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Eur J Cardiothorac Surg 2008;34:857-863. doi:10.1016/j.ejcts.2008.05.045
Copyright © 2008, European Association for Cardio-thoracic Surgery. Published by Elsevier. All rights reserved.
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Right arrow Coronary disease

Therapeutic angiogenesis using naked DNA expressing two isoforms of the hepatocyte growth factor in a porcine acute myocardial infarction model

Kwang Ree Choa, Jae-Sung Choib, Woong Hahnc, Dong Sik Kimc, Jin Sik Parkd, Dong Soo Leee, Ki-Bong Kimf,*

a Department of Thoracic and Cardiovascular Surgery, Cheju Halla General Hospital, Republic of Korea
b Department of Thoracic and Cardiovascular Surgery, DongGuk University International Hospital, Republic of Korea
c ViroMed Co., Ltd., Republic of Korea
d Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea
e Department of Nuclear Medicine, Seoul National University Hospital, Seoul, Republic of Korea
f Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital, 28, Yeongeon-dong, Jongno-gu, Seoul 110-744, Republic of Korea

Received 15 December 2007; received in revised form 14 May 2008; accepted 14 May 2008.

* Corresponding author. Tel.: +82 2 2072 3482; fax: +82 2 747 5245. (Email: kimkb{at}snu.ac.kr).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: We evaluated the potency of therapeutic angiogenesis using intramyocardial injection of naked DNA expressing two isoforms of hepatocyte growth factor (pCK-HGF-X7) in a porcine myocardial infarction model. Methods: Four weeks after left anterior descending coronary artery ligation, 14 pigs were allocated to pCK-Null (negative control, n = 7) or pCK-HGF-X7 (n = 7) treatment groups. Gated myocardial single photon emission computed tomography was performed 4 and 8 weeks following coronary ligation. The effect of pCK-HGF-X7 on capillary density in the gene-injected myocardium was examined by histological analysis using alkaline phosphatase staining. Results: Segmental myocardial perfusion of the underperfused area (≤70%) from coronary ligation increased in the pCK-HGF-X7 group (p = 0.051), without significant differences in changes over time between the two groups (p = 0.54). Systolic wall thickening (p = 0.001), left ventricular end-diastolic (p = 0.045) and end-systolic volumes (p = 0.009), and left ventricular ejection fraction (p = 0.041) changed in both groups without significant differences in changes over time between the two groups. The increase in the left stoke volume was higher in the pCK-HGF-X7 group than in the pCK-Null group (p = 0.008). Histological analysis showed that capillary density was significantly higher in the pCK-HGF-X7 group than the pCK-Null group (p < 0.001). Conclusion: Intramyocardial injection of pCK-HGF-X7 induced significant angiogenesis at infarct-border zone, and increased the left ventricular stroke volume probably caused by reverse remodeling process.

Key Words: Gene therapy • Angiogenesis • Coronary artery disease


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Despite recent innovative techniques in coronary artery bypass surgery or percutaneous coronary intervention, more than 10% of symptomatic patients with coronary artery disease cannot be completely revascularized [1]. For patients with ischemic heart disease who experience incomplete revascularization or are unable to be revascularized, therapeutic angiogenesis was introduced as a possible remedy. Vascular endothelial growth factor (VEGF) has been studied in ischemic heart disease. Although it demonstrated increased myocardial perfusion in various clinical trials, VEGF failed to meet the goal of therapeutic angiogenesis, consistent myocardial functional improvement [2,3]. Recently, hepatocyte growth factor (HGF), initially purified and cloned as a potent mitogen of hepatocytes [4], has increasingly been used due to its potent mitogenic, motogenic, angiogenic, and anti-apoptotic effects in various cell types after binding of the membrane tyrosine kinase receptor encoded by c-met proto-oncogene [5]. HGF produced more ideal vessels in the ischemic zone by promoting the migration of vascular smooth muscle cells as well as endothelial cell proliferation [6]. HGF was also demonstrated to attenuate apoptotic cell death at the infarct border zone in left ventricular remodeling after myocardial infarction [7]. Moreover, the HGF-c-met system in the myocardium showed a direct protective effect from ischemic injury on cardiomyocytes [8,9]. These promising effects of HGF led us to adopt this factor as a tool for therapeutic angiogenesis. A gene encoding human HGF is located at chromosome 721.1. It is comprised of 18 exons and 17 introns, and simultaneously expresses HGF consisting of 728 amino acids and deleted HGF (dHGF) consisting of 723 amino acids via alternative splicing, which occurs between exon 4 and 5, resulting in the deletion of 5 amino acids (FLPSS) [10,11].

In this study, we injected a genomic-cDNA hybrid of the HGF gene (HGF-X7) that expressed HGF and dHGF simultaneously with high efficiency into a plasmid DNA vector system (pCK) for therapeutic angiogenesis in a porcine myocardial infarction model. The pCK-null vector was used as a control. The aim of this study was to evaluate the therapeutic effects induced by intramyocardial injection of pCK expression vector containing the HGF-X7 gene (pCK-HGF-X7) in terms of (1) the potency of angiogenesis and (2) myocardial functional improvement.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animal care
All animals received humane care in compliance with the European Convention on Animal Care. The study was conducted in accordance with the animal care and guidelines of the Institutional Animal Care and Use Committee of the Seoul National University Hospital.

2.2 Plasmid DNA
pCK has been described in detail previously [12]. The HGF-X7 construct was generated as follows: HGF gene fragment 3 (HGF-F3) and HGF-F8 were amplified by polymerase chain reaction (PCR) from the genomic DNA of human HepG2 cells (ATCC, HB-8065, US). HGF-F1 and HGF-F4 were amplified by RT-PCR from human placenta total RNA (Clontech, CA, US). The amplified HGF fragments were each inserted into the PCR product cloning site of the pGEM-T easy plasmid (Promega, WI, US) to obtain pGEM-T easy-HGF-F1, HGF-F3, HGF-F4, and HGF-X8, respectively. Further, DNA fragments containing cDNAs of the HGF gene (cHGF) and the deleted HGF gene (dHGF) were amplified from human placenta cDNA. PCR primers were: 5'-ggatccacgcgtagcagcaccatgtgggtgaccaaa-3' and 5'-ggatcctctagattacttcagctatgactgtggtac-3'. The amplified cDNAs were cloned into the pGEM-T easy plasmid, resulting in pGEM-T easy-cHGF and pGEM-T easy-dHGF, respectively. About 1100 bp of the HGF-XhoI fragment was obtained from HGF cDNA by XhoI digestion. Following sequence confirmation, the HindIII/BamHI fragment of pGEM-T easy HGF-F1, which contains HGF-F1, was cloned into the HindIII/BamHI site of the pCK, resulting in pCK-F1. Subsequently, the MluI/BamHI DNA fragment of pGEM-T easy-HGF-F3, which contains HGF-F3, was cloned into the MluI/BamHI site of pCK-F1, resulting in pCK-F13M. The MluI restriction site of vector pCK-F13M was substituted with an HgaI restriction site by employing a site-directed mutagenesis kit (Stratagene, CA, US) to obtain pCK-F13. Further, plasmid pGEM-T easy-HGF-F4 was treated with BamHI/XbaI to obtain HGF-F4. pCK-F13 was treated with BamHI/XbaI, and HGF-F4 was inserted to obtain pCK-F134, respectively. The plasmid pGEM-T easy-HGF-F8 was treated with BamHI/XhoI to obtain HGF-F8. pCK-F134 was treated with BamHI/XhoI, and then HGF-F8 was inserted to obtain pCK-F1384. Finally, about 1100 bp of the HGF-XhoI fragment was cloned into the XhoI site of pCK-F1384, resulting in pCK-HGF-X7 (Fig. 1 ).


Figure 1
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  		Fig. 1. Structure of pCK-HGF-X7 pCK contains not only a promoter and enhancer of the immediate-early (IE) gene of the human cytomegalovirus (HCMV) but also its entire 5'-untranslated region upstream from the initiation codon of the IE gene. A genomic-cDNA hybrid of the HGF gene, HGF-X7, was inserted into the junction of exon2 and pA, resulting in pCK-HGF-X7. Abbreviations: pCMV, promoter and enhancer of the HCMV IE gene; I, exon 1; wavy line, intron; II, untranslated region of exon 2; pA, poly A tract of bovine growth hormone gene; ColE1, Escherichia coli origin of replication; Kan r., kanamycin resistance gene.

 
All the expression constructs were purified using an EndoFree plasmid Maxi or Giga prep kit (Qiagen, CA, US), dissolved in 0.9% NaCl, diluted to 1–5 µg/µl, and stored at –20 °C prior to use.

2.3 Animal surgery
Of the 20 male Yorkshire swine (31.5 ± 2.1 kg), 14 survived for the 8 week duration of the study period. Pigs were sedated and anesthetized with an intramuscular injection of ketamine hydrochloride (20 mg/kg) and xylazine hydrochloride (2 mg/kg). After intravenous injection of thiopental sodium (10 mg/kg) and vecuronium bromide (0.1 mg/kg), pigs were intubated and mechanically ventilated with 0.5% enflurane at the time of surgery. Under continuous monitoring of electrocardiography, oxygen saturation, and femoral arterial pressure, a left thoracotomy was performed through the fourth intercostal space. After entering the pericardial space, the distal third of the LAD artery (just distal to the second diagonal branch) was encircled with 5-0 polypropylene. After intravenous injection of lidocaine hydrochloride (1 mg/kg), ischemic preconditioning was performed. Preconditioning consisted of two 3-min occlusions, each followed by a 5-min period of reperfusion. After the ischemic preconditioning, the LAD just distal to the second diagonal branch was ligated. Additional lidocaine (1 mg/kg) was injected intravenously immediately after the ligation. Infarction was confirmed by visible blanching of the region at the time of ligation. The pericardium was repaired and a 28 Fr chest tube was inserted in the pleural cavity. The wound was closed in layers. After the animal regained self-respiration, chest tube removal and extubation were performed in the operating room. Intermittent intramuscular injections of ketoprofen (100 mg) were used for pain control. After 4 weeks of LAD ligation, gated myocardial single photon emission computed tomography (SPECT) was performed using 99mTc-methoxyisobutylisonitrile (MIBI). After the myocardial SPECT was performed, the pigs were transported to the operating table for gene injection. Under the same anesthesia used during coronary ligation, we utilized the opposite pleural space to approach the heart. This approach was helpful in avoiding lung injury from severe adhesion formation. The ligated coronary artery was identified from the suture material. Following complete dissection of the heart, animals were randomized to either negative control (pCK-Null) (n = 7) group or pCK-HGF-X7 (n = 7) group. All animals in the pCK-Null group received direct intramyocardial injection via a 27 gauge needle of 1 mg of pCK-Null vector into the infarction border zone. A total volume of 1 ml was injected into five separate areas. All the animals in the pCK-HGF-X7 group received the same dose and volume of the gene injection along the infarction border zone. During those injections, no ventricular arrhythmias occurred at all. The first injection point between the ligated LAD and the diagonal branch was marked with a metal ring for later tissue harvesting. The wound was then repaired in layers and the pig was recovered for another 4 weeks. After performing the follow-up myocardial SPECT, pigs were sacrificed with intravenous injection of thiopental sodium (120 mg/kg) and potassium chloride (20 meq). The heart was harvested and tissue samples (1 cm x 1 cm) were collected at the five gene injection sites for alkaline phosphatase staining of vascular endothelial cells.

2.4 Myocardial SPECT
After 4 h of fasting, pigs were sedated with intramuscular injection of ketamine hydrochloride (20 mg/kg) and xylazine hydrochloride (2 mg/kg). A MIBI-gated myocardial SPECT was performed through use of a dual-head gamma camera equipped with low-energy-high-resolution collimator (Forte, ADAC Laboratories, Milpitas, CA, US). Thirty-two step-and-shoot images were acquired with intervals of 3° for 25 s per step. For gating, 16 frames per cardiac cycle with a pre-fixed R-R interval and 40% windows were used. The reconstructed images were analyzed with an automatic quantifying software package (AutoQUANT, ADAC Laboratories, Milpitas, CA, US) without manual intervention. For regional analysis of myocardial perfusion and systolic wall thickening, a 20 segment model was adopted. In each segment, rest perfusion quantified by uptake of 99mTc-MIBI was expressed as the percentage of the maximal uptake. Segmental systolic wall thickening was also quantified and expressed as the percentage of end-diastolic thickness on gated images. Under-perfused segments from the LAD ligation were defined as segments showing rest perfusion <70%. Left ventricular volumes (end-systolic, end-diastolic, stroke volumes) and ejection fraction were also measured by quantitative gated SPECT.

2.5 Assessment of angiogenesis by alkaline phosphatase staining
Four weeks after the gene treatment, 5 specimens were obtained from the gene injection points along the infarct border zone and placed into tissue blocks using O.C.T. compound (Miles Inc, Elkhart, IN, US). The blocks were sliced into 5 µm sections with a cryocut microtome (HM550, MICROM International, Germany). Endothelial cells of the capillaries in the tissue sections were identified by alkaline phosphatase staining, using indoxyl-tetrazolium (Sigma, St. Louis, MO, US). Capillary density was quantitatively analyzed under a microscope at 200x magnification and presented as the number of capillaries per 0.6 mm2 in a blinded manner (Image-Pro® PLUS version 4.1, Media Cybernetics, Silver Spring, MD, US). It was then compared between the treatment groups.

2.6 Statistical analysis
Statistical analysis was performed using a statistical software package (SPSS version 11.0; SPSS Inc; Chicago, IL, US). To compare the changes in the myocardial perfusion, systolic wall thickening, left ventricular volumes and ejection fraction between the two groups, repeated measures ANOVA (analysis of variance) was done. Unpaired Student's t-test was done to compare the capillary densities between the two groups. All values were expressed as a mean ± one standard deviation, and a p < 0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Animal surgery
Of those 20 pigs, 4 pigs died immediately after the coronary ligation in the operating room. Ventricular fibrillation that could not be resuscitated was the cause of those mortalities. One pig died of sepsis before performing the follow-up myocardial SPECT, and another pig died of ventricular fibrillation during redo-surgery after 4 weeks of LAD ligation.

3.2 Regional myocardial perfusion and systolic wall thickening
Regional myocardial perfusion of the underperfused segments were not changed in either the pCK-Null group (43.8 ± 14.5% vs 43.4 ± 15.1%) or the pCK-HGF-X7 group (40.6 ± 14.9% vs 42.8 ± 15.8%) (p = 0.051). Although a small tendency toward increased perfusion was observed in the pCK-HGF-X7 group, the changes were not different between the two groups (p = 0.54) (Fig. 2 ). Systolic wall thickening significantly changed in both the pCK-Null group (27.2 ± 14.6% vs 30.4 ± 15.4%) and the pCK-HGF-X7 group (26.1 ± 17.9% vs 37.6 ± 13.0%) (p = 0.001). However, there were no significant differences in changes between the two groups (p = 0.25) (Fig. 3 ).


Figure 2
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  		Fig. 2. Effect of pCK-HGF-X7 on myocardial perfusion of MIBI-SPECT. Myocardial perfusion after gene injection was significantly improved in the pCK-HGF-X7 group while it remained stable in the pCK-Null group. Data are expressed as the mean ± one standard deviation bar.

 

Figure 3
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  		Fig. 3. Effect of pCK-HGF-X7 on systolic wall thickening of MIBI-SPECT. Regional systolic wall thickening after gene injection was significantly improved in the pCK-HGF-X7 group while it remained stable in the pCK-Null group. Data are expressed as the mean ± one standard deviation bar.

 
3.3 Left ventricular volumes and ejection fraction
Left ventricular end-diastolic volumes (LVEDVs) were significantly changed in both the pCK-Null group (60.1 ± 10.3 ml vs 68.1 ± 12.3 ml) and the pCK-HGF-X7 group (69.4 ± 12.4 ml vs 69.1 ± 15.7 ml) (p = 0.045) (Fig. 4 ). Left ventricular end-systolic volumes (LVESVs) were also significantly changed in both the pCK-Null group (29.9 ± 11.0 ml vs 34.4 ± 12.91 ml) and the pCK-HGF-X7 group (34.1 ± 10.8 ml vs 27.9 ± 8.6 ml) (p = 0.009). However, these changes were not different between the two groups (p = 0.84) (Fig. 4). Resulting from those volume changes, the increase in the left ventricular stroke volume was higher in the pCK-HGF-X7 group (36.0 ± 2.5 ml vs 41.6 ± 8.1 ml) than in the pCK-Null group (30.3 ± 5.1 ml vs 33.7 ± 2.6 ml) (p = 0.008). Left ventricular ejection fraction also changed in both the pCK-Null group (51.6 ± 11.5% vs 50.9 ± 9.9%) and the pCK-HGF-X7 group (51.6 ± 8.0% vs 60.4 ± 5.6%) (p = 0.041), without significant differences between the two groups (p = 0.29) (Fig. 5 ).


Figure 4
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  		Fig. 4. Effect of pCK-HGF-X7 on left ventricular volumes of MIBI-SPECT. The increase in end-diastolic volume in the pCK-Null group suggested that this animal model had effectively developed left ventricular remodeling (A). The increase in stroke volume (C) in the pCK-HGF-X7 group was mainly attributable to decreased end-systolic volume (B), while it was from increased end-diastolic volume in the pCK-Null group. These findings represented the improvement of myocardial contractility in the pCK-HGF-X7 group.

 

Figure 5
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  		Fig. 5. Effect of pCK-HGF-X7 on left ventricular ejection fraction of MIBI-SPECT. As a result of improved myocardial perfusion and attenuation of remodeling process, left ventricular ejection fraction was significantly improved from 52% to 60% in the pCK-HGF-X7 group.

 
3.4 Micro-capillary density
Alkaline phosphatase staining revealed the highest number of vascular endothelial cells per 0.6 mm2 in the pCK-HGF-X7 group. The pCK-HGF-X7 group had 355.0 ± 90.8 cells/mm2 compared with 189.4 ± 77.0 cells/mm2 in the pCK-Null group (p < 0.001) (Fig. 6 ).


Figure 6
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  		Fig. 6. Effect of pCK-HGF-X7 on capillary density in a porcine myocardial infarction model. The angiogenic potency of the pCK-HGF-X7 group was much higher than the pCK-Null group. Capillary density was quantitatively analyzed under a microscope at 200x magnification and presented as the number of capillaries per 0.6 mm2. The length of the scale bar represented 900 µm. Data are expressed as the mean ± one standard deviation. * p < 0.001 (vs pCK).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
This study demonstrated three major findings in terms of therapeutic angiogenesis. First, direct intramyocardial injection of pCK-HGF-X7 in a porcine model four weeks after myocardial infarction resulted in significantly enhanced angiogenesis at infarct border zone, without demonstrating the increased myocardial perfusion at infarcted area. Second, intramyocardial injection of pCK-HGF-X7 in the border zone of infarction attenuated postinfarction ventricular remodeling by improved left ventricular stroke volume. Third, intramyocardial injection of pCK-HGF-X7 tended to improve left ventricular systolic function by the enhanced angiogenesis and the attenuation of left ventricular remodeling.

Human clinical trials using the VEGF gene in peripheral arterial occlusive diseases have demonstrated promising results of improved vessel formation followed by perfusional increase [13,14]. In an animal model of ischemic heart disease, improved myocardial perfusion after VEGF gene therapy has also been demonstrated [15,16]. Phase I clinical studies in coronary artery disease showed symptomatic improvement with or without increased myocardial perfusion [2,3]. However, convincing evidence of improving global myocardial function after VEGF gene therapy in humans has been lacking until now. Along with the negligible effect of VEGF on myocardial functional improvement and side effects of angioma-like structure formation or myocardial edema from the increased membrane permeability [17,18], skepticism has been raised over the use of VEGF as a mechanism for therapeutic angiogenesis.

In a previous study using the same animal model, the regional myocardial perfusion and wall motion score evaluated with attenuation-corrected ECG-gated SPECT were significantly improved by the injection of VEGF gene [19]. However, when we re-analyzed the VEGF effect on myocardial perfusion by using attenuation-uncorrected ECG-gated SPECT analysis, myocardial perfusion did not increase significantly despite the increased angiogenesis caused by VEGF genes. In the present study, we did not perform attenuation correction because the benefit of attenuation correction in the resting-only study at LAD territory, where diaphragmatic interference was minimal, might be negligible; nevertheless, the beneficial effects of HGF on myocardial perfusion were prominent. We speculate that increased myocardial perfusion by HGF would be higher than by VEGF. If attenuation correction was also used in the present study, the increased myocardial perfusion in the pCK-HGF-X7 group might even be higher than this uncorrected result.

In the present study, pCK-HGF-X7 injection resulted in a significant increase in regional myocardial perfusion and systolic wall thickening at the underperfused (<70%) segments from coronary artery ligation when analyzed by paired Student's t-test. If repeated measures ANOVA test was performed after adjusting the time variables, however, the changes were not statistically significant when compared with the control group. Based on a report concerning reproducibility of systolic wall thickening by myocardial SPECT [20], the amount of improvement (15.1%) in the present study could be regarded as significant. In analyzing the myocardial SPECT data, we selected over 40 segments in each group, which comprised of infarcted area as well as infarct border zone. As all injection points were at infarct border zones, the increased capillary density at border zone might not be enough to increase the myocardial perfusion at infracted area. A gradual increase in left ventricular end-diastolic volumes after 4 and 8 weeks in the pCK-Null group revealed that this animal model effectively developed post-infarction left ventricular remodeling. The decrease in LVEDV in the pCK-HGF-X7 group, although statistically insignificant, could be regarded as an attenuating effect of HGF on left ventricular remodeling. This effect was more prominent in the changes in LVESV. The LVESV decreased in the pCK-HGF-X7 group and increased in the pCK-Null group, although it was not significantly different between the two groups. Resulting from enhanced systolic wall thickening and decreased LVESV, the stroke volume significantly increased in the pCK-HGF-X7 group. This finding implied that improvement in left ventricular systolic function in the pCK-HGF-X7 group might be derived from the attenuating effect of left ventricular remodeling (Fig. 4). When we re-analyzed the SPECT data of previous study using VEGF gene [19], the decrease in LVESV was not demonstrated after the VEGF therapy. This finding correlated with the report demonstrating the negligible effect of VEGF gene on post-infarction remodeling process [21]. Although the changes were not different between the two groups, a significant change in left ventricular systolic function expressed as left ventricular ejection fraction (from 51.6% to 60.4%, p = 0.041) was demonstrated in the pCK-HGF-X7 group. The 8.8% increase in this study was significant in comparison with two standard deviations (5.3%) of the diagnostic value reported in the reproducibility study of myocardial SPECT [22].

By the combined effect of direct facilitation of endothelial cell migration and proliferation along with smooth muscle cell proliferation, neo-vessel formation produced by HGF was predicted as a more ideal form of vessel than just the tubal mixture of endothelial cells produced by VEGF. Another mechanism of HGF in angiogenesis has been shown to be the indirect pathway that promoted the transcription of the VEGF gene in higher levels of the cell signal transduction pathway [17]. HGF has also been known to have additive endothelial cell proliferating action when used with fibroblast growth factor but not with VEGF [23]. When we compared microcapillary density in this study with the result of our previous study using pCK-VEGF165 [19], higher endothelial cell density in the pCK-HGF-X7 group was demonstrated, which might be from the potent angiogenic effect of HGF, not simply through the facilitation of the VEGF transcription. A direct protective effect of HGF on cardiomyocytes as well as anti-apoptotic effect has also been reported in a rat ischemia–reperfusion model. Antibody blocking of HGF after myocardial infarction increased the area of infarction and mortality. Moreover, the serum of the rat with myocardial infarction had a direct protective effect on cultured cardiomyocytes, but the protective effect was truncated after neutralization of HGF [24]. In a small animal model of myocardial infarction, expression of the HGF receptor (c-Met) mRNA was increased after use of HGF, followed by an increase in HGF protein. The increased angiogenesis, along with the anti-apoptotic effect of HGF, resulted in better myocardial function than in animals that did not receive the HGF gene [7]. The HGF-c-met system in cardiomyocytes has been demonstrated to recruit the strong anti-apoptotic Bcl-Xl receptor and anti-necrotic ERK receptor on the cell membrane following myocardial infarction. These receptors were involved in the direct protective effect of HGF on cardiomyocytes [24].

In the present study, we confirmed the attenuating effect of the naked DNA expressing two isoforms of HGF in postinfarction ventricular remodeling by measuring ventricular volumes. Enhanced angiogenesis at the border zone of infarction and the direct protective effect of the naked DNA expressing two isoforms of HGF on cardiomyocytes could possibly produce these results. Another important aspect in therapeutic angiogenesis is the timing of the gene application after myocardial infarction. In one small animal study, early (within 6 days) application of HGF after myocardial infarction protected left ventricular function, preventing left ventricular remodeling for 8 weeks [9]. Interestingly, the ratio of the HGF level (infarct-territory/non-infarct territory) at 4 weeks after reperfusion therapy in acute myocardial infarction correlated inversely with left ventricular dysfunction, and positively with left ventricular ejection fraction in a human study. Therefore, the level of HGF behaved as an opposite biochemical marker to BNP (brain-type natriuretic peptide) in left ventricular remodeling [8].

Although the present study demonstrated that pCK-HGF-X7 increased capillary density at infarct border zone and decreased the remodeling process by increasing stroke volume in a large animal model, there are limitations that must be recognized. First, it raised the question of whether the timing of gene injection (4 weeks after infarction) in this study was appropriate. Based on serum levels, HGF peaks 48 h after myocardial infarction [25]. The protective effect could be maximal just before or immediately after the infarction. However, the level of HGF in the infarct territory 4 weeks after infarction was still too high to prevent any later remodeling [8]. Because remodeling was effectively achieved in the pCK-Null group after 4 weeks of coronary artery ligation, we adopted 4 weeks to use as the appropriate injection time in order to expect the angiogenic effect as well as the anti-remodeling effect of the naked DNA expressing two isoforms of HGF. Second, although changes of the LVEDV, LVESV, and LVEF were significant in each group, these changes failed to be statistically significant. This might result from small sample size in this large animal study. Third, the dose of 1 mg divided in 5 injection sites might be not enough for effective therapeutic angiogenesis. A large scale dose-increasing study should be warranted.


   Footnotes
 
{star} This work was supported by grants from Seoul National University Hospital (04-2004-064-0), The Korean Society of Circulation (06-2004-149-2), ViroMed Limited, Republic of Korea (06-2004-047-0), and the Korean Ministry of Commerce, Industry and Energy (10020770).


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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