Previous studies have shown the beneficial effects of the hepatocyte growth factor (HGF) gene on myocardial perfusion and infarction size but not on the regional strain in relationship to global left ventricular function. A noninvasive magnetic resonance (MR) study was performed to determine the effect of a new HGF gene, VM202, expressing two isoforms of HGF, on regional and global left ventricular function. Pigs (8/group) were divided into three groups: 1) controls without infarction; 2) reperfused, infarcted controls; and 3) infarcted, treated (1 h after reperfusion) with VM202 injected at eight sites. Cine, tagging, and delayed enhancement MR images were acquired at 3 and 50 ± 3 days after infarction. At 50 days, ejection fraction in infarcted, treated animals increased (38 ± 1% to 47 ± 2%, P < 0.01) to the level of controls without infarction (52 ± 1%, P = 0.16) but decreased in infarcted controls (41 ± 1% to 37 ± 1%, P < 0.05). Two-dimensional strain improved in remote, peri-infarcted, and infarcted myocardium. Furthermore, the infarction size was smaller in infarcted, treated animals (7.0 ± 0.5%) compared with infarcted controls (13.2 ± 1.6%, P < 0.05). Histopathology showed a lack of hypertrophy in myocytes in peri-infarcted and remote myocardium and the formation of islands/peninsulas of myocytes in infarcted, treated animals but not in infarcted controls. In conclusion, the plasmid HGF gene caused a near complete recovery of ejection fraction and improved the radial and circumferential strain of remote, peri-infarcted, and infarcted regions within 50 days. These beneficial effects may be explained by the combined effects of a speedy and significant infarct resorption and island/peninsulas of hypertrophied myocytes within the infarcted territory but not by compensatory hypertrophy. The combined use of cine and tagging MR imaging provides valuable information on the efficacy of gene therapy.
- gene therapy
- heart failure
- magnetic resonance imaging
an accurate assessment of regional and global left ventricular (LV) function by imaging is clinically important in ischemic heart disease. Ejection fraction (EF) and end-systolic volume (ESV) are important predictors of survival and are used to manage therapy (51). Echocardiography is the most widely used technique for noninvasive imaging of LV function because of its efficacy, relatively low cost, portability, and widespread availability. The limitations of two-dimensional echocardiography include the need for adequate acoustic windows, operator dependence, and the use of geometric assumptions in computing LV volumes. Gated single-photon emission-computed tomography (SPECT) is also used for noninvasive imaging of regional and global LV function, but it has relatively low spatial and temporal resolution. Although echocardiography and SPECT are the most popular modalities for imaging the heart, magnetic resonance (MR) imaging (MRI) is more sensitive for the evaluation of LV function in patients and is used as a reference method (2, 6, 17).
Gene and stem-cell therapy are under investigation as new therapies to prevent the deterioration of LV function after myocardial infarction (36, 43). Hepatocyte growth factor (HGF) has been under investigation because of its combined angiogenic (29) and antifibrotic effects (1, 47, 48). Several studies have recently shown the beneficial effects of HGF gene therapy in animal models of ischemic heart disease (7, 19–21, 23, 24, 42). The effect of different gene therapies on EF has been shown, but the mechanism for the increase in EF has not been defined (10, 39, 44). Investigators have attributed the increase in regional function to 1) compensatory hypertrophy of remote myocardium (44) and 2) viable myocytes within the infarct (13, 39).
LV volumes, EF, regional wall thickness, systolic wall thickening (a measure of radial strain), and mass can be measured using cine MRI. Furthermore, tagging MRI provides detailed information on circumferential and peak strain during the cardiac cycle (11, 12, 25, 32, 33).
Recently, a new plasmid (VM202) expressing two isoforms of the HGF gene became available and was tested (39). The effects of this novel therapy on regional LV function (systolic wall thickening-radial strain and circumferential strain) and global LV function (LV volumes, EF, and mass) have not been previously determined using serial MRI. Accordingly, we performed a noninvasive MR study to determine whether gene therapy expressing two isoforms of the HGF gene could improve regional and global LV function in a swine model of reperfused myocardial infarction. Histopathology was used to confirm the effects of therapy at the cellular level.
The study conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No 85-23, Revised 1996) and was approved by the Institutional Committee on Animal Research. Pigs (n = 24, weight 30–40 kg) were premedicated by ketamine (20 mg/kg, Ketaset; Fort Dodge Laboratories; Fort Dodge, IA), xylazine (2 mg/kg; Anased; Lloyd; Shenandoah, IA), and atropine (0.04 mg/kg; Phoenix; St. Joseph, MO), anesthetized using a mixture of 1.8–2.5% isoflurane (IsoFlo; Abbott; North Chicago, IL) and oxygen, and intubated as previously described (37). The animals (n = 24) were divided into three groups: 1) controls without infarction (n = 8); 2) infarcted controls (n = 8); and 3) infarcted, treated animals with a gene expressing two isoforms of HGF (VM202) (n = 8). After thoracotomy, the pericardium was opened and the left anterior descending coronary artery (LAD) was identified and occluded for 2 h distal to the first diagonal branch, followed by reperfusion. In group 3, the HGF gene (2 mg in 4 ml physiological saline solution) was slowly injected into four peri-infarction sites and four infarction sites as previously described (39). The chest was closed layer by layer, and the animals were allowed to recover for 3 days before the first imaging session. Four additional animals died during the LAD occlusion/reperfusion.
VM202 (ViroMed; Seoul, Korea) was used for HGF gene therapy. VM202 is a genomic-cDNA hybrid of the human HGF gene, which expresses two isoforms of HGF, namely HGF and dHGF by way of alternative splicing. For the construction of VM202, HGF-X7 was inserted into the pCK DNA vector. The components of the plasmid HGF gene (VM202) and their function have been described in earlier publications (39, 41).
A 1.5-T MR clinical scanner (Philips Medical Systems) was used in all animals. Noninfarcted control animals were imaged once and infarcted animals twice 3 ± 0 days (acute phase) and 50 ± 3 days (chronic phase) after the surgical procedure. In short, cine, tagging, and delayed enhancement (DE) MR images were obtained of the left ventricle (18).
MRI protocols and parameters.
After the initial scout imaging to obtain the short- and long-axis imaging planes, a steady-state free precession cine sequence was used to image the whole LV in the short-axis view. Imaging parameters for the cine images were as follows: repetition time/echo time/flip angle = 3.5 ms/1.75 ms/70°; slice thickness = 10 mm, no slice gap; field of view (FOV) = 25 × 25 cm; matrix size = 160 × 152; and heart phases = 16. Cine images were used for the assessment of LV volumes, EF, wall thickness and thickening, and LV mass. A tagged turbo-field echo planar (TF-EPI) sequence, obtained in the short-axis view covering the whole left ventricle, was used. Imaging parameters for the tagged cine images were as follows: repetition time/echo time/flip angle = 35 ms/6.1 ms/25°; slice thickness = 10 mm, no slice gap; FOV = 24 × 24 cm; matrix size = 128 × 45; heart phases = 16; EPI factor = 11; tag technique = complementary spatial modulation of magnetization with vertical and horizontal tag orientation obtained in one breath hold; and line spacing = 8 mm. Delayed enhancement (DE) MRI (IR-GRE) in the short-axis view was used to locate and size the infarct so that the functional assessment could be performed in infarcted, peri-infarcted, and remote myocardium. DE-MRI was acquired 20 min following 0.15 mmol/kg of Gd-DOTA administration (Guerbet Group; Paris, France). The imaging parameters were repetition time/echo time/flip angle = 5 ms/2 ms/15°; slice thickness = 3 mm, no slice gap; FOV = 26 × 26 cm; matrix size = 256 × 162; and the inversion time was chosen to null normal myocardium (270–325 ms).
Global function was assessed as LV volumes, EF, and LV mass by manual delineation of the endocardial and epicardial contours at end systole and end diastole in cine images, using the freely available software Segment v1.6 (16) (http://segment.heiberg.se). LV volumes and mass were normalized to body weight to compare the results at the acute (32.3 ± 0.5 kg) and chronic phase (48.4 ± 1.4 kg).
Regional LV function was quantified in three consecutive apical, mid, and basal short-axis slices through the infarcted myocardium using the centerline method. In each of these images, a spoke wheel was placed in the center of the cavity, dividing the myocardium into eight regions starting with region 1 at the posteroseptal groove and continuing clockwise (Fig. 1). The quantification of regional thickening was performed in 960 regions (8 per slice; 3 slices; 16 animals imaged twice, and 8 animals imaged once). The peri-infarcted region was defined as 5 mm adjacent to the infarct in the anterior wall.
Cine MRI was used to determine systolic and diastolic wall thickness. Regional systolic wall thickening was determined as (end-systolic wall thickness − end-diastolic wall thickness)/(end-diastolic wall thickness)·100. Tagged MR images in the same three short-axis planes as for wall thickening were used for strain analysis. The circumferential strain was calculated for all regions, but only the remote, peri-infarct, and infarcted regions are shown for clarity. Midwall eulerian circumferential shortening was measured using the HARP software (Diagnosoft, Palo Alto, CA) (12, 32, 33), and peak circumferential strain was calculated. Peak strain values are negative during systole in normal myocardium because they express circumferential shortening. Infarct transmurality was calculated as the percentage of hyperenhanced myocardium in eight segments each for the apical, mid, and basal slices used for regional function analysis by Segment v1.6 (9).
The infarcted animals were euthanized after the second imaging session (50 ± 3 days after infarction), and the hearts were excised. The LV was sliced into 10-mm short-axis slices, weighed, and stained by 2% triphenyltetrazolium chloride (TTC) for confirmation of infarction. Tissue samples were obtained from the infarcted, peri-infarcted, and remote normal myocardium and embedded in paraffin, sliced (5 μm), and stained with hematoxylin and eosin and Masson's trichrome. Myocyte diameters were measured at ×400 magnification using a calibrated reticule in the ocular of the microscope (Olympus). The myocyte diameters were measured at the nuclear level from transversely cut cells in islands/peninsulas of myocytes within the scar tissue, peri-infarcted, and remote normal myocardium.
Continuous variables are presented as means ± SE. Wilcoxson matched pairs test was used to compare LV volumes, LV EF, and peak systolic strain at the acute and chronic phase in the same group of animals. The Mann-Whitney test was used to compare LV volumes, LV EF, and peak systolic strain between the groups. Two-tailed unpaired Student's t-test was used to compare myocyte diameter and two-tailed paired Student's t-test within the groups. A value of P < 0.05 was considered statistically significant.
Global LV Function on Cine MR
EF in infarcted animals at 3 days did not differ between infarcted, treated (38 ± 1%) and infarcted, control animals (41 ± 1, P = 0.13) (Fig. 2). Both groups had significantly lower EF compared with controls without infarction (52 ± 1%, P < 0.001 compared with both groups). However, at 50 ± 3 days after infarction, the EF of the infarcted, treated animals had increased to 47 ± 2% (P < 0.01 compared with 3 days, and P = 0.16 compared with controls without infarction) and decreased to 37 ± 1% in the infarcted controls (P < 0.05 compared with 3 days). At 50 days, the EF of infarcted, treated animals was markedly higher than in infarcted controls (47 ± 2% vs. 37 ± 1%, P < 0.001). The increase in EF in infarcted, treated animals over time was associated with a decrease in end-diastolic volume (EDV) (2.15 ± 0.10 vs. 1.73 ± 0.10 ml/kg, P < 0.05) and ESV (1.33 ± 0.07 vs. 0.92 ± 0.08 ml/kg, P < 0.01) (Fig. 2). In infarcted controls, ESV increased from 1.36 ± 0.04 to 1.51 ± 0.05 ml/kg (P < 0.05), but EDV was unchanged (2.35 ± 0.05 vs. 2.41 ± 0.09 ml/kg; P = 0.64) (Fig. 2). The correlations between true infarct size measured by histochemical TTC and global MR functional parameters are shown in Fig. 3. The correlations show that EF decreased with increasing true infarct size (r = 0.64, y = −1.0x + 50.7, P = 0.007) and that EDV (r = 0.66, y = 0.069x + 1.43, P = 0.0055) and ESV (r = 0.72, y = 0.0615x + 0.647, P = 0.0017) increased with increasing true infarct size with TTC. Normalized LV mass did not differ between the infarcted, treated and the infarcted, control groups at 3 days (2.43 ± 0.09 vs. 2.65 ± 0.09 g/kg, P = 0.08) or 50 days (2.16 ± 0.08 vs. 2.32 ± 0.10 g/kg, P = 0.23). Normalized LV mass in infarcted, treated animals at 50 days was not significantly different compared with controls without infarct (2.0 ± 0.1 g/kg, P = 0.10); however, normalized LV mass in infarcted controls were significantly higher (P < 0.05).
Regional LV Function on Cine MR
MR images at end-diastolic and end-systolic phases are shown in Fig. 4. Systolic wall thickening was decreased in the acute phase in infarcted and peri-infarcted regions compared with controls without infarction (Fig. 5). However, there was also a trend, but not significant, toward decreased systolic wall thickening in remote myocardium compared with controls without infarction. At 50 days, systolic wall thickening in remote myocardium in infarcted, treated animals did not differ significantly compared with controls without infarction. This was in contrast to the remote myocardium of infarcted controls, which had lower wall thickening, resulting in LV dysfunction. Wall thickness of the remote myocardium in the basal, mid, and apical part was similar between the infarcted, treated (8.1 ± 0.3, 7.5 ± 0.3, and 6.8 ± 0.2 mm, respectively) and the infarcted, control groups (9.0 ± 0.3, 8.3 ± 0.4, and 7.0 ± 0.6 mm, respectively) 50 days after infarction (P = 0.12, 0.33, and 0.87, respectively).
Regional LV Function on Tagging MR
Circumferential strain differed between the groups throughout the cardiac cycle as demonstrated in Fig. 6. Strain values are negative during systole in normal myocardium because they express circumferential shortening. The peak strain of the remote, peri-infarct, and infarcted regions for the basal, mid, and apical slices for all animals are shown in Fig. 7. At 3 days, the peak strain was not significantly different between controls without infarction and the infarcted, control and the infarcted, treated animals in remote myocardium. On the other hand, peak strain was significantly lower in the peri-infarcted (P < 0.01) and infarcted (P < 0.01) regions in both the infarcted, treated and the infarcted, control animals compared with control animals without infarction. At 50 days, the peak strain was still significantly lower in peri-infarcted and infarcted regions (P < 0.001) in infarcted, control animals compared with control animals without infarction. A small, but significant, increase in peak strain could be seen in the infarcted regions in the infarcted controls (P < 0.05). However, the increase in peak strain was more pronounced in the peri-infarcted (P < 0.05) and infarcted regions (P < 0.001) in the infarcted, treated animals. Moreover, the peak strain in the infarcted region of treated animals occurred late in the cardiac cycle compared with normal animals. Unlike infarcted, control animals, there was a significant increase (P < 0.01) in peak systolic strain in basal and apical slices of remote myocardium at 50 days in infarcted, treated animals.
Multislice MR images from infarcted, control and infarcted, treated animals are shown in Fig. 8. The infarcts were seen as hyperenhanced regions in the LV anteroseptal wall in all animals. As previously reported (39), the decrease in infarct size was greater in the infarcted, treated animals compared with infarcted controls. At 50 days, the infarct decreased in both infarcted, treated (16.5 ± 1.8% to 7.0 ± 0.5% of LV mass, P < 0.05) and infarcted, control animals (16.6 ± 1.1% to 13.2 ± 1.6% LV mass, P < 0.05) but to a greater extent in treated animals (P < 0.05). The transmurality of the infarction in the three slices used for cine and tagging analysis are shown in Fig. 9. Over time, the transmurality of the infarction decreased in both groups; however, the decrease was greater in the infarcted, treated animals. Also, an endocardial rim of viable tissue was seen in the basal part of the infarct (Fig. 9).
Histochemical TTC and histological staining confirmed the formation of infarction and scar tissue in animals that underwent occlusion of the LAD. Myocardial TTC sections confirmed the lower transmurality of the infarction seen on MRI in the infarcted, treated animals compared with the infarcted controls (Fig. 10). In the infarcted, treated animals, viable myocardium was interspersed with infarcted tissue and, in the basal slice of the infarction, an endocardial rim of viable myocardium could be seen both on MR and TTC images. There was a high correlation between the quantification of infarct size with MR and TTC (r = 0.92, y = 0.94x + 1.3, P < 0.001, Fig. 3).
Histopathology showed that the myocyte diameter was significantly smaller in the peri-infarcted (21 ± 1 μm) and remote myocardium (19 ± 1 μm) of the infarcted, treated animals compared with the infarcted controls (26 ± 1 and 25 ± 1 μm, respectively; P < 0.001 for both) (Fig. 10). In the infarcted, treated animals, the peninsulas and islands/peninsulas of viable myocytes were present within the infarct (Fig. 10). The diameter of these myocytes was 29 ± 1 μm, significantly larger than peri-infarcted and remote myocardium of the infarcted, treated animals (P < 0.001 for both).
The main findings are that 1) intramyocardially transferred plasmid HGF gene (VM202) caused near complete recovery of EF within 50 days of infarction; 2) the therapy prevented LV remodeling associated with infarction; 3) the improvement in regional and global function in treated animals is most likely attributed to the speedy and significant resorption of infarction and island/peninsulas of hypertrophied myocytes within the infarcted territory; 4) treated animals showed no evidence of compensatory hypertrophy in remote myocardium as shown in cine MR measurement of LV mass, wall thickness, or microscopic measurement of myocyte diameter compared with the infarcted, control animals; and 5) the complimentary use of cine and tagging MRI provides valuable information on the efficacy of gene therapy in ischemic heart disease; thus it can be used for noninvasive assessment of gene or cell therapy.
The importance of circumferential strain and systolic wall thickening (radial strain), using myocardial tagging and cine MRI, in patients with myocardial infarction or ischemic cardiomyopathy has been recently demonstrated (4, 14). Cine or tagging MRI, however, is often used in the context of an integrated MR examination, where MR sequences for imaging myocardial anatomy, perfusion, function, and infarction can all be readily used and provide complimentary information (14, 26, 35). Accordingly, in our previous study, we reported the beneficial effects of intramyocardial injection of the HGF gene (VM202) on myocardial perfusion and angiogenesis (39). The current MR study is a continuation of the previous work where the impact of intramyocardial injection of the HGF gene on the changes in regional and global LV function (remodeling indexes), LV mass, and natural infarct resorption were investigated. In the current study, the EF, systolic wall thickening, and strain were significantly reduced in all infarcted animals (n = 16) compared with controls without infarction (n = 8) on the third day after infarction. At 50 days, the infarcted, treated animals showed marked improvement in EF, systolic wall thickening, and circumferential strain compared with the infarcted controls. The improvement of EF and the decline in LV volumes indicate the prevention of LV remodeling by HGF gene therapy. Interestingly, peak systolic strain of infarcted myocardium in treated animals occurred late in the cardiac cycle, a contraction pattern associated with ischemic, but viable, myocardium (5, 46). The improvement in wall thickening and strain seen in treated animals can be attributed to 1) the speedy infarct resorption in treated animals resulting in a nontransmural infarction and 2) the presence of viable myocyte islands/peninsulas within the scar tissue. The improvement in LV function, however, was not linked to compensatory hyperfunction or hypertrophy in remote myocardium as shown by the lack of increase in LV mass, wall thickness, or cellular diameters on histology. These findings differ from previous findings using intracoronary injection of adenoviral gene-transferred FGF-5 to hibernating myocardium (28, 44), where the investigators observed a massive increase in LV mass at 2 (29% increase) and 4 wk (55% increase) after intracoronary FGF therapy.
Previously, we have shown the beneficial effects of intramyocardial injection of adeno-associated viral vector-encoding vascular endothelial growth factor (AAV-VEGF) gene into peri-infarcted and infarcted myocardium, using the same animal model, surgical procedure, and MRI protocol (18, 40). Intramyocardial injection of the AAV-VEGF gene into swine myocardium decreased infarction size, increased regional perfusion, and increased regional function in treated animal compared with infarcted controls. However, the AAV-VEFG gene did not increase the global function EF over time or cause the formation of viable islands/peninsulas in the infarcted myocardium. Furthermore, in a canine study, Ferrarini et al. (10) used the AAV-VEGF gene and found that the intramyocardially injected gene improved regional but not global function and that the therapy enhanced the formation of viable myocardium in the infarcted region. Similar findings were observed in sheep (50). Unlike AAV-VEGF gene therapy (10, 18, 40), the HGF gene in the present MR study showed a substantial improvement in both regional and global function over time and prevented the LV remodeling associated with myocardial infarction (8, 34). Therefore, the effects of HGF gene therapy on the restoration of LV function are more pronounced compared with VEGF gene therapy, and in this respect our findings support the notion of using HGF gene in the future design of clinical studies (29).
In the current study, the HGF gene caused a significant reduction in infarct size and transmurality. In a clinical MR study, Kim et al. (22) found that the transmural extent of infarction is predictive of post-revascularization functional recovery independent of LV function. They also found a close correlation between the transmural extent of infarction and a lower likelihood of functional recovery.
Unlike FGF or VEGF, HGF is characterized by its antifibrotic effect (1, 47, 48). The antifibrotic mechanism of HGF has been shown to be mediated through 1) the inhibition of synthesis of extracellular matrix via the inhibition of TGF-β expression and 2) the stimulation of degradation of extracellular matrix through the activation of matrix metalloprotease-1 and urokinase-like plasminogen activator (47, 49). Previous studies have shown that HGF acts on a molecular level by reducing the production of oxygen free radicals during ischemia and reperfusion, which could trigger necrosis (7), and enhances the expression of Bcl-2 (7, 15) and Bcl-XL (31), known inhibitors of apoptosis. These antifibrotic and antiapoptic effects of HGF gene treatment may explain the greater decrease in infarct transmurality in the infarcted, treated group compared with the infarcted controls in our study.
The angiogenic properties of HGF have been shown to be explained by a mitogenic action as well as an accelerated regeneration of the endothelial cells (30). Morishita et al. (30) found that HGF stimulates the migration of vascular smooth muscle cells to the sprouting new vessels. The effect on vascular smooth muscle cells may explain why HGF, as opposed to VEGF, does not increase vascular permeability and, as a consequence, does not cause oedema formation (29). A recent study showed for the first time that postischemic myocardial dysfunction (stunning) results from myofibrillar edema (3).
The mechanisms of formation of islands/peninsulas of viable myocytes in the peri-infarcted region may be explained by findings in a recent rat study by Gonzales et al. (13). They found that the administration of IGF-1 and HGF modified cardiac progenitor cells behavior. The study showed myocardial regeneration from cardiac progenitor cells forming myocytes, coronary arterioles, and capillaries. Interestingly, the investigators stated that “clusters of regenerated myocytes replaced foci of myocardial damage.” Thus the presence of viable myocytes at the peri-infarcted region can be explained by HGF rejuvenating cardiac progenitor cells to form new viable myocytes.
In the present study, intramyocardial injections of the HGF gene were performed at eight sites to cover most of the ischemic territory to ensure fair distribution of the therapy. Furthermore, the size of VM202 is relatively low (7,376 base pairs), and the gene is therefore expected to distribute by diffusion transport in the tissue. The intramyocardial delivery approach in an open-chest model has been previously used (13). We recognized that the inferoseptal wall cannot be reached using open-chest models; therefore, we recently developed a percutaneous transendocardial route for gene delivery (38).
Limitations of the present study are the lack of 1) measurement of gene expression (HGF protein), 2) placebo injections, and 3) noninvasive visualization of new collateral vessels. The gene expression was not in the scope of the present MR study. It should be noted, however, that the expression of HGF protein in plasma and tissue has been well documented in several earlier studies (1, 7, 27) and that individual levels of angiogeneic protein are quite variable (45).
In conclusion, intramyocardially transferred plasmid HGF gene caused almost a complete recovery of EF within 50 days of infarction; prevented LV remodeling; and increased radial (systolic wall thickening) and circumferential strain of remote, peri-infarcted, and infarcted regions. This may be explained by the combined effects of a speedy and significant infarct resorption and island/peninsulas of hypertrophied myocytes within the infarcted territory but not by compensatory hypertrophy. The combined use of cine and tagging MRI provides valuable information on the efficacy of gene therapy.
This work was supported by a National Heart, Lung, and Blood Institute Grant RO1-HL-07295 and a gift from ViroMed (Seoul, Korea).
We thank Loi Do for excellent technical support.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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