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Eur J Cardiothorac Surg 2002;22:753-761
© 2002 Elsevier Science NL
a Division of Cardiovascular Surgery, Albert-Ludwigs-University of Freiburg, School of Medicine, Freiburg, Germany
b Division of Surgical Research, Albert-Ludwigs-University of Freiburg, School of Medicine, Freiburg, Germany
Received 22 April 2002; received in revised form 12 August 2002; accepted 21 August 2002.
* Corresponding author. Tel.: +49-761-270-2818; fax: +49-761-270-2550
e-mail: lutter{at}ch11.ukl.uni-freiburg.de
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Abstract |
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 Top Abstract 1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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Key Words: Coronary disease • Transmyocardial laser revascularization • Growth factors • Arteriogenesis • Angiogenesis • Perfusion • Contractility
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1. Introduction |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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The delivery of pro-angiogenic factors may enhance this neovascularization, and direct growth factor transfer may provide a means of effective delivery to the target myocardium. Basic fibroblastic growth factor (FGF-2) is a monomeric polypeptide that induces endothelial cell and smooth muscle cell proliferation in vitro and angiogenesis in vivo [9]. It has been implicated in the regulation of blood vessel homeostasis and proliferation, undoubtedly part of a complex cascade that involves interactions with other factors, their receptors, and their signal transduction pathways [10]. In addition, our clinical experience with FGF-1 indicated formation of new vascular networks which bypass distal stenoses of coronary arteries after intramyocardial application [11].
The aim of the present study was to determine whether the intramyocardial administration of exogenous human recombinant FGF-2 surrounding each laser channel could trigger a coordinated response, resulting in accelerated angio- and arteriogenesis, and therefore improved myocardial blood flow and enhanced contractility in a porcine model of chronically myocardial ischemia.
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2. Material and methods |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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2.1. Experimental model
To mimic clinical coronary artery disease, we employed a model of chronic myocardial ischemia [4,12], (Fig. 1)
. In the first operation, an operative stenosis of the left anterior descending artery (LAD) was created. One week later (second operation), the animals were studied by analyzing different parameters (see parameters below). Afterwards, pigs were randomly assigned to one of four experimental groups. After 12 weeks (third operation), the animals were reanalyzed (same parameters as before) and sacrificed [4].
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2.3. Second operation
Through a re-minithoracotomy, the pericardium was opened and reexposed 7 days after the onset of chronic ischemia under the same conditions as described above. The monitoring was performed, as reported elsewhere [3,4,12].
Once angiography and UTT flow probe data confirmed the presence of a severe LAD stenosis with blood flow reduction, see above; baseline measurements of segmental myocardial shortening were recorded and colored microspheres were injected as described below for examination of regional perfusion. Animals were designated to one of four groups. To define the area at risk the LAD was occluded for 10 s prior to treatment. The pigs received therapy or were left untreated (see Section 2.4). The thorax was closed, and the pigs were allowed to recover.
2.4. Experimental groups
2.4.1. Ischemic control group
After re-minithoracotomy during the second operation, parameters were assessed, but the hearts did not receive any therapy (n=7).
2.4.2. TMLR group
Pigs received the same treatment as the control group, except for additional treatment by TMLR during the second operation. They were treated by creating one laser channel (1 mm in diameter) per cm2 in the area at risk, as reported elsewhere [3,4]. An 800-Watt carbon dioxide laser (PLC Medical Systems, Franklin, MA) was used to produce transmyocardial channels discharging 34–40 J over a pulse width of 33–46 ms. Channels were confirmed by transesophageal echo and by pulsatile flow during systole. There were 14–16 TMLR sites per heart (n=7).
2.4.3. FGF-2 group
The animals received the same treatment as the control group excepting additional treatment with 30 equidistant transmyocardial injections of 500 µg recombinant human basic fibroblastic growth factor (rhFGF-2) (Labgen, NatuTec Inc., Frankfurt, Germany) in the area at risk (n=6).
2.4.4. FGF-2+TMLR group
In this combined group, pigs were treated with the same regimen (14–16 TMLR sites per heart) as the TMLR-group; however, these animals received additional treatment by 500 µg rhFGF-2 application during the second operation. They underwent TMLR with two equidistant transmyocardial injections surrounding each TMLR site (n=6).
2.5. Operation 3
Twelve weeks later, the animals underwent a sternotomy. The animals were reassessed similarly to the second operation including a coronarography (to verify the high-graded LAD-stenosis, 
90%) [4,12] and sacrificed. The hearts were removed and cut into 5 mm transversal sections. Samples were taken from the ischemic area as well as from the remote myocardium (posterior wall) and fixed in paraformaldehyde.
2.6. Parameters
2.6.1. Regional myocardial blood flow
Regional perfusion of the LAD and left circumflex artery (LCx) territory was measured by fluorescent microspheres (Molecular Probes, Eugene, OR) based on the arterial reference sample technique, as previously described [12,14]. The microsphere suspensions (15±0.1 µm; density of 1.07 g/ml) were injected into the left atrium under stable hemodynamic conditions so that no baseline differences (second operation) between the experimental groups were revealed at rest. The reference samples were withdrawn from the common carotid artery over a 2 min period at a rate of 10 ml/min starting 5 s prior injection of microspheres. Microspheres were injected at rest and under stress in all animals at the second (before randomization) and third operation (Harvard apparatus, South Natick, MA). Therefore, dobutamine was infused for 10 min with a dose of 20 µg/kg per min IV.
2.6.2. Regional contractility
Assessment of segmental myocardial shortening (SMS) was performed at rest and under stress (dobutamine 20 µg/kg per min IV) in all animals using ultrasonic crystals (Transonic Systems Inc, Ithaca, NY), as previously described [4,12]. SMS was analyzed at two different times: after 1 week chronic ischemia (second operation, baseline), and after 3 months ischemia (third operation).
2.6.3. Histochemical assessment
Computerized, planimetric analysis to determine the total area of the LV, the area of ischemia and necrosis by the triphenyltetrazolium chloride technique was performed at the end of study, as described elsewhere [3,4].
2.6.4. Immunohistology and vessel counting
Myocardial samples from the ischemic area were fixed in 4% formaldehyde in a phosphate buffer immediately after removal and embedded in paraffin. Sections (5 µm thickness) were pretreated with methanol, H2O2, and pepsin (Sigma, Taufkirchen, Germany), and immunohistochemical double-stainings were performed according to standard protocols, after blocking with the appropriate serum. Endothelial cells were identified by successive incubation with anti-Von Willebrand Factor (DAKO, Hamburg, Germany), biotinylated swine anti-rabbit F(ab')2 fragment (DAKO), and Streptavidin-biotinylated alkaline phosphatase-complex (DAKO), and Fast Blue (Sigma). For staining of smooth muscle cells, anti-Smooth muscle actin (DAKO), biotinylated rabbit anti mouse F(ab')2 fragment (DAKO), and Streptavidin-biotinylated Horseradish Peroxidase-complex (DAKO) were used followed by development with DAB (Sigma). Counting of capillaries (400-fold enlargement) and arterioles (200-fold enlargement) was performed by a trained observer blinded to the experimental conditions. In sections of TMLR-treated animals, counting was performed in the channel area (area 1) and within a distance of at least one visual field from laser channels (area 2). In sections of non-TMLR-treated hearts, counting was performed in the ischemic territory. Arterial structures with more than three layers of smooth muscle cells were considered arteries and were excluded. For each animal, forty visual fields from different sections were counted. Slides were taken from representative sections, scanned, and post-processed using CorelDraw.
2.7. Statistical analysis
Data were analyzed based upon; Wilcoxon's signed rank test to compare paired data, and Mann–Whitney U-test to compare unpaired data for non-normally distributed data, as appropriate (SPSS-vers.10.01). In contrast, a two-way analysis of variance for repeated measurements was used for normally distributed data. Results are expressed as mean±standard deviation (SD). A P-value less than 0.05 was considered statistically significant. Statistics were analyzed and reviewed by a statistician (M.O.) from the Division of Medical Biometrics, University Freiburg.
Only data were used from animals who survived the entire 13-week observation period and suffered no apparent infarction.
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3. Results |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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The entire study group included 41 animals. Shortly after the LAD stenosis was established four pigs died due to intractable ventricular fibrillation. Six additional animals were excluded due to no observed severe LAD-stenosis (<90%) in the angiography at the second operation. After randomization into the experimental groups at the second operation two control and two pigs of the TMLR group died of intractable ventricular fibrillation. Another animal from the ischemic control group died 1 week after the second operation. All other animals (n=26) survived until euthanasia without postoperative complications, had a high graded LAD stenosis at the second and third operation (without differences between experimental groups) and were used in analysis (n=7 in the ischemic, n=7 in the TMLR, n=6 in the FGF-2 and n=6 in the combined group).
Measurements of heart rate, left ventricular end-diastolic pressure, mean arterial and left atrial pressure, ECG changes, segmental myocardial shortening, perfusion at rest and all additional parameters revealed no statistically significant difference between the four study groups during the second operation (P=ns).
3.2. Regional myocardial blood flow (RMBF)
One week after induction of chronic ischemia, regional myocardial blood flow at rest in the LCx territory (remote myocardium) of the four study groups averaged 0.81±0.24 ml/min per g. In contrast, RMBF in the ischemic LAD territory (Fig. 2)
was reduced comparably and averaged 0.37±0.10 ml/min per g (P=0.0065 versus LCx territory). Therefore, the residual blood flow into the LAD territory averaged 
45% of the rest flow in the LCx territory of the four ischemic groups; there was no difference in the rest- and stress perfusion between all experimental groups in each of the territories (P=ns) excepting the FGF-2-group which showed higher RMBF values under stress compared to at rest (P=0.028) and control (P=0.038) at baseline.
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In contrast, blood flow to the LCx territory was unchanged after 3 months compared to after 1 week, averaging 0.99±0.24 ml/min per g at rest and 2.33±0.91 ml/min per g under stress in the control, TMLR- and FGF-2+TMLR-group (no difference in comparison with baseline, P=ns), whereas the FGF-2-group demonstrated an increase at rest (P=0.028 versus baseline, P=0.01 versus ischemic control and P=0.0048 versus TMLR).
3.3. Regional contractility
Shortly after inducing stenosis, a deterioration of the SMS in the LAD territory was observed at rest and under stress and compared to baseline and LCx values in the first operation in all study groups (data not shown, P<0.001).
One week after creation of severe LAD stenosis (Fig. 3) , a reduction of the regional contractility in the LAD territory was observed at rest and under stress in all experimental groups (P<0.001) compared to normal values of healthy hearts (100% SMS corresponds to normal contractility before stenosis).
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Regional contractility of the remote myocardium did not reveal differences between baseline (1-week) and 3 months values (P=ns) excepting the values of the TMLR group which were increased under stress conditions after 3 months (P=0.017 versus baseline, P=0.01 versus control, P=0.01 versus FGF-2+TMLR).
3.4. Histochemical assessment
Histochemical analysis indicated that the total area of the LV, area at risk and necrosis in all study groups were not significantly different (P=ns). Only very small areas of necrosis were found in several animals of all experimental groups (P=ns).
3.5. Angiogenesis and arteriogenesis
Myocardial sections from the ischemic area were double-stained for von Willebrand factor and smooth muscle actin. Capillaries and arterioles were subsequently counted (Fig. 4)
. Chronic myocardial ischemia resulted in a significant decrease of number of capillaries after 3 months of ischemia compared to values of myocardium from healthy pigs (data not shown, P=0.008). Injection of FGF-2 did not increase the number of capillaries (P=ns) or arterioles (Fig. 4) compared to ischemia (P=ns). The non-channel territories of the FGF-2 groups and the TMLR group did not differ from each other (Fig. 4a, P=ns). Nevertheless, laser groups demonstrated higher numbers of capillaries in the channel area (Fig. 4a, P=0.001 for both groups). There was no evidence of angioma formation macroscopically as well as microscopically.
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4. Discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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Our lab did not use an ameroid constrictor due to the contraction-flow mismatch that often occurs after complete occlusion and subsequent coronary collateralization before treatment [16].
This experimental study demonstrated that CO2-laser revascularization with the intramyocardial application of human recombinant FGF-2 in chronically ischemic myocardium improved regional myocardial blood flow in the area at risk under rest and stress conditions after 3 months of ischemia. In contrast, a change of perfusion compared to baseline (1 week ischemia) was not observed in the ischemic control group (Fig. 2). Additionally, the sole FGF-2 group also demonstrated higher RMBF values at rest (but not under stress) compared to the baseline, control and TMLR groups, whereas RMBF in the TMLR group did not change.
After a 3-month observation period, the FGF-2+TMLR- and FGF-2-groups’ regional contractility recovered and demonstrated higher values at rest in the LAD territory. In contrast, regional contractility of the ischemic control- and TMLR group was unchanged from initial values (Fig. 3). Regional contractility of the remote myocardium was unchanged in all groups, except in the TMLR group, which indicated higher SMS values under stress conditions after 3 months which could be part of a remodelling process in the LCx territory after TMLR application in chronically ischemic myocardium [17].
TMLR alone as well as the combination of FGF-2 injections with TMLR caused an increase in the number of capillaries and arterioles only in the channel area compared to untreated ischemia. We did not observe vessel growth in the adjacent area of laser channels (with or without FGF-2 therapy). This observation has been already shown by Mueller and colleagues in their experimental porcine study [18]. Scars of TMLR channels exhibit an increased vascular density in comparison with scar tissue of myocardial infarction, which does not extend into their immediate vicinity [18,19].
4.1. Experimental studies
RMBF analysis in this study indicated that regional myocardial blood flow in the FGF-2- and FGF-2+TMLR-group was greater than that in the control group, mirroring the angiogenetic process that is enhanced by FGF-1 [9–11,16] and TMLR [6,7,17,18]. These results are consistent with our findings with regard to the enhancement of regional contractility. Nevertheless, an improvement of regional blood flow in the TMLR-only group was not observed in the area at risk which supports our TMLR results in chronically ischemic myocardium [4,12,19] in which TMLR is only able to enhance microperfusion. Nevertheless, the combined therapy of TMLR and FGF-2 was synergistic, because the FGF-2 therapy alone was of limited efficacy (FGF-2 application showed an improvement of perfusion only at rest) compared to the combined therapy.
4.2. Study limitations
This experiment has three main limitations. First, the FGF-2-group showed higher RMBF values under stress, but not at rest compared to control at baseline, whereas no difference in the rest- and stress-perfusion between all the other experimental groups in each of the territories were analyzed. Second, the simple delivery of a single factor may be insufficient to achieve an optimal therapeutic effect [10,16]. Nevertheless, TMLR with its controlled locally myocardial inflammation [8,18] stimulates this broader range of pro-angiogenic responses [6,7,12,17,18] which is needed for the stimulation of both capillary and larger vessel growth [9–11,18]. Third, our results with an experimental model in originally healthy procine myo-cardium (with one-vessel disease) cannot be transferred directly to chronic injury of human myocardium with three-vessel disease.
4.3. Therapeutic angio- and arteriogenesis
For therapeutic treatment in myocardial ischemia, the most frequently used growth factors have been members of the FGF [20–22] and vascular endothelial growth factor (VEGF) families. Both VEGF and FGF can induce angiogenesis and arteriogenesis with new capillaries and arteries being formed, whereas FGF is superior in vitro in inducing arteriogenesis in ischemic myocardium. One reason could be that FGF induces vascular growth by binding to receptors at the surface of the endothelial cells, and by binding to receptors located on other cell types, e.g. vascular smooth muscle cells [20,21]. In contrast, VEGF binds to receptors which are mainly located on the endothelial cells. It has not been shown in vivo so far, that FGF can induce arteriogenesis [11] more convincingly than VEGF in ischemic myocardium.
4.4. FGF-2
Basic fibroblast growth factor is a potent angiogenic growth factor that is currently under study (experimentally and clinically, [11,20]). The angiogenic efficacy of FGF-2 was confirmed in numerous animal models [11,20–22] and in a few clinical studies [11,20].
The combination of TMLR with the local administration of a specific growth factor, FGF-2 or VEGF121, as has been demonstrated, is synergistic [22,23]. These above-described effects in this study would not have been achieved by simple injection of either a single protein [22] or gene encoding for its production [13].
4.5. Clinical implications
As we have already shown in a clinical study, single intramyocardial application of FGF-1 proved to be effective in patients with chronically ischemic myocardium in conjunction with coronary artery bypass grafting (CABG), because FGF-1 induced formation of new vascular networks bypassing distal stenoses of coronary arteries, which could not be reached with bypass grafts alone [11]. Of course, these results cannot be directly compared with those of this experimental model [4,12], we used the combination of FGF-2 and TMLR.
However, every patient undergoing CABG may also be a candidate for angiogenic and transmural laser therapy of myocardial areas where, due to small vessel disease, conventional therapy (CABG) is unfeasible to revascularize chronically ischemic myocardium. Additional clinical trials will be necessary.
In conclusion, the data presented in this experimental study provide evidence that intramyocardial application of FGF-2 with the combined CO2-laser revascularization in chronically ischemic myocardium increases regional myocardial blood flow at rest and under stress, and improves regional contractility under rest conditions.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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