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Ann Thorac Surg 2003;76:1246-1251
© 2003 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, Stanford, California USA
b Department of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California, USA
Accepted for publication April 21, 2003.
* Address reprint requests to Dr Robbins, Falk Research Building, 2nd Floor, Stanford University School of Medicine, Stanford, CA 94305-5247, USA
e-mail: robbins{at}stanford.edu
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Abstract |
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METHODS: Twenty pigs underwent left circumflex artery occlusions. After 3 weeks, the animals received myocardial injections of adenovirus expressing ß-gal (n=6), DEL-1 (n=7), or VEGF (n=7). At 7 weeks, animals were assessed for both function and coronary flow and compared with baseline measurements.
RESULTS: Regional wall motion index and global ejection fraction showed deterioration in function in the ß-gal group and no change in the VEGF and DEL-1 groups between the treatment and harvest time points. Preload recruitable stroke work suggested functional improvement in the VEGF group (35.8 ± 8.6 vs 56.4 ± 17.8, p = 0.033). The increase in the DEL-1 group was not statistically significant (27.3 ± 9.8 vs, 40.2 ± 19.4, p = 0.067). The ß-gal group exhibited minimal change (30.7 ± 14.8 vs 35.9 ± 12.1, p = 0.96). Regional blood flow as assessed by fluorescent microspheres was improved under stress conditions in the VEGF group (1.00 ± 0.15 vs 1.15 ±0.22, p = 0.03).
CONCLUSIONS: Treatment with VEGF led to a modest improvement in regional blood flow and cardiac function in previously ischemic myocardial tissue.
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Introduction |
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Vascular endothelial growth factor (VEGF) is a potent and well-characterized mitogenic angiogenic factor. It is endogenously upregulated during periods of cardiac ischemia [2], and therefore may be an important regulator of angiogenesis under physiologic and pathologic conditions. Vascular endothelial growth factor targets its tyrosine kinase receptors almost exclusively on endothelial cells, which are then stimulated to migrate, proliferate, and aggregate into vessel-like structures [3]. Its exogenous administration has been found to improve collateral blood flow and function in ischemic myocardium [4]. Certain deleterious side effects, however, have also been described, such as vascular tumor growth [5], formation of hyperpermeable (leaky) vessels [6], and the possible counter-productive promotion of atherosclerotic plaques [7].
The investigation of alternative, novel angiogenic factors is therefore desirable. Developmental endothelial locus-1 (DEL-1) is a recently characterized extracellular matrix protein that may play an important role in vascular remodeling [8, 9]. Unlike the tyrosine kinase pathways initiated by the VEGF receptors, DEL-1 targets the 
Vß3 integrin receptor important to endothelial cell interactions with the extracellular matrix. Integrins are also known to regulate many of the biological events important to angiogenesis, such as cell migration, proliferation, and differentiation [10].
The purpose of this study was to compare the angiogenic effects of adenovirally mediated gene transfer of VEGF and DEL-1 as well as the negative control ß-galactosidase (ß-gal) in a porcine model of cardiac ischemia. Both function as well as blood flow parameters were assessed to determine the efficacy of the two treatment strategies.
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Material and methods |
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Porcine model of cardiac ischemia and intramyocardial gene delivery
Thirty-two female Yorkshire swine (mean weight, 28 ± 3.6 kg) were purchased from Pork Power Farms (Turlock, CA). Animals were treated in a humane manner, in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication No. 86-23, revised 1985). Anesthesia consisting of 6 mg/kg intramuscular telazol and 1% to 2% isoflurane inhalational anesthesia was administered before each intervention. The study was conducted over a 7-week period per animal and consisted of three separate phases.
Phase i (stent placement)
A 5 F femoral arterial line was inserted. Under fluoroscopic guidance, a pigtail catheter was advanced into the left ventricle via the left carotid artery. Thirty milliliters of contrast medium was delivered over 3 seconds with a power injector, and left ventriculogram images were captured in the left anterior oblique (LAO) and right anterior oblique (RAO) projections. The catheter was then withdrawn across the mitral valve into the left atrium. To determine regional blood flow (RBF), 2 x 106 15 µM fluorescent microspheres (Molecular Probes, Eugene, OR) were delivered in a quick bolus through the pigtail, and a reference blood sample was drawn simultaneously at a rate of 6 mL/minute over 90 seconds from the femoral artery [12].
The catheter was then placed through the ascending aorta into the ostium of the right and left coronary arteries. Baseline angiograms were performed in the LAO/Cranial and RAO/Caudal projections. An 8-mm copper stent (Pulse Systems, Concord, CA) was then placed over a guidewire to a selected target region in the proximal left circumflex artery (LCX). Copper induces neointimal hyperplasia in vessel walls, contributing to a gradual narrowing over time [13]. A final angiogram was performed to confirm a patent stent, after which an additional 5 x 105 microspheres of a different color were slowly injected into the LCX to help define the future area of myocardium at risk.
Phase II (direct myocardial injections)
After 3 weeks, animals returned to the catheterization lab and underwent repeat coronary angiography, left ventricular angiography, and microsphere delivery as described above. After these studies, the pigs were transported to the operating suites, and a left anterolateral thoracotomy was performed through the fourth intercostal space. The pericardium was opened and the lateral wall of the heart was exposed. The pigs were randomized to receive injections of VEGF (n=7), DEL-1 (n=7), or ß-galactosidase (n=6) expressing adenovirus in a blinded fashion to 10 separate sites within the area at risk in their myocardium. A precalibrated device (Microheart, Mountain View, CA) was used to deliver the individual 100-µL injections of a 5 x 109 pfu/mL solution at a set depth of 5 mm.
Phase III (terminal studies)
At 7 weeks, the animals underwent the same studies performed at phase II, except at this time, median sternotomy was performed and microsphere delivery was repeated both under rest and stress conditions with the hearts atrially paced at 180 beats per minute. After all studies, the hearts were harvested.
Assessment of left ventricular function
During the initial thoracotomy and again at time of harvest, sonomicrometry crystals were used to evaluate left ventricular function. The inferior vena cava (IVC) was mobilized and secured with a silastic vessel loop. After ventriculotomy, a 3 F Millar catheter (Millar Instruments, Inc, Houston, TX) was inserted into the left ventricular chamber for pressure measurements. Two 2-mm sonomicrometry crystals were then placed intramuscularly 2 to 3 cm apart within the ischemic region in the apical-basilar axis. Paired crystal tracings were measured using SonoLab v.2.2.0 (Sonometrics Corporation, Ontario, CA). Ventilation was held and 10-second tracings of crystal dimension, arterial pressure, and left ventricular pressure were obtained. Inferior vena cava flow was then obstructed by temporarily occluding the silastic loop and a second 10-second pressure dimension tracing was recorded. The crystals were removed and their locations marked with 7-0 Prolene sutures for future reference.
At time of cardiac procurement, a similar procedure was performed through the median sternotomy. The Prolene marking sutures from the previous surgery were identified and crystals were placed at these sites to allow for accurate intraanimal comparisons. Rest and IVC occlusion pressure measurements were then recorded as above.
Raw data files from SonoLab were analyzed by an observer blinded to treatment groups to determine the regional preload recruitable stroke work index (PRSW) for each animal. The PRSW was derived as the slope of pressure-dimension loops measured at end-systole and, therefore, in our calculations, was a unitless measure.
Regional myocardial blood flow
At time of cardiac procurement, the left ventricle was sectioned into base, mid, and apical portions along the short axis and then further divided into eight zones circumferentially as previously described [14]. Processing of the tissues and counting of the microspheres were conducted in blinded fashion by Interactive Medical Technologies (Irvine, CA). Regional blood flows were calculated according to the following equation: Qi(mL/min) = (fli/flref) · R(mL/min), where Qi=flow to each segment of myocardium, fli=fluorescence count of each piece, flref=fluorescence count of reference blood flow, and R(mL/min)=reference blood withdrawal rate. Regional blood flow measures from specimens within the area at risk were standardized to flow measured from tissues in the right ventricle (outside the area at risk) to eliminate interanimal variations in microsphere delivery.
Angiographic analysis
Coronary angiograms were evaluated in a blinded fashion and assigned a thrombolysis in myocardial infarction (TIMI) grade of coronary flow (0 = no flow; 1 = faint slow filling without opacification of the distal vessel; 2 = slow filling of the entire vessel length; 3 = brisk normal flow) [15], Rentrop score of collateral index (0 = no collaterals; 1 = faint filling of side branches without filling of the main branch; 2 = partial filling of the main vessel; and 3 = complete filling of the main vessel) [16], and percentage diameter stenosis (0% to 100%). Calculation of global ejection fraction (GEF) from left ventriculograms using image analysis software (Sanders Data Systems, Palo Alto, CA) was also performed. Regional wall motion index (RWMI) in the area at risk was assessed by visualization of the ventriculograms and assigned scores ranging from: 0 = dyskinesis; 1 = akinesis; 2 = severe hypokinesis; 3 = hypokinesis; 4 = mild hypokinesis; to 5 = normal wall motion [17].
Assessment of adenoviral gene expression
To assess gene expression in this model, sepearate pigs (n=3) were injected in similar fashion as described above with ß-galactosidase expressing adenovirus (Advß-gal). Each injection site was marked with a Prolene suture and the pigs were allowed to survive for 3, 7, and 14 days (n=1 each). Hearts were then harvested and tissue was sectioned around the identifying prolene sutures. Specimens were fixed in 0.1% glutaraldehyde and incubated in staining solution containing the X-gal substrate (Gibco, Rockville, MD) at 37°C for 4 hours. Tissues were then cut into 5-µm sections and photographed.
Statistical analysis
Rank order data was evaluated by the Kruskal-Wallis test statistic with its reference distribution generated by permutations to adjust for small sample sizes. Bonferroni corrections were made for multiple comparisons. All intraanimal parametric comparisons were made with paired Student's t test. Values of p less than 0.05 were considered significant.
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Results |
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Coronary angiography
The copper stent model resulted in 100% occlusion of the LCX in 17 of the surviving 20 pigs (85%) (Fig 1).
The 3 animals with incomplete occlusions were equally divided between the ß-gal, DEL-1, and VEGF groups, and consisted of 90, 90, and 99% stenoses, respectively.
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Animals with incomplete stent occlusions did not develop collateral vessels (Fig 2). Also, no statistically significant differences were seen in Rentrop score of collateral vessels in animals between time points 3 and 7 weeks in each of the three groups.
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Comment |
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Like other investigators, we chose adenovirally mediated gene transfer to provide a continuous release of angiogenic factors to myocardium at risk [18]. Experiments with Advß-gal in this study indicated that there was expression of this reporter gene for at least 1 week after intramyocardial injections.
Coronary occlusion in this model was induced by intraluminal copper stent placement. By creating a process of gradual neointimal hyperplasia, the development of ischemic myocardium was allowed to occur in much the same manner as with the more commonly utilized ameroid constrictor model. One potential benefit of the copper stents is the ability to place them percutaneously, thus obviating the need for thoracotomy. This decreased the amount of scar tissue and potential distortion of anatomy and cardiac function seen at the 3-week treatment phase when the adenoviral injections were administered.
Developmental endothelial locus-1 interacts with the Vß3 integrin receptors, unlike the tyrosine kinase receptor pathways employed by VEGF and fibroblastic growth factor (FGF). The integrins comprise a large family of membrane surface receptors that promote attachment of endothelial cells to the extracellular matrix (ECM), thus allowing their entry into the cell cycle. This attachment to the ECM also appears integral to the overall survival of these stimulated cells [19]. Currently, there are human trials investigating the clinical potential of integrin antagonists to promote tumor shrinkage in cancer patients. To our knowledge, the current study represents the first use of an Vß3 ligand to initiate therapeutic angiogenesis.
Developmental endothelial locus-1 appears to have a slightly less robust effect on cardiac function than VEGF. This is seen both with respect to the contractility measure of preload recruitable stroke work as well as with the assessment of global ejection fraction. This may infer that the angiogenic effect of VEGF is stronger. The potent nature of VEGF is well known and has been demonstrated in models where excessive induction via gene therapy led to the development of unregulated, immature, leaky vessels with enlarged lumens [20]. It is thus possible that the VEGF-treated hearts had more flow by virtue of the fact that their angiogenesis bordered on a dose-dependent angioma formation [21]. Whether such a response exists with DEL-1 remains to be seen in future dose-response studies; however, it is conceivable that angiogenesis that occurs secondary to integrin-mediated assembly of a cytoskeleton with binding of the extracellular matrix [19] may result in a more stable, albeit less robust vessel formation.
Improvements in cardiac function with angiogenic factor treatment in this study must be reconciled with the lack of increased collateral vessels seen on angiography. This may be explained by the caliber of angiogenically formed vessels. In one study, new vessels formed in the peripheral vasculature documented luminal diameters of only 200 µM that were visible as just a "blush" on angiogram [22]. Perhaps, a more relevant indicator of myocardial flow consistent with the development of this type of microcapillary circulation is thus obtained through the use of fluorescent microspheres [23].
Analysis of microsphere data in this experiment indicated that the potential benefit of angiogenic therapy was apparent only when comparing flow under conditions of stress (cardiac pacing) with rest. These results are consistent with similar findings by another group of observers, who found that regional blood flow was significantly improved in FGF-treated hearts under stress conditions only [24, 25]. One reason for this finding may be that collateral vessel formation in porcine hearts appears to progress early from the time of coronary occlusion and may already be well underway by the 7-week time point utilized in this study [26]. Thus, differences in regional blood flow that are difficult to detect under rest situations at this time point may become apparent only with stress.
One logical extension of the current studies would be the combination of integrin ligands such as DEL-1 with the growth factors VEGF or FGF. Carmeliet has questioned whether a single angiogenic factor alone is sufficient for clinical effect and whether factors administered in combination either simultaneously or sequentially may be more efficacious and have less toxicity [27]. As noted above, excessive VEGF administration can lead to a disorganized vascular state. It is possible that upregulating both growth as well as cell attachment factors may have a synergistic effect on angiogenesis, thus allowing decreased risk of any one particular angiogen [19].
Conclusions
Treatment of ischemic hearts with VEGF resulted in improved myocardial function in this swine model, as assessed by global ejection fraction, regional wall motion index, and preload recruitable stroke work. Developmental endothelial locus-1 treatment led to mixed results, with relative improvements in the first two parameters only. Functional improvements in our model were not corroborated by detectable differences in collateral index or coronary perfusion on angiography. Regional blood flow, however, indicated that coronary flow was improved under stress situations for the VEGF group. These data provide the framework for future studies designed to assess the dose-dependent efficacy of DEL-1-mediated angiogenesis as well as its potential use in combination with other angiogenic factors in the treatment of otherwise incurable ischemic heart disease.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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This article has been cited by other articles:
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  H.-K. V. Ho, J. J. Jang, S. Kaji, G. Spektor, A. Fong, P. Yang, B. S. Hu, R. Schatzman, T. Quertermous, and J. P. Cooke Developmental Endothelial Locus-1 (Del-1), a Novel Angiogenic Protein: Its Role in Ischemia Circulation, March 16, 2004; 109(10): 1314 - 1319. [Abstract] [Full Text] [PDF]
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