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Published online before print March 23, 2007, 10.1148/radiol.2432060928
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(Radiology 2007;243:451-460.)
© RSNA, 2007

Innovations

Adeno-associated Viral Vector–Encoding Vascular Endothelial Growth Factor Gene: Effect on Cardiovascular MR Perfusion and Infarct Resorption Measurements in Swine1

Maythem Saeed, DVM, PhD, David Saloner, PhD, Alastair Martin, PhD, Loi Do, BS, Oliver Weber, PhD, Philip C. Ursell, MD, Alexis Jacquier, MD, Randall Lee, MD, PhD, and Charles B. Higgins, MD

1 From the Departments of Radiology (M.S., D.S., A.M., L.D., O.W., A.J., C.B.H.), Pathology (P.C.U.), and Medicine/Cardiology (R.L.), University of California San Francisco, 513 Parnassus Ave, HSW 207 B, San Francisco, CA 94134-0628. Received May 29, 2006; revision requested July 31; revision received September 22; accepted October 26; final version accepted November 15. Supported by a grant from National Institutes of Health (RO1HL07295). A.J. supported by the French Radiological Society, 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
 References
 
Purpose: To prospectively determine in swine the effects of cardiac-specific and hypoxia-inducible vascular endothelial growth factor (VEGF) expression gene on angiogenesis and arteriogenesis by using cardiovascular magnetic resonance (MR) imaging for evaluation of infarct resorption and left ventricular (LV) function.

Materials and Methods: The investigation conformed to U.S. National Institutes of Health guidelines. Twelve pigs with reperfused infarcts were studied with cardiovascular MR 3 days and 8 weeks after surgery. In six pigs, adeno-associated viral (AAV) vector–encoding VEGF (AAV-VEGF) gene was injected at eight sites 1 hour after reperfusion. Six pigs served as controls. Cardiovascular MR measurements of perfusion, area at risk, infarct size, and LV function were used in evaluation of the therapy. Hematoxylin-eosin, Masson trichrome, and biotinylated isolectin B4 stains were used to assess regional vascular density. Two-way Student t test was used to determine significant differences between means.

Results: AAV-VEGF had no effect on cardiovascular MR perfusion or infarct size measurements 3 days after infarction. At 8 weeks, the therapy increased infarct resorption, perfusion, and vascular density and prevented deterioration of ejection fraction in treated animals. These changes were associated with a significantly greater reduction in extent of enhanced region in treated (18.6% of LV surface area ± 1.5 [standard error of mean] to 9.8% ± 1.1) than in control animals (17.7% ± 1.8 to 14.5% ± 1.5, P = .028). Histopathologic findings in treated animals showed increased capillary and arterial density in infarct and periinfarct regions. These new vessels were active and thin-walled compared with thick-walled vessels of control animals.

Conclusion: AAV-VEGF improves cardiovascular MR measurement of regional myocardial perfusion and LV function.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

Editor's note: In January 2006 (From the Editor), I announced a new section in Radiology—Innovations. Under this banner, we wish to publish original research that may possibly have far-reaching implications. Authors interested in having their manuscripts considered for Innovations should first read the Editorial to learn of more specifics. Regarding the manuscript by Saeed et al, positive comments we received concerning its publication in Innovations included: "The results of this study are indeed impressive," "It is significant work," and "the graphic abilities of cardiovascular MR to guide and monitor cardiac angiogenesis will be very important in clinical applications."

—Anthony V. Proto, MD, Editor

The discovery of vascular growth factors that stimulate angiogenesis and arteriogenesis has opened a new avenue for treatment of cardiovascular diseases. Various techniques have been used to promote the growth of new blood vessels in hearts with ischemic damage: surgical or percutaneous myocardial laser revascularization; angiogenic growth factor therapies; and, more recently, cell-based therapies. However, results of clinical studies (1–4) have not shown sustained benefits of these therapies. The inadequate results may be attributed to suboptimal delivery to the area of damage, lack of specificity of therapy, and suboptimal expression in relation to the status of tissue. Results of studies (5,6) have shown that the newly developed adeno-associated viral (AAV) vector for delivery of the vascular endothelial growth factor (VEGF) gene is expressed only in myocardium subjected to ischemia. Unlike other adenoviral vectors, the AAV vectors are nonpathogenic (7,8) and generate minimal inflammation (9,10).

Cardiovascular magnetic resonance (MR) imaging can allow evaluation of the effectiveness of new therapies. Cardiovascular MR imaging can be used to (a) quantify global and regional left ventricular (LV) function; (b) identify the presence, location, and extent of acute and chronic infarcts (11,12); (c) predict improvement in contractile function after revascularization in patients (13); (d) provide an accurate and reproducible method for quantifying LV mass and volumes (14); and (e) provide an optimum modality for monitoring infarct size and LV function over time because of excellent interstudy reproducibility (14,15).

To our knowledge, the effects of AAV vector–encoding VEGF gene (AAV-VEGF) on cardiovascular MR measurements of perfusion, extent of area at risk (AAR), and infarct resorption have not been investigated. We hypothesized that intramyocardial injection of AAV-VEGF produces differences in AAR, time course of infarct resorption, vascular density, and LV ejection fraction after infarction. Thus, the purpose of our study was to prospectively determine in swine the effects of a cardiac-specific and hypoxia-inducible VEGF expression gene on angiogenesis and arteriogenesis by using cardiovascular MR imaging for evaluation of infarct resorption and LV function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Guerbet Group (Paris, France) supplied the contrast agent gadoterate meglumine (Dotarem). The authors had control over the data and information submitted for publication.

AAV-VEGF
The AAV vector for delivery of the VEGF gene was provided by Yuet Kan, MD (University of California San Francisco). Briefly, the cardiac myosin light-chain 2v promoter and the hypoxia response element to mediate cardiac-specific and hypoxia-inducible VEGF expression were used in our study. The myosin light-chain 2v promoter is a contractile protein and is abundant in slow-twitch skeletal and cardiac muscles. The promoter was cloned between nine copies of the hypoxia-response element isolated from the erythropoietin and human VEGF165 complementary DNA in an AAV vector to generate AAV-VEGF (6). The promoter was used to derive genes for a human VEGF isoform, VEGF165, and LacZ. Approximately 1011 copies of the AAV-VEGF vector were mixed with 1010 copies of AAV-LacZ. A total of 1011 copies of AAV-VEGF was injected directly into myocardium by using a 30-gauge needle in strict sterile conditions.

Surgical Procedure
Our investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (publication no. 85–23, revised 1996). Twelve pigs (Hampshire; BRK Power Farms, Turlock, Calif) were premedicated by using ketamine (20 mg per kilogram of body weight; Ketaset, Fort Dodge Labs, Fort Dodge, Iowa), xylazine (2 mg/kg; Anased Injection, Lloyd Labs, Shenandoah, Iowa), and atropine (0.04 mg/kg; Phoenix Labs, St Joseph, Mo) and anesthetized by using a mixture of isoflurane (1.5%–2.0%) and oxygen. The body weights of the control and treated animals were 30–36 kg at 3 days and 47–57 kg at 8 weeks. There was no significant difference in body weight between control (31.6 kg ± 0.9 [standard error of the mean]) and treated (33.2 kg ± 0.4) animals at 3 days.

A middle thoracotomy was performed, and the heart was suspended in a pericardial cradle. At the midventricular level, 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 length of plastic tubing to form a snare, which was tightened to achieve coronary artery occlusion (16). Regional ischemia was confirmed by the development of cyanosis, a lack of contractility, and the inversion of the S-T wave within 1 minute of coronary occlusion. The left anterior descending coronary artery was occluded for 2 hours; this was followed by a 1-hour reperfusion prior to administration of the therapeutic agent (17). In six randomly selected animals, AAV-VEGF was slowly (1 minute) injected in four infarcted and four periinfarcted sites (total of eight sites in each animal) (M.S.). The therapeutic agent in a mixture of 500 µg AAV-VEGF and 500 µg AAV-LacZ dissolved in 0.5 mL phosphate buffered saline. The other six animals served as controls and underwent precisely the same pathologic intervention. Arterial blood pressure, oxygen tension, and heart rate were continuously measured during surgery and imaging.

Cardiovascular MR Imaging
Cardiovascular MR imaging was performed (A.M., O.W.) 3 days and 8 weeks after surgery. Electrocardiographically gated cardiovascular MR imaging was performed with a 1.5-T imager (Intera; Philips Medical Systems, Best, the Netherlands). A five-element phased-array cardiac coil was wrapped around the chest. The following pulse sequences were performed.

Cine cardiovascular MR imaging.—Image loops were acquired in contiguous short-axis sections covering the heart. A balanced steady-state free precession sequence was performed to measure LV chamber volumes and mass. Imaging parameters were as follows: repetition time msec/echo time msec, 8/5; section thickness, 10 mm; spacing, 0; flip angle, 20°; field of view, 24 x 24 cm; matrix size, 256 x 128; number of signals acquired, two; and number of phases, 16 (14).

First-pass contrast material enhancement.—A first-pass perfusion gradient-echo sequence was performed to assess regional perfusion and the extent of AAR, which were measured on three short-axis sections. Parameters were as follows: 3.0/1.5; field of view, 260 mm; matrix size, 144 x 145; flip angle, 20°; and temporal resolution, 270 msec per image. A 6-F angiographic catheter was placed in the femoral vein. The volume of MR contrast medium used was determined by using body weight (0.2 mL/kg). The rate of injection was 3 mL/sec with a power injector (Spectris MRI Injector; Medrad, Indianola, Pa). Saline solution (10 mL) was used as a flush. The first-pass images were obtained 5–6 seconds after injection of 0.1 mmol/kg gadoterate meglumine. This sequence allowed the acquisition of 210 images over the course of 70 seconds. Cardiovascular MR images and triphenyltetrazolium chloride (TTC)–stained slices were visually matched (M.S., L.D., A.J.) on the basis of anatomic landmarks, including LV shape, papillary muscle shape, and location and insertion point of the right ventricle and LV (18). The investigators were blinded as to whether the cardiovascular MR images were in AAV-VEGF–treated or control animals.

Delayed contrast enhancement.—Short-axis images encompassing the heart were acquired 20 minutes after gadoterate meglumine injection by using an inversion-recovery gradient-echo sequence. This sequence was performed to measure the extent of enhanced regions. Parameters were as follows: 4.4/2.1; section thickness, 10 mm; spacing, 0; flip angle, 15°; field of view, 24 x 24 cm; and matrix size, 152 x 134 (12). The optimal inversion times for nulling remote normal (posterior LV wall) myocardium were between 270 and 325 msec. Software (MRVision, version 1.67, 2005; MRVision, Menlo Park, Calif) was used to measure the extents of AAR and infarcts.

Cardiovascular MR Analysis
Morphometric analysis was performed as described in a previous report (18). LV volumes were measured by tracing endocardial and epicardial surface areas during systole and diastole (M.S., L.D., A.J., A.M.) by using software (ImageJ 1.36, 2005; National Institutes of Health, Bethesda, Md, available at http://www.nih.gov). The investigators who performed these analyses by consensus were blinded as to whether the images were in treated or control animals. Each volume was computed as the sum of volumes on short-axis view sections. LV mass on cardiovascular MR images was calculated by multiplying myocardial volume by a specific gravity of cardiac muscle (1.05 mg/mL). The extents of AAR and infarcts were calculated as percentages of LV mass.

The AAR was identified on first-pass perfusion images as a region with signal intensity –2.0 standard deviations of that of normal myocardium (13,18). The coordinates of the regions of interest (35–45 pixels) were placed in the center between the epicardium and endocardium and the LV chamber to avoid the volume-averaging effect. Changes in regional signal intensity during first-pass imaging were presented in arbitrary units. Myocardial infarcts were defined on delayed cardiovascular MR images when signal intensity was greater than 2.0 standard deviations of that of normal myocardium (mean signal intensity of entire free LV wall); this was a constant reference in all animals with regard to size and placement (13).

Postmortem Evaluation
Animals were euthanized at 8 weeks, and hearts were removed. The LV was dissected, weighed, sliced (10 mm), and stained with TTC to delineate scar tissue. Slices were used for histopathologic evaluation (M.S.). The extent of enhanced area on cardiovascular MR images was measured as a percentage of LV surface area by using software (ImageJ 1.36, 2005; National Institutes of Health) (19). The use of an identical approach in the measurement of abnormal zones is an important consideration in the intermodality comparison of cardiovascular MR images and TTC-stained slices.

Histopathologic Evaluation
Samples were obtained from remote normal myocardium, periinfarcted myocardium, and scar tissue (P.C.U., M.S., A.J., R.L.) and were fixed in 10% buffered formalin and embedded in paraffin. Several sections (5 µm) were stained with hematoxylin-eosin, Masson trichrome, and biotinylated Bandeiria simplicifolia isolectin B4 (Vector Laboratory, Burlingame, Calif) (20). The pathologist (P.C.U.) was blinded as to whether the sample was from an AAV-VEGF–treated or a control animal. Capillary (<10 µm) and arteriole (10–100 µm) densities (capillaries or vessels per square millimeter) were measured in the sampled regions. Vascular density was measured in 10 fields at x400 magnification for capillaries and x200 magnification for arterioles. The ratio of vessel diameter to lumen diameter was calculated (P.C.U., A.J., R.L., M.S.).

Statistical Analysis
Reported cardiovascular MR data were collectively acquired and analyzed by six investigators (M.S., A.M., L.D., O.W., A.J., D.S.). Data were expressed as means ± standard errors of the mean. A two-way Student t test (StatView, version I-5.0; SAS Institute, Cary, NC) was used to determine significant differences between the means of the following parameters: extent of area with perfusion deficit, extent of infarcts, LV volumes, LV mass, ejection fraction, signal intensity–time curve, and vascular density. Results of basic retrospective power calculation of all significantly different results in our study ranged from .54 to .97. The null hypothesis was rejected if P was less than .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
MR Perfusion Index
For both AAV-VEGF–treated and control animals, at the time when normal myocardium demonstrated peak signal enhancement on first-pass cardiovascular MR contrast medium–enhanced images, the AAR demonstrated hypoenhancement (Figs 1, 2). At 3 days, the enhancement of normal myocardium and the AAR, as well as the extent of the AAR, were not significantly different between the groups (39.4% of LV mass ± 1.7 in treated and 41.2% of LV mass ± 2.3 in control animals, P = .3, unpaired t test) (Figs 3, 4). The maximum upslopes of the first-pass perfusion curves of the AAR were also not significantly different (170 msec ± 17 in treated and 167 msec ± 23 in control animals, P = .92).


Figure 1
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  		Figure 1: First-pass perfusion MR images show AAR (black arrowheads) and delayed MR images show extent of enhanced regions (white arrowheads) in two control animals. Left: Images show 3-day-old infarcts. Right: Images show 8-week-old infarcts. Color sections from histochemistry show true infarcts at postmortem evaluation (white arrows). (TTC stain; one-third of original magnification.)

 

Figure 2
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  		Figure 2: First-pass perfusion MR images show AAR (black arrowheads) and delayed MR images show extent of enhanced regions (white arrowheads) in two AAV-VEGF–treated animals. Left: Images show 3-day-old infarcts. Right: Images show 8-week-old infarcts. Color sections from histochemistry show true infarcts at postmortem evaluation (white arrows). (TTC stain; one-third of original magnification.)

 

Figure 3
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  		Figure 3: Graphs of first-pass perfusion dynamics of LV blood, remote myocardium, and AAR. Left: Control animals (n = 6). Right: AAV-VEGF–treated animals (n = 6). Treated animals showed better perfusion than control animals at 8 weeks, as reflected by magnitude of signal intensity increase and maximum upslope of curves in AAR. a.u. = arbitrary units, Gd-DOTA = gadoterate meglumine.

 

Figure 4
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  		Figure 4: Graphs of comparison of percentage change in signal intensities of normal myocardium and AAR at 3 days and 8 weeks after infarction. Left: Control animals (n = 6). Right: AAV-VEGF–treated animals (n = 6). Bottom: Difference reached significance (P = .001) in treated compared with control animals. Maximum upslopes of curves in AAR were 225 msec ± 19 in control and 166 msec ± 16 in treated animals (P = .038). Gd-DOTA = gadoterate meglumine.

 
At 8 weeks, the AAR in treated animals showed an increase in signal intensity of 86% when compared with the AAR in control animals during the first pass of contrast medium (Fig 4). The extent of the AAR in treated animals (19.6% of LV mass ± 2.0) was significantly different from that in control animals (32.8% of LV mass ± 2.0, P = .001, unpaired t test). Unlike at 3 days, at 8 weeks, the maximum upslopes of the first-pass perfusion curves of the AAR were significantly different (166 msec ± 16 in treated and 225 msec ± 19 in control animals, P = .038) (Fig 4).

Extent of Enhanced Region
At 3 days, the extent of enhanced region on delayed cardiovascular MR images was comparable between treated (18.6% of LV surface area ± 1.5) and control animals (17.7% of LV surface area ± 1.8, P = .7, unpaired t test) (Fig 5). In both groups, the extent of enhanced region was significantly smaller than the AAR (P = .001).


Figure 5
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  		Figure 5: Graphs of extents of infarcts, as percentage of LV surface area, in control (n = 6) and treated (n = 6) animals at delayed cardiovascular MR imaging at 3 days and 8 weeks and TTC histochemical staining at 8 weeks. Enhanced regions at 8 weeks (white columns) were significantly smaller in both groups than those at 3 days (black columns). However, the decline in extent of infarcts was significantly greater in treated than in control animals at cardiovascular MR imaging and TTC staining (striped columns). * = P value of .01, {dagger} = P value of .04.

 
At 8 weeks, both groups showed significant (P = .04) decline in the extent of enhanced regions compared with that at 3 days (Fig 5). However, the extent of enhanced region was significantly smaller in treated animals (9.8% of LV surface area ± 1.1) than in control animals (14.5% of LV surface area ± 1.5, P = .03). The true infarct size at TTC staining was also smaller in treated animals (9.5% of LV surface area ± 0.7) than in control animals (13.8% of LV surface area ± 1.3, P = .04). A 32% increase in infarct resorption was observed in treated animals compared with that in control animals. At cardiovascular MR imaging, LV mass in treated animals was not significantly different from that in control animals at 3 days (86.6 g ± 2.1 vs 85.9 g ± 2.3, respectively, P = .90) or at 8 weeks (101.8 g ± 2.2 vs 107.7 g ± 3.5, respectively, P = .19). LV mass–to–body weight ratios showed no significant differences between treated and control animals (2.62 g/kg ± 0.1 vs 2.6 g/kg ± 0.2 at 3 days, P = .7, and 1.92 g/kg ± 0.1 and 2.03 g/kg ± 0.1 at 8 weeks, P = .2, respectively).

LV Function
At 3 days, cine cardiovascular MR images showed no significant difference in end-diastolic and end-systolic volumes between treated (2.12 mL/kg ± 0.05 and 1.27 mL/kg ± 0.05, respectively, P = .12) and control animals (2.24 mL/kg ± 0.05 and 1.31 mL/kg ± 0.04, respectively, P = .54). At 8 weeks, there was a significant difference in end-diastolic volume and end-systolic volume between treated (1.80 mL/kg ± 0.09 and 1.06 mL/kg ± 0.05, respectively) and control animals (2.3 mL/kg ± 0.10, P = .004, and 1.52 mL/kg ± 0.07, P = .003, respectively).

AAV-VEGF preserved LV function, as shown by the lack of significant change in the ejection fraction in treated animals (40.3% ± 0.9 at 3 days vs 41.0% ± 0.7 at 8 weeks, P = .49) (Fig 6). In contrast, the ejection fraction deteriorated over the course of 8 weeks in control animals (41.4% ± 0.7 at 3 days vs 36.1% ± 0.6 at 8 weeks, P = .001, paired t test) and was significantly lower than that in treated animals (P = .003, unpaired t test). Statistical power in these experiments was derived from relative changes in regional myocardial perfusion, delayed enhancement, and LV function within the same animal and among different groups. Intraobserver variability was ±2.1%.


Figure 6
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  		Figure 6: Graphs of changes in LV ejection fraction. Top: Control animals (n = 6). Bottom: AAV-VEGF–treated animals (n = 6). Control animals had progressive deterioration in ejection fraction 8 weeks after infarction. AAV-VEGF prevented decline in ejection fraction in treated animals subjected to same intervention. ** = P less than .01 compared with that at 3 days after infarction (paired t test), {dagger} = P less than .01 compared with that of control animals (unpaired t test).

 
Hemodynamic Measurements
The heart rates were 103 beats per minute ± 5 in treated animals and 99 beats per minute ± 4 in control animals at 3 days and 88 beats per minute ± 2 and 95 beats per minute ± 4 at 8 weeks (P = .60–.78), respectively. Mean arterial pressures in treated animals were 80 mm Hg ± 5 at 3 days and 81 mm Hg ± 4 at 8 weeks (P = .88) and 83 mm Hg ± 5 and 80 mm Hg ± 4 (P = .39), respectively, in control animals. Intramyocardial injection of AAV-VEGF caused no significant change in systolic (94 mm Hg ± 2 to 90 mm Hg ± 3, P = .29) or diastolic blood pressures (54 mm Hg ± 2 to 57 mm Hg ± 3, P = .42). There was no significant difference in arterial O2 saturation between 3 days (97% ± 2) and 8 weeks (96% ± 3, P = .79) in treated animals compared with that in control animals (97% ± 2 at 3 days vs 96% ± 3 at 8 weeks, P = .79).

Histopathologic Findings
Hematoxylin-eosin–stained slices demonstrated numerous new arterioles and capillaries in scar tissue and periinfarcted myocardium of treated animals (Fig 7). There was no evidence of angiogenesis or arteriogenesis in the thin layer of viable subendocardium or in remote normal myocardium. The sites of injection were recognized by the presence of abundant hemosiderin denoting the needle track. Masson trichrome stained the dense collagen blue, which indicated the presence of scar tissue. The biotinylated isolectin B4 stain localized vascular endothelial cells with brown reaction product. Normal remote myocardium showed numerous and uniformly distributed microvessels coursing in parallel with the myocytes but no new vessel formation. Sparse remodeled blood vessels (with thick vascular walls and small lumina) coursed in the scar tissue of control animals. The stain also showed the haphazard orientation of intact vessels in the scar tissue when compared with that in the remote myocardium.


Figure 7
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  		Figure 7: Micrographs of representative infarcts in, A, C, E, control animals and, B, D, F, AAV-VEGF–treated animals. A, B, At low magnification, infarct in control animal shows no appreciable neovascularization. A, Remodeled thick-walled blood vessels (arrows). B, AAV-VEGF–treated animal contains numerous vessels (arrowheads) in linear array representing injection needle track. C, D, At high magnification, control infarct has a few scattered remodeled thick-walled vessels (arrows), while AAV-VEGF–treated infarct contains numerous thin-walled vessels (arrowheads) filled with blood, suggesting active blood vessels. E, F, Biotinylated isolectin B4 stain localizes all vessels with brown reaction product. Sparse vessels in control infarct (arrows) and numerous vessels in AAV-VEGF–treated infarct (arrowheads) were demonstrated with lectin stain. I = infarct, LV = left ventricular cavity. A, B, Calibration bars = 1000 µm. C, D, E, F, Calibration bars = 200 µm. (A, B, C, D, Masson trichrome stain; E, F, immunoperoxidase methods with biotinylated isolectin B4 stain.)

 
Treated animals had a greater number of new thin-walled arterioles (38 vessels per square millimeter ± 2) than did control animals (6 vessels per square millimeter ± 2, P = .001), which is an increase of 533%. Treated animals had a capillary density of 1065 capillaries per square millimeter ± 78, whereas control animals had a capillary density of 576 capillaries per square millimeter ± 50 (P = .004), which is an increase of 86%. The number of thick-walled arterioles in the scar tissue was significantly smaller in treated animals (8 vessels per square millimeter ± 1, P = .001) than in control animals (17 vessels per square millimeter ± 1). In addition, the ratio of the arteriolar diameter to the lumen diameter was better in treated animals (3.3 ± 0.1) than in control animals (8.2 ± 0.5, P = .001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
The major findings of our study are that (a) delivery of AAV-VEGF to the infarct and periinfarct regions induced angiogenesis and arteriogenesis; (b) the therapy reduced the extents of AAR and infarct, as measured on first-pass perfusion and delayed cardiovascular MR images, which suggests better perfusion and greater infarct resorption; (c) AAV-VEGF preserved the ejection fraction and limited the increase in LV volumes at 8 weeks; and (d) cardiovascular MR imaging can depict time course effects of gene therapies in infarcted myocardium.

AAV-VEGF
Results of clinical and animal studies (21–25) have indicated that exogenous angiogenic factors can induce angiogenesis in infarcted myocardium and improve cardiac function. Concerns regarding the use of this type of modulating therapy in ischemic heart disease include (a) delivery of angiogenic growth factors in sufficient concentration to the damaged area, (b) limiting expression of the growth factor to specified tissue, and (c) the fact that optimal expression is dependent on the status of the tissue (such as ischemia). The major complications associated with prolonged and high-level expression of the VEGF gene include hemangioma (26) and VEGF expression in tissues other than the heart (27–29). Therefore, in a recent study, Su et al (10) modulated the expression of VEGF by using the cardiac myosin light-chain promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF (AAV-VEGF) expression in mice. In our study, we used the same AAV-VEGF in a swine model and found evidence of sufficient gene expression, as reflected by increases of 86% and 533% in capillary and arterial densities, respectively. These findings confirmed results of previous studies in the mouse heart (5,6). The effectiveness of gene transfer to infarcted myocardium is highly dependent on the type of vector used (30) and the delivery route of the genetic materials (7,29). The main advantage of the AAV vector is that it has not been associated with any human disease. Being a naturally defective virus (30), AAV provides attractive safety features.

Results of a recent study (31) have shown that VEGF gene therapy promotes the growth of capillaries, arteries, and veins, which leads to better perfusion. Similar results were obtained in our study. The few blood vessels seen in the scar tissue of control animals had thick walls and small lumina (32), which may be attributed to an altered fibrous matrix of the vascular wall and isoform shift of actin in vascular smooth muscle (33–35). The numerous blood vessels seen in the scar tissue, but not in the thin viable subendocardium, of treated animals confirm the high specificity of the AAV vector in the expression of VEGF in scar tissue (5,6).

Myocardial Perfusion and LV Function
Cardiovascular MR measurement of first-pass gadolinium-based dynamics indicated apparent improvement in perfusion of the AAR by 86% in treated animals. The increase in the AAR signal intensity, however, can be attributed to several factors: (a) increase in blood flow to the territory, (b) increase in regional blood volume, and (c) increase in efflux of the contrast medium into the interstitial space in vasodilation status (36) and/or increase in gap space in the capillaries (37) as a result of the expression of VEGF.

The finding of a greater number of blood vessels filled with blood in periinfarct myocardium and scar tissue in treated animals than in control animals offers a feasible explanation for active vessels and increased perfusion. This confirms results of a previous study (38) that revealed revascularization of infarcted myocardium when a human bone marrow–derived angioblast was used as a source of VEGF. The produced VEGF reduced LV remodeling and improved cardiac function.

Infarct Resorption
The difference in resorption between occluded and reperfused infarct has been demonstrated by using contrast-enhanced cardiovascular MR imaging (39). Our cardiovascular MR study results demonstrated infarct resorption in both control and treated animals. The 32% increase in infarct resorption in VEGF-treated animals can be attributed to the compact nature of scar tissue, better perfusion of the AAR (39,40), cardiomyocyte cytokinesis (19,41,42), and activation of macrophages that have important roles in removing necrotic tissue (43). Laguens et al (41,42) found that VEGF has mitogenic effects on adult swine cardiomyocytes and increases the number of myocytes by 22%. Infarct resorption has been well documented in patients (44–46), and the degree of resorption is comparable to that in swine (19). In species with good collateral vessels (45), such as dogs, the resorption of infarcts is very high (79%) (39) compared with that in other species with poor collateral vessels, such as swine (25%) (19) and humans (27%–32%) (43,44,47).

Direct delivery of myocardial therapeutic agents with cardiovascular MR guidance is in an early stage (48–50), but our approach seems to be the next logical step in local delivery of therapeutic agents because it provides greater retention of therapeutic agents by selectively hitting the target with fewer systemic problems (51,52).

Study Limitations
Intramyocardial injection of therapeutic agents by using thoracotomy is limited to the anterior and free wall. It was found that the infarct always extended to the septal wall, which was not surgically accessible with our transepicardial delivery method. Using a catheter-based MR-guided method to deliver therapy to the septum would be desirable (44–46). Another limitation of our study was that we did not inject AAV-LacZ in control animals. The scope of our cardiovascular MR study was not to prove VEGF gene expression by using AAV-VEGF or by using plasmid AAV-LacZ but to prove the effect of expressed VEGF on infarcts. It should be noted that VEGF expression was previously reported (10) in vitro (rat skeletal myocytes and mouse fibroblasts with the vector) and in vivo (mouse hearts) by using AAV-VEGF. VEGF was expressed only in ischemic myocardium and not in other organs (such as the liver, kidney, spleen, gonads, or diaphragm) with measurable VEGF expression (10). The number of animals used in our study is in line with the sample size in another VEGF gene study (53).

In conclusion, we have provided evidence, by using noninvasive cardiovascular MR imaging, that AAV-VEGF improves regional myocardial perfusion and preserves LV function. Our study results highlight the potential benefit of AAV-VEGF in infarct resorption. Enhanced angiogenesis, arteriogenesis, perfusion, and infarct resorption are probable contributors to the salutary effects seen with AAV-VEGF in infarcted myocardium.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   ACKNOWLEDGMENTS
 
Special thanks to the Guerbet Group for providing gadoterate meglumine.


   FOOTNOTES
 

Abbreviations: AAR = area at risk • AAV = adeno-associated virus • AAV-VEGF = AAV vector–encoding VEGF • LV = left ventricle • TTC = triphenyltetrazolium chloride • VEGF = vascular endothelial growth factor

See Materials and Methods for pertinent disclosures.

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, M.S., L.D., A.J.; experimental studies, M.S., D.S., A.M., L.D., O.W., P.C.U., A.J.; statistical analysis, M.S.; and manuscript editing, all authors


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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