Cardiovascular Research

Cardiovascular Research 1998 40(2):272-281; doi:10.1016/S0008-6363(98)00136-9
© 1998 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Lopez, J. J Articles by Simons, M. Search for Related Content PubMed PubMed Citation Articles by Lopez, J. J Articles by Simons, M. Social Bookmarking          What's this?

Copyright © 1998, European Society of Cardiology

VEGF administration in chronic myocardial ischemia in pigs

John J Lopez^a,b, Roger J. Laham^a,b, Alon Stamler^c, Justin D Pearlman^a,d, Stuart Bunting^f, Aaron Kaplan^e, Joseph P Carrozza^b, Frank W Sellke^c and Michael Simons^a,b,*

^aAngiogenesis Research Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
^bCardiovascular Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
^cDepartment of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
^dDepartment of Radiology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
^eLocal Med, Palo Alto, CA, USA
^fGenentech, Inc., South San Francisco, CA, USA

^* Corresponding author. Tel.: +1 (617) 667 5364; Fax: +1 (617) 972 5201; e-mail: msimons@bidmc.harvard.edu

Received 1 December 1997; accepted 6 April 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Previous investigations have shown the effectiveness of sustained intra- or extravascular administration of vascular endothelial growth factor (VEGF) in chronic myocardial ischemia in improvement of left ventricular function. The present investigations were undertaken in order to evaluate efficacy of a single bolus or local intracoronary delivery. Methods: Yorkshire pigs underwent placement of a left circumflex artery ameroid occluder. Three weeks later the animals were randomized to treatment with VEGF (20 µg) accomplished by local intracoronary delivery system (InfusaSleeve^TM, n=10), intracoronary bolus infusion (n=7) or by epicardial implantation of an osmotic delivery system (n=7). An additional group of animals received intracoronary administration of saline and served as a control (n=9). Three weeks after initiation of therapy, the animals were evaluated with regard to myocardial perfusion and global as well as regional ventricular function. Results: All three VEGF treatment groups but not the control animals demonstrated a significant increase in the left-to-left (but not right-to-left) collateral index, myocardial blood flow (pre-therapy LCX vs. LAD (average of all groups): 0.76±0.35 vs. 0.96±0.38 ml*min^–1*g^–1, p=0.03; post-therapy: LCX vs. LAD: 1.16±0.39 vs. 1.15±0.28 ml*min^–1*g^–1, p=NS) and coronary vasodilatory reserve 3 weeks after growth factor administration. The observed increase in VEGF-induced perfusion correlated with improvement in regional ventricular function in all VEGF-treated groups (pre-therapy vs. post-therapy: i.c. VEGF 20±5.1 vs. 33±4.8; local VEGF 16±2.8 vs. 33.6; pump VEGF 17±3.8 vs. 34±4.9 p<0.05 for all) but not control animals (21±3.3 vs. 27±5.8, p=NS). Conclusion: Single intracoronary delivery (intravascular bolus or local delivery) of VEGF is effective in stimulating physiologically significant angiogenesis in porcine model of chronic myocardial ischemia.

KEYWORDS Angiogenesis; VEGF; Myocardial ischemia; Growth factors; Magnetic Resonance Imaging

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Therapeutic approaches to management of chronic myocardial ischemia traditionally include manipulations designed to reduce myocardial oxygen demand or to increase blood supply to compromised territories by providing new (coronary artery bypass grafting) or restoring old (coronary angioplasty) pathways for blood flow. A potential alternative to these approaches may include an attempt to induce growth and development of new collateral vessels [1, 2]. Recently, a number of studies have evaluated the therapeutic potential of administering various angiogenic growth factors in chronic myocardial ischemia [3–7]. Several of these studies have demonstrated improved collateral blood flow and myocardial function in the setting of chronic ischemia after vascular endothelial growth factor (VEGF) administration [5, 7]although a recent study suggested a lack of therapeutic efficacy of VEGF in these settings [8]. However, all of these studies typically employed a prolonged form of VEGF delivery, relying either on continuous intracoronary or extracoronary infusions or repeat intra-arterial administration. These methods of cytokine delivery, however, raise a number of practical issues for application to human trials. Clearly, a one time, single intracoronary administration of the growth factor would have significant advantages over repeat dosing and/or continuous infusions given the significant vasoactive properties of VEGF [9–11].

In evaluating different forms of intracoronary delivery, a distinction needs to be made between local delivery and intracoronary bolus administration. The former approach creates a local extravascular depot of the growth factor, while the latter relies on the ability of capillary beds and/or cardiac tissues in the ischemic territory to retain the growth factor. To date there is no convincing evidence suggesting a beneficial effect with either local or intracoronary bolus administration of VEGF.

With these considerations in mind we set out to determine the ability of VEGF to improve myocardial blood flow and function in the setting of established coronary ischemia, to evaluate two forms of intracoronary VEGF delivery, and to compare them to a previously studied method of sustained-release periadventitial administration.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
All animal experiments in this study were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and the study protocol was approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee.

2.1 Ameroid constrictor model and study design
Yorkshire pigs (25–30 kg.) were instrumented with a size-matched ameroid constrictor (Research Instruments, Corvalis, OR) on the proximal circumflex coronary artery under general anesthesia as described previously [4, 5]. In addition, implantation of an osmotic polymer pump (Alza Corporation) containing heparinized saline (50 U heparin/2 ml saline) was carried out in animals randomized to pump VEGF delivery with tip of the pump catheter positioned over the adventitial surface of the LCX artery 1–2 cm distal to the ameroid occluder. In all animals, a set of colored microspheres was injected into the left atrium during transient LCX occlusion to enable subsequent determination of the of LCX territory at risk (see below). Postoperatively all animals were treated with antibiotics for 48 h and narcotic analgesics were used as needed.

Three weeks later, a period of time sufficient for ameroid closure and development of myocardial ischemia in this model [12, 13], the animals underwent a set of baseline studies carried out under general anesthesia with hemodynamic monitoring. Coronary angiography, performed through an 8F JR4 guiding catheter (Cordis Corp, Miami, FL) via a right femoral cutdown, in multiple LAO and RAO projections using ionic contrast, was employed to document LCX occlusion at the site of ameroid placement as well as to assess the extent of collateral circulation (collateral index) in this territory. A set of colored microspheres was injected into the left atrium to determine myocardial blood flow prior to initiation of VEGF therapy. In addition, magnetic resonance imaging was carried out to obtain quantitative measures of global and regional left ventricular function (ejection fraction, radial wall motion, regional wall thickening) as well as to assess regional myocardial perfusion using previously validated myocardial contrast density mapping [14].

Following this baseline evaluation, animals were assigned to one of four treatment groups: (1) VEGF pump delivery; (2) local intracoronary catheter-based VEGF delivery; (3) local intracoronary bolus VEGF injection; and (4) local intracoronary catheter-based saline delivery (control). All animals were pretreated with aspirin, bretylium (50 mg i.v.) and intravenous heparin (3000 U) prior to a coronary intervention. Animals randomized to local catheter-based VEGF underwent two directional intracoronary deliveries of 10 µg of recombinant human VEGF₁₆₅ (rhVEGF, Genentech, South San Francisco, CA) in a volume of 2 ml saline each using a 10 mm InfusaSleeve^TM catheter (Local Med, Palo Alto, CA) loaded over a 3.0 mmx20 mm angioplasty balloon, delivered over a 0.014 inch angioplasty guidewire. VEGF delivery was accomplished by first inflating the angioplasty balloon to 30 psi, and then delivering the cytokine through the local delivery catheter at 80 psi over 30 s. In all cases an attempt was made to deliver VEGF into the LCX artery proximal to the ameroid occluder, as well as into the left anterior descending (LAD) artery at the level of LCX ameroid occluder placement. In those animals in which a proximal LCX injection could not be carried out, the second dose of VEGF was delivered into the LAD at the same site as the original delivery. Animals randomized to the control group received two intracoronary deliveries of saline in a similar manner. Intracoronary bolus infusion of VEGF was accomplished by a manual injection of 20 µg of VEGF through an Ultrafuse-X dual lumen catheter (SciMed, Minneapolis, MN) into the proximal LAD artery carried out over 10 minutes. Animals randomized to pump VEGF therapy were taken to the operating room and, following repeat thoracotomy, the osmotic pump primed with heparinized saline was replaced with a pump containing 25 µg VEGF and 50 U heparin in 2 ml of phosphate-buffered saline. With 80% of the material delivered over 3 weeks by the pump, the final estimated delivered dose in each animal was 20 µg.

Three weeks after initiation of therapy, animals were brought back for a final evaluation. This consisted of repeat coronary angiography (collateral index assessment), microsphere coronary blood flow studies at rest and after administration of a maximal vasodilatory dose of intracoronary adenosine, and MRI studies to assess myocardial function and perfusion. At the end of these studies, animals were sacrificed by direct intracardiac administration of KCl. Myocardial samples were collected for determination of coronary flow in ischemic and normally perfused myocardium as previously described [4, 5].

2.2 Coronary angiographic evaluation
Evaluation of angiographic collateral density was performed by two experienced angiographers, blinded to treatment assignment. The ‘collateral index’ was assessed for left to left (LAD to LCX or LCX to LCX) and right to left (RCA to LCX) collaterals using a standard 4 point scale (0=no visible collateral vessels, 1=faint filling of side branches of the main epicardial vessel, without filling the main vessel, 2=partial filling of the main epicardial vessel, 3=complete filling of the main vessel) [15, 16]. Differences in score assignment between the observers were resolved by joint film review.

2.3 Myocardial blood flow
To determine the extent of the LCX territory at risk, a set of colored microspheres (15±0.1 µm diameter, Triton Technology Inc., San Diego, CA) was injected prior to the ameroid constrictor placement [5]with the LCX artery held transiently occluded. At the time of analysis, sections of the myocardium containing <10% of microspheres from this set were considered to belong to the LCX territory at risk.

For determination of regional myocardial blood flow 3 and 6 weeks after ameroid placement, an angiographic catheter was advanced into the left atrium retrograde through the mitral valve and a set of colored microspheres (10⁶) was forcefully injected after verification of catheter placement. Reference blood samples were withdrawn using a syringe pump at a constant rate of 4 ml/min through the femoral artery. In addition to resting blood flow determination at the time of final study, an additional set of microspheres was injected after maximal coronary vasodilation was achieved by adenosine infusion (1.25 mg/kg/min) into the central circulation [17].

Following sacrifice, with the heart excised and the left ventricle dissected free of other structures, a trans-axial slice approximately 1 cm thick was cut at the mid- ventricular level. From this slice, eight radial samples were obtained and processed as previously described [4, 5]. Regional myocardial flow (expressed in ml min^–1g^–1) was calculated as [18]:

Coronary resistance prior to and during adenosine infusion was calculated as mean arterial pressure (mm Hg) divided by coronary blood flow (ml min^–1 g^–1). For data presentation, myocardial blood flow is given as a weighted average of flows in subendocardial, mid-myocardial, and subepicardial regions.

2.4 Magnetic Resonance (MR) imaging
MR imaging was performed in the body coil of a 1.5 Tesla whole-body Siemens Vision system. Baseline anatomic images were obtained by a turboFLASH technique [19]to identify coordinates for apical four-chamber, two-chamber, and short-axis views and to locate any areas of myocardial infarctions. Functional imaging was performed during breath-hold and the time series of images were then analyzed to define the size and severity of the contrast arrival deficit. MR image analysis was performed on a dedicated Sun Microsystems workstation using automated object recognition software to define myocardial borders, to correct misregistrations, and to explore at interactive speed the time intensity curves. From the endocardial border image, the floating centroid center of mass was determined with the aid of computer software and radials were extended to the epicardial border [20].

For assessment of myocardial perfusion and infarction size, the presence of myocardial blood flow was determined by measuring the intensity of the gadodiamide-enhanced signal in the different parts of the LV wall and generating a space-time map of myocardial perfusion as previously described [14]. Areas of myocardium demonstrating no increase in contrast intensity (defined as <25% increase in baseline value) were considered to have no significant perfusion and were taken to represent areas of infarction. The extent of such territories as well as territories demonstrating delayed contrast arrival was calculated as previously described [14]as a percent of total LV volume. In addition, the presence of myocardial necrosis was confirmed by visual macroscopic and histological analysis of myocardial tissues.

2.5 Statistics
All data are expressed as mean±SD. A p value of less than or equal to 0.05 was considered significant. Comparison of angiographic collateral density via the ‘collateral index’ was assessed among all groups by the Kruskall–Wallis test for non-parametric, ordinal data. Changes in angiographic collateral scores before and after treatment over the course of the study within a group were assessed by the Wilcoxon rank-sum test for paired ordinal non-parametric data. One way analysis of variance and Bonferroni-corrected t-tests were used for multiple group comparisons.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Forty animals underwent placement of ameroid constrictors. Two animals died in the immediate post-op period and another two died prior to 3 week base-line evaluation point. In all cases the cause of death appeared to be acute myocardial infarction due to premature LCX occlusion. Three animals died during surgery for VEGF pump placement due to surgical complications of repeat thoracotomy. The remaining thirty-three animals (local VEGF, n=10, intracoronary bolus VEGF, n=7, pump VEGF, n=7, control, n=9) completed the entire study. MR data were not available in two control animals due to data acquisition problems.

3.1 Coronary angiography
All coronary angiograms were analyzed with blinding to the treatment group and the timing (first or second) of the angiogram. Disagreement in assignment of collateral index score was adjudicated by joint film review in 12.5% of all angiograms.

Analysis of changes in the extent of coronary collaterals (collateral index) and the origin (LAD or LCXLCX or RCALCX) of collateral vessels was carried out for each of four groups by comparing angiograms performed before and after therapy. There was a significant increase in collateral index for left-to-left (LADLCX or LCXLCX) collaterals for all three VEGF-treated but not control group (Fig. 1A, Wilcoxon signed rank test). However, there was no change in the number of RCALCX collaterals in any of the groups as assessed by the collateral index (Fig. 1B). Thus, administration of VEGF into the left coronary system resulted in a directional increase in collateral circulation regionally restricted to the left but not right coronary system suggesting a local effect of VEGF administration.

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Collateral index. Changes in the collateral index scores between initial (3 week pre-treatment) and follow-up (6 weeks post-treatment) angiograms for collaterals originating from the left anterior descending or left circumflex coronary arteries (Fig. 1A), or the right coronary artery (Fig. 1B) in pigs subjected to local delivery of VEGF (top left), intracoronary VEGF infusion (bottom left), control local delivery (top right), and VEGF pump implantation (bottom right). P-values are for Wilcoxon rank sum test for paired non-ordinal data.

 
3.2 Myocardial blood flow
To confirm the angiographic evidence of improved perfusion following VEGF therapy, regional myocardial blood flow in the LCX and LAD territories was analyzed immediately before and after initiation of VEGF therapy. In addition, coronary resistance in both the LCX and LAD beds was assessed during adenosine-induced maximal vasodilation at the time of final study.

Three weeks after implantation of ameroid occluders resting myocardial blood flow in the LCX territory was similarly reduced in all four groups and was significantly lower than flow in the LAD territory in all animals (LCX vs. LAD: 0.76±0.35 vs. 0.96±0.38 ml*min^–1*g^–1, p=0.03). At the same time, there were no differences between the groups with regard to both LCX (Fig. 2) and LAD (data not shown) flows. Three weeks following initiation of therapy, (6 weeks after ameroid placement), there was no significant differences among the groups with regard to LAD territory perfusion. Analysis of flow in the LCX territory showed that all three VEGF-treated groups demonstrated a significant increase in the resting blood flow compared to pre-treatment flow (paired t-test) while control animals showed no significant change in LCX perfusion (Fig. 2). Analysis of variance showed no difference between the three VEGF treatment groups with regard to LCX perfusion. Combining all VEGF groups together showed that VEGF treatment resulted in LCX perfusion that was equal to that seen in the LAD territory (LCX vs. LAD: 1.16±0.39 vs. 1.15±0.28 ml*min^–1*g^–1, p=NS). At the same time LCX flow in VEGF-treated animals was significantly higher than in controls (VEGF vs. control: 1.16±0.39 vs. 0.85±0.21 ml*min^–1*g^–1, p=0.04).

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of VEGF administration on coronary blood flow. Changes in the pre-treatment (black bars) and post-treatment (gray bars) microsphere-determined rest coronary blood flow (ml*min–1*g–1) for the left circumflex (Fig. 2A) and left anterior descending (Fig. 2B). Note increased rest coronary blood flow in all 3 VEGF treatment groups (Local, local VEGF delivery; i.c., intracoronary VEGF bolus delivery; pump, pump VEGF delivery) in the LCX but not the LAD territory. *p<0.05, post-treatment vs. pre-treatment (paired t-test).

 
To further assess the impact of VEGF treatment on coronary perfusion, coronary vasodilation was induced by administration of adenosine into the central circulation in the amount previously demonstrated to induce maximal coronary vasodilation [17]and maximal increase in coronary blood flow in this model [21]. Calculations of coronary resistance demonstrated an expected significant reductions in resistance in the LAD territory in all four groups (Fig. 3A). At the same time, LCX resistance following adenosine infusion significantly decreased in the three VEGF-treated groups but not in control animals (compared to pre-adenosine values; paired t-test, see Fig. 3B), suggesting the presence of impaired vasodilatory reserve in the LCX bed in control animals that was reversed by VEGF administration. The reductions in coronary resistance (and the increase in coronary blood flow) in these settings is somewhat lower than the 3.5–4 fold change usually seen in dogs due to a rapid drop in systemic blood pressure that limits coronary perfusion during colored microspheres injections. However, direct measurements of coronary blood flow during adenosine infusion in this model demonstrate on the average a 3.5 fold increase in coronary blood flow [21].

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of VEGF Administration on coronary flow reserve. Coronary resistance (mm Hg/ml*min-1*g-1) was determined prior to (dark bars) and following administration of maximally vasodilating dose of adenosine. A significant decline in the LAD territory resistance is seen all 4 groups (Fig. 3A) while only VEGF-treated groups showed a significant decline in coronary resistance in the LCX territory (Fig. 3B). (Local, local VEGF delivery; i.c., intracoronary VEGF bolus delivery; pump, pump VEGF delivery). *p<0.05 vs. control (ANOVA, Bonferroni t-test); ^p<0.05 post-adenosine vs. pre-adenosine (paired t-test).

 
In addition to coronary blood flow analysis, MR-based perfusion assessment of the myocardium was carried out at 3 and 6 week time points. The size of the zone of delayed contract arrival was similar in all groups 3 weeks after ameroid occluder implantation (control: 15±5.5, local VEGF: 22±6.4, i.c. VEGF: 18±13, pump VEGF: 16±8.3% LV volume, F=1.77, p=NS by ANOVA). However, at the time of final study all VEGF groups demonstrated a marked reduction in the size of delayed arrival zone while the control group showed no significant change (Fig. 4). Comparison of the size of the zone at the time of final study between the four groups demonstrated marked differences (F=5.17, p=0.006) with significant reductions (p<0.05 by Bonferroni t-tests) in animals treated with local intracoronary and pump administration of VEGF.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Magnetic resonance assessment of myocardial perfusion. The extent of myocardial perfusion determined as% left ventricular (%LV) myocardium showing delayed increase in contrast signal intensity on an MR perfusion study was assessed for all 4 groups 3 weeks after ameroid implantation (1st study, black bars) and following completion of assigned therapy (2nd study, gray bars). No significant differences in the extent of myocardial territories showing absent perfusion were observed between any of the groups at the time of initial study. However, at 6 weeks there was a significant reduction in the size of the delayed arrival zone in all VEGF-treated but not control groups. (Local, local VEGF delivery; i.c., intracoronary VEGF bolus delivery; pump, pump VEGF delivery). *p<0.05, paired-t-test; ^p<0.05, Bonferroni t-test, VEGF treatment group vs. control.

 
3.3 Left ventricular function
To assess the functional significance of VEGF-mediated improvement in myocardial blood flow, we employed MR imaging to assess global and regional myocardial function in all study animals. The porcine ameroid occlusion model is associated with the development of small areas of left ventricular myocardial necrosis in most animals [13]. To exclude the possibility that differences in infarct size could affect comparisons of ventricular function between different groups, the extent of myocardial necrosis in each group was assessed by MR imaging at the time of treatment initiation (3 weeks post-ameroid implantation) and at the time of final study (6 weeks), with myocardial infarction defined as areas of myocardium showing no increase in contrast signal intensity on perfusion imaging. Using this parameter, there was no significant differences in the extent of myocardial necrosis among any groups at either 3 weeks (control: 1.3±3.1, local VEGF: 1.6±3.5, i.c. VEGF: 3.0±7.3, pump VEGF: 3.0±4.8% LV volume, ANOVA, p=NS) or 6 weeks (control: 1.1±2.8, local VEGF: 1.7±3.5, i.c. VEGF 4.7±7.8, pump VEGF: 3.4±4.4% LV volume, ANOVA, p=NS).

Left ventricular ejection fraction determined at rest three weeks following occluder implantation was similar in all treatment groups and was within normal limits for this model of intubated closed-chest pigs (control, 46±2.6; local, 48±1.2%; intracoronary, 49±2.7%; pump, 49±2.1%; ANOVA, p=NS). Analysis of wall thickening in the anterior wall (LAD territory) likewise demonstrated no difference among the groups (data not shown). At the same time, there was evidence of impaired regional wall motion (% wall thickening) in the LCX territory in all animal groups (there was no difference between the groups) 3 weeks after ameroid occluder placement (% wall thickening, LCX vs. LAD: 18.16±10.53% vs. 47.32±4.14%, p<0.001).

Repeat evaluation of global and regional LV function was carried out 3 weeks following assignment to one of the VEGF or control groups. At that time, again there was no significant differences between the groups with regard to LV ejection fraction (control, 48±3.3%; local, 51±1.9%; intracoronary, 50±2.9%; pump, 50±1.9%; ANOVA, p=NS), and analysis of regional wall motion in the LAD territory demonstrated no changes from baseline (data not shown). There was a significant improvement in regional wall thickening in the LCX territory in all 3 VEGF groups compared to pre-treatment values while control animals demonstrated no such improvement (paired t-test, Fig. 5).

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of VEGF administration on regional wall thickening. MR-determined% regional wall thickening was assessed at 3 (1st study, black bars) and 6 (2nd study, gray bars) weeks after ameroid constrictor placement in the LCX (Fig. 5A) and LAD (Fig. 5B) territories all 4 groups. Note significant improvements in the LCX region in animals treated with all forms of VEGF delivery. *p<0.05, post-treatment vs. pre-treatment (paired t-test).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
A number of growth factors including several members of the fibroblast growth factor (FGF) family (FGF-1, FGF-2, FGF-5) as well as VEGF have been shown to stimulate angiogenesis in vitro and in vivo [3–7, 22–28]. However, given the typically long time course of new collateral development, most attempts to stimulate angiogenesis have employed various methods of prolonged growth factor delivery including gene therapy, continuous infusions, repeat injections or sustained release polymers to provide extended exposure to the desired cytokine. While substantial enhancement in collateral flow as well as in absolute coronary blood flow and left ventricular function were seen in these studies, such delivery methods present significant practical difficulties, including the need for multiple administrations or implantation of delivery devices. In contrast to these prolonged dosing schedules, beneficial effects of VEGF administration were seen in animal models of peripheral ischemia following single-bolus intraarterial infusion of VEGF [28]or plasmid-based gene therapy [29]. However, there have been no successful attempts to achieve physiologically significant improvement in coronary blood flow using a single dose VEGF regimen in setting of chronic myocardial ischemia. While the study of Hariawala et al. examined the effect of single bolus delivery of 500 µg of VEGF in pig ameroid model [10], the study was complicated by a 50% mortality in the treatment group (four of eight animals) secondary to refractory VEGF-induced hypotension thereby rendering the results less than conclusive.

With these considerations in mind, the present study was designed to assess the effectiveness of two forms of intracoronary delivery: single bolus intracoronary infusion and local catheter-based intracoronary delivery using a local delivery catheter, and to compare them to a previously studied local adventitial delivery system using an implantable osmotic polymer pump, with regard to stimulation of physiologically meaningful angiogenesis in a porcine model of chronic myocardial ischemia.

To study these different means of VEGF delivery, we initiated therapy three weeks after placement of ameroid constrictors. This study design allowed us to document the ameroid occlusion prior to initiation of therapy and to obtain serial data with regard to changes in coronary flow and left ventricular function in all study animals. Furthermore, this study protocol avoided potential complications associated with initiation of therapy prior to the occluder placement, given the theoretical potential of heparin-binding growth factors such as VEGF to influence the rate of constrictor closure and thereby affect the final outcome.

We found that ameroid constrictors were occluded in all study animals 3 weeks after placement. Comparison of left ventricular infarct sizes as well as global and regional left ventricular function parameters showed no differences among the four groups prior to initiation of treatment. Furthermore, the size of myocardial infarcts in all 4 groups (1.2 to 5%) were within the range reported for this model [13]. Likewise, there were no significant differences in absolute coronary flow values or the extent of MR-determined perfusion in either LCX or LAD territories between the groups. We conclude, therefore, that all 4 groups were closely matched with regard to coronary flow, left ventricular function and infarction size prior to initiation of VEGF delivery.

Implantation of ameroid occluders resulted in decreased resting blood flow in the LCX compared to the LAD territory in all animals that was accompanied by reduced left ventricular wall thickening and radial wall motion. The subsequent recovery of these parameters suggests the presence of hibernating myocardium in this model. The presence of chronically reduced blood flow in the LCX territory in control animals correlates with previously observed increases in expression of VEGF [5]as well as VEGF and bFGF receptors [30], all known to be sensitive to ischemia/hypoxia induction of expression [2], in the LCX but not in normal (LAD) myocardium seen as long as 8 weeks after ameroid implantation.

VEGF administration by either local, intracoronary or extravascular delivery was associated with a significant improvement in the coronary flow in the circumflex territory at 3 weeks following initiation of therapy compared to flow values prior to treatment, while control animals showed no such improvement. Similar changes in coronary flow in the chronically ischemic myocardium at rest were seen following bFGF administration in the same model [26]. Additional evidence of VEGF-enhanced perfusion comes from examination of coronary resistance following adenosine infusion. As expected, there was a substantial decline in resistance in the LAD territory in all animals. At the same time, there was no significant reduction in LCX resistance in control animals suggesting decreased vasodilatory reserve in this territory. In contrast, VEGF therapy was associated with restoration of adenosine-induced vasodilation in the LCX territory thus demonstrating improvement in vasodilatory reserve. MR analysis of tissue perfusion demonstrated marked reduction in the size of the zone of delayed contrast arrival in VEGF-treated but not control animals.

In accord with microsphere-and MR-based determinations of improved tissue perfusion, angiographic analysis of collateral density demonstrated a significant increase in the number of visible collateral vessels in all three VEGF groups but not in control animals. Interestingly, while there was an increase in the number of left-to-left collaterals, there was no increase in the number of right-to-left collaterals. It is not clear whether this spatial inhomogeneity of collateral development is the result of growth factor administration into the left coronary system, the relative proximity of LAD and LCX but not LCX and RCA coronary branches in porcine hearts or some other unknown effect.

In addition to improvement in angiographic collateral index, and myocardial blood flow, VEGF therapy was associated with significant recovery of regional left ventricular wall motion in the LCX territory thus documenting functional benefit of cytokine-augmented angiogenesis in this single dose treatment regimen.

This demonstration of VEGF efficacy in improving coronary flow and function in myocardial ischemia further supports our prior observation demonstrating the beneficial effect of VEGF pump delivery in a similar model [5], as well as studies of Banai et al. in a dog ameroid constrictor model [7]and single bolus VEGF administration in peripheral circulation model of chronic ischemia [28]. However, these findings contrast with a recent study that found no beneficial effects of repetitive left atrial boluses of VEGF in a canine model of myocardial ischemia [8]. Potential explanations for this difference in outcome include handling of the growth factor, presence of adequate amounts of heparin to prevent VEGF binding to tubing, timing of growth factor delivery, as well as fundamental physiological differences between the extensively collateralized dog and substantially less collateralized pig ischemia models. The latter point is perhaps particularly relevant given that previous studies using the same porcine ameroid model have shown gradual improvements in control animals progressing to almost complete restoration of resting myocardial blood flow and function due to spontaneous collateral development [31]. The time course of these changes however, is considerably longer than could be monitored in the present study, suggesting that VEGF therapy accelerates and perhaps augments endogenous angiogenesis.

We did not observe any significant differences between the three forms of VEGF delivery employed in this study. InfusaSleeve^TM, a local delivery catheter used in this study, achieves significant deposition of material into the arterial wall with moderate (6 atm) pressure inflation [32]. Higher pressure inflations (30 atm) as used in this study, results in periadventitial and intramyocardial deposition of the material; however the efficiency of this delivery system still remains rather low (<2% of the total load, M. Simons and A. Kaplan, unpublished observations). The use of a local delivery device is potentially associated with a risk of local arterial complications (thrombosis, dissection, subacute closure) and a long-term risk of restenosis secondary to injury-induced neointimal proliferation, although none of these complications were observed in the current study. The potential benefit of local administration is the ability to achieve a desirable effect with a smaller dose of VEGF thus minimizing hemodynamic effects associated with intracoronary bolus delivery [21].

It is important to consider several limitations of the study. First, the statistical analysis of inter-group comparison is based on post-hoc combination of control and heparin groups. Second, three VEGF pump animals that died during pump replacement may have had especially deficient collateral supply thus contributing to a selection bias. Finally, degree of coronary vasodilation in response to adenosine seen in this study was somewhat less than typically observed in similar settings. To some extent this difference may be attributable to general anesthesia and a somewhat low mean blood pressure in our study.

In summary, we observed significant improvements in myocardial blood flow and regional myocardial function following initiation of VEGF therapy in the setting of fully established chronic myocardial ischemia in a porcine model. Thus single bolus intracoronary or local VEGF administration may prove to be useful therapeutic strategies for treatment of myocardial ischemia.

Time for primary review 43 days

	Acknowledgements

 
Supported in part by NIH grants HL-46716 (FWS), HL-53793 (MS) and a grant from Genentech, Inc. Dr. Lopez and Dr. Simons were also supported by the Clinical Investigator Training Program, Beth Israel Hospital-Harvard/MIT Health Science and Technology, in collaboration with Pfizer, Inc.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Folkman J, Shing Y. Angiogenesis. J Biol Chem. (1992) 267:10931–10934.[Free Full Text]
2. Ware J.A, Simons M. Angiogenesis in ischemic heart disease. Nature Med. (1997) 3:158–164.[CrossRef][Web of Science][Medline]
3. Yanagisawa-Miwa A, Uchida Y, Nakamura F, et al. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science. (1992) 257(5075):1401–1403.[Abstract/Free Full Text]
4. Harada K, Grossman W, Friedman M, et al. Basic fibroblast growth factor improves myocardial function in chronically ischemic porcine hearts. J Clin Invest. (1994) 94(2):623–630.[Web of Science][Medline]
5. Harada K, Friedman M, Lopez J.J, et al. Vascular endothelial growth factor administration in chronic myocardial ischemia. Am J Physiol. (1996) 270(5 Pt 2):H1791–H1802.[Medline]
6. Unger E.F, Banai S, Shou M, et al. Basic fibroblast growth factor enhances myocardial collateral flow in a canine model. Am J Physiol. (1994) 266(4 Pt 2):H1588–H1595.[Web of Science][Medline]
7. Banai S, Jaklitsch M.T, Shou M, et al. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. (1994) 89(5):2183–2189.[Abstract/Free Full Text]
8. Lazarous D.F, Shou M, Scheinowitz M, et al. Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury. Circulation. (1996) 94:1074–1082.[Abstract/Free Full Text]
9. Ku D.D, Zaleski J.K, Liu S, Brock T.A. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol. (1993) 265(2 Pt 2):H586–H592.[Web of Science][Medline]
10. Hariawala M.D, Horowitz J.J, Esakof D, et al. VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts. J Surg Res. (1996) 63(1):77–82.[CrossRef][Web of Science][Medline]
11. Yang R, Thomas G, Bunting S, et al. Effects of vascular endothelial growth factor on hemodynamic and cardiac performance. J Cardiovasc Pharmacol. (1996) 27:838–844.[CrossRef][Web of Science][Medline]
12. Longhurst J, Ordway G, Buja L. Evaluation of coronary native and coronary collateral pressure gradients in the conscious dog. Am J Cardiovasc Pathol. (1987) 1:79–90.[Medline]
13. Roth D, Maruoka Y, Rogers J, White F, Longhurst J, Bloor C. Development of coronary collateral circulation in left circumflex Ameroid-occluded swine myocardium. Am J Physiol. (1987) 253(5 Pt 2):H1279–1288.[Web of Science][Medline]
14. Pearlman J.D, Hibberd M.G, Chuang M.L, et al. Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nat Med. (1995) 1(10):1085–1089.[CrossRef][Web of Science][Medline]
15. Rentrop K.P, Cohen M, Blanke H, Phillips R.A. Changes in collateral channel filling immediately after controlled coronary artery occlusion by an angioplasty balloon in human subjects. J Am Coll Cardiol. (1985) 5(3):587–592.[Abstract]
16. Fujita M, Sasayama S, Asanoi H, Nakajima H, Sakai O, Ohno A. Improvement of treadmill capacity and collateral circulation as a result of exercise with heparin pretreatment in patients with effort angina. Circulation. (1988) 77(5):1022–1029.[Abstract/Free Full Text]
17. Carroll S.M, White F.C, Roth D.M, Bloor C.M. Heparin accelerates coronary collateral development in a porcine model of coronary artery occlusion. Circulation. (1993) 88:198–207.[Abstract/Free Full Text]
18. Kowallik P, Schulz R, Guth B.D, et al. Measurement of regional myocardial blood flow with multiple colored microspheres. Circulation. (1991) 83(3):974–982.[Abstract/Free Full Text]
19. VanRugge F.P, Botreel J.J, Van der Walls E.E, et al. Cardiac first pass myocardial perfusion in normal subjects assessed by sub-second Gd-DPTA enhanced MR imaging. J Comp Assisted Tomogr. (1991) 15:959–965.
20. Pearlman J, Hogan R, Wiske P, Franklin T, Weyman A. Echocardiographic definition of the left ventricle centroid; analysis of methods for centroid calculation from a single tomogram. J Am Coll Cardiol. (1990) 16(4):986–992.[Abstract]
21. Lopez J, Laham R.J, Carrozza J.C, et al. Hemodynamic effects of intracoronary VEGF delivery: evidence of tachyphylaxis and NO dependence of response. Am J Physiol. (1997) 273:H1317–H1323.[Web of Science][Medline]
22. Bauters C, Asahara T, Zheng L.P, et al. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol. (1994) 267:H1263–H1271.[Web of Science][Medline]
23. Bauters, C. Asahara, T. Zheng, L.P. et al. Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg. 1995; 21(2): 314-24; discussion 324-5.
24. Banai S, Jaklitsch M.T, Casscells W, et al. Effects of acidic fibroblast growth factor on normal and ischemic myocardium. Circ Res. (1991) 69(1):76–85.[Abstract/Free Full Text]
25. Baffour R, Berman J, Garb J.L, Rhee S.W, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg. (1992) 16(2):181–191.[CrossRef][Web of Science][Medline]
26. Lopez J.J, Edelman E.R, Stamler A, et al. Basic fibroblast growth factor in a porcine model of chronic myocardial ischemia: a comparison of angiographic, echocardiographic and coronary flow parameters. J Pharmacol Exp Ther. (1997) 282:385–390.[Abstract/Free Full Text]
27. Takeshita S, Pu L.Q, Stein L.A, et al. Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation. (1994) 90:II228–II234.[Medline]
28. Takeshita S, Zheng L.P, Brogi E, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest. (1994) 93(2):662–670.[Web of Science][Medline]
29. Isner J, Walsh K, Symes J, et al. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. Circulation. (1995) 91:2687–2692.[Free Full Text]
30. Sellke F.W, Wang S.Y, Stamler A, et al. Enhanced microvascular relaxations to VEGF and bFGF in chronically ischemic porcine myocardium. Am J Physiol. (1996) 271:H713–H720.[Medline]
31. White F, Carroll S, Magnet A, Bloorm C. Coronary collateral development in swine after coronary artery occlusion. Circ Res. (1992) 71(6):1490–1500.[Abstract/Free Full Text]
32. Gottsauner-Wolf M, Jang Y, Penn M.S, et al. Quantitative evaluation of local drug delivery using the InfusaSleeve catheter. Cathet Cardiovasc Diagn. (1997) 42(1):102–108.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				R. C. Scott, J. M. Rosano, Z. Ivanov, B. Wang, P. L.-G. Chong, A. C. Issekutz, D. L. Crabbe, and M. F. Kiani Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function FASEB J, October 1, 2009; 23(10): 3361 - 3367. [Abstract] [Full Text] [PDF]

				J. Zhang, L. Ding, Y. Zhao, W. Sun, B. Chen, H. Lin, X. Wang, L. Zhang, B. Xu, and J. Dai Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction Circulation, April 7, 2009; 119(13): 1776 - 1784. [Abstract] [Full Text] [PDF]

				G. Wu, J. S. Rana, J. Wykrzykowska, Z. Du, Q. Ke, P. Kang, J. Li, and R. J. Laham Exercise-induced expression of VEGF and salvation of myocardium in the early stage of myocardial infarction Am J Physiol Heart Circ Physiol, February 1, 2009; 296(2): H389 - H395. [Abstract] [Full Text] [PDF]

				K. A. Horvath and Y. Zhou Transmyocardial Laser Revascularization and Extravascular Angiogenetic Techniques to Increase Myocardial Blood Flow Card. Surg. Adult, January 1, 2008; 3(2008): 733 - 752. [Full Text]

				L. Ye, H. Kh. Haider, S. Jiang, R. S. Tan, R. Ge, P. K. Law, and E. K.W. Sim Improved angiogenic response in pig heart following ischaemic injury using human skeletal myoblast simultaneously expressing VEGF165 and angiopoietin-1 Eur J Heart Fail, January 1, 2007; 9(1): 15 - 22. [Abstract] [Full Text] [PDF]

				S. Mieno, M. Boodhwani, R. T. Clements, B. Ramlawi, N. R. Sodha, J. Li, and F. W. Sellke Aging is associated with an impaired coronary microvascular response to vascular endothelial growth factor in patients J. Thorac. Cardiovasc. Surg., December 1, 2006; 132(6): 1348 - 1355. [Abstract] [Full Text] [PDF]

				P. Oettgen Regulation of Vascular Inflammation and Remodeling by ETS Factors Circ. Res., November 24, 2006; 99(11): 1159 - 1166. [Abstract] [Full Text] [PDF]

				S. U. Lee, J. J. Wykrzykowska, and R. J. Laham Angiogenesis: Bench to Bedside, Have We Learned Anything? Toxicol Pathol, January 1, 2006; 34(1): 3 - 10. [Abstract] [Full Text] [PDF]

				R. J. Laham, M. Post, M. Rezaee, L. Donnell-Fink, J. J. Wykrzykowska, S. U. Lee, D. S. Baim, and F. W. Sellke TRANSENDOCARDIAL AND TRANSEPICARDIAL INTRAMYOCARDIAL FIBROBLAST GROWTH FACTOR-2 ADMINISTRATION: MYOCARDIAL AND TISSUE DISTRIBUTION Drug Metab. Dispos., August 1, 2005; 33(8): 1101 - 1107. [Abstract] [Full Text] [PDF]

				N. M. Degabriele, U. Griesenbach, K. Sato, M. J. Post, J. Zhu, J. Williams, P. K. Jeffery, D. M. Geddes, and E. W. F. W. Alton Critical appraisal of the mouse model of myocardial infarction Exp Physiol, July 1, 2004; 89(4): 497 - 505. [Abstract] [Full Text] [PDF]

				L. Ye, H. K Haider, S.-J. Jiang, and E. K. Sim Therapeutic Angiogenesis Using Vascular Endothelial Growth Factor Asian Cardiovasc Thorac Ann, June 1, 2004; 12(2): 173 - 181. [Abstract] [Full Text] [PDF]

				K. Ueyama, G. Bing, Y. Tabata, M. Ozeki, K. Doi, K. Nishimura, H. Suma, and M. Komeda Development of biologic coronary artery bypass grafting in a rabbit model: Revival of a classic concept with modern biotechnology J. Thorac. Cardiovasc. Surg., June 1, 2004; 127(6): 1608 - 1615. [Abstract] [Full Text] [PDF]

				G. C. Hughes, S. S. Biswas, B. Yin, R. E. Coleman, T. R. DeGrado, C. K Landolfo, J. E. Lowe, B. H. Annex, and K. P. Landolfo Therapeutic angiogenesis in chronically ischemic porcine myocardium: comparative effects of bFGF and VEGF Ann. Thorac. Surg., March 1, 2004; 77(3): 812 - 818. [Abstract] [Full Text] [PDF]

				G. von Degenfeld, P. Raake, C. Kupatt, C. Lebherz, R. Hinkel, F. J. Gildehaus, W. Munzing, A. Kranz, J. Waltenberger, M. Simoes, et al. Selective Pressure-Regulated retroinfusion of fibroblast growth factor-2 into the coronary vein enhances regional myocardial blood flow and function in pigs with chronic myocardial ischemia J. Am. Coll. Cardiol., September 17, 2003; 42(6): 1120 - 1128. [Abstract] [Full Text] [PDF]

				T. D. Henry, B. H. Annex, G. R. McKendall, M. A. Azrin, J. J. Lopez, F. J. Giordano, P.K. Shah, J. T. Willerson, R. L. Benza, D. S. Berman, et al. The VIVA Trial: Vascular Endothelial Growth Factor in Ischemia for Vascular Angiogenesis Circulation, March 18, 2003; 107(10): 1359 - 1365. [Abstract] [Full Text] [PDF]

				H. Su, J. Arakawa-Hoyt, and Y. W. Kan Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model PNAS, July 9, 2002; 99(14): 9480 - 9485. [Abstract] [Full Text] [PDF]

				R. J. Laham, M. Simons, J. D. Pearlman, K. K. L. Ho, and D. S. Baim Magnetic resonance imaging demonstrates improved regional systolic wall motion and thickening and myocardial perfusion of myocardial territories treated by laser myocardial revascularization J. Am. Coll. Cardiol., January 2, 2002; 39(1): 1 - 8. [Abstract] [Full Text] [PDF]

				S. B. Freedman and J. M. Isner Therapeutic Angiogenesis for Coronary Artery Disease Ann Intern Med, January 1, 2002; 136(1): 54 - 71. [Abstract] [Full Text] [PDF]

				F. L. Celletti, P. R. Hilfiker, P. Ghafouri, and M. D. Dake Effect of human recombinant vascular endothelial growth factor165 on progression of atherosclerotic plaque J. Am. Coll. Cardiol., June 15, 2001; 37(8): 2126 - 2130. [Abstract] [Full Text] [PDF]

				M. Simons Therapeutic coronary angiogenesis: a fronte praecipitium a tergo lupi? Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H1923 - H1927. [Full Text] [PDF]

				X. Xu, J. Li, M. Simons, J. Li, R. J. Laham, and F. W. Sellke Expression of vascular endothelial growth factor and its receptors is increased, but microvascular relaxation is impaired in patients after acute myocardial ischemia J. Thorac. Cardiovasc. Surg., April 1, 2001; 121(4): 735 - 742. [Abstract] [Full Text] [PDF]

				M. J. Post, R. Laham, F. W. Sellke, and M. Simons Therapeutic angiogenesis in cardiology using protein formulations Cardiovasc Res, February 16, 2001; 49(3): 522 - 531. [Abstract] [Full Text] [PDF]

				S. E. Epstein, S. Fuchs, Y. F. Zhou, R. Baffour, and R. Kornowski Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards Cardiovasc Res, February 16, 2001; 49(3): 532 - 542. [Abstract] [Full Text] [PDF]

				K. Sato, T. Wu, R. J. Laham, R. B. Johnson, P. Douglas, J. Li, F. W. Sellke, S. Bunting, M. Simons, and M. J. Post Efficacy of intracoronary or intravenous VEGF165 in a pig model of chronic myocardial ischemia J. Am. Coll. Cardiol., February 1, 2001; 37(2): 616 - 623. [Abstract] [Full Text] [PDF]

				R. J. Laham, N. A. Chronos, M. Pike, M. E. Leimbach, J. E. Udelson, J. D. Pearlman, R. I. Pettigrew, M. J. Whitehouse, C. Yoshizawa, and M. Simons Intracoronary basic fibroblast growth factor (FGF-2) in patients with severe ischemic heart disease: results of a Phase I open-label dose escalation study J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2132 - 2139. [Abstract] [Full Text] [PDF]

				M. Simons, R. O. Bonow, N. A. Chronos, D. J. Cohen, F. J. Giordano, H. K. Hammond, R. J. Laham, W. Li, M. Pike, F. W. Sellke, et al. Clinical Trials in Coronary Angiogenesis: Issues, Problems, Consensus : An Expert Panel Summary Circulation, September 12, 2000; 102 (11): e73 - e86. [Abstract] [Full Text] [PDF]

				I. Zachary, A. Mathur, S. Yla-Herttuala, and J. Martin Vascular Protection : A Novel Nonangiogenic Cardiovascular Role for Vascular Endothelial Growth Factor Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1512 - 1520. [Abstract] [Full Text] [PDF]

				C. Operschall, L. Falivene, J.-P. Clozel, and S. Roux A new model of chronic cardiac ischemia in rabbits J Appl Physiol, April 1, 2000; 88(4): 1438 - 1445. [Abstract] [Full Text] [PDF]

				A. Haunstetter and S. Izumo Toward Antiapoptosis as a New Treatment Modality Circ. Res., March 3, 2000; 86(4): 371 - 376. [Full Text] [PDF]

				R. Kornowski, S. Fuchs, M. B. Leon, and S. E. Epstein Delivery Strategies to Achieve Therapeutic Myocardial Angiogenesis Circulation, February 1, 2000; 101(4): 454 - 458. [Abstract] [Full Text] [PDF]

				H. Su, R. Lu, and Y. W. Kan Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart PNAS, December 5, 2000; 97(25): 13801 - 13806. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Lopez, J. J Articles by Simons, M. Search for Related Content PubMed PubMed Citation Articles by Lopez, J. J Articles by Simons, M. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics