmol/L Pipes, 0.1 mol/L NaCl, 2 mmol/L MgCl2; Sigma-Aldrich, Steiheim, Germany and 0.1% Triton X 100) in PBS for 30 minutes at 4°C. After two washes in PBS-FCS nuclei were stained with anti-NF-κB antibody at 5 µg/ml for 45 minutes at 4°C. Mouse anti-NF-κB p65 monoclonal antibody (Santa Cruz, USA) recognizes epitopes mapping to the C amino-acid terminus of mouse NF-κB p65. After two washes in PBS-FCS nuclei were incubated for 45 min-utes at 4°C with fluorescein isothiocyanate (FITC)-con-jugated rabbit anti-mouse anti-Ig antibody fragments (1/50; Santa Cruz, CA, USA). After washings in PBS- FCS nuclei were analyzed by flow cytometry on a FACSC Caliber (BD Biosciences, San Jose, CA, USA). Fluorescence intensity of single nuclei was detected at random. A total of 104 events were recorded for each sample.


Data analysis was performed using the Statictical Pack-age for Social Science (SPSS 11.5). All data are pre-sented as mean±standard deviation (SD). One way analysis of variance (ANOVA) was used for multiple group comparison. If a significant F value was obtained, further comparisons were determined with least signifi-cant difference (LSD) test. Significance was accepted at the level of P<0.05.

3. Results

3.1. MTT Assay for Cell Viability

The results from the MTT assay indicated that in the control group the viability of cardiac myocytes were (87.3±11.4)%. In the A/R group the cell viability dropped to (35.4±6.9)%. However, the cell viability was improved in the HS+A/R group and the pCDNA


J. C. LIU ET AL. 10

HSP70 + A/R group. These data showed that HS stress and HSP70 gene transfection could lessen the injury from the A/R procedure and improve the cell viability (Figure 2).

3.2. Analysis for Biochemistry Detection

As shown in the table the activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS+A/R group and the pCDNA HSP70+A/R group clear decreases in activity were observed. There were statisti-cally significant differences between these groups. It showed that HS stress and HSP70 gene transfection could inhibit the elevation of activity of CPK and LDH in-duced by A/R injury.

3.3. TEM for Ultrastructural Analysis

The cells subjected to TEM examination were analyzed for ultrastructural differences. The irregular pattern of muscular fibril was observed in cardiac myocytes of the A/R group. The sarcoplasmic reticulum was extended to the vacuole and the mitochondria were swollen and mis-

shapen. However, in the HS+A/R group and the pCDNA HSP70+A/R group the muscular fibril was arranged regularly (Figure 3A) and the structure of the sarcoplas-mic reticulum and mitochondria were normal. No ap-parent differences in the microscopic appearance were seen between the HS+A/R (Figure 3B) and pCDNA HSP70 + A/R groups (Figure 3C).

3.4. Expression of HSP70 Gene in Cardiac Myocytes

To determine the expression of the HSP70 gene in cardiac myocytes, RT-PCR and Western analysis were per-formed. In the control group, there was no expression of the HSP70 gene. There was a slight upregulation for ex-pression of HSP70 in the A/R group and a clear increase in the expression of the HSP70 gene was observed by RT-PCR in the HS+A/R group and pCDNA HSP70+A/R group compared with the A/R group (Figure 4). In-creased expression of HSP70 protein was confirmed by Western blot analysis. Compared with the A/R group, an obvious increase in the HSP70 protein was found in the

Figure 2. Effect of various treatments on viable cell in rat’s cultured ventricular myocytes (n=8, mean±SD) (*P>0.05, vs con-trol group; #P<0.01, vs A/R group)

Figure 3. Representative TEM photographs showing for ultrastructure of cardiac myocytes. A: A/R group. B: HS + A/R group. C: pcDNA HSP70 + A/R group


J. C. LIU ET AL. 11

Table 1. Activity of CPK and LDH (IU/L)

*P<0.01, compared with A/R group

HS+A/R group and in the pCDNA HSP70+A/R group. In addition, there was a significant difference in expression of the HSP70 gene between the HS+A/R group and the pCDNA HSP70+A/R group (Figure 5). These data show that the HSP70 gene could be induced by heat shock stress and gene transfection by liposome. And more gene expression could be induced by gene transfection.

3.5. Analysis for activity of NF-κB

High activity of NF-κB (5.76±0.64) was detected in the A/R group. Compared with the control group, there was a significant difference (1.72±0.31; P<0.01). It showed that A/R injury could lead to an obvious increase in ac-tivity of NF-κB. But in the HS+A/R group, there was a statistically significant decrease in the activity of NF-κB compared with the A/R group (3.11±0.52 vs 5.76±0.64, P <0.01). The same statistically significant difference was also observed in the pCDNA HSP70 + A/R and A/R groups (2.83±0.49 vs 5.76±0.64, P<0.01 ). These data indicate that HS stress and HSP70 gene transfection could inhibit the increased of activity of NF-κB induced by A/R injury.

4. Discussion

The present study provided evidence that upregulation of HSP70 by a mild heat shock attenuates anoxia- reoxy-generation. Furthermore, transfection experiments di-rectly showed that elevated expression of HSP70 alone was sufficient for this protection. Using neonatal rat car-diac myocytes, we provided evidence that upregulation of HSP70 by gene transfection attenuates A/R injury.

The important role of HSP70 in protecting the heart against ischemia-reperfusion has been clearly shown by many experiments. Using a protocol that mimics clinical donor heart preservation, Jayakumar et al3 demonstrated that overexpression of HSP70 in whole hearts led to the protection of mitochondrial respiratory function and ventricular function after prolonged cold cardioplegic arrest. Wischmeyer et al14 showed that administration of glutamine could significantly reduce the deleterious

changes in myocardial metabolism and preserve cardiac output after reoxygeneration, which was owed to the enhanced expression of HSP70 induced by glutamine. However, the protective effects may involve many path- ways15,16. To confirm the direct effect of HSP70 on car-diac protection gene transfection was adopted to modify neonatal rat cardiac myoctyes in our study and the results were excited.

The development of techniques to introduce exoge-nous DNA into mammalian somatic cells has opened up the possibility of treating inherited and acquired diseases at the genetic level. Gene transfer strategies involve viral and nonviral techniques. The concerns with viral tech-niques are that some viruses might disturb the host DNA transcription and synthesis, regain their pathogenic activ-ity or generate immune responses17,18. Nonviral gene transfer techniques are attractive alternatives because they produce fewer side effects19. Lipofection, which is liposome-mediated transfection, is one such technique. In the present study the HSP70 gene was transfected into cardiac cells successfully by lipofection. This result was confirmed by RT-PCR analysis and Western blot analy-sis.

In the present study the pCDNA HSP70 was trans-fected into neonatal rat cardiac cells by lipofection and the overexpression of HSP70 mRNA and protein were achieved. An A/R injury model was used to test HSP70 protection: cell viability was improved, the activity of LDH and CPK were attenuated and the cell ultrastructure was nearly normal. There was no difference in cardiac cell protection between heat shock stress and HSP70 gene transfection. It showed that the effect of cardiac protec-tion could be achieved by HSP70 gene transfection. And it also indicated that the effect of heat shock stress on cardiac protection is mediated by overexpression of HSP70 gene.

Ischemia and reperfusion injury is a complex phe-nomenon directly associated with inflammatory changes. The traditional studies on the mechanisms of cardiac injury induced by A/R focused on the direct effect of toxic substances including proinflammatory cytokines,


J. C. LIU ET AL. 12

Figure 4. Effect of various treatments on expression of HSP70 mRNA in rat’s cultured ventricular myocytes (n=8, mean±SD). A: representative electrophoretic diagram of agarose gels for HSP70 mRNA. B: densitometric fold of A/R. (*P <0.01, vs A/R group; #P <0.01, vs HS+A/R group)

chemokines and adhesion molecules. However, a recent study shows a role of these factors in gene regulation of NF-κB in cardiac injuries. NF-κB is an inducible eu-karyotic transcription factor that normally exists in an inert cytoplasmic complex bound to inhibitory proteins of the IκB family and is induced by a variety of patho-genic stimuli. Zhang et al20 showed in their study that NF-κB was markedly increased in the ischemia- reperfu-sion injury heart. And they demonstrated that the cardio-protective effects of pentoxifylline against I/R injury were due to reduction in the activation of NF-κB. It has been demonstrated that exposure to a number of chemi-

cal inducers not only leads to HSP induction but also to inhibition of NF-κB21. In the present study, the activity of NF-κB were detected in each group and an obvious reduction in the activation of NF-κB was confirmed in HS+A/R group and in the pCDNA HSP70 + A/R group. The results indicated that cardioprotective effect of HSP70 may be due to inactivation of NF-κB. In summary, we demonstrated that overexpression of HSP70 alone by gene transfection leads to the protection for cardiac myocytes against anoxia-reoxygeneration. These cardio-protective effects were related to the reduction in activa-tion of NF-κB.


J. C. LIU ET AL. 13

Figure 5. Effect of various treatments on expression of HSP70 protein in rat’s cultured ventricular myocytes (n=8, mean±SD). A: representative Western blots for HSP70

protein. B: densitometric fold of A/R (*P <0.01, vs A/R group; #P <0.01, vs HS+A/R group)

References

[1] Amrani M, Corbett J, Allen NJ, O’Shea J, Boateng SY, May AJ, et al. Induction of heat-shock proteins enhances myocardial and endothelial functional recovery after prolonged cardioplegic arrest. Ann Thorac Surg 1994; 57:157-160.

[2] Currie RW, Karmazyn M, Kloc M, Mailer K. Heat-shock response is associated with enhanced postischemic ven-tricular recovery. Circ Res 1988; 63: 543-549.

[3] Jayakumar J, Suzuki K, Khan M, Smolenski RT, Farrell A, Latif N, et al. Gene therapy for myocardial protection transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ische-mia-reperfusion injury. Circulation 2000; 102 Suppl III: 302-306.

[4] Simpson S, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating non

myocardial cells. Circ Res 1982; 50: 101-116.

[5] Koyama T, Temma K, Akera T. Reperfusion-induced contracture develops with a decreasing [Ca2+]: in single heart cells. Am J Physiol 1991; 261: H1115-H1122.

[6] Xu MF, Wang YG, Kyoji H, Ayub A, Ashraf M. Calcium preconditioning inhibits mitochondrial permeability tran-sition and apoptosis. Am J Physiol Heart Circ Physiol 2001; 280: H899-H908.

[7] Mizuguchi H, Nakagawa T, Nakamishi M, Imazu S, Na-kagawa, S, Mayumi T. Efficient gene transfer into mam-malian cells using fusogenic liposomes. Biochem Bio-phys Res Commun 1996; 218: 402-407.

[8] Gomez LA, Alekseev AE, Aleksandrova LA, Brady PA, Terzic A. Use of the MTT assay in adult ventricular car-diomyocytes to assess viability: effects of adenosine and potassium on cellular survival. J Mol Cell Cardiol 1997; 29: 1255-1266.


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[9] Eng LF, Lee YL, Murphy GM, Yu AC. A RT-PCR study of gene expression in a mechanical injury model. Prog Brain Res 1995; 105: 219-229.

[10] Dix DJ, Rosario-Herrle M, Gotoh H, Mori C, Goulding EH, Barrett CV, et al. Developmentally regulated expres-sion of HSP

70-2 and a HSP

70-2/lacZ transgene during

spermatogenesis. Dev Biol 1996; 174: 310-321.

[11] Gershoni JM. Protein blotting: a manual. Methods Bio-chem Anal 1988; 33: 1-12.

[12] Vetterlein F, Schrader C, Volkmann R, Neckel M, Ochs M, Schmidt G, et al. Extent of damage in ischemic, non-reperfused, and reperfused myocardium of anesthetized rats. Am J Physiol Heart Circ Physiol 2003; 285: H755-H765.

[13] Cognasse F, Sabido O, Béniguel L, Genin C, Garraud O. A flow cytometry technique to study nuclear factor-kappa B (NFκB) translocation during human B cell activation. Immunol Lett 2003; 90: 49-52.

[14] Wischmeyer PE. Glutamine and heat shock protein ex-pression. Nutrition 2002; 18: 225-228.

[15] Benjamin IJ, McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and dis-ease. Circ Res 1998; 83: 117-132.

[16] Samali A, Orrenius S. Heat shock proteins: regulators of stress response and apoptosis. Cell Stress Chaperones 1998; 3: 228-236.

[17] Nazir SA, Metcalf JP. Innate immune response to adeno-virus. J Investig Med 2005; 53: 292-304.

[18] McConnell MJ, Imperiale MJ. Biology of adenovirus and its use as a vector for gene therapy. Hum Gene Ther 2004; 15: 1022-1033.

[19] Khan TA, Sellke FW, Laham RJ. Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia. Gene Ther 2003; 10: 285-291.

[20] Zhang M, Xu YJ, Saini HK, Turan B, Liu PP, Dhalla NS. Pentoxifylline attenuates cardiac dysfunction and reduces TNF-α level in ischemic-reperfused heart. Am J Physiol Heart Circ Physiol 2005; 289: H832-H839.

[21] Thanos D, Maniatis T. NF-kappa B: a lesion in family values. Cell 1995; 80: 529-532.




Surgical Outcome and Clinical Follow-up in Patients with Symptomatic Myocardial Bridging

ABSTRACT

From 1997 to 2006, 37 463 patients received selective coronary angiography in the Fuwai Cardiovascular Hospital, Beijing, China. Of these, 484 patients had angiographic diagnosis of myocardial bridging. Of the 484 patients, 35 un-derwent surgery for treatment of myocardial bridging with significant systolic arterial compression. Among the surgical treatment patients, 24 presented with other cardiac disorders, and the remaining 11 symptomatic patients with isolated myocardial bridging were included in the follow-up study. Keywords: myocardial bridging, myocardial ischaemia, myotomy, coronary artery bypass grafting, coronary

angiography

1. Introduction

Muscle overlying the intramyocardial segment of an epicardial coronary artery is termed a myocardial bridg-ing, and it is generally confined to the middle segment of the left anterior descending artery (LAD)1. Traditionally, myocardial bridging is considered a benign condition2,3, but it may be associated with myocardial ischaemia4, acute coronary syndromes and sudden death5. Surgery has been attempted in selected patients6,7. However, there is little information about the prognosis of patients with this anomaly, especially the outcome of surgical treat-ment for myocardial bridging. Therefore, we analyzed the clinical, angiographic and functional followup after surgical treatment in patients with isolated symptomatic myocardial bridging.

2. Methods

2.1. Patient Selection

From 1997 to 2006, 37 463 patients received selective coronary angiography in the Fuwai Cardiovascular Hos-pital, Beijing, China. Cardiac catheterization was per-formed with the use of a standard Judkins technique and images were obtained in multiple views, in which sys-

tolic compression of the anterior descending coronary artery could be observed clearly in every patient. Of these, 484 patients had angiographic diagnosis of myo-cardial bridging. Out of the 484 patients, 35 underwent surgical treatment of myocardial bridging with signifi-cant systolic arterial compression. The patients who had a diagnosis of moderate or severe aortic stenosis, moder-ate or severe left ventricular hypertrophy of any origin or hypertrophic cardiomyopathy were excluded. Patients with coronary artery disease, understood as fixed steno-sis of more than 50% at some point of the coronary net-work, were also excluded. From these 35 patients, 8 men and 3 women with myocardial bridging but without other cardiac diseases were included in the followup study. The coronary angiographies and medical histories of these 11 patients were reviewed.

2.2. Follow-up

Follow-up was carried out by telephone and completed with a review of the medical histories of the patients who had new admissions and medical records at the hospital. Clinical evaluation was also accomplished by direct in-terview of the patients at clinic visits. Clinical symptoms,

X. H. HUANG ET AL. 16

daily activities without chest pain or anginal symptoms, current medical treatment, and events experienced (myocardial infarction, need to repeat coronary angiography or revascularization) were analyzed throughout the follow-up period.

3. Results

The 37 463 patients with selective coronary angiographic analysis included 484 with myocardial bridging; a prevalence of 1.3%. Of these 484 patients, 35 received surgical treatment for myocardial bridging with systolic compression of the left anterior descending artery. Among the surgically treated patients, 24 presented with other cardiac disorders: hypertrophic cardiomyopathy in 3 cases, coronary heart disease in 3, and valve and other cardiac diseases in 18. We only studied the remaining 11 patients with isolated myocardial bridging. All of their coronary angiographies revealed myocardial bridging in the middle segment of LAD with systolic compression ≥75% (ranging from 75% to 90%).

The mean age of the male patients was 48.4 years (SD=8.9 years). Four patients had arterial hypertension and 6 were active smokers at the time that coronary an-giography was performed. The clinical presentation was typical angina in 10 patients and atypical chest pain in 1. Two patients had previous anterior myocardial infarction. ECG stress testing showed significant ST segment de-pression or T wave inversion from the anterior leads during exercise in six of the 11 patients, while five had a positive Tc-99m Sestamibi SPECT (single photon emis-sion computed tomography) test with filling defects dur-ing stress, which were reversible at rest. Ventricular function was normal in all of these patients. Surgical myotomy was performed in 3 patients and CABG in 8. Eight patients were operated on with an off-pump ap-proach and 3 patients with a cardiopulmonary bypass technique after medial sternotomy. Conversion to on pump CABG surgery was necessary in 1 patient because of perforation of the right ventricle. The left internal mammary artery (LIMA) was the graft used in all the patients with CABG. The acute clinical success rate was 100% with respect to the absence of myocardial infarc-tion, death or other in-hospital complications. Complete recovery was achieved in the postoperative period in all patients.

All of the patients were followed up clinically. The median follow-up was 35.3 months (range: 6 to 120 months). Nine patients were free from symptoms and one of them continued taking beta-blockers. The re-maining 2 patients with myotomy had symptoms of atypical chest pain. One of them received coronary an-giography again and no stenosis was found at two years

after operation; while exercise testing was performed in the other patient and revealed no evidence of myocardial ischaemia. Both of them were taking beta-blockers and symptoms were controlled. No myocardial infarction or other major adverse cardiac events were encountered in these patients during follow-up.

4. Discussion

Myocardial bridging was recognized at autopsy in 1737 and first described angiographically in 1960. The current best method for diagnosing myocardial bridging is selec-tive coronary artery angiography and the typical an-giographic finding is systolic narrowing of an epicardial artery. The incidence of myocardial bridging reported in angiographic studies ranged from 0.5% to 40%. The lim-ited frequency of myocardial bridging observed an-giographically is in contrast with that of autopsy studies, which have reported a frequencies of 5% to 86%1. In the present study of selective coronary angiography with more than 37 000 patients, the prevalence of myocardial bridging is 1.3%. Variation between autopsy and an-giography may in part be attributable to small and thin bridges causing little compression. New imaging tech-niques like intravascular ultrasound, intracoronary Dop-pler ultrasound and electron beam tomography and mul-tislice CT8 as noninvasive imaging techniques can be used for diagnosis of functional and morphological status of bridging.

Though bridging is considered as a harmless anatomi-cal vessel malformation in most cases, the intramural course of certain portions in the left anterior descending coronary artery may cause myocardial ischaemia and infarction4. The mechanism by which myocardial bridg-ing causes myocardial ischaemia is not very clear but associated with decreased coronary flow reserve9. Indeed, intravascular ultrasonography and Doppler imaging have demonstrated that vessel compression during systole is followed by a delay in the increase in luminal diameter during diastole, thus affecting the predominant phase of coronary perfusion, especially during episodes of tar-chycardia10,11. In the present follow-up study, at baseline before operation, all of the patients had angina and two had myocardial infarction of the anterior wall.

In symptomatic cases, there are several therapeutic options available. The medications of choice are beta-blockers10,12, whose mechanism of action is through a negative inotropic and chronotropic effect. Sometimes medical treatment is insufficient and other interventions are required. The efficacy and long term outcome of stent implantation in symptomatic myocardial bridging is still controversial13, though several studies have demon-strated that stenting can prevent external compression of


X. H. HUANG ET AL. 17

bridged coronary artery segments, with increase in lu-minal diameter and improve symptoms. Haager et al14 evaluated the results of coronary stenting in 11 patients with symptomatic myocardial bridging of the central portion of the LAD. Followup angiography at 7 weeks demonstrated mild to moderate or severe stent stenosis in 5 patients; revascularization was repeated in 4 patients. High restenosis15,16 or major periprocedural complica-tions17, including perforation of the artery have been re-ported in other studies. Thus, the rate of restenosis of stent implantation in myocardial bridging has been too high to generally recommend this approach in sympto-matic patients.

Surgical treatment with dissection of the overlying myocardium (myotomy)6,7 or coronary artery bypass grafting18 is limited to patients with symptoms that per-sist despite medical treatment. Surgical myotomy, first reported by Binet et al, abolishes clinical symptoms and is associated with reversal of local myocardial ischaemia and an increase in coronary flow6. However, this treat-ment strategy carries certain risk. The unpredictable in-tramural course of the coronary artery may require deep incision of the ventricular wall, potentially leading to subsequent perforation of the right ventricle7,19. There is little information about the prognosis of surgical treat-ment for symptomatic myocardial bridging.

In the present study, for all 11 patients, isolated myo-cardial bridging had caused more than 75% compression of the middle portion of the anterior descending coronary artery and was refractory to oral medication. Myotomy was performed in 3 patients and CABG in 8 patients. The LIMA was the graft used in all of the patients. One patient suffered a right ventricular perforation that was successfully repaired by change to on pump CABG. The others had no serious surgical complications and they were discharged from the hospital uneventfully. During the middle- and long-term follow-up of 3 years (range: 6 to 120 months), 2 had atypical chest pain and were con-trolled by medical treatment. The other 9 patients were symptom free without the need for further invasive di-agnostic studies. No myocardial infarction or other major adverse cardiac events occurred during follow-up study. Therefore, surgery either by myotomy or CABG is rela-tively safe and is an option for the treatment of selected patients with symptomatic myocardial bridging. Addi-tional studies are needed to investigate which patients should be selected for surgical therapy and to assess the long term prognosis of the surgical treatment.

There were several limitations in this study. Patients were included in the study based on a coronary an-giography made before the study was designed, so there was no established protocol for the coronary angiogra-phy. Not every patient received intracoronary nitroglyc-

erine at the time of angiography in our study, thus the prevalence could be underestimated. Additionally after operation during followup, only one of our patients un-derwent coronary angiography again.

In conclusion, myocardial bridging, a congenital ves-sel anomaly, is a relatively common angiographic find-ing20.

Although usually a benign condition, myocardial

bridging can occasionally generate clinically important complications. Medication is the initial therapy for symptomatic patients. Surgical myotomy or CABG should be limited to patients who are refractory to oral medication. Benefits with low operative risk and excel-lent middle- and long-term results can be achieved by surgical relief of myocardial ischaemia due to systolic compression of intramyocardial coronary arteries. When surgery is chosen, the decision to perform myotomy or CABG is made on a case by case basis. In arteries that take a very deep course through the septum, approaching the right ventricular subendocardium and surgical expo-sure of the intramuscular coronary artery may be difficult, CABG is preferred to myotomy and the LIMA is the best choice of graft.

5. Conclusions

Myocardial bridging is a relatively common an-giographic finding. Surgical myotomy or CABG should be limited to patients who are refractory to oral medica-tion. Surgical relief of myocardial ischaemia due to sys-tolic compression of intramyocardial coronary arteries can be accomplished with low operative risk and excel-lent middle- and long-term results.

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18

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[12] 12. Nair CK, Dang B, Heintz MH, Sketch MH. Myocar-

dial bridges: effect of propranolol on systolic compres-sion. Can J Cardiol 1986; 2: 218-221.

[13] 13. Berry JF, von Mering GO, Schmalfuss C, Hill JA, Keresky PA. Systolic compression of the left anterior de-scending coronary artery: a case series, review of the lit-erature, and therapeutic options including stenting. Catheter Cardiovasc Interv 2002; 56: 58-63.

[14] 14. Haager PK, Schwarz ER, Dahl J, Klues HG, Reffel-mann T, Hanarath P. Long term angiographic and clinical follow up in patients with stent implantation for sympto-matic myocardial bridging. Heart 2000; 84: 403-408.

[15] 15. Wang NF, Pan H, Tong GX. The evaluation on stent implantation efficacy of myocardial bridge and severe atherosclerosis lesions in the segments proximal to the myocardial bridge. Chin J Cardiol (Chin) 2005; 33: 684-686.

[16] 16. Yan HB, Wang J, Zhu XL, Gao H, Li N, Hui A. Fre-quency of infarct-related artery with myocardial bridging in patients with ST-elevation myocardial infarction and its impact upon percutaneous coronary intervention. Chin Med J 2006; 119: 539-543.

[17] 17. Broderick TM, Kerelakes DJ, Whang DD, Toltzis RJ, Abbottsmith CW. Myocardial bridging may predispose to coronary perforation during rotational atherectomy. J In-vasive Cardiol 1996; 8: 161-163.

[18] 18. Pratt JW, Michler RE, Pala J, Brown DA. Minimally invasive coronary artery bypass grafting for myocardial muscle bridging. Heart Surg Forum 1999; 2: 250-253.



T3/T4 Thoracic Sympathictomy and Compensatory Sweating in Treatment of Palmar Hyperhidrosis

ABSTRACT

Compensatory sweating (CS) is one of the most common postoperative complications after thoracic sympathectomy, sympathicotomy or endoscopic sympathetic block (ESB) for palmar hyperhidrosis. This study was conducted to examine the relevance between CS and the sympathetic segment being transected in the surgical treatment of palmar hyper-hidrosis, and thus to detect the potential mechanism of the occurrence of CS. The rates of occurrence and severity of CS are lowered with the lower sympathetic chain being transected Keywords: thoracic sympathicotomy, palmar hyperhidrosis, compensatory sweating

1. Introduction

Compensatory sweating (CS) is one of the most common postoperative complications after thoracic sympathec-tomy, sympathicotomy or endoscopic sympathetic block (ESB) for palmar hyperhidrosis. Severe CS in some cases has an adverse effect on patients’ quality of life, but the exact mechanism of this complication is not clear. This randomized prospective study was designed to in-vestigate the correlation between the sympathetic tran-section level and postoperative CS in patients with pal-mar hyperhidrosis. One hundred and sixty-three patients with palmar hyperhidrosis treated from October 2004 to June 2006 were randomly divided into two groups: in-tercostal video-mediastinoscopic thoracic sympathi-cotomy at T3 level in 78 patients and at T4 level in 85 patients..

2. Methods

Of the 163 patients, 96 were male and 67 were female, the age of the patients was from 14 to 52 years with a median of 28.3 years. All patients had excessive sweat-ing in hands as the chief complaint. The severity of sweating was graded into mild, moderate and severe lev-els. Systemic or secondary hyperhidrosis was excluded before randomization. ECG and chest X-ray examination were performed routinely. The basic data of T3 (78 pa-tients ) and T4 (85) groups are shown in Table 1.

2.1. Anesthesia

The procedure was performed under single-lumen in-tubated general anesthesia, by which a low volume ven-tilation or block of ventilation for 2−3 minutes was taken to collapse the lung lobe, then the thoracic sympathetic trunk could be clearly identified in parallel to the verte-bral column. Saturation of blood oxygen (SO2) was care- fully monitored during the operation. After re-ventilation when SO2 was less then 0.80, the operation was paused until SO2 reached the normal level.

2.2. Intercostal Video-mediastinoscopic Sympathicotomy

A 2.0−2.5 cm incision was made between the second and third rib at armpit, then a video-mediastinoscope was inserted into the thoracic cavity to identify the T3 sym-pathetic ganglion by inspection and palpation. Normally, the rib that can be seen at the top of the cavity is the second, and the first rib is often covered by surrounding fat tissue. Then an electrocautery hook was inserted to isolate and cut the sympathetic chain at the level of T3 or T4 according to preoperative randomization. An ablation area around the cutting site of the sympathetic chain was extended with a range of 2 cm along the rib to destroy the nerve fibers of Kuntz completely. Video-mediastinoscope was removed at the end of the procedure, and the

J. YANG ET AL. 20

suction tube was aspirated while the lung was reinflated and continuous positive pressure was exerted for a few seconds. Then the suction tube was removed quickly and the incisions were closed with absorbable suture. No closed chest drainage was performed. Chest X-ray and ECG were performed on day 1 after the operation1,2.


The chi-square test and radit analysis were used in ana-lyzing the result through statistical software SPSS 11.0 for Windows. A P value less than 0.05 was considered statistically significant.

3. Results

3.1. Operative Results

There was no conversion to open techniques. Neither perioperative mortality nor serious complications such as cardiac arrythmia or arrest were observed during the op-eration. The median duration of the operation was 30 minutes (range, 20 to 40 minutes). Palmar hyperhidrosis was cured in all patients shortly after sympathicotomy with warm and dry hands, and the temperature at the palm increased by 1.5−3°C compared with that before sympathectomy. No bradycardia or Horner’s syndrome was encountered. Hospital stay for all patients ranged from 1 to 2 days. All patients resumed their normal life and work within 2 weeks after discharge from the hospi-tal.

3.2. Follow-up

Phone-call or letter follow-up were available for 163 patients after sympathicotomy, 8 of them lost contact. The period of follow-up lasted for 3−24 months. The mean duration was 13.8 ± 6.2 months. No recurrence of palmar hyperhidrosis was observed. Information of postoperative sweating over the body was collected for analysis. The severity of CS was classified into three levels according to the definition described in the litera-ture3: mild: a little more sweating than before, no influ-ence on normal daily activities; moderate: more sweating than before, a mild influence on normal daily activities, which embarrasses the patient; severe: obvious sweating with severe influence on normal daily activities, which makes the patient regret for receiving sympathicotomy.

3.3. CS

CS with different severity occurred immediately or at day 1 after sympathicotomy (Table 2). The most com-mon sites of CS included the back and chest. The un-common sites included the waist buttock, and thigh. CS occurred not more common in patients with T4 sympa-

thicotomy than in those with T3 sympathicotomy (P<0.05). No severe CS was seen in all patients. Mild CS was not significantly different between T4 and T3 sym-pathicotomy group (P>0.05), but less moderate CS oc-curred in patients with T4 sympathicotomy than in T3 patients (P<0.05).

4. Discussion

CS is a phenomenon of a greater amount of sweating elsewhere in the body after treatment for hyperhidrosis4,5. It has an effect on patients’ satisfaction to the surgery.

4.1. Mechanism of CS

Up to the present, the mechanism of CS after sympathi-cotomy remains poorly understood, and there is no ac-ceptable explanation for it. The concept of “compensa-tion” described in the 1970s indicates that a greater amount of sweating elsewhere in the body compensates for the lack of sweating in the treated body area in order to maintain sweating balance of the whole body6,7. However, recent data are conflicting with this theory. One is that there is no CS after the treatment with botulinum toxin for palmar hyperhidrosis8.

Although the exact mechanism is still unknown, the occurrence of CS is believed to be a result of disturbance of the sympathetic system after surgery. After comparing different surgical approaches for hyperdirosis, Lin et al9

concluded that destruction of the nerve reflex arch be-tween the sympathetic trunk and hypothalamus is re-sponsible for excessive sweating elsewhere in the body after sympathicotomy. It’s a disorder of imbalanced regulation of sweating of the body. They suggested that preservation of the negative afferent tone to the hypo-thalamus be the key technique for the prevention of postoperative CS. This hypothesis is so far the most sat-isfactory explanation for CS after sympathicotomy and is already verified by other clinical studies.

4.2. Influence of CS on Human Body

Patients’ lifestyle with CS will not be affected3. Only after repeat enquiries many patients may complain of more sweating elsewhere in their body after surgery, without effect on their normal life. This condition is called mild CS (minor), normally consisting of the ma-jority of cases with CS. In the present study, the mild CS accounted for 47.4% in T3 group and 37.6% in T4 group respectively. In some patients excessive sweating occurs at some new sites of the body after surgery and the new hyperhidrosis has an adverse effect on patients’ lifestyle. This condition is so called compensatory hyperhidrosis, with which the patient has a decreased but acceptable satisfaction to the procedure. This kind of CS is classi


J. YANG ET AL.


21

Table 2. CS after T3/ T4

sympathicotomy

fied as moderately severe. In the present study the mod-erate CS accounted for 23.1% in T3 group and 7.1 % in T4 group respectively. In a few cases, unfortunately, se-vere excessive sweating occurs elsewhere in the body, making the patient feel anguished and embarrassed, even regret for having the sympathicotomy done. This condi-tion is called severe CS. No a single case of severe CS was found in the present study.

4.3. Correlation Between CS and the Level of Thoracic Sympathicotomy

The correlation between CS and surgical approaches, particularly the level of thoracic sympathetic chain transaction is a hot topic for investigation in this field in recent years. According to the anatomy and physiology of the upper thoracic sympathetic chain, limiting de-struction area or lowering the transection level of the sympathetic chain will result in a localized desympathe-tization, which would be a theoretical basis for the pre-vention of CS10,11. This hypothesis is supported by clini-cal findings12,13. For example, a comparative analysis by Yoon et al14,15 showed that CS after T3 sympathicotomy (16.73%) was significantly less than that after T2− T3 sympathicotomy (45.8%), but with almost the equal therapeutic rate of 100% for palmar hyperhidrosis under the two procedures. Comparison of T2− T4 sympathi-cotomy with T4 sympathetic clipping for the treatment of palmar hyperhidrosis16 showed that T4 sympathetic clip-ping resulted in a significantly less CS but an equal therapeutic effect on hyperhidrosis. At present more and more surgeons agree that the preservation of T2 ganglion and sympathetic segment above can lead to a major re-duction of CS, particularly embarrassing and disabling CS, which is also supported by the results of the present study17,18.

Whether lowering sympathetic transection level from T3 down to T3 can reduce further CS or not is worth in-vestigating since there is no randomized clinical trial

about it. The results of the present randomized clinical trial showed that the incidence of moderate CS was sig-nificantly lower in T4 sympathicotomy group than in T3 group (P<0.05), although there was a similar incidence of mild CS in the two groups (P>0.05). This finding in-dicates that lowering the thoracic sympathetic transection level further can significantly reduce both incidence and severity of CS. Single T4 sympathetic transection or clipping as a popular surgical procedure for palmar hy-perhidrosis has been accepted by more and more sur-geons19,20.

In conclusion, these clinical findings are helpful to better understand dominant characteristics of the upper thoracic sympathetic chain and the exact mechanism of CS after sympathicotomy although this side effect can not be avoided completely at the moment.

References

[1] Yang J, Wang J, Tan JJ, Ye GL, Gu WQ. The prelimi-nary experience of intercostal video-mediastinoscopy in cliinical application. Chin J Thorac Cardiovasc Surg (Chin) 2004; 20: 148-150.

[2] Yang J, Wang J, Tan JJ, Ye GL, Gu WQ, Liu YG. Pro-cedure modification for sympathicotomy untilized in the treatment of palmar hyperhidrosis. Chin J Thorac Cardiovasc Surg (Chin) 2005; 21: 377-378.

[3] Licht PB, Pilegaard HK. Severity of compensatory sweating after thoracoscopic sympathectomy. Ann Tho-rac Surg 2004; 78: 427-431.

[4] Riet M, Riet M, Smet AA, Kuiken H, Kazemier G, Bon-jer HJ. Prevention of compensatory hyperhidrosis after thoracoscopic sympathectomy for hyperhidrosis. Surg Endosc 2001; 15: 1159-1162.

[5] Gossot D, Kabiri H, Caliandro R, Debrosse D, Girard P, Grunenwald D. Early complications of thoracic endo-

J. YANG ET AL. 22

scopic sympathectomy: a prospective study of 940 pro-cedures. Ann Thorac Surg 2001; 71: 1116-1119.

[6] Adar R, Kurchin A, Zweig A, Mozes M. Palmar hyper-hidrosis and its surgical treatment: a report of 100 cases. Ann Surg 1977; 186: 34-41.

[7] Chou SH, Kao EL, Li HP, Lin CC, Huang MF. T4 sym-pathectomy for palmar hyperhidrosis: an effective ap-proach that simultaneously minimizes compensatory hy-perhidrosis. Kaohsiung J Med Sci 2005; 21: 310-313.

[8] Krogstad AL, Skymne A, Pegenius G, Elam M, Wallin BG. No compensatory sweating after botulinum toxin treatment of palmar hyperhidrosis. Br J Dermatol 2005; 152: 329-333.

[9] Lin CC, Telaranta T. Lin-Telaranta classification: the importance of different procedures for different indica-tions in sympathetic surgery. Ann Chir Gynaecol 2001; 90: 161-156.

[10] Liu YG, Shi XZ, Yu EH, Wang J. Applicative anat-omy study of upper thoracic sympatheotic trunk for clinical sympathictomy. Chin J Thoracic Cardiovasc Surg (Chin) 2005; 21: 75-77.

[11] Neumayer C, Zacherl J, Holak G, Jakesz R, Bischof G. Experience with limited endoscopic thoracic sympathetic block for hyperhidrosis and facial blushing. Clin Auton Res 2003; 13 Suppl 1: I52-57.

[12] Ramsaroop L, Singh B, Moodley J, Partab P, Pather N, Satyapal KS. A thoracoscopic view of the nerve of Kuntz. Surg Endosc 2003; 17: 1498.

[13] Licht PB, Pilegaard HK. Gustatory side effects after tho-racoscopic sympathectomy. Ann Thorac Surg 2006; 81:

1043-1047.

[14] Yoon DH, Ha Y, Park YG, Chang JW. Thoracoscopic limited T-3 sympathicotomy for primary hyperhidrosis: prevention for compensatory hyperhidrosis. J Neurosurg 2003; 99(1 Suppl): 39-43.

[15] Yazbek G, Wolosker N, de Campos JR, Kauffman P, Ishy A, Puech-Leao P. Palmar hyperhidrosis-which is the best level of denervation using video-assisted thoraco-scopic sympathectomy: T2 or T3 ganglion? J Vasc Surg 2005; 42: 281-285.

[16] Neumayer C, Zacherl J, Holak G, Fugger R,Jakesz R,Herbst F, et al. Limited endoscopic thoracic sympa-thetic block for hyperhidrosis of the upper limb: reduc-tion of compensatory sweating by clipping T4. Surg En-dosc 2004; 18: 152-156.

[17] Ahn SS, Wieslander CK, Ro KM. Current developments in thoracoscopic sympathectomy. Ann Vasc Surg 2000; 14: 415-420.

[18] Doolabh N, Horswell S, Williams M, Huber L, Prince S, Meyer DM, et al. Thoracoscopic sympathectomy for hy-perhidrosis: indications and results. Ann Thorac Surg 2004; 77: 410-414.

[19] Lin CC, Wu HH. Endoscopic T4-sympathetic block by clamping (ESB4) in treatment of hyperhidrosis palmaris et axillaries-experiences of 165 cases. Ann Chir Gynaecol 2001; 90: 167-169.

[20] Choi BC, Lee YC, Sim SB. Treatment of palmar hyper-hidrosis by endoscopic clipping of the upper part of the T4 sympathetic ganglion. Preliminary results. Clin Auton Res 2003; 13 Suppl 1: I48-51.




Therapeutic Strategy for Traumatic Instability of Subaxial Cervical Spine

-

ABSTRACT

A simple, safe and effective therapeutic strategy for traumatic instability of the subaxial cervical spine, as well as its prognostic assessment, is still controversial. The therapeutic options for 83 patients of traumatic instability of the subaxial cervical spine, whose average age was 35 years, were determined, according to the Allen-Ferguson classifica-tion, general health and concomitant traumatic conditions, neurological function, position of compression materials, concomitant traumatic disc herniation/damage, concomitant locked-facet dislocation, the involved numbers and posi-tion, and the patients’ economic conditions. An anterior, posterior or combination approach was used to decompress and reconstruct the cervical spine. No operations with an anterior-posterior-anterior approach were performed. The best surgical strategy should be determined by the type of subaxial cervical injury, patients’ general health, local pathological anatomy and neurological function. Keywords: cervical instability, disease classification, surgical strategy

1. Introduction

Traumatic instability of the subaxial cervical spine, a common type of injury, includes fracture or dislocation of the spine, as well as ligament damage. It often causes damage to the spinal cord or nerve root. Therapeutic op-tions include decompression of the neural elements, re-constructing or recovering the normal anatomical align-ment of the spine, which leads to immediate stability. Although surgery is the choice of most doctors, there is still no agreement on many correlative factors, such as deciding operation time, surgical approach, sequence/ level, internal fixator, and dealing with concomitant local pathological situations (traumatic disc herniation/damage, locked-facet dislocation). In addition, the outcomes of its prognosis and evaluation show considerable differences. Another issue is whether an anterior or posterior ap-proach to reconstruction is the better option for those patients who do not need a particular surgical approach or sequence, due to general or local pathological condi-tions. Our orthopaedics department has retrospectively visited 83 cases from January 1998 to December 2006.

2. Methods

2.1. Patients

There were 83 patients (59 male and 24 female), with an average age of 35 years (range 18–66 years), and the time between injury and seeking medication was 1.5–4 days. Main injury levels were: C3–4 (10 cases), C4–5 (28 cases), C5–6 (40 cases), C6–7 (10 cases), C7–T1 (3 cases), more than two main levels (C4–5+C5–6) (7 cases). Causes of injury were: traffic accident (49 cases), falling (24 cases), being hit by a heavy object (7 cases), and other causes (3 cases). Concomitant injuries were: traumatic brain injury (6 cases), rib fracture and he-mopneumothorax (5 cases), and limb fracture (9 cases). According to the Allen-Ferguson classification, the in-jury types were as follows: 33 cases with distrac-tion-flexion (including 23 with fracture-dislocation with locked facet), 29 with compression-flexion, 12 with ver-tical compression, six with compression-extension and three with compression-lateral. Among the 23 cases of fracture-dislocation with locked-facet, there were 17 with normal neurological functions or some neurological dysfunction, six with complete spinal cord injury (SCI) (including six with single locked-facet, 17 with double

P. CAO ET AL. 24

locked facet, 13 with facet or neural arch fractures; and seven, nine, five and two with C4–5, C5–6, C6–7 and C7–T1 fracture-dislocations, respectively). All patients had full radiological examinations (static/dynamic X-ray and MRI/CT), neurological assessment (American Spinal Cord Injury Association (ASIA) neurological function assessment, ASIA motion function scale assessment), functional grade assessment (Japanese Orthopaedics As-sociation (JOA) grade), and visual analog scale (VAS) assessment before and after surgery. Radiographic as-sessments included the following: degree of cervical kyphosis (based on Cobb angle), degree of vertebral body translation, disc height ratio, fusion process of the operated levels, as well as looseness and subsidence of the internal fixator. Among the 83 patients, after exclud-ing those who needed a particular surgical approach or sequence due to general or local pathological conditions, 42 who were reconstructed with either a single anterior or posterior approach were included in this retrospective study.

2.2. Therapeutic Options

Patients were subjected to ASIA neurological function assessment and full radiological examinations (static/ dynamic X-ray, and MRI/CT); some correlative compli-cations were dealt with. Large doses of methylpredniso-lone were given to patients with SCI. All patients were treated with skull or Glisson tong traction.

Given that patients were generally in a stable condi-tion, different therapeutic strategies and sequences were practised based on the injury type and local pathological conditions. We used early and continued closed skull traction-reduction to treat fracture-dislocation with locked facet in patients with distraction-flexion-type frac-tures, under the guidance of X-ray and neurological function grade assessment. When any neurological func-tion deterioration or disability of reduction or intolerance of traction occurred, we performed different approaches to decompress and reconstruct the cervical spine. This

depended on whether there was concomitant disc herni-ation and damage to the dislocated levels. Posterior re-duction and fixation were performed in patients with no concomitant disc herniation; in those with concomitant disc herniation, we performed direct anterior reduction and fixation or a three-stage strategy (The first step was anterior decompression, followed by posterior reduction and fixation, and finally, anterior reconstruction). When closed reduction was successful, we continued with skull traction (some patients were given a further MRI scan), and chose an appropriate date to carry out reconstruction using an anterior, posterior or combination approach (Figures 1 and 2).

In patients with other types of injury, individual op-erations were adapted according to the location and level of injuries, general and concomitant diseases (for exam-ple, osteoporosis), location and number of spinal levels affected by the injury, and the economic conditions and needs of the patient. Patients who had injury to the pos-terior tension band of the cervical spine, those who were of relatively old age, and those with osteoporosis or mul-tiple-level cervical injuries, needed posterior-approach surgery. Patients with anterior column compression needed anterior-approach decompression-reconstruction operations. Some patients needed a combined approach.

In the anterior approach operation, we used discec-tomy or corpectomy to perform decompression of the injured spine by the Smith-Robinson method, then the auto-iliac bone or titanium mesh was implanted with anterior locked-cervical plate fixation (CSLP, Synthesis, USA; Zephir/Orion plate, Medtronic, USA; Slim-lock/ Codamn plate, Depuy, USA); Posterior-approach opera-tions were mainly performed using reduction and fixa-tion techniques. If infold fracture fragment of lamina or lateral mass, compression of the neural elements, or concomitant cervical spinal canal stenosis occurred, we performed total laminectomy or intervertebral foramen dissection decompression, using lateral mass fixators (Axis/Vertax, Medtronic; Cervifix, Synthesis, USA).

Figure 1. A: C6-7 fracture-dislocation with facet-locked occurred. B: C6-7 traumatic disc herniation occurred. C: Closed traction-reduction was successful. D: Anterior fusion and fixation was practised at an appropriate date.


P. CAO ET AL. 25

Figure 2. A: C5-7 fracture-dislocation with instability occurred. B: Combined anterior and posterior-approach operation occurred.

Table 1. Patient characteristics and functional scores before and after operations (Mean value)

Table 2. Pre- and post-operative functional scores and patient characteristics according to surgical approach (Mean value)

*The difference between the anterior and posterior groups was significant (P <0.05)


P. CAO ET AL.


26

2.3. Postoperative Follow-up

Patients were given antibiotics and vitamin B-12 for nerve recovery, and instructed to wear a cervical collar for 2–3 months. We visited the patients regularly and recorded their ASIA neurological function grades, ASIA mobility function index scores, JOA scores, and VAS scores. Radiological assessment included the following:

Cobb angle, vertebral body translation, disc height ra-tio, fusion process of the operated levels, as well as looseness and subsidence of the internal fixator.


Statistical analysis was performed by Student’s t test and P <0.05 was considered significant.

3. Results

All cases were followed up for an average of 3 years and 9 months (range, 3 months to 6 years and 4 months). There were 16 cases with complete SCI, 39 with incom-plete SCI, and 28 cases with normal neurological func-tion. Twenty-eight patients had anterior cord syndrome, five had Brown-Sequard syndrome, and 22 had central cord syndrome. There were 46, 28 and 9 cases treated with anterior, posterior and combination operations, re-spectively. There were no operations using an ante-rior-posterior-anterior approach. Before surgery, the av-erage JOA and VAS scores were 11.2 (0–17) and 7.8 (1–10), respectively. At the final assessment, the JOA and VAS scores improved to 15.3 (1–17) and 2.6 (0–6) respectively. The average ASIA motor score was 53.5 (0–100) before operation and 67.8 (11–100) at the final follow-up. For incomplete SCI, the average ASIA neu-rological function score was improved by 1–2 levels. Patients with complete SCI had no neurological recovery, but nerve root function recovered to a different extent. The average Cobb angle, vertebral body translation, and disc height ratio were +21.8° (+46 to –2.1°), 6.0 mm (posterior translation 0.6 mm, anterior translation 8.3 mm), and 71% (38%–89%) respectively, before surgery, and –2.9° (+2.4 to –4.1°), 0.35 mm (0–2.2 mm), and 96% (82%–145%), respectively, at final follow-up (Ta-ble 1). Forty-two patients were treated with either single anterior or posterior approach reconstruction (Table 2). Fusion was achieved in all patients. The complications were as follows: one patient with internal fixator loose-ness and esophagus fistula; four with titanium mesh sub-sidence; five with temporary recurrent laryngeal nerve injury; and two with superficial infection.

4. Discussion

Traumatic instability of the subaxial cervical spine is a

common injury that often causes severe neurological disability. Although the general therapeutic principles of spinal surgery are followed, namely, to decompress the neural elements, reconstruct or recover normal anatomi-cal alignment of the spine, and acquire immediate stabil-ity, specific therapeutic methods and strategies need to be individually tailored according to general and local pathological situations.

Some authors1,2 believe that cervical dynamic X-ray at an early stage can help overcome the false-positive re-sults of MRI. Although cervical dynamic X-ray is eco-nomic and convenient, patients’ protective spasm and potential neurological risks limit its early use. Therefore, we only used cervical dynamic X-ray under close sur-veillance when MRI could not be practised, or when subaxial cervical spinal instability could not be deter-mined.

Although there are different opinions,3-6 giving pa-tients with cervical cord injury high-dose methylpredni-solone at an early stage can reduce secondary injury to the spinal cord. In addition, we followed the NASCIS5 therapeutic option so as to retain as much function as possible in the spinal cord and nerve roots. Theoretically, quickly decompressing the cervical spinal cord and re-constructing or recovering the normal anatomical align-ment of the cervical spine is beneficial for promoting nerve function recovery, and this has been supported by the results of animal experiments.7,8

However, due to a lack of randomized double-blind and prospective clinical trials, there is still controversy concerning the operation time in patients who have cervical cord injuries.7-9 Be-cause of the common concomitant limb and visceral in-juries, as well as the preoperative diagnosis and prepara-tion periods, we think it is difficult to finish an emer-gency operation within 6–8 hours. Moreover, emergency operations increase blood loss and perioperative compli-cations. Therefore, it is reasonable and feasible to per-form an early, but non-emergency operation on patients with cervical cord injuries, and to adopt the strategy of proper operation time in patients who have subaxial cer-vical spinal injury without neurological disability.

Treatment of subaxial cervical spine fracture-dislocation with concomitant locked facet is still controversial, especially in cases in which there is disc herniation in front of the dislocated cervical cord, but there is normal neurological function or only partial dysfunction.10,11 The greatest risk in closed or posterior unlocking reduction lies in catastrophic deterioration of neurological function if there is major traumatic disc herniation before the op-eration.12

Therefore, decompression, reduction and re-construction of the subaxial cervical spine using an ante-rior approach has been suggested by many authors.13-17

It is believed that this option can reduce neurological

P. CAO ET AL. 27

function deterioration and cervical axial pain induced by the trauma of surgical exposure, and it has the advantage of acquiring fixation levels that are as low as possible.

We are opposed to closed or reduction under general anesthesia, and disagree with the three-stage therapeutic strategy of Vital et al.18

We believe that MRI before treatment has great significance in establishing the spinal cord compression status (level, direction, materials and nature of the compression) and injury to the surrounding soft tissue, in order to determine if the cervical spine is stable, although it is possible to delay the course of re-duction to some extent and increase the potential risks during examination.19 We think that if the patient is in a conscious and cooperative status, a close and dynamic examination of the neurological function grade and cer-vical anatomic realignment can, to the greatest extent, reduce neurological deterioration, and that continued traction can, to the greatest extent, eliminate patients’ protective muscle spasm, which is good for reduction. If neurological function deteriorates during traction- reduc-tion, excess traction of injured levels occurs, severe and refractory neck or upper limb pain appear discontinuing traction, or resistance against the traction weight cannot be maintained during skull traction, we use surgical re-duction and fixation instead of closed traction-reduction.16,20 If closed traction-reduction is successful, we continue skull traction and determine an appropriate time to carry out internal fixation and fusion, so as to finish the operation under the best conditions for both the sur-geon and the patient. The operation approach is deter-mined by the results of MRI before and after reduction. The location of the materials causing compression, the stability status of the injured spinal units, assessment of bone mass and bone structure, and the patient’s general condition and economic status should all be taken into consideration. An anterior approach operation is the first choice, and when necessary, operations using the poste-rior approach, combined approach, or anterior-posterior- anterior approach can be used.

Although there are different methods of classifying traumatic subaxial cervical fracture-dislocation,21-23 we think that the Allen-Ferguson classification system21 is useful for deciding the correct therapeutic strategy. We determined specific operation types according to the fol-lowing criteria: location and extent of the injury, general condition and concomitant injury status (for example, osteoporosis), injury level and number of lesions, and the patient’s economic status. Patients who had injuries to the posterior tension-band of the cervical spinal cord were of relatively old age, and those who had osteoporo-sis or multiple-level cervical injuries needed poste-rior-approach operations; patients with anterior column compression needed anterior-approach decompres-

sion-reconstruction operations; and some patients needed combination approach surgery. Brodke et al24 compared two groups of prospective randomized cases who un-derwent anterior or posterior operations, excluding some patients with decompression or reduction-fixation through special approach. Among our 83 cases, 46 were treated with an anterior approach, 28 with a posterior approach, and 9 with a combined approach. If we ex-cluded those who needed a particular operation approach or surgical sequence, due to general or local pathological conditions, 42 cases who underwent either a single ante-rior or posterior approach reconstruction were included. With the improvement in cervical spine fixation equip-ment, if we exclude those operations performed using a special approach for decompression and reduction-reconstruction, there is not much difference between ante-rior- and posterior-approach operations. However, ante-rior-approach operations have the advantages of fewer posture changes, simple surgical exposure, less traumatic bleeding and fixed levels in the operation, as well as lower occurrence of postoperative cervical axial pain. In addition, with the improvement in surgical skills, we can realize decompression and recovery of normal spinal height and physiological curvature at the same time. However, posterior-approach operations retain their value when certain conditions exist, such as invaginated fracture of lamina or zygapophysial joint, development of cervical canal stenosis, refractory locked-facet dislo-cation or old subaxial cervical injury, multiple-level cer-vical injuries, serious osteoporosis, or poor general health. In addition, although posterior cervical pedicle screws can provide the strongest three-dimensional sup-port, posterior cervical lateral mass screws are still re-garded as the best choice if technical requirements and subsequent surgical risks are taken into consideration. Combined-approach operations are usually performed in cases of serious subaxial cervical spinal instability, those requiring long segment reconstruction, severe osteoporo-sis, serious injury to the cervical-thoracic junction, and some irreversible locked-facet dislocation caused by spinal cord compression, which results from serious an-terior column fracture or disc herniation.10,11

We carried out a functional assessment during fol-

low-up, by adopting the JOA and ASIA neurological assessments and functional grades. Before operation, the average JOA scores and ASIA functional grades were 11.2 (0–17) and 53.5 (0–100), respectively. At the final assessment, they improved to 15.3 (1–17) and 67.8 (11–100), respectively. For incomplete SCI, the average ASIA neurological function scale was improved by 1–2 levels. Patients with complete SCI had no neurological recovery, but their nerve root function recovered to dif-ferent extents.


P. CAO ET AL. 28

The spine trauma study group25 has stated that the un-dermentioned radiological parameters can be used as a follow-up index before and after the following opera-tions: cervical kyphosis (Cobb angle), vertebral body translation, disc height ratio, maximal spinal canal com-promise and spinal cord compression, facet fracture fragment size, and percentage facet subluxation. The average Cobb angle, vertebral body translation, and disc height ratio were +21.8° (+46 to –2.1°), 6.0 mm (poste-rior translation 0.6 mm, anterior translation 8.3 mm), and 71% (38%–89%), respectively, before operation, and –2.9° (+2.4 to –4.1°), 0.35 mm (0–2.2 mm), 96% (82%–145%), respectively, at final follow-up, which indicated that both normal cervical physiological lordosis was recovered and disc height or vertebral body align-ment was reconstructed. Fusion was achieved in all pa-tients and neurological functions were improved at dif-ferent levels, although few cases had internal fixator looseness and esophageal fistula, titanium mesh subsi-dence, and superficial infection. The average VAS grades decreased from 7.8 (1–10) before operation to 2.6 (0–6) after operation.

In conclusion, based on a full application of modern spinal fixation (cervical anterior self-locked plate/ante-rior column supporting structure, posterior lateral mass screws/pedicle screws), as well as imaging techniques and cervical injury classification, the best surgical strat-egy is determined by the subaxial cervical injury type, patients’ general health, local pathological anatomy and neurological function. Individual tailoring of surgical treatment is the key to success.

References

[1] Horn EM, Lekovic GP, Feiz-Erfan E, Sonntag VKH, Theodore

[2] N. Cervical magnetic resonance imaging abnormalities not predictive of cervical spine instability in traumatically injured patients. J Neurosurg (Spine 1) 2004; 1: 39-42

[3] Hadley MH. Radiographic assessment of the cervical spine in symptomatic trauma patients. Neurosurgery 2002; 50: S36-S43

[4] Bracken MD, Shepard MJ, Collins WF, Holford TR, Baskin DS, Eisenberg HM et al. Methylprednisolone or naloxone treatment after acute spinal cord injury: one-year follow-up data. Results of the second national acute spinal cord injury study. J Neurosurg 1992; 76: 23-31.

[5] Bracken MB, Shepard MJ, Holford TR, Summers LL, Aldrich EF, Fazl M, et al. Methylprednisolone or tirilazad mesylate administration after acute spinal cord injury: 1

year follow up. Results of the third national acute spinal cord injury randomised controlled trial. J Neurosurg 1998; 89: 699-706.

[6] Short DJ, Ei Masry WS, Jones PW. High close methyl-prednisolone in the management of accure spinal cord injury: a systematic review from a clinical perspective. Spinal Cord 2000; 38: 273-86.

[7] Timothy J, Towns G, Girn HS. Cervical spine injuries. Curr Orthop 2004; 18: 1-16

[8] Fehlings MG, Sekhon LH, Tator C. The role and timing of decompression in acute spinal cord injury: what do we know? What should we do? Spine 2001; 16: S101-S110.

[9] Fehlings MG, Tator CH. An evidence-based review of decompressive surgery in acute spinal cord injury: ra-tionale, indications, and timing based on experimental and clinical studies. J Neurosurg (Spine 1) 1999; 91: 1-11.

[10] Albert TJ, Kim DH. Timing of surgical stabilization after cervical and thoracic trauma. J Neurosurg (Spine 3) 2005; 3: 182-190.

[11] Wiseman DB, Bellabarba C, Mirza SK, Chapman JR. Anterior versus posterior surgical treatment for traumatic cervical spine dislocation. Curr Opin Orthop 2003; 14: 174-181.

[12] Kwon BK, Beiner J, Grauer JN, Albert TJ. Ante-rior/posterior operative reduction of cervical spine dislo-cation: techniques and literature review. Curr Opin Or-thop 2003; 14:193-199.

[13] Eismont FJ, Arena MJ, Green BA. Extrusion of an in-tervertebral disc associated with traumatic subluxation or dislocation of cervical facets. J Bone Joint Surg Am 1991; 73: 1555-1560.

[14] Tribus CB. Cervical disk herniation in association with traumatic facet dislocation. Tech Orthop 1994; 9: 5-7.

[15] Yuan W, Jia LS, Chen DY, Liu Y, Mao ZG. Surgical treatment of lower cervical fracture and dislocation by anterior approach. Chin J Spine Spinal Cord (Chin) 2001; 11: 23-25.

[16] Ordonez BJ, Benzel EC, Naderi S, Weller SJ. Cervical facet dislocation: techniques for ventral reduction and stabilization. J Neurosurg 2000; 92: 18-23.

[17] Reindl R, Ouellet J, Harvey EJ, Berry G, Arlet V. Ante-rior reduction for cervical spine dislocation. Spine 2006; 31: 648-652.

[18] Payer M, Schmidt MH. Management of traumatic bilat-eral locked facets of the subaxial cervical spine. Contemp Neurosurg 2005; 27: 1-4.

[19] Vital JM, Gille O, Senegas J, Pointillart V. Reduction


P. CAO ET AL.


29

technique for uni- and biarticular dislocations of the lower cervical spine. Spine 1998; 23: 949-954.

[20] Hart RA, Vaccaro AR, Nachwalter RS. Controversies in spine: cervical facet dislocation: when is magnetic reso-nance imaging indicated? Spine 2002; 27: 116-118.

[21] Hadley HN. Initial closed reduction of cervical spine fracture-dislocation injuries. Neurosurgery 2002; 50: S44-S50.

[22] Allen BL, Ferguson RL, Lehmann TR. A mechanistic classification of closed, indirect fractures and dislocations of the lower cervical spine. Spine 1982; 7: 1-28.

[23] Steinmann JG, Anderson PA. Subaxial cervical spine

fractures with internal fixation: The posterior approach. Semin Spine Surg 1996; 8: 35-48.

[24] Brodke DS, Harris M. Subaxial cervical trauma: Evalua-tion and management options. Semin Spine Surg 2001; 13: 128-141.

[25] Brodke DS, Anderson PA, Newell DW, Grady MS, Chapman JR. Comparison of anterior and posterior ap-proaches in cervical spinal cord injuries. J Spinal Disord Tech 2003; 16: 229-235.

[26] Bono CM, Vaccaro AR, Fehlings M, Fisher C, Dvorak M, Ludwig S, et al. Measurement techniques for lower cer-vical spine injuries. Spine 2006; 31: 603-609.



Original Article Inhibitory Effect of Ginsenoside Rg3 on Ovarian Cancer Metastasis

ABSTRACT

Ginsenosides are main components extracted from ginseng, and ginsenoside Rg3 is one of the most important parts. Ginsenoside Rg3 has been found to inhibit several kinds of tumor growth and metastasis. The present study was under-taken to investigate the effect of ginsenoside Rg3 on human ovarian cancer metastasis and the possible mechanism. The experimental lung metastasis models of ovarian cancer SKOV-3 and the assay of tumor-induced angiogenesis were used to observe the inhibitory effects of Rg3 on tumor metastasis and angiogenesis. The effect of Rg3 on invasive ability of SKOV-3 cells in vitro was detected by Boyden chamber, and immunofluorescence staining was used to recognize the expression of matrix metalloproteinase 9 (MMP-9) in SKOV-3 cells. Ginsenoside Rg3 can significantly inhibit the me-tastasis of ovarian cancer. The inhibitory effect is partially due to inhibition of tumor-induced angiogenesis and de-crease of invasive ability and MMP-9 expression of SKOV-3 cells. Keywords: ginseng, neoplasm, metastasis, angiogenesis, ovarian cancer

1. Introduction

Ginseng is a medicinal herb widely used in Asian coun-tries for its wide spectrum of medicinal effects such as tonic, immunomodulatory, adaptogenic, and anti-aging activities.1These effects are attributed to the triterpene glycosides known as ginsenosides and Rg3 is one kind of the ginsenosides. The molecular formula of ginsenoside Rg3 is C42H72O13 and its molecular weight is 784.30. Ginsenoside Rg3 can inhibit catecholamine secretion, protect cultured cortical cells from glutamate-induced neurodegeneration, and anti-contraction of vascular smooth muscle.2-4 Researchers found that ginsenoside Rg3 can also resist tumor. For example, it inhibited inva-sion and metastasis of B16 melenoma without impairing cell growth, and proliferation of tumor cells combined with cyclophosphamide.5-7 However, the effect of ginse-noside Rg3 on human ovarian cancer has not been iden-tified and the mechanism of its anti-tumor is unknown.

The present study was to assess the effects of ginse-noside Rg3 on angiogenesis and metastasis produced by human ovarian cancer and on the invasion and the matrix

metalloproteinase 9 (MMP-9) expression of SKOV-3 cells in vitro.

2. Methods

2.1. Drug and Reagents

Ginsenoside Rg3 was provided by Department of Or-ganic Chemistry of Preclinical Medicine of Jilin Univer-sity. Its purity is more than 99.5%. Athymic mice were purchased from the Department of Experimental Ani-mals of Jilin University. Boyden chamber and matrigel were purchased from BD Company, USA.

2.2. Cell Culture

Human ovarian cancer SKOV-3 cell line and mouse NIH3T3 fibroblast cell were obtained from the Tumor Research Department of Jilin Province and maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS).

T. M. XU ET AL. 31

Table 1. Inhibitory effect of ginsenoside Rg3 on experimental lung metastasis and angiogenesis caused by ovarian cancer

*P <0.01, **P <0.001 compared with control group

2.3. Assay for Experimental Lung Metastasis

In 40 mice, each was injected with SKOV-3 cells (2×105) into the lateral tail vein. The mice were randomly di-vided into 4 groups (n=10 for each group): Rg3 groups (0.3, 1.0 and 3.0 mg/kg) and control group. Ginsenoside Rg3 was injected intraperitoneally at daily doses (0.3, 1.0 and 3.0 mg/kg) to the tumor-bearing mice the next day after tumor inoculation. The mice were killed 20 days after the inoculation. The lungs were fixed in Bouin’s solution and the lung tumor colonies were counted under a dissecting microscope.

2.4. Cell Invasion Assay

The invasive activity of SKOV-3 cells was assayed in Boyden chambers. The upper surface of the filters in Boyden chambers was precoated with 60 µl of matrigel. The SKOV-3 cells were harvested, washed 3 times and re-suspended to a final concentration of 1×106/ml in RPMI1640. Then the cells were divided into 3 groups after pretreatment with ginsenoside Rg3 of different concentrations (0, 2.5 and 5.0 µg/ml) at 37°C for 30 minutes. 200 µl of each cell suspension was added to the upper compartment of the chamber and 200 µl of condi-tioned medium of NIH3T3 cells to the lower compart-ment. After 5-hour incubation, the cells on the upper surface of the filters were removed by wiping with cot-ton swabs and the filters were fixed with FAA solution and stained with hematoxylin and eosin. The cells that had invaded through the matrigel and filters into the lower surface were manually counted under a micro-scope at a magnification of ×400. Each assay was per-formed in triplicate. The data were expressed as the number of invaded cells/field.

2.5. Assay for Tumor-induced Angiogenesis

In 20 athymic mice, each was inoculated intradermally with SKOV-3 cells (5×105) on the back. They were ran-domly divided into 4 groups (n=5): Rg3 groups (0.3,

1.0 and 3.0 mg/kg) and control group. Ginsenoside Rg3 at various doses was injected intraperitoneally to the mice 1, 2, 3, 4 and 5 days after tumor inoculation. Two days later, the mice were sacrificed and the skin was separated from the underlying tissues. Angiogenesis was

quantified by counting the number of vessels oriented toward the tumor mass under a dissecting microscope.

2.6. MMP-9 Immunofluorescence Studies

The SKOV-3 cells that were seeded onto 24-well plates and treated with 0, 2.5 and 5.0 µg/ml of ginsenoside Rg3 for 24 hours were used for immunofluoresecent staining. First, the cells were washed briefly with phosphate buff-ered saline three times and then fixed in 4% phosphate buffered paraformaldehyde for 30 minutes. Second, they underwent permabilization with the addition of phos-phate-buffered 0.1% Triton X-100 for 10 minutes and then were incubated in 1% bovine serum albumin for 30 minutes all at room temperature. Third, the cells were incubated with mouse anti-human MMP-9 monoclonal antibody (1: 100 dilution) and then with goat anti-mouse fluorescein isothiocyanate (FITC) antibody respectively for 1 hour at 4°C. Finally, the coverslips were mounted and sealed for examination under a confocal microscope.


Measurement data were evaluated by one-way analysis of variance (ANOVA) for multiple group comparisons, and the LSD test for two-group comparisons. Ranked data were evaluated by the Kruskal-Wallis test for multi-ple group comparisons and the Nemenyi test for two-group comparisons. P <0.05 was considered statis-tically significant.

3. Results

3.1. Effect of Ginsenoside Rg3 on Experimental lung Metastasis of SKOV-3 cells

As shown in Figure 1, after intraperitoneal injection of ginsenoside Rg3 at the doses of 0.3, 1.0 and 3.0 mg/kg, the number of tumor colonies in the lung was lower than that of the control group (P <0.01). The results indicate that ginsenoside Rg3 can significantly inhibit the lung metastasis of SKOV-3 cells.

3.2. Inhibitory Effect of Ginsenoside Rg3 on Tumor-induced Angiogenesis

The number of vessels oriented toward the tumor mass in the three groups that the tumor-bearing mice were given


T. M. XU ET AL. 32

intraperitoneal injection of ginsenoside Rg3 at the doses of 0.3, 1.0 and 3.0 mg/kg was less than that of the control group (Table). This indicates that ginsenoside Rg3 may inhibit ovarian tumor-induced angiogenesis.

3.3. Cell invasion Assay

After the SKOV-3 cells were treated with ginsenoside Rg3 at three concentrations of 0, 2.5 and 5.0 µg/ml, the number of the cells invading through matrigel and filters into the lower surface was 157.3±29.4, 110.8±25.6 and 92.5±18.4 respectively. Among the three groups, the group of 0 µg/ml ginsenoside Rg3 was control group. Statistical analysis illustrated that the number of cells invading filters of the ginsenoside groups was less than

that of the control group (P <0.001, Figure 1), and that ginsenoside Rg3 can depress the ability of invading of SKOV-3 cells.

3.4. Assay of MMP-9 Immunofluorescence

The SKOV-3 cells were pretreated with ginsenoside Rg3 at doses of 2.5 and 5.0 µg/ml for 24 hours and im-munofluorescence staining was used to recognize the expression of MMP-9 in these tumor cells. Figure 2 shows that cytoplasmic fluorescence intensity in the SKOV-3 cells was depressed as the dose of ginsenoside Rg3 increased. This result shows that ginsenoside Rg3 can depress the expression of MMP-9 in the SKOV-3 cells.

Figure 1. Inhibitory effect of ginsenoside Rg3 on the invasion of SKOV-3 cells. The SKOV-3 cells were pretreated with gin-senoside Rg3 at doses of 2.5 and 5.0 µg/ml for 30 minutes and then were added to Boyden chambers. After 5-hour incubation, the cells that had invaded through the matrigel and filters were manually counted. A: control group; B: 5.0 µg/ml ginsenoside Rg3 group (HE, original magnification ×400)


T. M. XU ET AL. 33

Figure 2. Immunofluorescence staining of MMP-9 antibody in SKOV-3 cells. The SKOV-3 cells were pretreated with ginse-noside Rg3 at doses of 2.5 and 5.0 µg/ml for 24 hours and immunofluorescence staining was used to recognize the expression of MMP-9. A: control group; B: 2.5 µg/ml ginsenoside Rg3 group; C: 5.0 µg/ml ginsenoside Rg3 group (original magnifica-tion ×400)


T. M. XU ET AL.


34

4. Discussion

Ovarian cancer is a severe disease threatening the health and life of women. The 5-year survival rate of patients with such disease remains low after conventional treat-ment.8 Thus it is necessary to develop new agents and methods for a cure.

With the development of Chinese herbal medicine, researchers are increasingly interested in detecting anti-tumor components in Chinese herbal medicine. Ginsenoside Rg3, a saponin extracted from ginseng, was found to have a high anti-tumor effect. It inhibited an-giogenesis of Lewis lung carcinoma,9 invasion and me-tastasis of intestinal adenocarcinoma and B16 melenoma,5,6 and proliferation of prostate cancer cell.10,11

The present study demonstrated that ginsenoside Rg3 can also inhibit the metastasis caused by ovarian cancer, which will benefit patients with ovarian cancer.

The ability to invade tissues and establish colonies at remote sites is a definite characteristic of malignant neo-plasms. As a complex cascade it is affected by many factors. Angiogenesis is a key step in tumor invasion and metastasis.12 Since massive formation of blood vessels at the tumor site increases the opportunity for tumor cells to enter the circulation, the suppression of angiogenesis will decrease the metastasis of malignant tumor. In this study, the number of vessels oriented toward the tumor mass after intraperitoneal injection of ginsenoside Rg3 at the doses of 0.3, 1.0 and 3.0 mg/kg was less than that of the untreated group, indicating that ginsenoside Rg3 may inhibit ovarian tumor-induced angiogenesis, and subse-quently decrease the metastasis of ovarian cancer.

In addition, tumor invasion and adhesion to an ex-tracellular matrix and basement membrane components are important events in the process of tumor metastasis. In this study, matrigel, an analogue of basement mem-brane, was used to determine the effect of ginsenoside Rg3 on invasion of ovarian cancer cells in vitro. Because the tumor cells adhere to matrigel, then invade and cross the matrigel, we can evaluate the invasion of tumor cells by the number of cells passing the matrigel. We observed that the number of the SKOV-3 cells invading through the matrigel and filters into the lower surface decreased after they were treated with ginsenoside Rg3. This indi-cates that ginsenoside Rg3 can prevent the invasion of ovarian cancer cells.

However, angiogenesis as well as tumor invasion and metastasis depend heavily on the controlled interactions between the cells and the extracellular matrix (ECM).13

These interactions are mediated by integral membrane proteins and extracellular proteinases. Extracellular pro-teolysis plays an important role in many aspects, includ-ing basement degradation and cell migration/ extracellu-

lar matric invasion, and is mediated by metallopro-teinases (MMPs) and serine proteinases.14,15

MMPs belong to a family of zinc-dependent endopep-tides.

They secrete as inactive pro-enzymes and are acti-vated by partial proteolytic cleavage.16 MMP-9 or the dominant MMPs released by most endothelial cells and tumor cells appear to play an important role in degrada-tion of basement membrane type VI collage and other matrixproteins.17-19 Therefore, MMP-9 is regarded as marker of tumor invasion and metastasis, and the sup-pression of its expression may inhibit malignant tumor invasion and metastasis.20 The results of the present study demonstrated that ginsenoside Rg3 depressed the secretion of ovarian cancer cells, which may be the channel by which ginsenoside Rg3 inhibits the metasta-sis of ovarian cancer.

In conclusion, ginsenoside Rg3 can significantly in-hibit the metastasis of ovarian cancer. The inhibitory effect is partially due to the inhibition of tumor-induced angiogenesis and decrease of the invasive ability, which may be related to the depression of MMP-9 expression of ovarian cancer cells.

References

[1] Zhang Y, Takashina K, Saito H, Nishyama N. Anti-aging effect of DX-9386 in senescence accelerated mouse. Biol Pharm Bull 1994; 17: 866-868.

[2] Tachikawa E, Kudo K, Nunokawa M, Kashimoto T, Ta-kahashi E, Kitagawa S. Characterization of ginseng saponin ginsenoside-Rg3 inhibition of catacholamine se-cretion in bovine adrenal chromaffin cells. Biochem Pharm 2001; 62: 943-951.

[3] Kim YC, Kim SR, Markelonis GJ, Oh TH. Ginsenoside Rb1 and Rg3 protect cultured rat cortical cells from glu-tamate-induced neurodegeneration. J Neurosci Res 1998; 53: 426-432.

[4] Kim ND, Kang SY, Kim MJ, Park JH, Schini-Kerth VB. The ginsenoside Rg3 evokes endothelium-independent relaxation in rat aortic ring: role of K

+ channels. Eur J

Pharmacol 1999; 367: 51-57. [5] Mochizuki M, Yoo YC, Matsuzawa K, Sato K, Saiki I,

Tono-oka S, et al. Inhibitory effect of tumor metastasis in mice by saponin, ginsenoside-Rb2, 20(R)- and 20(S)-ginsenoside-Rg3, of red ginseng. Biol Pharm Bull 1995; 18: 1197-1202.

[6] Xu TM, Xin Y, Cui MH, Jang X, Gu LP. Inhibitory effect of ginsenoside Rg3 combined with cyclophosphamide on growth and angiogenesis of ovarian cancer. Chin Med J 2007; 120: 584-588.

[7] Zhang Q, Kang X, Zhao W. Antiangiogenic effect of

T. M. XU ET AL. 35

low-dose cyclophosphamide combined with ginsenoside Rg3 on Lewis lung carcinoma. Biochem Biophys Res Commun 2006; 342: 824-828.

[8] Workman P, Johnston PG. Genomic profiling of cancer: what next? J Clin Oncology 2005; 23: 7253-7256.

[9] Zhang Q, Kang X, Zhao W. Antiangiogenic effect of low-dose cyclophosphamide combined with ginsenoside Rg3 on Lewis lung carcinoma. Biochen Biophys Res Commun 2006; 342: 824-828.

[10] Kim HS, Lee EH, Ko SR, Choi KJ, Park JH, Im DS. Ef-fects of ginsenosides Rg3 and Rh2 on the proliferation of prostate cancer cells. Arch Pharm Res 2004; 27: 429-435.

[11] Keum YS, Han SS, Chun Ks, Park KK, Park JH, Lee SK, et al. Inhibitory effects of the ginsenoside Rg3 on phorbol ester-induced cyclooxygenase-2 expression, NF-kappaB activation and tumor promotion. Mutat Res 2003; 523-524: 75-85.

[12] Kumar S, Witzig TE, Timm M, Haug J, Wellik L, Fonsecs R, et al. Expression of VEGF and its receptors by myeloma cells. Leukemia 2003; 17: 2025-2031.

[13] Bussolino F, Mantovani A, Persico G. Molecular mecha-nisms of blood vessel formation. Trends Biochem Sci 1997; 22: 251-256.

[14] Pepper MS, Montesano E, Mandriota SJ, Orci L, Vassalli JD. Angiogenesis: a paradigm for balanced extracellular

proteolysis during cell migration and morphogenesis. Enzyme Protein 1996; 49: 138-162.

[15] Pepper MS. Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol 2001; 21: 1104-1117.

[16] Nagase H, Woessner JF Jr. Matrix metalloproteinases. J Biol Chem 1999; 274: 21491-21494.

[17] Zucker S, Mirza H, Conner CE, Lorenz AF, Drews MH, Bahou WF, et al. Vascular endothelial growth factor in-duces tissue factor and matrix metalloproteinase produc-tion in endothelial cells: Conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 1998; 75: 780-786.

[18] Fisher C, Gilbertson-Beadling S, Powers EA, Petzold G, Poorman R, Mitchell MA. Interstitial collagenase is re-quired for angiogenesis in vitro. Dev Biol 1994; 162: 499-511.

[19] Zucker S, Conner C, DiMassimo BI, Ende H, Drews M, Seiki M, et al. Thrombin induces the activation of proge-latinase A in vascular endothelial cells: physiologic regu-lation of angiogenesis. J Biol Chem 1995; 270: 23730-23738.

[20] Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer me-tastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 1991; 64: 327-336.




Association of CALCA Genetic Polymorphism with Essential Hypertension

ABSTRACT

Calcitonin gene-related peptide (CGRP) is the predominant neurotransmitter in capsaicin-sensitive sensory nerves. Participation of CGRP in hypertension is one of the most extensively studied topics in the field. There is growing evi-dence to the effect that CGRP is associated with essential hypertension (EH). The aims of this study were to pinpoint whether single nucleotide polymorphisms (SNPs) in the genes coding for CALCA were associated with EH susceptibil-ity in a Hunan Han population. A total of 293 subjects with EH and 208 controls were enrolled in the study. Genomic DNA was extracted from peripheral blood leucocytes by a phenol-chloroform method. The CALCA T-692C was geno-typed using a restriction fragment length polymorphism method. CALCA genetic polymorphism is associated with EH susceptibility. Carriers of at least one C allele at the polymorphic site CALCA T-692C showed increased risk for EH. Keywords: hypertension, calcitonin gene-related peptide, single nucleotide polymorphism

1. Introduction

Epidemiologic studies have reported that essential hy-pertension (EH) is prevalent, affecting approx 20%–25% of the adult population in the world, becoming a major worldwide public health issue.1,2 Hypertension can in-crease the risk of coronary heart disease, stroke and con-gestive heart failure. EH is considered to be a multifacto-rial, complex disease. Family and twin studies have shown that about 80% EH is determined by genetic fac-tors interacting with environmental factors.3-6 Human calcitonin gene-related peptide (CGRP), a very potent vasodilating neuropeptide, includes alpha-CGRP (CALCA) and beta-CGRP (CALCB). The growing evi-dence from animal and human studies indicates that CGRP may play an important role in the initiation, pro-gression and maintenance of hypertension via the altera-tions in its synthesis and release.

The human CALCA gene is located on chromosome 11p15.2-p15.1 and codes for both calcitonin and al-pha-CGRP through alternative RNA splicing of gene transcripts, which spans approximately 3.8 kb, and con-tains five exons. Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation, and they may contribute to the individual sus-ceptibility to EH. Many studies have demonstrated that

SNPs in genes may not only affect the expression or ac-tivities of the enzymes or proteins but are associated with the risk of EH; such as the genes for

angiotensinogen,7 endothelial NO synthase8 and CYP3A5.9 The aims of this study were to discover a polymorphism in the 5’ flanking region of the CALCA gene in Chinese subjects and to assess the association between this gene and EH. To test this hypothesis, we performed genotyping analyses for the CALCA T-692C polymorphism in a population-based case-control study of the Chinese Han population.

2. Methods

2.1. Clinical Materials

From December 2005 to August 2006, eligible patients for this population-based case-control study of EH were recruited at our community program of EH prevention and cure in Hunan province. They were genetically un-related ethnic Han Chinese and were from Hunan

province in middle China. There were 293 EH patients (male/female ratio=1.53). EH was diagnosed based on the following criteria: seated systolic blood pressure (BP) ≥140 mmHg or diastolic BP ≥90 mmHg on three occa-

X. L. LUO ET AL. 37

sions within 2 months after the first BP reading. Patients diagnosed as having secondary hypertension were ex-cluded. A total of 208 normotensive age-matched healthy individuals (male/female ratio =1.70) served as control subjects. All control subjects had systolic BP <140 mmHg and diastolic BP <90 mmHg. The study was ap-proved by the ethnic committee of Xiangya Hosptial.

2.2. Genetic Studies

Based on the information obtained from the NCBI SNP database, we chose a SNP CALCA T-692C that had a minor allele frequency of more than 10%. Blood samples were collected from all participants and genomic DNA was extracted from the peripheral blood leukocyte pellet by the standard method with proteinase K digestion and followed by phenol-chloroform extraction and ethanol precipitation. The CALCA genotypes were detected by the polymerase chain reaction (PCR) restriction fragment length polymorphism assay with primers of 5’CG CATCTGTACCTTGCAACT3’ (forward) and 5’TCAA ATT CCCGCTCACTTTA3’ (reverse). The 20 μl PCR mixture consisted of approximately 100 ng of genomic DNA, 40 pmol of each primer, 40 pmol dNTP, 2 μl 10×PCR buffer, and 2 U Taq DNA polymerase (Sangon, China). The PCR profile consisted of an initial melting step of 94 ℃ for 5 minutes, 38 cycles of 94°C for 50 seconds, 57 ℃ for 50 seconds and 72°C for 1 minute, and a final extension step of 72°C for 10 minutes. The 636 bp PCR product was then digested overnight by restriction enzyme of 1 U PshA I (TaKaRa, Japan). The -692TT genotype produced a single 636 bp fragment, the -692CC genotype produced two fragments of 235 bp and 401 bp, and the heterozygous -692CT genotype produced three fragments of 636 bp, 235 bp and 401 bp. Genotyping was performed without knowing the case/control status of the subjects. Twelve DNA samples of EH cases were randomly selected to have sequencing analysis and the

results were 100% concordant with the restriction frag-ment length polymorphism (RFLP) results.


All continuous variables were expressed as mean ± stan-dard deviation. Differences in selected demographic characteristics, selected variables and frequencies of the genotypes and alleles of CALCA between the cases and controls were evaluated using the chi-square test and/or Student’s t test. The association of CALCA genotypes with EH risk were estimated by computing the odds ra-tios (ORs) and their 95% CIs from multiple Logistic re-gression analyses with adjustment for gender, age, smoking history, dyslipoproteinemia, family history of premature cardiovascular disease and obesity. The Hardy-Weinberg equilibrium was tested by a chi-square test to compare the observed genotype frequencies with the expected frequency among the control subjects. Stratification analysis was performed according to dif-ferent groups of selected variants. All the statistical analyses were performed with Statistical Package for the Social Science (SPSS) 15.0. All the P values were two sided and with the significant levels of P <0.05.

3. Results

Human CGRP includes two subtypes, alpha- and beta-CGRP (CALCA and CALCB), and CALCB shows high sequence homology with CALCA. In the National Center for Biotechnology Information (NCBI) single nucleotide polymorphism (SNP) database (http://www. ncbi.nlm.nih.gov/projects/SNP/), 34 Chinese SNPs at CALCA (NT_009237，REGION: complement (137672 88.13791073)) and CALCB (NT_009237, REGION: complement (13872387.. 13897418)) have been recorded, as shown in Tables 1 and 2.

Table 1. Eleven SNPs loci of Chinese CALCA gene

*The SNP rs number in dbSNP database. #The SNP locus of CALCA gene in this research


X. L. LUO ET AL. 38

Table 2. Twenty-three SNPs loci of Chinese CALCB gene

The characteristics of the 293 EH cases and 208 con-trols included in the study were summarized in Table 3. The mean age was 50 years (±12 years) for cases and 49 years (±11 years) for controls, which was adequately matched on age as suggested by the Student’s t test (P=0.560). Compared with the control subjects, the EH cases had a significantly higher family history of EH (P <0.001), and had a significantly higher serum TBIL (P <0.001) and BS (P=0. 215). Moreover, as shown in Ta-ble 3, serum HDL was significantly lower in EH cases (P <0.001).

The observed genotypes for this polymorphism among the control subjects were both in Hardy-Weinberg equi-librium. The genotype distributions of CALCA T-692C in the cases and controls and their association with EH are shown in Table 4. For the CALCA T-692C poly-morphism the genotype frequencies of TT, TC and CC were 85.1%, 14.9% and 0 in the controls, respectively, which were significantly different from the EH cases (71.7% TT , 26.9% TC and 1.4% CC) (χ2=13.734,

P=0.001). Logistic regression analyses revealed that compared with the TT genotype, the adjusted OR of EH for subjects carrying TC and combined genotypes TC +CC were 1.942 (95% CI: 1.216–3.103) and 2.093 (95% CI: 1.317–3.326), respectively. The frequencies of the C allele was 14.85% in EH group, and 7.45% in control group, the prevalence of C alleles in the EH subjects and controls were significantly incomparable (P <0.001). In addition, the risk of EH associated with premature car-diovascular disease (adjusted OR=9.114, 95% CI: 2.105–39.455, P=0.003).

4. Discussion

It is generally believed that the neuropeptide CGRP plays an integral role in the pathophysiology of hyper-tension. In experimental animal models, strong evidence supports the hypothesis that reduction both in alpha- and beta-CGRP could contribute to blood pressure eleva-tion.10 Up to now, differences between normal and hy-pertensive patients in the concentration of plasma CGRP


X. L. LUO ET AL. 39

Table 3. Distributions of select variables in EH cases and controls

BMI: body mass index, TBIL: total bilirubin, BUN: blood urea nitrogen, Cr: creatinine, BS: blood glucose, TC: total cholesterol, TG: triglyceride, HDL: high-density lipoprotein, LDL-C: low-density lipoprotein cholesterol

Table 4. Frequency distributions of the CALCA T-692C polymorphism among

the EH cases and controls and their association with EH

*Adjusted by gender, age, smoking history, dyslipoproteinemia, family history of premature cardiovascular disease and obesity. Frequency distributions of TT, TC, CC and C alleles between control and EH groups were found significantly different (χ2=23.434, P <0.001)

are uncertain. Previous studies have shown that plasma CGRP concentrations were significantly lower in hyper-tensive patients and preeclamptic pregnancies than in normotensive controls.11,12 The present study demon-strates that calcium intake and anti-hypertension drugs enhance the release or response of endogenous CGRP, resulting in a decrease in blood pressure.13 In addition, intra-arterial CGRP infusions have been reported to in-duce vasodilation in a dose-dependent manner in the human forearm, increase forearm blood flow ratio and lower blood pressure.14,15 These results are consistent

with the hypothesis that the decrease in CGRP synthesis and release contributes to elevated blood pressure. In contrast, other studies have shown increased plasma CGRP levels in patients with hypertension and shown a significant positive correlation between systolic and dia-stolic blood pressure.16 The increase in CGRP levels, or the enhancement of vascular sensitivity response to CGRP, plays a beneficial compensatory depressor role in the development of hypertension. Some functional stud-ies reported that CALCA gene knockout accelerated hy-pertension-induced heart and kidney damage.17 The


X. L. LUO ET AL. 40

systolic blood pressure of CALCA gene knockout mice has been reported to be significantly elevated.18 Re-cently, CALCA genetic polymorphisms show a possible association with Parkinson’s disease, ovarian cancer and bone mineral density, suggesting that polymorphisms in the CALCA gene have functional significance.19-21 Therefore, we reasoned that polymorphisms of CALCA genes may contribute to EH susceptibility in the general population. However, there has been only one report dealing with the relationship between the CALCA gene variants and EH. In a study in Japanese a novel 2-bp mi-crodeletion polymorphism was discovered in intron 1 of the CALCA and showed that the CALCA gene could be the susceptibility gene of EH.22 In our population-based case-control study of EH, we investigated, for the first time, the association of SNP (CALCA T-692C) with the risk of EH in a Han population in Hunan province of middle China. We found that CALCA T-692C was asso-ciated with EH risk, suggesting that this variant may play a role in the etiology of EH. However, these results are preliminary because of the limited sample size in the subgroup populations and need for validation in a further larger studies. Although the main findings of current study were positive and encouraging, several limitations of this study need to be addressed. First, we can not rule out the possibility that other, as yet unidentified, altera-tions in genes involved in cell cycle and DNA replication influence the risk of developing EH. In addition, because of the populationbased design of the study, these results may not be generalizable to the general population. Ge-netic polymorphisms often vary between ethnic groups. Furthermore, the sample size of the EH cases was not large enough to detect a small effect from very low penetrance genes or SNPs. In conclusion, our study pro-vides evidence for association of the CALCA T-692C variant genotypes with risk of EH in this study popula-tion. A large prospective study is needed to verify our findings, particularly in subjects with a family history of EH.

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[18] Gangula PR, Zhao H, Supowit SC, Wimalawansa SJ, Dipette DJ, Westlund KN, et al. Increased blood pressure in alpha-calcitonin gene-related peptide/calcitonin gene knockout mice. Hypertension 2000; 35: 470-475.

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