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

Induction of chronic myocardial ischemia by implantation of a reduction stent into the left anterior descending artery (LAD)

A coronary polytetrafluoroethylene-covered stent-graft (Jomed, Germany) of 13 mm length was placed on a 1.5 mm diameter needle, and a ligature was placed around the middle of the stent (Fig. 1A). Thereafter, the stent was crimped on a percutaneous transluminal angioplasty-balloon (20 mm length/3.0 mm diameter) (Fig. 1B) and implanted (14 atm) into the proximal LAD in all 52 pigs. The stent remained unexpanded at the site of the ligature, resulting in an hourglass-shaped stenosis (Fig. 1B). The degree of stenosis calculated from the cross-sectional area at the site of the ligature (1.8 mm²) and at the expanded edges of the stent (7.1 mm²) was 75%. An antiplatelet and anticoagulation regimen consisting of clopidogrel 75 mg PO, ASA 100 mg PO, and reviparine 0.25 ml SC was administered daily for 28 days.

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Figure 1

Induction of chronic myocardial ischemia using a reduction stent. (A)A stent graft is ligated in the center. (B)The stent is crimped on balloon and inflated at 14 atm; it remains unexpanded at the site of the ligature, resulting in an hourglass-shaped stenosis. (C)Postmortem angiogram at day 28. The arrowindicates the location of the reduction stent, which is totally occluded. Collaterals are visible connecting the proximal left anterior descending artery (LAD) and diagonal branches to the distal LAD.

After seven days, the degree of LAD stenosis at the site of the stent was reexamined (fluoroscopy). If the stent was occluded, pigs were excluded from further analysis (n = 5). In all other pigs, hemodynamics and left ventricular (LV) function (LV end-systolic and end-diastolic pressures, dp/dt_max, dp/dt_min) were measured using a microtip catheter (Millar). Cardiac output was determined by thermodilution.

Application of FGF-2

Seven days after implantation of the reduction stent and after fluoroscopic control of the patency of the stent, pigs were treated by retroinfusion of saline (controls, group A, n = 7) or 150 μg FGF-2 (retroinfusion, group B, n = 7). In group C (n = 7), pigs were treated with antegrade intracoronary infusion of 150 μg FGF-2 into the LAD.

In groups A and B, a 6F 4-lumen retroinfusion catheter was selectively placed into the anterior cardiac vein (12–14). Pressure-regulated retroinfusion of arterial blood without suction was carried out for 30 min with a preset retroinfusion pressure of about 20 mm Hg higher than the systolic coronary venous occlusion pressure, resulting in a mean retroinfusion pressure of 58 ± 9 mm Hg. Heparinized saline solution containing 150 μg FGF-2 (Peprotech, Germany) was continuously added to the retroinfused arterial blood throughout the 30-min treatment period in group B, whereas heparinized saline solution alone was added in the controls (group A). In group C, heparinized saline solution containing 150 μg FGF-2 was continuously infused during 30 min into the LAD proximal to the reduction stent.

In six additional experiments, the binding of ¹²⁵iodine-labeled FGF-2 was studied to compare antegrade and retrograde application. Fibroblast growth factor-2 was radiolabeled using the chloramine T-catalyzed iodination method similar to that previously described in detail (15). Specific radioactivity of the iodinated FGF-2 was 11 μCi/μg. [¹²⁵I]Fibroblast growth factor-2 retained full biological activity in a proliferation assay (data not shown). In six pigs with similar high-grade stenosis seven days after implantation of the reduction stent, 30 μg [¹²⁵I]FGF-2 and 120 μg unlabeled FGF-2 were applied during 30 min, either by antegrade infusion (LAD) or by pressure-regulated retroinfusion (anterior cardiac vein). Forty-five minutes after application, the pigs were euthanized, and transmural samples from the targeted LAD territory and the nontargeted circumflex artery (CX) territory and different organs were harvested and measured using a γ-counter (LKB, Sweden).

Regional myocardial blood flow

Regional myocardial blood flow was measured using fluorescent labeled microspheres (16). Briefly, 1 × 10⁷microspheres were injected into the left atrium (Molecular-Probes) for each measurement. Reference blood samples were withdrawn from the abdominal aorta at a rate of 4.1 ml/min.

In seven pigs used to establish the reduction-stent model, regional myocardial blood flow was studied at rest and during adenosine-induced flow reserve (80 μg·kg⁻¹·min⁻¹intracoronary) using different colors of microspheres seven and 28 days after implantation of the reduction-stent graft.

Regional myocardial blood flow at rest and during pacing (110 beats/min) was determined at day 28 after implantation of the reduction stent in groups A, B, and C. In all pigs, two transmural myocardial samples were obtained from the proximal and distal ischemic LAD territory and one sample from the CX territory after euthanasia. Samples were divided into endocardial, midmyocardial, and epicardial probes. Fluorescence was measured using a sample processing unit (16).

Regional myocardial function

Subendocardial segment shortening was determined by sonomicrometry (HSE, Germany) (17). After sternotomy, three pairs of 5-MHz ultrasonic crystals were implanted in two defined areas of the ischemic LAD territory (1 cm and 3 cm distal to the reduction stent) and in the CX territory.

Capillary density

The central slice of each transmural myocardial probe was used for assessment of capillary density. Alkaline phosphatase staining was performed (18)and separately analyzed by two investigators blinded to the groups.

Collateral score

An in vivo angiogram was performed prior to thoracotomy (Exposcop 8000, Ziem, Germany). For assessment of collateral arteries, a postmortem angiogram was obtained (Coroskop HIP, Siemens, Germany) by flushing the coronaries of the excised heart with saline and 0.2 mg glyceryl-trinitrate, followed by injection of 10 ml contrast agent into the left coronary artery (100 mm Hg). In the postmortem angiogram, collateral arteries connecting the CX, the proximal LAD, or the first diagonal branch with the distal LAD were counted by two separate investigators blinded for the groups (19).

Assessment of infarct size and viability

In seven pigs used to establish the reduction-stent model, infarct size was determined at 28 days by tetrazolium-chloride staining followed by slicing of the LV (14)using computer based planimetry (SigmaScan-Pro). In groups A, B, and C myocardial infarction (MI) was determined at day 28 by morphometric assessment and by histology after standardized sampling of nine representative transmural probes (0.5 cm in diameter) of the entire LAD territory (1 cm, 2 cm, and 3 cm distal to the reduction stent as well as 0.5 cm, 1.5 cm, and 2.5 cm perpendicular to the LAD). Each sample was cut on a microtome (15 μm sections; hemalaun-eosine staining) and assessed for the presence of MI.

Fluorodeoxyglucose (FDG)-positron emission tomography (PET)

Dynamic PET was performed in three pigs of the control group using a whole-body scanner (CTI/ECAT 951R/31; Siemens, Germany). Twenty mCi of ¹³N-labeled ammonia (¹³NH₃) were administered intravenously (30 s). Dynamic PET acquisition was performed using varying frame durations (12 × 10 s, 6 × 30 s, and 3 × 300 s). Thirty minutes later 10 mCi of ¹⁸FDG were injected and dynamic image acquisition was repeated. A threshold of <50% of the maximum pixel count density was used for defining areas with significant reduction in ¹³NH₃uptake (perfusion defect). Further processing and analysis comprised myocardial rate of glucose uptake in ¹⁸FDG study.

Statistical analysis

All data were analyzed using SPSS. Data are presented as mean ± standard error of the mean (SEM). The investigators performing the experiments at day 28 were blinded for the assigned treatment groups at day seven. Differences between groups A, B, and C were assessed with ANOVA. In case of statistically significant differences with ANOVA, two group comparisons were performed using Mann-Whitney test. A p value <0.05 was considered statistically significant.

Tissue binding of [¹²⁵i]FGF-2

To compare tissue binding of [¹²⁵I]FGF-2 after antegrade and retrograde delivery, radioactivity was determined in myocardial samples harvested 45 min after application at day seven in six pigs. After antegrade application, calculated FGF-2 retained in the targeted LAD territory was about 4.5 ± 1% (n = 3). After selective pressure-regulated retroinfusion, binding of FGF-2 in the targeted LAD territory was increased twofold to about 11 ± 2% (n = 3) (Fig. 4); thus, binding of [¹²⁵I]FGF-2 was increased 15- to 30-fold in the LAD territory compared with in the CX territory. After retrograde administration, FGF-2 binding was significantly lower in the lung (8,954 ± 1,860 vs. 128,140 ± 2,509 cpm/g, p < 0.05) but was not statistically different in the liver (84,148 ± 3,813 vs. 105,684 ± 27,521 cpm/g) and in the spleen (54,276 ± 6,059 vs. 82,716 ± 27,497 cpm/g).

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Figure 4

Tissue binding of ¹²⁵iodine-labeled fibroblast growth factor-2 45 min after retroinfusion into the anterior cardiac vein (hashed bars, n = 3) or antegrade application into the left anterior descending artery (LAD) (black bars, n = 3). CX = circumflex artery.

Collateral score and capillary density

At day 28, the number of visible collateral arteries at postmortem angiography was higher in the retroinfusion group B (5.0 ± 1.3; p < 0.05) compared with the control group A (2.95 ± 0.4) and with the antegrade group C (3.3 ± 0.3). Similar results were obtained with regard to capillary density (capillaries/muscle fiber) being higher in group B (1.45 ± 0.2; p < 0.05) than in group A (1.0 ± 0.17) and group C (1.05 ± 0.15).

Regional myocardial blood flow

At day 28, regional myocardial blood flow at rest in the LAD territory was significantly higher in pigs treated with retroinfusion of FGF-2 (group B) than in group A and in group C (Fig. 5): 1.07 ± 0.14 ml·min⁻¹·g⁻¹vs. 0.66 ± 0.07 ml·min⁻¹·g⁻¹, vs. 0.72 ± 0.17 ml·min⁻¹·g⁻¹. Of note, regional myocardial blood flow in the ischemic LAD territory was significantly lower than in the CX territory in all groups (Table 2). There was a gradient from proximal to distal probes and from the epicardial to the endocardial layers within the LAD territory in all groups (Table 2). In pigs treated by retroinfusion of FGF-2 (group B), this difference was significant between the proximal and distal endocardial probes (1.23 ± 0.15 vs. 0.69 ± 0.15 ml·min⁻¹·g⁻¹). Under the condition of increased oxygen demand during pacing (110 beats/min), regional myocardial blood flow in the LAD territory slightly increased in pigs of group B (retroinfusion of FGF-2) but was unchanged in group A and group C (Fig. 5).

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Figure 5

Regional transmural myocardial blood flow at day 28 in the left anterior descending artery (LAD) territory. Left panel= at rest; right panel= during pacing. Group A= controls; group B= retroinfusion of fibroblast growth factor-2; group C= antegrade delivery. *p < 0.05.

Regional myocardial function

The impact of FGF-2 treatment at day seven on regional myocardial function at day 28 was studied at rest and during pacing. Substantial preservation of regional myocardial function at day 28 was observed within the LAD territory after retroinfusion of FGF-2 at day seven in group B (Fig. 6). The efficacy, however, decreased with the distance to the reduction stent. In the proximal ischemic LAD territory (1 cm distal to the reduction stent), subendocardial segment shortening was well preserved in the retroinfusion group B (18.5 ± 4.1% [% end-diastolic length]) in contrast to controls (5.7 ± 2.9%) (Fig. 6, top panel). Similar results were obtained with regard to pacing induced functional reserve. In the distal LAD territory, regional myocardial function at rest was compromised in all groups (Fig. 6, bottom panel). During pacing, dyskinesia developed in groups A and C, in contrast to group B (Fig. 6, bottom panel).

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Figure 6

Regional myocardial function at day 28. White bars(group A) = controls; black bars(group B) = retroinfusion of fibroblast growth factor-2; hashed bars(group C) = antegrade delivery. Top panel= proximal ischemic left anterior descending artery (LAD) territory; bottom panel= distal ischemic LAD territory.

Global LV function

Global LV function was similar at day seven in all groups (Table 1). Twenty-one days after treatment, however, LVEDP increased in the control group A (16.5 ± 2.1 mm Hg) and was significantly higher than in the retroinfusion group B (10.8 ± 1.6 mm Hg). Antegrade infusion of FGF-2 (group C) was also associated with higher LV end-diastolic pressure (14.3 ± 1.5 mm Hg), though less pronounced than in controls.

Infarct size

At day 28 after implantation of the reduction stent, the extent of MI was not different in groups A, B, and C determined by morphometric and histological assessment. In nine representative probes of the LAD territory assessed from each pig, transmural necrosis was found in 0.9 ± 0.2 probes of group A, 1.0 ± 0.2 probes of group B, and 0.8 ± 0.2 probes of group C. Focal necrosis was seen in 1.9 ± 0.3 probes of group A, 1.9 ± 0.4 probes of group B, and 1.9 ± 0.3 probes of group C.

Chronic myocardial ischemia induced by implantation of a reduction stent

A novel catheter-based approach was used in this study instead of surgical placement of an ameroid constrictor (5,19)for induction of chronic myocardial ischemia. Using a percutaneously implanted reduction stent, a highly reproducible degree of LAD stenosis was achieved acutely and the degree of stenosis increased from about 72% to about 91% at seven days. Thus, regional myocardial blood flow in the LAD territory was moderately reduced at rest and flow reserve was significantly reduced (Fig. 2A). Adenosine stress was used to assess flow reserve and to compare it with the data known from the well-established ameroid constrictor model (5,19,20). Similar to the ameroid constrictor model, the time point of complete occlusion of the coronary artery was not exactly defined after implantation of the reduction stent. As the patency of the stents at seven days was a prerequisite for the inclusion in the treatment groups, total occlusion of the reduction stent occurred between day seven and day 28 (Fig. 1C).

The extent of MI at day 28 in controls was similar to the ameroid constrictor model applied in the CX in the pig (20). In contrast to the ameroid constrictor model (21,22), myocardial blood flow at rest was significantly reduced in controls at day 28 (Fig. 2B). Accordingly, regional myocardial function at rest was also impaired in the LAD territory, in line with the definition of hibernating myocardium and in agreement with the findings in a pig model of chronically hibernating myocardium (23,24). The presence of hibernating myocardium at day 28 was also confirmed in an exemplary fashion by the increased FDG uptake in the under-perfused LAD territory (Fig. 3).

Angiogenic effects induced by selective pressure-regulated retroinfusion of FGF-2

Selective pressure-regulated retroinfusion into the coronary veins was used for the first time to deliver recombinant angiogenic growth factors into ischemic myocardium, in line with the concept of prolonging contact time of drugs and gene vectors (12–14). After retroinfusion of FGF-2 (day seven), the number of collateral arteries visible at postmortem angiography (day 28) was significantly increased compared with in controls and with antegrade delivery. Although collateral blood flow in pigs is very low in acute ischemia (25), the development of collateral arteries has been described in case of severe chronic ischemia (19,26). Hence, the development of angiographically visible collateral arteries was enhanced after retroinfusion of FGF-2.

Histology also showed increased capillary density in the LAD territory 21 days after retroinfusion of FGF-2. Furthermore, short-term treatment (30 min) by retroinfusion of FGF-2 clearly enhanced tissue binding compared with antegrade delivery (Fig. 4). About 5% of the applied radioactivity was detected in the LAD territory after antegrade delivery of [¹²⁵I]FGF-2, similar to results obtained in a dog model (26). Retroinfusion resulted in an about twofold increase in tissue binding of FGF-2 compared with antegrade delivery, indicating successful targeting of FGF-2 to the ischemic myocardium. Because tissue binding of FGF-2 was determined only once in our study (45 min after application), we cannot exclude different redistribution kinetics with both modes of delivery.

Selective retroinfusion led to a 15- to 30-fold increase in tissue binding of [¹²⁵I]FGF-2 in the targeted ischemic LAD territory compared with in the non-ischemic CX territory. Nevertheless, systemic redistribution was present with both delivery methods, especially to the lungs, the liver, and the spleen. However, there were no abnormal macroscopic or histologic findings, such as hemangioma formation, in adjacent or remote organs.

Effects on regional myocardial blood flow and function

The impact of arteriogenesis and angiogenesis on regional myocardial blood flow and function was determined 21 days after treatment. Of note, regional myocardial blood flow was reduced under resting conditions in all three groups, indicating the presence of ischemia in the LAD territory (Fig. 5). The level of regional myocardial ischemia in the LAD territory, however, was different in the three groups. This difference was not explained by an imbalance in the extent of MI between the three groups, which was ruled out by morphometric and histological determination.

As expected from the increase in collaterals and the density of capillaries, regional myocardial blood flow at rest was higher in pigs treated by retroinfusion of FGF-2. Improved regional myocardial blood flow in the LAD territory of pigs treated by retroinfusion of FGF-2 was accompanied by a significant preservation of regional myocardial function, as segment shortening in the proximal LAD territory was substantially higher at rest and during increased oxygen demand. Increased oxygen demand by atrial pacing was used instead of adenosine-induced flow reserve to detect functional relevant recovery of hibernating myocardium. Thus, for the distal LAD territory, differences in segment shortening between the groups were detected during pacing, showing that regional dyskinesia was prevented only after retroinfusion of FGF-2 (Fig. 6). In contrast to the proximal LAD territory, the preservation of regional myocardial blood flow in the distal LAD territory was not sufficient to prevent myocardial dysfunction at rest, which is reasonable for a decrease in subendocardial blood flow to 0.69 ± 0.15 ml·min⁻¹·g⁻¹in pigs (27).

Although there was a trend toward improved regional myocardial blood flow and function in pigs treated by antegrade infusion, this effect did not reach statistical significance, which is in agreement with observations in dogs after single antegrade injection of FGF-2 (3). A limitation of the present study, however, is that treatment groups were not randomly assigned.

Conclusions

We characterized a new pig model with percutaneous induction of chronic myocardial ischemia and with preliminary data indicating the development of hibernating myocardium in the LAD territory. As catheter-based access to the cardiac veins is not hampered, this model is suitable for studying the effects of angiogenic proteins, such as FGF-2 by retrograde delivery into the cardiac veins. Selective pressure-regulated retroinfusion of FGF-2 significantly improved regional perfusion and function, suggesting that retrograde FGF-2 delivery increased tissue binding and targeted FGF-2 to vessel segments most receptive and responsive to them (28,29). The preservation of regional myocardial blood flow and function, however, was still incomplete after the short-term (30 min) retrograde treatment. Whether higher concentrations of FGF-2 or longer retroinfusion periods might enhance the efficacy has not been addressed in this study. Furthermore, the combined application of FGF-2 with other angiogenic proteins might be a promising approach based on reports of their synergistic effects (18,30).