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Ann Thorac Surg 2003;75:1283-1287
© 2003 The Society of Thoracic Surgeons

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Original article: cardiovascular

Gene transfer of hepatocyte growth factor improves angiogenesis and function of chronic ischemic myocardium in canine heart

^a First Department of Surgery, Osaka University Medical School, Osaka, Japan

Accepted for publication October 21, 2002.

^* Address reprint requests to Dr Sawa, First Department of Surgery, Osaka University Medical School, Yamada-oka 2-2, Suita, Osaka 565, Japan
e-mail: sawa{at}surg1.med.osaka-u.ac.jp

	Abstract

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BACKGROUND: Hepatocyte growth factor (HGF) induces angiogenesis in myocardium. In the present study, its effects in chronic ischemic myocardium were tested.

METHODS: Four weeks after left anterior descending coronary artery ligation in canine hearts, HVJ-liposome containing either human HGF gene (160 µg; HGF group, n = 7) or nothing (control group, n = 6) was directly injected into ischemic myocardium. Four weeks after gene transfection, the thickness fraction (TF), an index of regional myocardial contractility (assessed by epicardial pulse-Doppler crystals), the myocardial perfusion flow (assessed by color microspheres), and the capillary density (assessed by immunostaining of vessels) were evaluated in ischemic myocardium.

RESULTS: Thickness fraction (percent of nonischemic myocardium) was significantly improved in the HGF group (80 ± 15 from 52 ± 16 of pregene; p < 0.05) whereas it was not changed in the control group (52 ± 10 from 50 ± 8 of pregene). The perfusion flow (% of nonischemic myocardium) was significantly improved in the HGF group (98 ± 17 from 51 ± 14 of pregene; p < 0.05) while it was not changed in the Control group (58 ± 13 from 62 ± 18 of pregene). The capillary density was significantly higher in the HGF group (894 ± 211/mm²; p < 0.05) than that in the control group (511 ± 127/mm²).

CONCLUSIONS: Gene transfection of HGF improved angiogenesis, thereby improved regional myocardial function and perfusion in chronic ischemic myocardium. It indicates a potent therapeutic value of HGF gene transfection for chronic ischemic heart diseases such as myocardial infarction.

	Introduction

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Rapid progress in the gene therapy field has led to the creation of a therapeutic strategy for ischemic heart diseases that is termed "therapeutic angiogenesis" [1]. This strategy is designed to promote the development of supplemental collateral blood vessels by using angiogenic growth factors that will constitute endogenous bypass conduits around occluded native arteries, and improve blood supply of ischemic myocardium. Several growth factors (VEGF, FGF, for example) were proposed as candidates for this therapy. Clinical studies have reported the efficiency of this new strategy in the patients with coronary artery diseases [2, 3].

We have focused on a novel potent angiogenic agent, hepatocyte growth factor (HGF) [4]. It has been reported that gene transfer of HGF can promote angiogenesis in normal and infarct myocardium in rat hearts [5]. However, there is no direct evidence for whether this angiogenesis could improve regional contractility and perfusion of chronic ischemic myocardium. From the clinical standpoint, the myocardial perfusion and contractility of the target area are the most important outcome of therapeutic angiogenesis and first evidence for dividing functional vessels from nonfunctional angioma formation.

In this study, we used the canine chronic myocardial ischemia model to investigate the effects of human HGF gene transfection on regional myocardial function, perfusion and angiogenesis of ischemic myocardium.

	Material and methods

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Animal model and experimental protocol
A model of chronic myocardial ischemia was created by left anterior descending coronary artery (LAD) ligation in adult beagles (n = 13; 9.0 ± 2.0 kg). All animal care procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 86 to 23, revised 1985). Beagles were anesthetized with ketamine (8 mg/kg, intramuscularly) and pentobarbital (30 mg/kg, intravenously), then intubated and ventilated (Ventilator 710, Siemens-Elema AB, Solna, Sweden). Anesthesia was maintained with inhalation of Sevoflurane (2% v/v). After a small thoracotomy was done through the left fifth intercostal space, the pericardium was incised and tended. Ligation was performed at the middle portion of LAD just below first diagonal branch by a 2-0 surgical suture. The pericardium and chest were then closed and the animal was allowed to recover.

Four weeks after LAD ligation, the left thoracotomy was reopened. Two sonomicrometer crystals (WT-20; Crystal Biotech, Matec Instruments, Northborough, MA) were permanently fixed by surgical suture on the surface of Left ventricle (LV). One of them was on the ischemic myocardium in the anterior wall; another one was on the nonischemic myocardium in the posterior wall (Fig 1). A 5F micromanometer (MPC-500; Millar, Houston, TX) were inserted into the LV from the LV apex to measure LV instantaneous pressure and dp/dt. A flare-tipped catheter was inserted into the left atrium for the injection of color microsphere (Triton Technology, Nottinghamshire, UK). Another catheter was inserted percutaneously into the left femoral artery for microsphere reference blood withdrawal. After pregene data were collected and microspheres were given, vector administration was performed as described later. All three catheters were removed; the pericardium and chest were then closed. One animal that received human HGF gene was killed at 4 days after gene transfection for analysis of human HGF expression in myocardial tissue.

View larger version (36K): [in this window] [in a new window]   Fig 1. Schematic of experimental design. Vector administration was performed at four sites ("T" marks) in the ischemic area. The regional contractile function was measured by sonomicrometer crystals at the two sites as indicated.

Preparation of vectors and gene transfection
Human HGF cDNA was inserted into the Not I site of the pUC-SR expression vector [6]. HVJ-liposome was prepared as previously described [7]. HVJ-HGF-liposome (containing 160 µg HGF gene) or HVJ-liposome in HEPES-buffered saline solution (1 mL) was introduced into the myocardium by direct intramyocardial injection using a 26G needle. It was injected randomly at four sites (4 x 0.25 mL) in the ischemic myocardium. Beagles received the human HGF gene-encoding vector in the HGF group (n = 7) or empty vector in the Control group (n = 6).

Definition of ischemic myocardium
The myocardium that is surrounding the scar tissue and 0.5 cm wide was defined as ischemic myocardium. The scar tissue was identified at visual bases. The scar tissue size was 2.5 ± 1.0 cm² and there was no statistically significant difference between the groups. A sonomicrometery crystal was fixed on ischemic myocardium just beside the borderline of scar tissue. Vectors were injected in the ischemic myocardium near the borderline on both sides of the crystal (Fig 1). They were 0.5 cm and 1 cm apart from the crystal. Surgical sutures were left as a marker at the sites of injection.

HGF assay in plasma and tissue samples
Venous blood was obtained at daily bases during the first week after gene transfection and once a week thereafter. Plasma samples were collected and stored. Human HGF in cardiac tissue and plasma was measured by means of enzyme-linked immunosorbent assay (ELISA) using antihuman HGF monoclonal antibody (Institute of Immunology, Tokyo, Japan) as described previously [8].

Evaluation of myocardial function
The LV end-systolic pressure (ESP), end-diastolic pressure (EDP) and dP/dt were measured and calculated from LV pressure wave curves. The heart rate (HR) was measured from ECG records. Regional myocardial function was assessed by the epicardial crystal pulsed-Doppler technique [9, 10] using sonomicrometer crystals. Absolute wall thickeness of ischemic and nonischemic myocardium and LV pressure and dp/dt were recorded continuously and analyzed by using computer software (MacLab, version 3.5/c; ADInstruments Ltd, Oxfordshire, UK). Dobutamine was infused intravenously in a dose of 20 µg · kg^-1 · min^-1 for 10 minutes by an infusion pump (Model 210; KD Scientific Inc., Boston, MA) before collecting the data at stress. The LV diastolic and systolic wall thickeness were measured at the onset and the end of systole, as defined by the initial upward and peak negative deflections of the LV dp/dt tracings, respectively. Thickeness fraction (TF) is calculated as follows: [(end-systolic wall thickeness - end-diastolic wall thickness) / end-diastolic wall thickness] x 100%. Measurements of wall thickeness were made during a 5-second apnea. The percent TF was calculated as follows: (TF of ischemic myocardium / TF of nonischemic myocardium) x 100%.

Evaluation of regional myocardial perfusion
Regional myocardial perfusion was measured by color microsphere technique [11, 12]. The 15-µm plastic microspheres (4.5 x 10⁶ spheres in 1.5 mL saline) was infused within 10 seconds while reference blood was withdrawn at a rate of 6 mL/min by a pump (Model 210, KD Scientific). Microsphere amount was measured by spectrophotometry method. Myocardial tissue and blood samples were analyzed for absorbency using an UV-visible recording spectrophotometer (UV-160A; Shimadzu Inc., Tokyo, Japan). Regional myocardial perfusion flow (Qm) was computed using the formula Qm = Qr*Cm/Cr, where Qr is the reference flow rate (6 mL/min), Cm indicates the absorbency from the myocardial tissue sample, and Cr indicates the absorbency from the reference blood sample. Each perfusion flow was normalized for the weight of the myocardial tissue sample. The percent Qm was calculated as follows: (Qm of ischemic myocardium / Qm of nonischemic myocardium) x 100%.

Evaluation of angiogenesis
The extent of vascularity was examined by measurements of the number of capillaries in light microscopic sections retrieved from ischemic and nonischemic myocardium. Myocardial tissue sections were used in the Antifactor VIII vessel immunostaining with the Dako Enhanced Polymer One-step Staining kit (Dako Inc., Tokyo, Japan). Sections were cut at a thickeness of 5 µm and fixed in acetone at 4°C for 10 minutes. The antibody was rabbit polyclonal antihuman factor VIII associated antigen. The sections were incubated with the antibody at room temperature for 30 minutes. Then, the peroxidase was visualized by DAB followed by incubation with DAB-enhancing solution (Dako). Computer programed appraisal (MacScope, version 2.51; Sensory Arts &amp; Science, Lederach, PA) of vascular diameter and numbers was performed at different cutoff sections. A total of ten different fields from each sample were randomly selected, and the numbers of capillaries were counted to determine the capillary density (number/mm²). Vessels in diameter less than 10 µm were counted as capillary.

Statistical analysis
All values are expressed as mean ± standard deviation. Comparisons (%TF, %Qm, dP/dt, HR, ESP, and EDP) were done by ANOVA and Bonferroni post hock test. Others were compared using Student’s t test. A p value less then 0.05 was considered statistically significant.

	Results

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Human HGF expression in the heart and blood
A marked expression of human HGF was detected at the injection sites at 4 days after gene transfection in the heart that received HGF gene. The highest value was 10.05 ± 0.28 ng/g tissue wet weight although HGF was not detected (< 0.01 ng) in the other parts of myocardium ( 1.5 cm from the injection sites) and scar tissue. In contrast, human HGF was not detected in the myocardial tissue samples of both groups at four weeks after gene transfection. The plasma level of human HGF was not detectable (< 0.01 ng) in blood samples of both groups throughout the experiment.

Global and regional cardiac function
Table 1 illustrates the global and regional cardiac function before and 4 weeks after gene transfection. The HR and ESP were not significantly changed. The EDP was significantly reduced in the HGF group, whereas it was not changed in the control group; dP/dt was not significantly changed in the HGF group, whereas it significantly declined in the control group; and %TF was significantly improved in the HGF group either at rest or under Dobutamine stress, whereas it was not changed in the control group.

Immunostaining of vessels
Figure 2 illustrates the ischemic myocardium with immunostaining of vessels. The capillary density of the ischemic myocardium (at the injection sites) was significantly higher in the HGF group (894 ± 211/mm²; p < 0.05) than that in the control group (511 ± 127/mm²). The capillary density of the nonischemic myocardium was not significantly different between the HGF group (964 ± 92/mm²) and the control group (1164 ± 161/mm²). The capillary density of the ischemic myocardium was about 94% of normal value in HGF group and 43% in the control group.

View larger version (52K): [in this window] [in a new window]   Fig 2. Representative images from ischemic myocardium (bar = 50 µm) illustrating increased number of small vessels with endothelium stained for factor VIII in the hepatocyte growth factor group (A) than that in the control group (B).

	Comment

Top Abstract Introduction Material and methods Results Comment References

Hepatocyte growth factor (HGF) is a potent angiogenic agent [4], possesses mitogenic, motogenic, and morphogenic effects through its own specific receptor, c-Met, in various types of cells including myocytes [13]. Recent studies reported that HGF and c-Met are upregulated in ischemic myocardium of animal and human hearts [14–16]. The evidence that only ischemic, not normal myocytes, express c-Met indicates an important role of cardiac HGF/c-Met system in ischemic myocardium. We reported the protective effect of HGF against ischemic injury in myocardium achieved by gene transfection of HGF or supplement of HGF in rat models [17, 18]. In this experiment, we demonstrated the angiogenic effect of HGF with gene transfection in the canine model.

Considering the angiogenic gene therapy in clinical trials, the only candidates for gene therapy are those patients with diffuse coronary artery disease that have nongraftable chronic ischemic myocardium. It has been reported that gene transfer on around occluded native artery improved cardiac function in animal models [19]. However, we do not believe that gene transfection could create large arteries except capillaries. We prefer to apply the gene on ischemic myocardium so that it improves capillary network between ischemic and nonischemic myocardium and within the ischemic myocardium, which we believe that to be more efficient than creating capillaries around occluded arteries. Most patients receive surgical therapy over several hours or days after a heart attack event. Therefore, this study was designed to perform the gene transfer of HGF at 4 weeks after ligation of coronary artery when the hearts had stable infarction, chronic ischemic myocardium, and stable cardiac function similar to the patient characteristics of ischemic heart disease.

The canine model of coronary artery ligation is widely used in acute and chronic studies [20]. Because the canine heart is rich with preformed coronary artery collaterals, LAD ligation created only a small infarction (< 10% of LV surface) with mild remodeling of heart in our study. The reason why we used LAD ligation, not ameroid constriction, and waited for 4 weeks to apply the gene was to give the ischemic myocardium a time long enough for opening all preformed collaterals and completing the natural capillary growth after ischemic insult so that we could exclude those factors from our results. The perfusion flow data from the control group proved this hypothesis is correct. Hepatocyte growth factor (HGF) gene transfection partly attenuated the decline of the global cardiac function (dP/dt). It might be the reason that only a small part of ischemic myocardium was functionally improved and this improvement was not enough to change the global performance. However, as a pilot study, our data provided the evidence that the performance of ischemic myocardium could be improved by HGF gene transfection.

We found that human HGF expression is limited an area within 2-cm around the injection site. Scar tissue did not express human HGF. A part of nonischemic myocardium surrounding the ischemic area expressed human HGF (up to 2 ng/g) at 4 days after gene transfection, but in contrast, neither the vessel density nor the perfusion flow was altered in similar nonischemic myocardium 4-weeks after gene transfection. It indicated that the diffusion distance of HGF from transfection site is limited to 2 cm. Its angiogenenic effect was limited to more smaller area depend on concentration gradient. Our data suggests that transfection sites may not be apart more than 2 cm in future clinical trials.

We did not find any expression of human HGF in blood throughout the experiment. Although we could not exclude the possibility of vector leakage into blood stream, it was hard to find out the unwanted infection sites in other organs and what kind of regional effect was actually happen at that sites when a relatively small amount of leaked vector was distributed to whole body. We, at least, could say that transfection of HGF gene was limited to the target area and the blood HGF concentration would not reach a hazardous level by this dose and method. Still, it is hard to determine what kind of cell type is mostly likely infected by HGF gene and which cell type produces the most part of HGF. We expect it will be clarified in small animal models in future studies.

This study presents the effectiveness of HGF gene therapy in a coronary artery disease model by functional and pathologic evidence under similar clinical settings, but left several unresolved issues [1]. Due to lack of measurement techniques for canine HGF, changes and roles of endogenous HGF in this model is unclear [2]. The long-term outcome of the angiogenic effects of HGF is unclear.

If HGF gene therapy is proven to be safe and effective in clinical trials, it will be a valuable treatment for patients with diffuse ischemic heart disease not amenable to conventional surgical therapy. Our data raise an interest to investigate therapeutic value of the HGF genes or HGF recombinant proteins in clinical trials of therapeutic angiogenesis. Our study provides a new tool of therapeutic angiogenesis for the clinical practice.

	References

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