Comparison of cardioprotective effects using ramipril and DanShen for the treatment of acute myocardial infarction in rats

Life Sciences 73 (2003) 1413 – 1426 www.elsevier.com/locate/lifescie

Comparison of cardioprotective effects using ramipril and DanShen for the treatment of acute myocardial infarction in rats XinYan Ji a, Benny K.-H. Tan a, Yi Chun Zhu b, Wolfgang Linz c, Yi Zhun Zhu a,* a

b

Department of Pharmacology, National University of Singapore, Singapore The Key Laboratory of Molecular Medicine and Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, China c Disease Group, Cardiovascular Research, Aventis Pharma AG, Frankfurt, Germany Received 9 September 2002; accepted 26 February 2003

Abstract In the present study, we compared cardioprotective effects of DanShen (an extract from Salvia miltiorrhiza) and the angiotensin-converting enzyme inhibitor, ramipril, in rats. With both treatment regimens, DanShen- and ramipril similar effects were observed: (1) a higher survival rate, (2) a significant reduction of infarct size, (3) significantly lower ratios of heart weight to the body weight as well as the left and right ventricular weights to body weight. DanShen showed some unique effects in the following aspects: (1) higher activities of antioxidant defense enzymes such as superoxide dismutase (SOD), catalase (CAT), glutatione perioxidase (GSH-Px) and glutathione S-transferase (GST) in the liver of rats with acute myocardial infarction (AMI), (2) lower myocardial and hepatic TBARS values; (3) augmented VEGF mRNA expressions in the non-ischemic parts of rat hearts with AMI. These results were consistent with the findings of a slight increase in myocardial capillary density and the special distribution pattern of coronary blood vessels in DanShen-treated rats. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Salvia miltiorrhiza; DanShen; Ramipril; Acute myocardial infarction; Antioxidants; Infarct size

* Corresponding author. Department of Pharmacology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore. Tel.: +65-6-874-3676; fax: +65-6-873-7690. E-mail address: [email protected] (Y.Z. Zhu). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00432-6

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Introduction Salvia miltiorrhiza (DanShen), a popular Chinese herb, has been widely and successfully used mainly for angina pectoris, myocardial infarction (MI) and stroke (Ji et al., 2000). It has revolutionized the management of these diseases in Chinese societies. Ramipril, an angiotensin converting enzyme (ACE) inhibitor, has become an important medicine in prevention of cardiovascular diseases in developed countries (Zhu et al., 1998). ACE inhibitors have so far proved to be the most successful class of drugs in the treatment of left ventricular heart failure (LVF), reducing post-MI size, especially left ventricular volume, often increases and this is associated with deteriorating myocardial function and poor prognosis. Experimental evidence has indicated that this harmful remodeling could be inhibited by ACE inhibitors, which has led to great interest in their therapeutic potential (Stauss et al., 1994; Scholkens and Linz, 1990). Experimental studies showed that ACE inhibitors administered chronically before AMI might limit myocardial infarct size, improve cardiac function, and prevent cardiac hypertrophy (Zhu et al., 1998; Scholkens and Linz, 1990). Clinical studies have shown that ACE inhibitors reduce mortalities and improve symptoms and long-term outcome of AMI (AIRE Study Investigators, 1993; HOPE study Investigators, 2000). Compared to ramipril, there is very limited biochemical information available to demonstrate the mechanisms of DanShen’s cardio-protective effects. Herbal medicines like DanShen (Ji et al., 2000; Thomas, 2000), ginkgo biloba, garlic, SanQi (Panax notoginseng) (Dan and Andrew, 1993) and many others play an important role in the management of AMI and its complications. Among these herbs, DanShen is the most common herb used for preventing and treating angina pectoris and MI in Chinese societies (Ji et al., 2000). DanShen has at least 4 beneficial effects in angina or MI patients including sedative, antioxidant, and platelet effect and improved coronary microcirculation without significant known side effects. With regard to gene expression effects, no studies using treatment with DanShen have been done to investigate on its effects on myocardial vascular endothelium growth factor (VEGF). Whether the cardio-protective effects of DanShen are mediated by effects on or via the ReninAngiotensin System (RAS), including ACE, angiotensin receptor 1 (AT1) and receptor 2 (AT2) are also not known. Furthermore, there is no literature reporting comparative studies on the cardio-protective effects of DanShen and ramipril. In this study, we investigate the biochemical and molecular mechanisms of DanShen’s cardio-protective effects and compare such effects of with those of ramipril in rats with AMI.

Materials and methods Animals and treatment procedure Eighty-four Wistar rats were divided into 3 treatment groups and 1 sham group randomly as shown in Table 1. All the animals were housed under diurnal lighting conditions and allowed food and water ad libitum. For accuracy, the whole procedure was single blinded. The drugs were administered intra-peritoneally once daily. After one week of treatment, the rats underwent a permanent ligation of the left anterior descending coronary artery (LAD). This is a widely used procedure to induce AMI in animals (Stauss et al., 1994; Zhu et al., 2001). Treatment was continued for another 2 weeks after the surgery. At the end of

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Table 1 Groups of experiment rats Treatments (each n = 12)

Dose

Sham-operated DanShen Saline Ramipril

0.675 g/kg/day 6.75 ml/kg/day 1 mg/kg/day

the treatment period, all rats were sacrificed by decapitation. Hearts and livers were collected for further studies including antioxidant assays, morphological examination, coronary capillary density and molecular studies. Hepatic antioxidant assays SOD activity was determined by an improved method of Marklund and Marklund (1974); CAT was assayed by the amended method of Aebi (1984). For the assay of GSH-Px activity, it was determined by the amended method of Beutler (1984). GST was assayed by the method of Habig et al. (1974). Thiobarbituric acid-reactive substances (TBARs) in the heart and the liver of rats with AMI TBARs are the secondary products of lipid peroxidation. These secondary products are mainly aldehydes, the major compound being malondialdehyde (MDA). The determination of MDA content in hearts and livers was performed by the thiobarbituric acid (TBA) method of Mehta and Pepine (1978), which has been widely adopted as a sensitive method for assaying lipid peroxides in animal tissues (Ohkawa et al., 1979). Morphological examination of rat hearts with AMI The hearts collected from rats with AMI were stained by Tetrazolium-Blue and kept in 4% phosphate buffered paraformaldehyde for morphological examination. Myocardial infarction was distinguished by the different color tone, white for ischaemic myocardium and dark red for non-ischaemic myocardium. After weighing, the hearts were dissected into left and right ventricles, and the weight of each ventricle measured (the septum was included with the left ventricle). The infarcted area was judged from both epicardial and endocardial sides and outlined on paper, cut and weighed. The sizes of the left ventricle and the infarct area were evaluated by computer using Scion Image (Scion Inc., California, USA). The ratios of the total heart weight to body weight, the right ventricular weight to body weight and the infarcted area weight to heart weight were calculated. The infarct size was expressed as a proportion of the left ventricular size (Stauss et al., 1994). Coronary capillary density study on rat hearts with AMI The hearts were removed from rats with AMI, dipped in M-1 Embedding Matrix For Frozen Sectioning (LIP’SHAW, U.S.A), frozen in liquid nitrogen and stored at 80 jC.

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Using a Leica CM1800 Cryostat (Germany), myocardial sections were cut from the middle part of the frozen heart at 16 Am. Six to eight sections, each 80 Am apart, were obtained from each heart. HE staining of sections was performed to differentiate coronary capillaries, after which they were covered with crystal mount (Biomeda, CA, USA). Myocardial sections were examined under a microscope (ZEISS AX10SKOP-H, USA) (Xie et al., 1997). The process of RNA isolation Each of the hearts was separated into 3 parts: left, and right ventricles and intraventricular septum immediately after removal from rats. The same parts of 6 hearts from each group were mixed together as one sample for RNA isolation. In total, 12 final samples were obtained from the 3 treatment and sham groups. These samples were sham, saline-, DanShen- and ramipril-treated left ventricular samples; sham, saline-, DanShen- and ramipril-treated right ventricular samples; sham, saline-, DanShen- and ramipriltreated intraventricular septum samples (Zhu et al., 2000). Total RNA of the 12 samples was extracted according to manufacturer’s instructions and then stored in DEPC (diethyl pyrocarbonate) water at 80 jC for reverse transcriptase–polymerase chain reaction (RT–PCR). Reverse transcriptase–polymerase chain reaction (RT–PCR) Total RNA (5 Ag) of each of the 12 samples was reverse-transcribed into first-strand complementary DNA (fs cDNA) using oligo-dT primers (Gibco, BRL). The fs cDNA was amplified by polymerase chain reaction (PCR). The PCR was carried out in a total volume of 100 AL containing Tris–HCL 20 mmol, KCL 50 mmol, MgCl2 1.5 mmol, dNTP 0.2 mmol, 0.6 mmol of corresponding sense and antisense primers including h-actin, ACE, AT1, AT2 and VEGF respectively, and 2.5 units of Tag DNA polymerase (Promega, USA). The expression of the housekeeping gene, h-actin mRNA, served as an internal standard. PCR was run 30 cycles for h-actin, ACE, AT1 and AT2 receptor and 35 cycles for VEGF cDNA in a Perkin Elmer 9600 thermocycler. Three-step PCR of denaturing, annealing and extension reactions proceeded at 94 jC for 1 min, at 60 jC (h-actin), 64 jC (ACE), 57 jC (AT1-), 53 jC (AT2-) and 59 jC (VEGF) for 1 min, and at 72 jC for 1 min, respectively. The primer used for VEGF was: sense 5V-CCATGAACTTTCTG CTCTCTTG-3Vand anti-sense 5VGGTGAGAGGTCTAGTTCCCGA-3V.

Results General survival rate Survival rates during the period of study are shown in Fig. 1. In all treatment groups, the highest mortality occurred within 24 hrs of surgery. The survival rate at the end of the observation period for rats given DanShen, saline and ramipril were 68.9% (22/32), 50% (12/24) and 66.5% (16/24), respectively. The rates were not significantly different in the treatment groups possibly because the number of rats in each group was not sufficient to make a statistical difference.

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Fig. 1. Survival rates in the different treatment groups of AMI Rats. The rates are given for each group as the decimal fraction of the number of rats included in the study. The absolute numbers of rats that survived the whole experiment are given to the right of the graph.

Hepatic anti-oxidant assays As shown in Table 2, significantly higher hepatic CAT activity was observed in DanShen-treated rats than in saline-treated rats (P < 0.05, one-way ANOVA). There was no significant difference between DanShen- and ramipril-treated rats (P = 0.596, Bonferroni), or between saline- and ramipril- treated rats (P = 0.514, Bonferroni). Hepatic SOD activity was significantly higher in DanShen-treated rats than in both saline- and ramipril- treated rats (P < 0.001 for both, one-way ANOVA). However, no significant difference in SOD activity (P = 0.053, Bonferroni) was observed (Table 2) in saline-treated rats compared to ramipriltreated rats. Hepatic GSH-Px activity was significantly higher in DanShen-treated rats than in saline- and ramipriltreated rats (P < 0.001 for both, one-way ANOVA); Hepatic GSH-Px activity was significantly lower in ramipril-treated rats than in saline-treated rats (P < 0.001, Bonferroni) (Table 2). Hepatic GST activity (Table 2) was significantly higher in DanShen- treated rats than in saline- and ramipril- treated rats (P < 0.001 for both, one-way ANOVA); A significantly higher hepatic GST activity was also observed in saline-treated rats compared to ramipril- treated rats (P < 0.001, Bonferroni). Table 2 Anti-oxidant enzyme activities in the liver of DanShen-, saline- and ramipril- treated rats Enzyme (U/mg protein)

DanShen

CAT SOD GSH-Px GST

0.674 16.24 0.453 5.23

F F F F

Saline 0.015* 0.59** 0.013*** 0.114**

0.583 10.56 0.353 4.03

Ramipril F F F F

0.031 0.31 0.012 0.215

0.630 8.49 0.248 2.62

F F F F

0.019 0.67 0.012*** 0.105**

1. Values are mean F SEM (n = 6). All assays were performed in duplicate at 25 jC. 2. One unit of SOD is defined as the amount of enzyme necessary to inhibit the superoxide-dependent oxidation of 10mmol/L pyrogallol; 1 unit of catalase (CAT) defined as mmol H2O2 decomposed/min; 1 unit of glutathione peroxidase (GSH-Px) is defined as 1 Amol reduced nicotinamide adenine dinucleotide phosphate (NADPH) converted to NADP+/min; 1 unit of Glutathione S-transferase (GST) is defined as 1 Amol CDNB converted to CDNB-glutathione/min. * P < 0.05 compared with corresponding saline- and ramipril- treated group. ** P < 0.001 compared with corresponding saline- and ramipril- treated group. *** P < 0.0001 compared with corresponding saline- and ramipril- treated group.

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In conclusion, DanShen increased the activities of the antioxidant enzymes, SOD, CAT, GSH-Px and GST; while ramipril did not affect SOD and CAT activity, and suppressed the activities of GSH-Px and GST in the liver of rats with AMI. Thiobarbituric acid-reactive substances (TBARS) The cardiac and hepatic TBARS values (see Table 3) were significantly lower in DanShen-treated rats than in both saline- and ramipril- treated rats (P < 0.001 for both, one-way ANOVA). There was no significant difference in cardiac (P = 0.61, Bonferroni) and hepatic (P = 1, Bonferroni) TBARS values between saline- and ramipril- treated rats. Morphological examination of rat hearts with AMI Based on the examination of Tetrazolium-Blue stained hearts with AMI, a typical LAD ligationinduced myocardial necrosis was observed in DanShen- and ramipril-treated rats. However, in salinetreated rats with AMI, besides the typical necrotic band, a big portion of necrosis at the base of heart was also seen. Along the necrotic border, small blood vessels were observed in the hearts of DanShen-treated rats, but not in saline- and ramipril- treated rats. In the sham group, only a small portion of epimyocardial necrosis was seen (see Fig. 2). Infarct size Infarct size as a proportion of left ventricular size is shown in Fig. 3. Infarct size was reduced significantly in the hearts of both DanShen- and ramipril-treated rats compared to that in saline-treated rats (P < 0.001, one-way ANOVA); no significant difference was found in infarct size between DanShen- and ramipril-treated rats with AMI. Cardiac weight Cardiac weight was examined by determining the ratios (g/kg) of total heart weight (HW) to body weight (BW), left ventricular weight (LVW) to body weight and right ventricular weight (RVW) to body weight (Fig. 4). Table 3 TBARs content in the heart and liver of DanShen-, saline- and ramipril- treated rats with AMI Treatment

TBARs value (nmol/g wet weight) Heart

Liver

DanShen Saline Ramipril

202.23 F 1.77a 231.48 F 7.09 245.35 F 4.33c

320.23 F 4.22b 385.83 F 3.39 382.54 F 5.21

Values a P b P c P

are the mean F SEM (n = 6). Assays were performed in duplicate at 25 jC. < 0.001 (Bonferroni) DanShen-treated rats vs saline-treated rats. < 0.005 (Bonferroni) DanShen-treated rats vs saline-treated rats. < 0.001 (Bonferroni) ramipril-treated rats vs DanShen-treated rats.

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Fig. 2. Tetrazolinium-blue stained hearts of AM1 rats from different treatments. Note: White dark red colored zones represent ischaemic and non-ischaemic zones, respectively.

The body weights were not significantly different in the three treatment groups. The ratios of heart weight to the body weight, the left and right ventricular weight to body weight were significantly lower in DanShen- and ramipril-treated rats with AMI than that of saline-treated rats with AMI; there was no significant difference between DanShen- and ramipril-treated rats.

Fig. 3. The ratio of infarct size to left ventricular size in DanShen-, Saline- and ramipril-treated rats. Note: Infarct size as a ratio of the left ventricular size after LAD ligation in the different treatment groups. a p < 0.001 (Bonferroni) DanShen-treated rats vs saline-treated rats. b p < 0.005 (Bonferroni) ramipril-treated rats vs saline-treated rats. Each column represents the mean of six samples and the bar indicates the S.E.M. IS: infarct size; LV: left ventricle

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Fig. 4. The ratios of heart weight to body weight in DanShen-, Saline- and Ramipril-treated rats. a p < 0.001 (Bonferroni) DanShen-treated rats vs saline-treated rats. b p < 0.05 (Bonferroni) ramipril-treated rats vs saline-treated rats. Each column represents the mean of six samples and the bar indicates the S.E.M. HW: heart weight; BW: body weight.

Coronary capillary density and its distribution pattern Ischemic zone Total myocardial fibrosis was observed in the myocardial sections of DanShen- and saline-treated rat hearts with AMI, while apparent ‘‘islands’’ of surviving myocardium within the necrotic zone were found in the myocardial sections of ramipril-treated group. No blood vessels were seen in the ischemic zone of saline-treated rats with AMI; only a few small vessels were found in DanShen-treated rats with

Fig. 5. Vessel distribution patterns in ischemic zones of heart. Sections from DanShen-, Saline- and ramipril-treated Groups.

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AMI and some blood vessels were seen in surviving myocardial islands (areas at risk) in the ischemic zone of the myocardial section of ramipril-treated rats with AMI (see Fig. 5). Border zone The margin between ischemic and non-ischemic zone, (border zone) was found to be clear and sharp in the three groups (see Fig. 6). Since there are different definitions of ‘‘border zone’’, we define it as a gradient of histological or biochemical changes, composition or of function at the edge of an infarct (Robert and Braunwald, 1980). Vessels were not seen along the border zone in myocardial section of saline- and ramipril-treated rat hearts with AMI, but were observed along the border zone of DanShentreated rat hearts with AMI. Non-ischemic zone of the left ventricle Several big and small vessels were found in non-ischemic zones in myocardial sections of saline- and ramipril-treated rat hearts with AMI, while about double the amount of vessels, especially small vessels, were observed in the non-ischemic zone in myocardial sections of DanShen-treated rats (see Fig. 7). Capillary density in the non-ischemic zone Myocardial capillary density was significantly increased in DanShen- and ramipril-treated rats compared to that in saline-treated rats (P < 0.0001, one-way ANOVA) and a slight but significant

Fig. 6. Vessel distribution patterns along border zones of heart sections from DanShen-, Saline- and ramipril-treated groups.

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Fig. 7. Vessel distribution patterns in non-ischemic zones of heart sections from DanShen-, Saline- and ramipril-treated groups.

increase in myocardial capillary density was observed in DanShen-treated rats compared to ramipriltreated rats (P < 0.05, one-way ANOVA) (see Table 4). Furthermore, the distribution of blood vessels in these treatment groups appeared to be different. In the DanShen-treated group, the blood vessels appeared mainly in non-ischemic myocardium with an increased density along the border, while in the ramipril-treated group, the number of blood vessels showed a slight increase in the non-ischemic zone compared to the ischemic zone but without any increase in vessels along the border zone; in the saline-treated group, blood vessels predominantly presented in the non-ischemic zone while few blood vessels were observed in the ischemic and border zones. DanShen and renin-angiotension system (RAS) mRNA levels of ACE, AT1- and AT2-receptors could not be detected in the left and right ventricles and interventricular septum of rats with AMI 2 weeks after LAD surgery in all treatment and shamTable 4 Coronary capillary density in left ventricles of AMI rats given different treatments Treated groups

DanShen 292.65 F 4.71

Values a P b P c P

a,b

Saline

Ramipril

259.83 F 2.56

285.50 F 3.73c

are the mean F SEM (n = 6). < 0.001 (Bonferroni) DanShen-treated rats vs saline-treated rats. < 0.005 (Bonferroni) DanShen-treated rats vs ramipril-treated rats. < 0.001 (Bonferroni) ramipril-treated rats vs saline-treated rats.

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Fig. 8. Gel electrophoresis in 1.5% agarose gel of RT – PCR fragments of aˆ-actin (870 bp, upper section) and myocardial VEGF mRNA (704 bp, lower section). M: molecular weight marker; Lane 1: LV-Sham; 2: LV-DanShen; 3: LV-saline; 4: LV-ramipril; 5: RV-Sham; 6: RV-saline; 7: RV-ramipril; 8: RV-DanShen; 9: IP-DanShen; 10: IP-saline; 11: IP-ramipril; 12: IP-Sham, respectively. LV: the left ventricle, RV: the right ventricle, IP: the intraventricular septum.

operated groups. This could be that over-expression of senses in RAS were only observed in the early phase following AMI (Zhu et al., 2000). DanShen and VEGF mRNA expression VEGF mRNA expressions were detected in the three parts of DanShen-treated rat hearts with AMI, viz. the left and right ventricles and the intraventricular septum; VEGF mRNA expressions were only observed in the left ventricles of ramipril- and saline-treated groups on day 14 after AMI. Unfortunately, the expressions of VEGF mRNA were too weak to be quantified. No VEGF expressions were detected in the sham-operated group (See Fig. 8).

Discussion In present study, DanShen elicited a similar cardioprotective outcome in survival rate, IS reduction, myocardial hypertrophy and myocardial capillary density as ramipril. The increased survival rate, smaller IS, prevention and regression of ventricular hypertrophy are important end-points in the treatment of cardiovascular diseases (Zhu et al., 1997). DanShen increased the activities of antioxidant defense enzymes including SOD, CAT, GSH-Px and GST in the liver of AMI rats in this study. These indicate that DanShen can scavenge various free radicals effectively from different sites of antioxidant systems through enhancing the activities of the antioxidant enzymes in AMI rats including the hearts, because the increased enzymes in the liver circulate to the heart and other parts of the body through the circulation. Furthermore, the antioxidant enzymes in the heart were found to be enhanced by DanShen. The lower content of TBRS in the heart and liver indicates that DanShen could reduce lipid peroxidation in these rats with AMI. Lipid peroxidation is a free radical chain reaction, which is triggered by hydroxyl radical and leads to membrane breakdown to produce more free radicals. Hydroxyl radicals can attack and damage every molecule found in living cells (Von, 1987). DanShen could stop the chain reaction and reduce the production of free radicals in rats with AMI. This result is in line with similar studies on DanShen (Zhao et al., 1996; Cao et al., 1996; Albert et al., 1991). This study thus shows that DanShen scavenges free radicals through enhancing antioxidant defense enzymes and reduces free radical production by inhibiting lipid peroxidation. These could be major factors contributing to DanShen’s cardioprotective effects.

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On the other hand, there was no evidence to show that ramipril had any favorable effects on the antioxidant enzymes. In contrast, ramipril did not affect hepatic lipid peroxidation, but augmented myocardial lipid peroxidation in rats with AMI by inhibiting the activities of GSH-Px and GST (refer to Table 2), which are the antioxidant enzymes to remove H2O2. The surviving myocardium observed in ramipril-treated rats could be due to the opening of collateral vessels, since these have been shown to develop from angiogenesis in the ischemic myocardium (Charles et al., 1999). The possible reasons for ramipril-induced capillary proliferation are as follows: firstly, Zimmermann et al. (1999) demonstrated that VEGF mRNA levels were increased significantly when the ACE inhibitor ramipril was given at a blood pressure-lowering dose of 1 mg/kg/d as well as at a non-blood pressure lowering dose of 10 Ag/kg/d. Secondly, ACE inhibitors have been reported to reduce myocardial bradykinin degeneration, resulting in deletion of coronary vessels and thus increased myocardial blood flow (Mall et al., 1985). The bradykinin-mediated vasodilation and augmentation of myocardial blood flow may also contribute to capillary proliferation (Zhu et al., 1997). This may explain the finding of a reduced IS of ramipril-treated rats too. The increased number of blood vessels along the border zone and in the non-ischemic zone in DanShen-treated rat myocardium could be the result of capillary proliferation or opening of pre-existing capillaries. This could be a compensatory response to correct the imbalance between the perfusion capacity of coronary vessels and the need for oxygen and nutrients by the ischemic myocardium. Also, this was associated with a higher survival rate, reduced IS and myocardial hypertrophy in DanShentreated rats. The different patterns of vessel distribution indicated that the cardioprotective effects of DanShen and ramipril were mediated through different pathways. However, further studies need to be done to clarify the significance of the different patterns of blood vessel distribution in conferring myocardial protection. It could lead to the future use of DanShen in conjunction with, or as a safer alternative to, ACE inhibitors in the management of AMI. The present study also showed that both DanShen and ramipril did not affect RAS 14 days after AMI. This could be that over-expression of senses in RAS were only observed in the early phase following AMI (Zhu et al., 2000). Studies have shown that a time-dependent pattern of up-regulation of ACE, AT1 and AT2 receptor mRNA levels in rat heart is associated with the early myocardial remodeling process following an AMI and is independent of ACE inhibition (Reiss et al., 1993; Nio et al., 1995; Zhu et al., 1999). The time-dependent pattern of gene expression was as follows: the mRNA levels of AT1, AT2 in DanShen- and ramipril-treated rats began to rise markedly at 30 min, reached a peak 24 h after AMI, decreased over days 3 to 7, and were still higher than those of sham-operated rats. DanShen augmented VEGF mRNA expression in non-ischemic rat myocardium following AMI, especially in the right ventricle and the septum. The increased VEGF mRNA levels could contribute to coronary microvascular remodeling and myocardial repair after AMI. Although the VEGF expressions in this study were weak, we nevertheless found that the positive VEGF mRNA expressions in DanShen-treated rats were in line with the results of vessel distribution patterns in the heart sections from DanShen group. We may deduce that DanShen could stimulate myocardial revascularization in the non-ischemic part of the heart to compensate for the damaged heart function due to AMI. However, further studies on DanShen’s influence on myocardial ACE mRNA expression at the early stage of AMI will be needed to confirm whether DanShen affects the RAS; the demonstration of

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the time course of myocardial VEGF mRNA expressions after AMI will provide more information to help us better understand the mechanism by which DanShen increases myocardial capillary density and produces the particular blood vessel distribution pattern found in rat hearts with AMI. Since DanShen does not have potent and direct effects on RAS, it may be used in combination with ACE inhibitors. However, further studies on possible synergy between DanShen and ACE inhibitors need to be done.

Acknowledgements This study was supported by a research grant (R-184-000-044-731, PI: Y.Z. Zhu) of National Medical Research Council of Singapore. Y.Z. Zhu is a recipient of a Lee Kuan Yew research fellowship.

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