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Eur J Cardiothorac Surg 2003;23:74-80
© 2003 Elsevier Science NL
a Department of Cardiovascular Surgery, Albert-Ludwigs-University Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
b Institute of Pathology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
c Institute for Molecular Medicine, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Received 5 February 2002; received in revised form 16 September 2002; accepted 21 October 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: Transmyocardial laser revascularization • Vascular endothelial growth factor121 • Chronic myocardial ischemia
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1. Introduction |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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TMLR has been established as an alternative therapy for patients with otherwise intractable angina pectoris. It has been shown to improve the clinical status in these patients, but results with regard to perfusion and heart function remain contradictory [1]. We previously demonstrated that transmyocardial laser channels failed to achieve a short-term increase in regional myocardial blood flow to the ischemic porcine heart [2]. However, TMLR induces acute and chronic inflammation and may hereby stimulate the development of a collateral circulation [3].
The additional delivery of angiogenic factors in the form of protein or DNA to the ischemic target area may enhance this vessel development. Interestingly, there is evidence that TMLR enhances the expression of proteins encoded by intramyocardially injected naked plasmid DNA [4].
Vascular endothelial growth factor (VEGF) is a strong mitogen for vascular endothelial cells and therefore a major initiator of angiogenesis, its expression being determined by hypoxia [5]. Moreover, it mobilizes vascular endothelial progenitor cells from bone marrow [6]. It has been successfully employed in a number of animal studies and clinical trials [7,8].
The objective of our study was to determine whether combined therapy of TMLR with the intramyocardial injection of a naked VEGF121 DNA plasmid stimulates the development of a sufficient collateral circulation and is hereby able to resume cardiac function 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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All animal procedures were performed in compliance with the ‘Principles of Laboratory Animal Care’, the ‘Guide for the Care and Use of Laboratory Animals’, National Institute of Health publication 85-23, revised 1985, and the German ‘Law for Animal Protection’.
Pigs of ‘German Landrace’ weighing 24–30 kg were premedicated with an intramuscular injection of 0.2 mg/kg flunitrazepam and 7 mg/kg ketamine hydrochloride. An ear vein was cannulated and anesthesia was induced with 0.1 mg/kg flunitrazepam and 10 mg/kg ketamine hydrochloride by titrated intravenous injection. After muscular relaxation had been induced with 0.1 mg/kg i.v. vecuronium bromide, an endotracheal tube was inserted and mechanical ventilation begun. Anesthesia was maintained by continuous intravenous infusion of 0.06 mg/kg per h vecuronium, 0.004 mg/kg per h fentanyl dihydrogen citrate, 5.2 mg/kg per h propofol, and 0.04 mg/kg per h flunitrazepam. The same anestethic regimen was used for each of the three different surgical procedures that the animals underwent. Cefazolin (0.5 g i.v.) was given preoperatively.
Continuous electrocardiographic and pulse-oximetric monitoring was used throughout the procedure to ensure stable cardiac rhythm and setting. After maintenance of a stable left anterior descending artery stenosis for >60 min, the flow probe was removed and the chest was closed in layers with the pericardium partially closed. A control angiography was performed to verify the high-grade left anterior descending artery stenosis. The pigs were then allowed to recover in their intensive-care cages.
The animals were monitored daily by a veterinarian, her staff, and the surgical team. Pain medications were also given intramuscularly until the animals could ambulate without difficulty and exhibited normal activity. Until the third operation, 100 mg acetylic salicylic acid was administered daily.
2.1.1. First operation
The heart was exposed via an anterolateral minithoracotomy. The LAD was carefully dissected and isolated immediately distal to the bifurcation of the first diagonal branch (D1) over 1–2 cm to accept an ultrasonic transit time (UTT) flow probe (Transonic Systems, Inc, Ithaca, NY) recording downstream flow through the LAD.
A severe LAD stenosis (immediately distal to D1) was created proximal to the flow probe to produce an area at risk of about 25% of the left ventricular (LV) anterior free wall. Following the arterial puncture needle technique, the stenosis was established by gathering of the vessel wall with a monofil polypropylene 7/0 suture, which allows an exact dosage of the remaining blood flow. The blood flow distal to the stenosis was reduced to about 50% as assessed by UTT and coronarography. The wound was closed in layers, and the animals were allowed to recover.
2.1.2. 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. Once angiography and UTT flow probe data confirmed the presence of chronic ischemia (severe LAD stenosis with blood flow reduction, see above), segmental myocardial shortening was assessed at baseline. In addition, microspheres were injected 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 2.2). The thorax was closed, and the pigs were allowed to recover.
2.2. Experimental groups
2.2.1. TMLR
The pigs were treated by creating two laser channels (1 mm in diameter) per cm2 in the area of the LAD beyond the first diagonal branch, as reported elsewhere [2,9]. 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.
2.2.2. VEGF
The pigs received 2 mg human VEGF121 cDNA cloned into a mammalian expression vector under control of the cytomegaly virus (CMV) promotor (pCI-neo, Promega Inc., Palo Alto, CA), supplemented with 400 IE Heparin and diluted in 0.9% sodium chloride to a total volume of 2.8 ml. This amount was divided into 15 units and each injected transmyocardially across the ischemic area into the endo-, mid-, and epimyocardium.
2.2.3. VEGF121+TMLR
In this combined group, the pigs were treated with one laser channel per mm2 as described above; however, these animals received additional treatment by application of 2 mg human VEGF121 cDNA as described above with two equidistant intramyocardial injections adjacent to each TMLR site.
2.2.4. Ischemia
The pigs were operated on and assessed as described but were left untreated to serve as control group.
Fourteen of 51 animals received a stenosis but died before being designated to the experimental groups. Nine pigs were assigned to the ischemic group, ten to TMLR, nine to VEGF121, and nine to combined therapy. After placement in the experimental groups, one animal in the TMLR group, two in the VEGF121 group, and two in the combined group died before the third operation. All other animals (n=33) survived until the end of the study without postoperative complications and had a high grade LAD stenosis at the second and third operations. One pig each in the ischemic and the TMLR groups were excluded due to an apparent infarction after 3 months. Thus, the ischemic group and TMLR group consisted of eight pigs each, the VEGF121 and combined TMLR+VEGF121 groups numbered seven animals each.
2.3. Third operation
After 3 months, a sternotomy was performed. The animals were reassessed similarly to the second operation and sacrificed. The hearts were removed and cut into 5 mm transversal sections to obtain samples from the ischemic area for perfusion analysis as well as for immunohistology.
2.4. Parameters
2.4.1. Immunohistology and vessel counting
Four transmural myocardial samples were taken from across the ischemic area. They 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 trained observers blinded to the experimental conditions. In sections of TMLR-treated animals, counting was performed at a distance of at least one visual field from laser channels. Arterial structures with more than three layers of smooth muscle cells were considered arteries and were excluded.
For each animal, ten visual fields, randomly distributed evenly across the myocardium, were counted per section (40 visual fields per animal). Slides were taken from representative sections, scanned, and postprocessed using CorelDraw.
2.4.2. Regional myocardial blood flow (RMBF)
Regional perfusion of the LAD territory was measured using microspheres based on the arterial reference sample technique, as previously described [10,11]. The microsphere suspensions (15 µm) were injected into the left atrium under stable hemodynamic conditions. The reference samples were withdrawn from the internal carotid artery over a 2 min period at a rate of 10 ml/min starting 5 s before the injection of microspheres. Microspheres were injected in all animals during the second (before randomization) and third operations (Harvard apparatus, South Natick, MA). At the end of study, microspheres were retrieved from myocardial samples from all layers of the myocardium. The tissue was processed according to the manufacturers’ instructions. Results were calculated as mlxmin-1x100 g-1.
2.4.3. Segmental myocardial shortening (SMS)
Assessment of SMS was performed in all animals using ultrasonic crystals (Transonic Systems, Inc., Ithaca, NY). The probes were placed in the endomyocardium of the ischemic area at a distance of approximately 20 mm.
Segmental myocardial shortening (SMS) was calculated as follows:
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SMS were analyzed at three different times: before stenosis (first operation), after 1 week chronic ischemia (second operation, baseline), and after 3 months ischemia (third operation).
2.4.4. Statistical analysis
The absolute values (mean±SD) are presented in Table 1. SPSS 8.0 was used for statistical analysis. Due to considerable differences between the animals, each animal served as its own control for the parameters of perfusion and regional contractility. Changes in the area at risk were calculated within the groups using the Wilcoxon's signed rank test. The respective figures show the 3 months data as mean percentage ±SD of the 1 week values. Only the numbers of capillaries and arteries were compared between the groups employing the Mann–Whitney U-test because there are no pre-ischemic or baseline (1 week ischemia) data. A P-value less than 0.05 was considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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3.2. RMBF
Regional perfusion in the ischemic area was assessed using microspheres after 1 week ischemia before application of the therapy (baseline) and 3 months later. Only combined TMLR+VEGF121 [n=5] therapy rendered an improvement in RMBF compared to baseline (P=0.043), whereas there was no change in untreated hearts [n=8] or following either VEGF121 [n=6] or TMLR [n=8] treatment (P=0.263, P=0.345, P=0.161, respectively) (Fig. 3)
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After 3 months, SMS in untreated pigs [n=5] was decreased further (P=0.043 versus 1-week baseline). Contractility following TMLR treatment alone [n=7] remained unchanged (P=0.612 versus 1-week baseline). The application of combined VEGF121+TMLR [n=7] or VEGF121 [n=6] therapies resulted in an improvement compared to the baseline (VEGF121+TMLR, P=0.018, VEGF121, P=0.028). In addition, VEGF121+TMLR and VEGF121 were able to restore pre-ischemic regional contractility (VEGF121+TMLR, P=0.176, VEGF121, P=0.116, 3 months versus pre-ischemic), whereas the values for untreated ischemia and TMLR after 3 months ischemia were still lower (ischemia, P=0.043, TMLR, P=0.018) (Table 1, Fig. 4) .
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4. Discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionReferences |
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The development of an effective collateralization involves angiogenesis, which is the sprouting of new capillaries, and arteriogenesis, the development of arterial structures from small preexisting collateral vessels [12]. Whereas angiogenesis is stimulated mainly by ischemia-driven VEGF expression, arteriogenesis depends on an inflammatory environment [13] and is considered to be more important than angiogenesis due to higher perfusion capacity of arterial vessels.
The end-points of our experiment describe different parameters of the myocardium. The development of capillaries and arteries is a morphological criterion, whereas perfusion reflects the result of a successful collateralization. In contrast, segmental myocardial shortening represents a physiological function of the myocardium due to preserved or restored microperfusion, at least on the condition of perfusion-contraction matching in normal and hibernating myocardium. However, the comparison of all results reveals several incongruencies and indicates that vessel growth, enhanced regional perfusion, and improvement of myocardial function do not have to correspond directly.
Whereas the number of arterioles increased following TMLR and TMLR+VEGF121 therapy, better perfusion is rendered only by the combined therapy. This phenomenon might be explained by immediate effects of VEGF121. In addition to its mitogenic function for endothelial cells, VEGF has vasoprotective properties (reviewed by Zachary [14]) and is able to restore impaired vasomotor responses in microvessels [15]. These activities might supplement the arteriogenesis in the combined TMLR+VEGF121 group and could represent an advantage compared to TMLR alone, resulting in restored regional perfusion.
At the same time, restored contractility is achieved by VEGF121 and by combined treatment. A reason could be the expression of the VEGF receptors Flk-1 and Flt-1 in cardiomyocytes. Stimulation by VEGF is followed by activation of several mitogen-activated kinases [16] and of focal adhesion kinase [17] as well as by the upregulation of the gap junction protein connexin43 [18]. Thus, VEGF exerts cytoprotective and anti-apoptotic effects on cardiomyocytes and is involved in the maintenance of their intercellular signaling. These could be mechanisms protecting the cells in the early phase after VEGF gene therapy thereby recovering the regional contractility for a longer term.
The question remains, however, why arteriogenesis only is observed in the TMLR+VEGF121 group, since angiogenesis would be expected from VEGF121 participation. TMLR treatment is accompanied by a strong inflammative reaction with presence of an array of chemokines. It has been suggested that under these circumstances an inhibition of the VEGF signaling in vascular endothelial cells might take place, which could be conducted by alternatively spliced VEGF receptor forms [19,20].
A certain limitation of our studies is the small groups. Due to the intraoperative condition of the animals or to problems of raising or analyzing the data, not all parameters could be evaluated from each animal. Some additional information might have been obtained by examination of regional and systemic function under stress conditions.
Whereas many studies have scrutinized the effects of either TMLR or growth factors in chronic myocardial ischemia, there is only a small number of experiments concerning the use of TMLR in combination with growth factors. In 1996, Fleischer et al. [21] employed VEGF protein as well as profilin encoded by an adenovirus. In a non-ischemic porcine model, they found more severe inflammation than with TMLR alone, but no improved vascularity. In contrast, vessel growth as well as improvement of regional contractility by combined VEGF121+TMLR treatment could be achieved in the present study.
These results are supported by other researchers’ comparable observations: An increase in the number of arterioles (but also of capillaries) following combined treatment with TMLR and injection of bovine bone derived growth factor mixture was shown by Mueller et al. [22]. Sayeed-Shah et al. [4] recently reported the reversal of wall motion abnormalities 6 weeks after combined treatment with TMLR and a DNA plasmid coding for VEGF165 in porcine hearts.
Three questions remain at the back of one's mind: Firstly, how specific are the effects of TMLR, since vessel growth is also obtained by mere mechanical puncturing of the myocardium, which causes expression of growth factors and angiogenesis [23].
Secondly, how specific are the effects of the growth factors or are positive results only caused by the inflammation due to injection of proteins or DNA as suspected by Fleischer and coworkers [21]?
Thirdly, if growth factor application as an adjunct to TMLR works, which growth factor should be used? In addition to VEGF, a number of other growth factors, i.e. fibroblast growth factor-1 or -2, hepatocyte growth factor, or monocyte chemoattractant protein-1 have been shown to render therapeutic effects in chronic ischemia. Additionally, should the factors be used as protein or as a gene? Proteins can be applied at a set dosage, but are quickly eliminated in tissue or blood, whereas vector-encoded growth factors remain in the system for a longer time period but at varying concentrations. These questions have still to be investigated.
This study of chronic myocardial ischemia with a porcine model indicates a synergistic action of TMLR and VEGF121 gene therapy. Combined treatment alone achieved an increase in regional myocardial perfusion, which accompanied arteriogenesis and corresponded with the restoration of regional function.
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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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