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DOI: 10.1148/radiol.2451061077
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(Radiology 2007;245:196-205.)
© RSNA, 2007

Molecular Imaging

Injection of Adeno-associated Viral Vector–Encoding Vascular Endothelial Growth Factor Gene in Infarcted Swine Myocardium: MR Measurements of Left Ventricular Function and Strain1

Alexis Jacquier, MD, Charles B. Higgins, MD, Alastair J. Martin, PhD, Loi Do, BS, David Saloner, PhD, and Maythem Saeed, DVM, PhD

1 From the Department of Radiology, University of California San Francisco, 513 Parnassus Ave, HSW 207 B, San Francisco, CA 94134-0628. From the 2006 RSNA Annual Meeting. Received June 20, 2006; revision requested August 23; revision received October 25; accepted November 22; final version accepted February 12, 2007. Supported by grant RO1HL07295 from National Institutes of Health. A.J. is a research fellow supported by a postdoctoral research grant from the Société Française de Radiologie, Paris, France. Address correspondence to M.S. (e-mail: Maythem.Saeed{at}radiology.UCSF.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Purpose: To prospectively investigate the long-term effect of adeno-associated viral (AAV) vector–encoding vascular endothelial growth factor gene (VEGF) (AAV-VEGF) on left ventricular (LV) mass and volumes, as well as on regional contractility and circumferential strain, in a swine model of reperfused myocardial infarction.

Materials and Methods: All experimental procedures received approval from the institutional committee on animal research. Of 16 pigs subjected to reperfused myocardial infarction, six were treated, six were controls, and four died during the ischemic intervention. In six animals, cardiac-specific AAV-VEGF was injected into the periinfarcted and infarcted myocardium 1 hour after reperfusion. Magnetic resonance (MR) imaging was performed at 3 days and 8 weeks after infarction by using cine, tagged, and delayed enhancement (with gadoterate meglumine) sequences to measure global and regional LV function and infarct size. At postmortem examination, tissue samples stained with isolectin B4, Masson trichrome, and hematoxylin-eosin were used to characterize injured myocardium. Two-tailed Student t test was used for statistical analysis.

Results: Six treated animals showed no change in mean LV ejection fraction after 8 weeks (40.3% ± 0.9 [standard error of the mean] vs 41.0% ± 0.7) in contrast to a decrease measured in six control animals (41.4% ± 0.7 vs 36.1% ± 0.6, P < .001). AAV-VEGF improved wall thickening and circumferential strain in periinfarcted and remote myocardium. A greater reduction in gadoterate meglumine–enhanced infarct area was measured in treated animals (18.6% ± 1.5 of the LV mass at 3 days vs 9.8% ± 1.0 of the LV mass at 8 weeks, P < .001) compared with control animals (17.7% ± 2.0 vs 14.8% ± 1.0, P = .008). Findings at histopathologic evaluation indicated an increase in vascular density and a decrease in myocyte diameter in the periinfarcted myocardium of treated, compared with control, animals.

Conclusion: Angiogenesis and arteriogenesis induced by VEGF genes improved regional myocardial strain and wall thickening and preserved ejection fraction after infarction.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Revascularization of acute myocardial infarction has significantly reduced mortality; however, left ventricular (LV) remodeling and heart failure remain as chronic sequelae (1). The major structural changes in hearts subjected to coronary occlusion include the following: (a) replacement of necrotic myocardium with scar tissue (2), (b) hypertrophy of periinfarcted myocardium (3), and (c) vascular and cellular remodeling (4,5). Furthermore, LV remodeling modifies three myocardial compartments, namely, myocytes, extracellular matrix, and blood vessels. Sequential magnetic resonance (MR) imaging has demonstrated progressive LV dilatation and heart failure after infarction (6). Various treatments such as angiotensin-converting enzyme inhibitors (7), calcium blockers (8), and, more recently, vascular growth factors, genes (6), and cells (9) have been used to reduce or stabilize LV remodeling and improve LV function after acute infarction.

Researchers in several studies have demonstrated that vascular endothelial growth factor (VEGF), proteins or vector encoding the genes, is one of the most specific angiogenic growth factors (10–12). Results of some studies indicate that VEGF proteins and VEGF genes improve global LV function (11,12), but those of other studies were unable to confirm these results (13,14). The lack of beneficial effects in these studies could be attributed to insufficient dose, improper route of injection, or short plasma half-life of VEGF proteins. The purpose of this study was to prospectively investigate the long-term effect of adeno-associated virus (AAV) vector–encoding VEGF gene (AAV-VEGF) on LV mass and volumes, as well as on regional contractility and circumferential strain, in a swine model of reperfused myocardial infarction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
MR Contrast Medium
The manufacturer provided the extracellular MR contrast medium gadoterate meglumine (Dotarem; Guerbet Group, Paris, France), with a concentration of 0.5 mmol/mL, in 20-mL vials. No member of the Guerbet Group is part of the authorship or had control of the study data.

Animal Preparation
All experimental procedures received approval from the institutional committee on animal research and were performed in accordance with the National Institutes of Health guidelines for care and use of laboratory animals. Sixteen pigs that weighed 30–36 kg (Hampshire; BRK Power Farms, Turlock, Calif) were premedicated by using 20 mg per kilogram body weight of ketamine (Ketaset; Fort Dodge Labs, Fort Dodge, Iowa), 2 mg/kg xylazine (AnaSed; Lloyd Labs, Shenandoah, Iowa), and 0.04 mg/kg atropine (Phoenix Labs, St Joseph, Mo), anesthetized by using a mixture of 1.8%–2.5% isoflurane (IsoFlo; Abbott Laboratories, North Chicago, Ill) and oxygen, and intubated as described previously (15). Electrocardiogram, heart rate, arterial pressure, and oxygen saturation were constantly monitored. After thoracotomy, the pericardium was opened and the heart was suspended in a pericardial cradle. The middle portion of the left anterior descending coronary artery was dissected free from surrounding tissue and was encircled by a suture. The two ends of the suture were threaded through a plastic tubing, forming a snare, and were cross-clamped after tightening to achieve coronary artery occlusion. One investigator (M.S.) performed occlusion of the artery for 120 minutes to induce transmural infarct, and this procedure was followed by reperfusion (16).

Four animals died as a result of arrhythmias during occlusion or reperfusion but prior to therapeutic injection. Selection of treated or control animals was performed in a random fashion. In six animals, AAV-VEGF (human VEGF165 complementary DNA) was injected into the border and core of the ischemic area 1 hour after reperfusion. Eight injections of 500 µL each were performed, and a 2-cm gap was allowed between each injection to cover the entire ischemic area. Each injection of AAV-VEGF was delivered into the epicardium at 3-mm depth by using a polypropylene syringe and a stainless steel needle (30 gauge) by one investigator (M.S.). The other six pigs underwent the same surgical intervention but did not receive AAV-VEGF injection and served as control animals. After the coronary intervention, the pericardium and chest were closed and the animals were allowed to recover.

Adeno-associated Viral Vector–Encoding VEGF Gene
The vector–encoding VEGF gene was synthesized (Quiagen, Valencia, Calif) as previously described (17,18). Cardiac myosin light chain 2V promoter was used to derive genes for a human VEGF isoform, VEGF165 complementary DNA and LacZ. Approximately 1011 copies of the AAV-VEGF mixed with 1010 copies of AAV encoding for LacZ were injected in each animal (17).

MR Imaging
All imaging was performed by one investigator (A.J.M.) with a 1.5-T MR imager (Intera; Philips Medical Systems, Best, the Netherlands). A five-element phased-array cardiac coil was wrapped around the chest. At 3 days and 8 weeks after infarction, all 12 surviving animals were imaged after anesthesia was induced. Three MR sequences were used in the assessment of global and regional function and structure. Cine steady-state free precession sequences were performed on a short axis for regional and global LV functional analysis.

The following parameters were used: repetition time msec/echo time msec, 8/5; flip angle, 20°; section thickness, 10 mm; spacing, zero; field of view, 24 x 24 cm; matrix, 256 x 128; heart phases, 16. A short-axis tagged turbo field-echo echo-planar sequence was used for regional strain analysis. Imaging parameters were as follows: 35/6.1; flip angle, 25°; section thickness, 10 mm; field of view, 24 x 24 cm; matrix, 128 x 45; heart phases, 16; echo-planar imaging factor, 11; tag technique, complementary spatial modulation of magnetization; horizontal and vertical tag orientation in one breath hold; line spacing, 8 mm; image time, 18 seconds per section at a heart rate of 70 beats per minute. Inversion-recovery gradient-echo images were acquired 20 minutes after administration of a 0.1 mmol/kg dose of gadoterate meglumine. The imaging parameters were repetition time msec/echo time msec/inversion time msec, 4.4/2.1/270–325; flip angle, 15°; section thickness, 10 mm; spacing, zero; field of view, 24 x 24 cm; and matrix, 152 x 134. The inversion time was set to null the signal intensity of normal myocardium.

MR Image Analysis
LV volumes, ejection fraction, and mass were determined by tracking the endocardial and epicardial contours in a semiautomated fashion at the end-diastolic and end-systolic phases by using a software and hardware package (ViewForum; Philips Medical Systems). LV volume–body weight (milliliters per kilogram) and mass–body weight (grams per kilogram) ratios were used to account for the variations in body weight between 3 days (30–36 kg) after infarction and 8 weeks (47–57 kg) after infarction. Anatomic landmarks such as right ventricular insertion and papillary muscle location were used to coregister cine, tagged, and delayed enhancement images (16). Three consecutive short-axis sections (namely, apical, middle, and basal sections) were selected in cine and tagged sequences to cover the infarct area.

Systolic wall thickness and circumferential strain were analyzed at 3 days and at 8 weeks after infarction by three investigators (A.J., L.D., and M.S., with 4, 2, and 18 years of experience in cardiac MR imaging, respectively). For wall thickness analysis, a 0° starting point was defined at the posteroseptal groove for each cine sequence: Eight segments (45° each) of each section were numbered in a clockwise direction by using the software and hardware package mentioned before. At the workstation, we calculated the two-dimensional mean centerline of the eight LV segments (45° per segment) at the end-diastolic and end-systolic phases. A total of 576 segments (eight segments per section, three sections per heart, and 12 pigs imaged at 3 days and 8 weeks after infarction) were measured and included all animals at 3 days and 8 weeks. Percentage of systolic wall thickening was calculated as follows: [(Wes – Wed)/Wes]·100, where Wes is end-systolic wall thickness and Wed is end-diastolic wall thickness.

For strain analysis, the myocardium was divided into three regions, namely, infarcted (contrast material–enhanced area), remote normal (opposite wall of the infarct), and periinfarcted (rim) myocardium on the basis of delayed enhancement. By using the postprocessing software (HARP; Diagnosoft, Palo Alto, Calif), middle wall circumferential shortening and middle wall end-systolic eulerian circumferential shortening (peak systolic strain) were measured in these three regions (19). Peak systolic circumferential strain was expressed as the difference between end-systolic and end-diastolic dimension divided by a reference end-diastolic dimension and presented as a percentage value.

The middle wall circumferential shortening represented the value of the circumferential strain for each cardiac phase and was expressed as a percentage. Peak systolic strain values are typically negative during systole because they express circumferential shortening. Peak systolic strain values that were less negative indicate a diminished regional LV contractility. The extent of infarct, expressed as a percentage of LV mass, was measured from delayed enhancement MR images at 3 days and 8 weeks. A threshold value of +2 standard deviations above the mean signal intensity of normal myocardium was used to define the voxels consisting of infarcted myocardium. Cardiac MR measurements were performed in consensus for LV volumes, mass, visual matching, wall thickening, and strain (A.J., L.D., M.S.).

Postmortem Evaluation
The animals were sacrificed after the second imaging session at 8 weeks by using an overdose of sodium pentobarbital, and the hearts were excised. The LV was trimmed, weighed, and sliced transversely into 10-mm slices, the same thickness as MR image sections. The slices were stained by soaking them in 2% triphenyltetrazolium chloride for 10 minutes at 37°C to stain viable myocardium. Tissue samples were obtained from the infarcted, remote normal, and periinfarcted myocardium and fixed in 10% formalin. The samples were embedded in paraffin, sliced (5 µm), and stained with hematoxylin-eosin, Masson trichrome, and biotinylated Bandeiraea simplicifolia isolectin B4 (hereafter referred to as isolectin) stains (15).

Capillaries were defined at isolectin staining as vessels with diameters that were smaller than 10 µm with a single layer of endothelial (brown) cells. Capillary density was calculated on 10 fields at x400 magnification at isolectin staining in the infarcted, remote normal, and periinfarcted myocardium and was expressed in capillaries per square millimeter. Arterioles were defined at Masson trichrome staining as vessels with a lumen of 10–100 µm in diameter and a smooth muscle wall (20). Vascular counts were performed by using software (Openlab 4.0.2; Improvision, Coventry, England) on 10 fields at x200 magnification at Masson trichrome staining and expressed as a number of arterioles per square millimeter. The wall-lumen ratio was calculated by dividing the diameter of the whole vessel by the diameter of the lumen. The cardiomyocyte diameters in periinfarcted and remote normal myocardium were measured at the nuclear level from transversely cut cells (21). In each myocardial region, 20 fields were examined and 200 myocytes were measured per region (22). Analyses were performed with blinding to the identity of the animal group (A.J.).

Statistical Analysis
Because of the institutional constraint on animal numbers, we used the minimum number of animals required to obtain results that were statistically significant. Similar numbers of animals were used in a recent study in which another VEGF gene injected into myocardium was tested (11). All values are given as mean ± standard error of the mean. The two-tailed paired Student t test was used to compare LV volumes, LV ejection fraction, peak systolic strain, and extent in the area of delayed contrast enhancement at 3 days and at 8 weeks after infarction in the same group of animals. The two-tailed unpaired Student t test was used to compare LV volumes, LV ejection fraction, peak systolic strain, extent in the area of delayed contrast enhancement, vessel density, vessel diameter, and myocyte diameters between the two groups.

We performed a retrospective power analysis, and the statistical power was computed by using the significance level, actual difference, and sample size. Values of power analysis were assigned to all significant measurements. To produce P values that were adjusted for the multiple measurements of wall thickening gathered from each animal, generalized estimating equation analysis was used on the basis of time, image section (base, middle, and apex), and group of segments in each image section (infarcted myocardium [segments 2–4], normal myocardium [segments 6–8], and periinfarcted myocardium [segments 1 and 5]). Generalized estimating equation analysis was performed by using statistical software (Stata; Stata, College Station, Tex). All other statistical analyses were performed by using other software (StatView 5.0.1, SAS Institute, Cary, NC). A difference with P < .05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
LV Function
At 3 days, there was no significant difference in the end-diastolic and end-systolic volumes or LV ejection fraction between the control and treated animals (Figs 1, 2). Deterioration in mean ejection fraction was observed in the control animals over a period of 8 weeks, with 41.4% ± 0.7 (standard error of the mean) at 3 days versus 36.1% ± 0.6 at 8 weeks (P < .001); retrospective power analysis (RPA) yielded a power of 74%. In contrast, in the treated animals the mean ejection fraction remained stable during 8 weeks after infarction (40.3% ± 0.9 at 3 days vs 41.0 ± 0.7% at 8 weeks). Between 3 days and 8 weeks, the LV dilated in control animals, as shown by the significant increase in mean end-systolic volume from 1.31 mL/kg ± 0.04 to 1.52 mL/kg ± 0.07 (P = .04, RPA = 54% power) and a tendency toward an increase in mean end-diastolic volumes (2.24 mL/kg ± 0.05 to 2.36 mL/kg ± 0.10), without a significant difference, both of which suggested LV remodeling.


Figure 1
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  		Figure 1: Cine cardiac steady-state free precession MR images (8/5; flip angle, 20°; section thickness, 10 mm; matrix, 256 x 128; number of heart phases, 16) in middle myocardial short-axis view show changes in LV volumes and wall thickness during systole and diastole. A, Control animals. B, AAV-VEGF–treated animals. Three days after infarction, there was no difference in these parameters between groups. The difference between the groups was pronounced 8 weeks after infarction.

 

Figure 2
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  		Figure 2: Change in LV end-diastolic and end-systolic volumes and ejection fraction during 8 weeks. Left: Six control animals. Right: Six AAV-VEGF–treated animals. There was a significant decrease in end-systolic and end-diastolic volumes at 8 weeks in treated but not control animals. The ejection fraction remained the same at 8 weeks in treated animals. Differences with P = .007–.001 (*), compared with values obtained in control animals (unpaired two-tailed Student t test), and with P = .08 to < .001 (§), compared with values obtained at 3 days (paired two-tailed Student t test), were observed. Bold line = averaged data.

 
Conversely, in the treated animals there were decreases in mean end-systolic volume from 1.27 mL/kg ± 0.05 at 3 days to 1.06 mL/kg ± 0.05 at 8 weeks (P < .001, RPA = 94% power) and in mean end-diastolic volume from 2.02 mL/kg ± 0.05 at 3 days to 1.8 mL/kg ± 0.08 at 8 weeks (P < .001, RPA = 91% power). At 3 days, there was no significant difference in LV wall thickening between control and treated animals (Fig 3). At 8 weeks, treated animals showed a greater mean wall thickening value (28% ± 4) in the periinfarcted zone (segments 1 and 5), compared with that in control animals (10% ± 2, P < .001, RPA = 97% power). At 8 weeks, treated animals showed a greater mean wall thickening value of 34% ± 4 in remote myocardium (segments 6–8) compared with that in the control animals with a mean value of 19% ± 1 (P < .001, RPA = 97% power). There was no significant difference in wall thickening of the infarcted regions of the apical and middle sections between treated and control animals (segments 2–4) at 8 weeks.


Figure 3
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  		Figure 3: Changes obtained from cine MR images in segmental systolic wall thickening in six control animals (black bars) and six AAV-VEGF–treated animals (white bars). Left: At 3 days, there was no significant difference in LV wall thickening between control and treated animals. Right: At 8 weeks, there was a significant increase in wall thickening in the periinfarcted myocardium (segments 1 and 5) of the treated animals in the chronic phase. The improvement at 8 weeks was observed predominantly in the basal (top right) and middle (middle right) LV sections. A difference with P = .02 to < .001 (*), compared with values obtained in control animals by using generalized estimating equation analysis, was observed.

 
On tagged cine images, there was no significant difference in peak systolic strain between control and treated animals at 3 days. In the remote and periinfarcted myocardium, peak systolic strain was significantly greater in treated animals compared with control animals at 8 weeks (Figs 4, 5). In control animals, there was no substantial change in mean peak systolic strain during 8 weeks in the remote normal myocardium (–16.4% ± 0.7 at 3 days vs –15% ± 0.6 at 8 weeks) and periinfarcted myocardium (–7.6% ± 0.4 at 3 days vs –7.7% ± 0.5 at 8 weeks). In AAV-VEGF–treated animals, peak systolic strain increased by 10% in the remote normal myocardium, with a mean value of –16.8% ± 0.5 at 3 days versus –18.5% ± 0.5 at 8 weeks (P = .02, two-tailed paired Student t test; RPA = 64% power) and by 41% in the periinfarcted myocardium, with a mean value of –7.8% ± 0.5 at 3 days versus –11% ± 0.5 at 8 weeks (P = .02, two-tailed paired Student t test; RPA = 64% power) (Fig 5). These changes in strain were consistent with the changes in regional wall thickening in control animals and treated animals.


Figure 4
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  		Figure 4: Representative images (left and right sets) and middle wall circumferential shortening (middle plots), which signifies the value of the circumferential strain for each cardiac phase (n = 16) and is expressed as a percentage, at 8 weeks. Anatomic coregistration between images is observed. Top left and right: Inversion-recovery gradient-echo short-axis delayed contrast-enhanced MR images (4.4/2.1/270–325; flip angle, 15°; section thickness, 10 mm; matrix, 152 x 134). Middle left and right: Turbo field-echo echo-planar short-axis tagged cine MR images (35/6.1; flip angle, 25°; section thickness, 10 mm; matrix, 128 x 45; number of heart phases, 16; echo-planar imaging factor, 11; tag technique, complementary spatial modulation of magnetization; line spacing, 8 mm) delineate infarcted (A), periinfarcted (B), and remote (C) myocardium. Bottom left and right: Steady-state free precession short-axis cine MR images (8/5; flip angle, 20°; section thickness, 10 mm; matrix, 256 x 128; number of heart phases, 16). Middle: Corresponding graphs A–C show the difference in the circumferential strain profile in one treated animal compared with one control animal. Graph A, Infarcted myocardium. Graph B, Periinfarcted myocardium. Graph C, Remote myocardium. The strain value of the ninth phase represents the peak systolic strain.

 

Figure 5
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  		Figure 5: Mean peak systolic strain changes in control animals (black bars) and AAV-VEGF–treated animals (white bars) at 3 days (left) and 8 weeks (right). In the AAV-VEGF–treated animals, improvement in peak systolic strain was observed in the periinfarcted and remote myocardium at 8 weeks, compared with control animals. A difference with P = .01–.001, compared with values obtained in control animals (unpaired two-tailed Student t test) (*), was observed. Error bars represent standard error of the mean.

 
Myocardial Infarcts
Infarcts at 3 days and 8 weeks were substantially enhanced by using gadoterate meglumine (Fig 6). All infarcts were located in the anteroseptal wall of the LV, the territory of the occluded artery. The decrease in the area of delayed contrast enhancement was significantly greater in treated animals, with a mean value of 18.6% ± 1.5 of the LV mass at 3 days and of 9.8% ± 1.0 of the LV mass at 8 weeks (P < .001, RPA = 96% power) compared with the decrease in control animals, with a mean value of 17.7% ± 2.0 at 3 days and of 14.8% ± 1.0 at 8 weeks (P = .008, RPA = 76% power).


Figure 6
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  		Figure 6: Delayed contrast-enhanced inversion-recovery gradient-echo cardiac MR images (4.4/2.1/270–325; flip angle, 15°; section thickness, 10 mm; matrix, 152 x 134) in short- and long-axis views 20 minutes after administration of 0.1 mmol gadoterate meglumine show the changes in the size of infarcted areas (arrows) of myocardium in, A, control and, B, AAV-VEGF–treated animals. At 8 weeks, the area of myocardial delayed enhancement was smaller in treated animals.

 
Histopathologic Findings
Histochemical triphenyltetrazolium chloride and histopathologic staining confirmed the presence of transmural infarction in all animals (Fig 7). With triphenyltetrazolium chloride staining, mean infarct size was 13.8% ± 1.3 of the LV mass in control animals and 9.5% ± 0.7 of the LV mass in treated animals.


Figure 7
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  		Figure 7: Histopathologic findings of the periinfarcted myocardium 8 weeks after infarction. Left: Control animals. Right: AAV-VEGF–treated animals. Top: Tissue sections show that capillary density (arrowhead) was greater in the AAV-VEGF–treated animals, as observed with isolectin staining (original magnification, x400). Middle: Tissue sections show that arteriole density was also increased by the treatment, as observed with Masson trichrome stain (original magnification, x200 [right]). Thin-walled new arterioles (black arrows), compared with old thick-walled remodeled vessels (white arrow), were predominant in the treated animals. Bottom: Tissue sections show that periinfarcted myocardium myocytes (arrowheads) were smaller in the treated animals compared with those in the control animals, as shown with hematoxylin-eosin stain (original magnification, x400).

 
The three major histopathologic findings were as follows: (a) AAV-VEGF treatment mitigated the wall-lumen ratio of the arterioles, with a mean value of 3.2 ± 0.1 for treated animals versus 8.1 ± 0.4 for control animals (P = .002, RPA = 82% power) in the infarcted and periinfarcted myocardium. (b) With AAV-VEGF, an increase in the mean number of capillaries was observed in the treated animals, with a mean value of 1065 capillaries per square millimeter ± 22, compared with that in the control animals of 598 capillaries per square millimeter ± 10 (P < .001, RPA = 97% power). An increase in mean arteriole density was also observed in the treated animals, with a value of 46.3 arterioles per square millimeter ± 0.9, compared with the value in the control animals of 22.4 arterioles per square millimeter ± 0.8 (P = .001, RPA = 91% power) in the infarcted and periinfarcted myocardium. (c) Mean myocyte diameter was significantly smaller in the periinfarcted myocardium of treated animals (23.1 µm ± 0.1) compared with that in control animals (28.2 µm ± 0.4), with P < .001 and RPA yielding 97% power. On the other hand, in the normal myocardium, there was no difference in mean myocyte diameter of treated animals (15.3 µm ± 0.1) compared with that of control animals (15.2 µm ± 0.2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
The main findings of our study are as follows: (a) Intramyocardial injection of VEGF genes after infarction prevented LV remodeling and improved wall thickening and circumferential peak systolic strain. (b) Intramyocardial injection of VEGF genes caused a decrease in the area of delayed enhancement at 8 weeks. (c) Intramyocardial injection of VEGF genes promoted angiogenesis and arteriogenesis and reduced myocyte diameter in infarcted and periinfarcted myocardium.

Cardiac MR imaging combines the capability (a) to demarcate acute myocardial infarct and scarred tissue with delayed enhancement sequences (23); (b) to quantify LV end-diastolic volume, end-systolic volume, and ejection fraction with cine sequences (24); and (c) to quantify regional myocardial strain with tagged cine sequences (25). Our tagging technique previously has been validated in animal models (25) and applied in humans (26).

In control animals, myocardial infarction was accompanied by LV remodeling, which was evidenced by an increase in LV end-systolic and end-diastolic volumes and a decline in ejection fraction at 8 weeks. These findings are consistent with those in previous studies (3,4). In contrast, AAV-VEGF–treated animals showed a significant decrease in LV volumes and a stable ejection fraction. These findings are also in agreement with results in previous studies in rabbits (27) and dogs with the use of plasmid VEGF165 (11). The stability of global LV function can be attributed to the improvement in regional wall thickening and strain in periinfarcted and remote normal myocardium. Giordano et al (28) found in the cardiac VEGF–deficient mouse model a thinner LV wall and a lower LV ejection fraction compared with those in normal mice.

Rottbauer et al (29) found that VEGF increased myocardial contractility and coronary blood flow in embryonic hearts. Therefore, it is conceivable that the increase in VEGF expression improved myocardial function in adult hearts. AAV-VEGF did not change peak systolic strain or wall thickening in the infarct core, and this finding was probably caused by the transmural nature of the infarct at this site. Similar findings have been previously observed in humans (30) and dogs (31). The results of our study underline the importance of the periinfarcted myocardium in LV remodeling and as a target for VEGF injection. A recent clinical study demonstrated the critical role of periinfarcted myocardium in the prediction of mortality (32).

Intramyocardial injection of VEGF genes enhanced infarct resorption at 8 weeks, and this result suggests the involvement of VEGF in infarct healing. A progressive decrease in the infarct size over several weeks to months has been documented on delayed contrast-enhanced MR images obtained in humans and animals (33,34). This decline was attributed to infarct resorption (35). It has been shown that VEGF enhances wound healing by activating and recruiting macrophages, thus playing an important role in removing necrotic myocardium (36). Other investigators found that the greater decline in the infarcted area in treated animals could also be explained by myocyte proliferation. Laguens et al (21,22) found that VEGF has a mitogenic effect on adult swine myocytes and that it increases the number of myocytes by 22% in periinfarcted myocardium. Our study does not exclude the potential role of the vasodilatation or antiapoptotic properties of VEGF (37,38).

VEGF genes reduced the vascular remodeling process in infarcted and periinfarcted myocardium. Researchers in previous studies characterized vascular remodeling in infarcted myocardium in terms of a thicker arterial wall, a high wall-lumen ratio (16), and reduced coronary reserve (4). Thus, periinfarcted myocardium had the highest risk of further ischemic damage as a result of vascular remodeling (4). In our study, VEGF genes promoted the formation of both capillaries and arterioles in infarcted and periinfarcted myocardium, as shown previously in studies in swine and rodents (11,17). Formation of larger-caliber vessels, such as arterioles, allows greater blood flow compared with that allowed in the formation of microvessels (39).

Cardiac myocytes become hypertrophic after infarction in response to increased wall stress (40). In our study, myocyte diameter was significantly smaller in the periinfarcted myocardium of treated animals compared with that of control animals. The decrease in myocyte diameter after treatment could be attributed to the increase in regional perfusion and a decrease in wall stress (41). Other researchers (22) have speculated that periinfarcted cardiomyocytes in animals treated with plasmid VEGF genes are smaller than in those not treated and that the difference in size is caused by mitosis. Kramer et al (41) showed the correlation between cellular hypertrophy in periinfarcted regions and the mechanical dysfunction in sheep. It is possible that the increase in vascular density in periinfarcted myocardium contributes to recovery and protects LV function.

Our study had limitations. No MR imaging assessment of LV function was performed prior to infarction. However, data about baseline LV function have been reported in a study in which matched body weight was used (42). The postprocessing software used in our study does not allow through-plane systolic motion correction. Our evaluation of the decrease in infarct size as a function of time was based on delayed enhancement imaging and not on histochemical staining. However, Kim et al (43) found in large animals a close correlation between delayed enhancement and triphenyltetrazolium chloride staining. Control animals were subjected to the same surgical intervention (coronary occlusion and reperfusion) as treated animals but did not receive sham intramyocardial injections of saline or AAV encoding for LacZ to confirm that the latter has no angiogenic effect. However, several investigators documented the lack of angiogenic activity of AAV encoding for LacZ vector (17,44).

VEGF genes may be useful in the prevention of LV remodeling after infarct. Cardiac MR imaging is an effective technique for monitoring global and regional LV function after administration of gene or cell therapies.

In conclusion, angiogenesis and arteriogenesis induced by VEGF genes improved regional myocardial strain and wall thickening and preserved global ejection fraction after infarct. Long-term studies are needed to investigate the safety of VEGF genes.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 


   IMPLICATION FOR PATIENT CARE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
References
 


   FOOTNOTES
 

Abbreviations: AAV = adeno-associated virus • AAV-VEGF = AAV vector–encoding VEGF gene • LV = left ventricle • RPA = retrospective power analysis • VEGF = vascular endothelial growth factor

See Materials and Methods for pertinent disclosures.

See also Science to Practice in this issue.

Author contributions: Guarantor of integrity of entire study, M.S.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, A.J., M.S.; experimental studies, A.J.M., L.D., D.S., M.S.; statistical analysis, A.J., L.D., M.S.; and manuscript editing, all authors


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 

  1. Mahon NG, O'Rorke C, Codd MB, McCann HA, McGarry K, Sugrue DD. Hospital mortality of acute myocardial infarction in the thrombolytic era. Heart 1999;81(5):478–482. [Abstract/Free Full Text]
  2. Agocha A, Lee HW, Eghbali-Webb M. Hypoxia regulates basal and induced DNA synthesis and collagen type I production in human cardiac fibroblasts: effects of transforming growth factor-beta1, thyroid hormone, angiotensin II and basic fibroblast growth factor. J Mol Cell Cardiol 1997;29(8):2233–2244. [CrossRef][Medline]
  3. Maytin M, Colucci WS. Molecular and cellular mechanisms of myocardial remodeling. J Nucl Cardiol 2002;9(3):319–327. [CrossRef][Medline]
  4. Kalkman EA, van Haren P, Saxena PR, Schoemaker RG. Regionally different vascular response to vasoactive substances in the remodelled infarcted rat heart: aberrant vasculature in the infarct scar. J Mol Cell Cardiol 1997;29(5):1487–1497. [CrossRef][Medline]
  5. Weisman HF, Bush DE, Mannisi JA, Bulkley BH. Global cardiac remodeling after acute myocardial infarction: a study in the rat model. J Am Coll Cardiol 1985;5(6):1355–1362. [Abstract]
  6. Ripa RS, Jorgensen E, Wang Y, et al. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction (STEMMI) trial. Circulation 2006;113(16):1983–1992. [Abstract/Free Full Text]
  7. Pfeffer MA, McMurray JJ, Velazquez EJ, et al. Valsartan, captopril, or both in myocardial infarction complicated by heart failure, left ventricular dysfunction, or both. N Engl J Med 2003;349(20):1893–1906. [Abstract/Free Full Text]
  8. Lauerma K, Saeed M, Wendland MF, Derugin N, Yu KK, Higgins CB. Verapamil reduces the size of reperfused ischemically injured myocardium in hypertrophied rat hearts as assessed by magnetic resonance imaging. Am Heart J 1996;131(1):14–23. [CrossRef][Medline]
  9. Janssens S, Dubois C, Bogaert J, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006;367(9505):113–121. [CrossRef][Medline]
  10. Rutanen J, Rissanen TT, Markkanen JE, et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation 2004;109(8):1029–1035. [Abstract/Free Full Text]
  11. Ferrarini M, Arsic N, Recchia FA, et al. Adeno-associated virus-mediated transduction of VEGF165 improves cardiac tissue viability and functional recovery after permanent coronary occlusion in conscious dogs. Circ Res 2006;98(7):954–961. [Abstract/Free Full Text]
  12. Pearlman JD, Hibberd MG, Chuang ML, et al. Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nat Med 1995;1(10):1085–1089. [CrossRef][Medline]
  13. Kastrup J, Jorgensen E, Ruck A, et al. Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris A randomized double-blind placebo-controlled study: the Euroinject One trial. J Am Coll Cardiol 2005;45(7):982–988. [Abstract/Free Full Text]
  14. Stewart DJ, Hilton JD, Arnold JM, et al. Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment. Gene Ther 2006;13(21):1503–1511. [CrossRef][Medline]
  15. Saeed M, Lee RJ, Weber O, et al. Scarred myocardium imposes additional burden on remote viable myocardium despite a reduction in the extent of area with late contrast MR enhancement. Eur Radiol 2006;16(4):827–836. [CrossRef][Medline]
  16. Saeed M, Martin AJ, Lee RJ, et al. MR guidance of targeted injections into border and core of scarred myocardium in pigs. Radiology 2006;240(2):419–426. [Abstract/Free Full Text]
  17. Su H, Joho S, Huang Y, et al. Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts. Proc Natl Acad Sci U S A 2004;101(46):16280–16285. [Abstract/Free Full Text]
  18. Su H, Lu R, Kan YW. Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart. Proc Natl Acad Sci U S A 2000;97(25):13801–13806. [Abstract/Free Full Text]
  19. Osman NF, McVeigh ER, Prince JL. Imaging heart motion using harmonic phase MRI. IEEE Trans Med Imaging 2000;19(3):186–202. [CrossRef][Medline]
  20. Crottogini A, Meckert PC, Vera Janavel G, et al. Arteriogenesis induced by intramyocardial vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs. Hum Gene Ther 2003;14(14):1307–1318. [CrossRef][Medline]
  21. Laguens R, Cabeza Meckert P, Vera Janavel G, et al. Entrance in mitosis of adult cardiomyocytes in ischemic pig hearts after plasmid-mediated rhVEGF165 gene transfer. Gene Ther 2002;9(24):1676–1681. [CrossRef][Medline]
  22. Laguens R, Cabeza Meckert P, Vera Janavel G, et al. Cardiomyocyte hyperplasia after plasmid-mediated vascular endothelial growth factor gene transfer in pigs with chronic myocardial ischemia. J Gene Med 2004;6(2):222–227. [CrossRef][Medline]
  23. Simonetti OP, Kim RJ, Fieno DS, et al. An improved MR imaging technique for the visualization of myocardial infarction. Radiology 2001;218(1):215–223.[Abstract/Free Full Text]
  24. Semelka RC, Tomei E, Wagner S, et al. Interstudy reproducibility of dimensional and functional measurements between cine magnetic resonance studies in the morphologically abnormal left ventricle. Am Heart J 1990;119(6):1367–1373. [CrossRef][Medline]
  25. Lima JA, Jeremy R, Guier W, et al. Accurate systolic wall thickening by nuclear magnetic resonance imaging with tissue tagging: correlation with sonomicrometers in normal and ischemic myocardium. J Am Coll Cardiol 1993;21(7):1741–1751. [Abstract]
  26. Axel L, Dougherty L. Heart wall motion: improved method of spatial modulation of magnetization for MR imaging. Radiology 1989;172(2):349–350. [Abstract/Free Full Text]
  27. Bull DA, Bailey SH, Rentz JJ, et al. Effect of Terplex/VEGF-165 gene therapy on left ventricular function and structure following myocardial infarction: VEGF gene therapy for myocardial infarction. J Control Release 2003;93(2):175–181. [CrossRef][Medline]
  28. Giordano FJ, Gerber HP, Williams SP, et al. A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function. Proc Natl Acad Sci U S A 2001;98(10):5780–5785. [Abstract/Free Full Text]
  29. Rottbauer W, Just S, Wessels G, et al. VEGF-PLCgamma1 pathway controls cardiac contractility in the embryonic heart. Genes Dev 2005;19(13):1624–1634. [Abstract/Free Full Text]
  30. Bogaert J, Bosmans H, Maes A, Suetens P, Marchal G, Rademakers FE. Remote myocardial dysfunction after acute anterior myocardial infarction: impact of left ventricular shape on regional function—a magnetic resonance myocardial tagging study. J Am Coll Cardiol 2000;35(6):1525–1534. [Abstract/Free Full Text]
  31. Croisille P, Moore CC, Judd RM, et al. Differentiation of viable and nonviable myocardium by the use of three-dimensional tagged MRI in 2-day-old reperfused canine infarcts. Circulation 1999;99(2):284–291. [Abstract/Free Full Text]
  32. Yan AT, Shayne AJ, Brown KA, et al. Characterization of the peri-infarct zone by contrast-enhanced cardiac magnetic resonance imaging is a powerful predictor of post-myocardial infarction mortality. Circulation 2006;114(1):32–39. [Abstract/Free Full Text]
  33. Fieno DS, Shea SM, Li Y, Harris KR, Finn JP, Li D. Myocardial perfusion imaging based on the blood oxygen level-dependent effect using T2-prepared steady-state free-precession magnetic resonance imaging. Circulation 2004;110(10):1284–1290. [Abstract/Free Full Text]
  34. Choi CJ, Haji-Momenian S, Dimaria JM, et al. Infarct involution and improved function during healing of acute myocardial infarction: the role of microvascular obstruction. J Cardiovasc Magn Reson 2004;6(4):917–925. [CrossRef][Medline]
  35. Fieno DS, Hillenbrand HB, Rehwald WG, et al. Infarct resorption, compensatory hypertrophy, and differing patterns of ventricular remodeling following myocardial infarctions of varying size. J Am Coll Cardiol 2004;43(11):2124–2131. [Abstract/Free Full Text]
  36. Battegay EJ. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J Mol Med 1995;73(7):333–346. [Medline]
  37. Meeson AP, Argilla M, Ko K, Witte L, Lang RA. VEGF deprivation-induced apoptosis is a component of programmed capillary regression. Development 1999;126(7):1407–1415. [Abstract]
  38. Thomson BJ. Viruses and apoptosis. Int J Exp Pathol 2001;82(2):65–76. [CrossRef][Medline]
  39. Chiu RC. Therapeutic cardiac angiogenesis and myogenesis: the promises and challenges on a new frontier. J Thorac Cardiovasc Surg 2001;122(5):851–852. [Free Full Text]
  40. Mitchell GF, Lamas GA, Vaughan DE, Pfeffer MA. Left ventricular remodeling in the year after first anterior myocardial infarction: a quantitative analysis of contractile segment lengths and ventricular shape. J Am Coll Cardiol 1992;19(6):1136–1144. [Abstract]
  41. Kramer CM, Rogers WJ, Park CS, et al. Regional myocyte hypertrophy parallels regional myocardial dysfunction during post-infarct remodeling. J Mol Cell Cardiol 1998;30(9):1773–1778. [CrossRef][Medline]
  42. Kuehne T, Saeed M, Gleason K, et al. Effects of pulmonary insufficiency on biventricular function in the developing heart of growing swine. Circulation 2003;108(16):2007–2013. [Abstract/Free Full Text]
  43. Kim RJ, Fieno DS, Parrish TB, et al. Relationship of MRI delayed contrast enhancement to irreversible injury, infarct age, and contractile function. Circulation 1999;100(19):1992–2002.[Abstract/Free Full Text]
  44. Su H, Arakawa-Hoyt J, Kan YW. Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model. Proc Natl Acad Sci U S A 2002;99(14):9480–9485.[Abstract/Free Full Text]

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