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Eur J Cardiothorac Surg 2003;24:785-793
© 2003 Elsevier Science NL
a Department of Cardiovascular Surgery, University Hospital Freiburg, Freiburg, Germany
b Department of Cardiovascular Surgery, University of Schleswig Holstein, Campus Kiel, School of Medicine, Kiel, Germany
Received 12 December 2002; received in revised form 2 June 2003; accepted 16 June 2003.
* Corresponding author. Department of Cardiovascular Surgery, University of Schleswig Holstein, Campus Kiel, School of Medicine, Arnold-Heller-Str. 7, 24105 Kiel, Germany. Tel.: +49-431-597-4401; fax: +49-431-597-4402
e-mail: lutter{at}kielheart.uni-kiel.de
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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Key Words: Growth factors • Gene therapy • Coronary disease • Molecular biology
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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Interest is growing in the use of growth factors stimulating vessel growth for treating chronic myocardial ischemia, either alone or in combination with other procedures. Many studies of clinical and experimental nature, underlining the benefits of growth factor therapy, have been published [1,2]. Two of the factors most often used are vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2).
The search for the most effective growth factor acquires new importance in view of the recent discussion about the superiority of arteriogenesis over angiogenesis. Angiogenesis describes the sprouting of new capillaries, whereas arteriogenesis is characterized by active enlargement of pre-existing collateral vessels. This process is triggered by shear stress and depends on macrophage and monocyte activity [3]. Arteriogenesis is assumed to be more important than angiogenesis due to the higher transport capacity of arteriolar and arterial vessels compared to that of capillaries.
Whereas VEGF promotes mainly angiogenesis, FGF-2 also affects arteriogenesis. The third factor that we are interested in, the cytokine monocyte chemoattractant protein-1 (MCP-1), contributes to arteriogenesis by activating monocytes/macrophages.
Some studies already compared different cytokines and growth factors in rabbit hindlimb models, employing, for instance, MCP-1 and granulocyte-macrophage colony stimulating factor (GM-CSF) [4] or platelet-derived growth factor (PDGF)-BB and FGF-2 [5]. However, there are still no studies to our knowledge on ischemic myocardium that directly compare angiogenic and arteriogenic growth factors to determine which is the most effective in developing efficient collateral circulation.
DNA vectors for gene therapy are modified viruses or plasmids, the latter not infecting cells actively. Despite a lower transfection rate, the advantages of plasmids include the reduced risk for angiomas and no interference by pre-existing antiviral antibodies.
We employed a porcine model of chronic myocardial ischemia and injected plasmid DNA encoding VEGF, FGF-2, or MCP-1 or vector only intramyocardially. We evaluated the therapies after 3 months focusing on effects on regional and global cardiac function.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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2.2. In vitro experiments
2.2.1. Cell culture
Primary porcine cardiac fibroblasts were obtained from fresh perivascular connective tissue from a porcine heart. The samples were minced and single pieces placed into 8.7 cm2 cell culture dishes. The flakes were squeezed under a glass cover slip, and supplemented with Dulbecco's Modified Eagle Medium (DMEM; Cell Concepts, Germany) containing 10% fetal calf serum (FCS; Life Technologies), 200 mM L-glutamine (Cell Concepts), 200 IU/ml penicillin/streptomycin (Cell Concepts), 10 µg/ml minocycline (ICN), and 10 µg/ml amphotericin B (Bristol-Myers Squibb). Fibroblasts started to grow out of the tissue on the second day. Media was changed three times a week. To increase the yield, some cover slips were taken off after 2 weeks and placed into new dishes. After reaching sufficient confluency, cells were trypsinated, transferred into cell culture flasks, and subsequently fed with DMEM, supplemented with 10% FCS, 200 mM L-glutamine, and 100 IU/ml penicillin/streptomycin. Cells up to the 12th passage were used for experiments.
2.2.2. Transfection of primary cardiac fibroblasts
Primary cardiac fibroblasts were trypsinated, washed, and suspended in Dulbeccos’ phosphate buffered saline (DPBS) to a concentration of 3.5–4.0x106 cells per ml; 400-µl aliquots were incubated with 100 µg DNA in 4-mm electroporation cuvettes (Peqlab, Erlangen, Germany) for 10 min on ice, electroporated at 220 V and 500 µF (Gene Pulser II, Biorad, Hercules, CA), left on ice for another 10 min, suspended in 1-ml supplemented DMEM, and seeded on cell culture plates (Greiner, Fricken-hausen, Germany) containing supplemented DMEM.
2.2.3. Gene expression
2.2.3.1. VEGF-ELISA
Cells were transfected with VEGF121 or EGFP. After the times indicated, samples of cell culture supernatants were taken, stored at -80 °C, and analyzed using an ELISA (PromoCell, Heidelberg, Germany) according to the manufacturers instructions. The ELISA cross-reacts with porcine VEGF.
2.2.3.2. MCP-1—intracellular immunofluorescence microscopy
Four days after transfection with MCP-1 or vector, cells were trypsinated, washed in DPBS/1% FCS, fixed in formaldehyde 4% in DPBS, and washed again. The pellets were resuspended in permeabilization buffer (DPBS, 1% FCS, 0.1% saponin, 0.1% sodium acid) and incubated with a monoclonal mouse anti human MCP-1 antibody (BD PharMingen, Heidelberg, Germany) for 30 min on ice. After washing steps in permeabilization buffer, samples were incubated with a goat anti mouse FITC antibody for 30 min, washed again, and resuspended in permeabilization buffer. Fluorescence was visualized and representative images were recorded using a Zeiss fluorescence microscope. Additional controls were made omitting the primary antibody.
2.2.3.3. FGF-2—Western blotting
Cells were transfected with FGF-2 or vector. After the times indicated, supernatants were removed and centrifuged at 16 000 g, 4 °C for 10 min. The adherent cells were scraped off in DPBS with 0.5% protease inhibitor cocktail (Complete, Roche, Mannheim, Germany) and centrifuged at 16 000 g, 4 °C for 5 min. The pellets were pooled, washed once, and extracted using a Triton X-containing buffer with 1% protease inhibitor cocktail. The cell extracts were stored at -80 °C, separated on a 15% SDS page and blotted on PVDF (Roth, Karlsruhe, Germany). The membrane was blocked with 10 mM Tris, 100 mM NaCl, pH 7.5, 5% skim milk for 1 h at room temperature, incubated with a polyclonal rabbit anti human FGF-2 antibody (1:250 in blocking buffer, cross-reacts with porcine VEGF; NatuTec, Frankfurt/M., Germany) overnight at 4 °C, followed by a goat anti rabbit peroxidase conjugated antibody (1:3000 in blocking buffer; DAKO, Hamburg, Germany) for 1 h at room temperature, and subsequently developed using ECL (Amersham, Piscataway, NJ).
Equality of protein loading was confirmed by counterstaining the blotted gel with Coomassie Blue.
2.3. In vivo experiments
2.3.1. Surgical procedures
2.3.1.1. Animals and anesthesia
All animal procedures were carried out 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 and anesthetized by flunitrazepam and ketamine; relaxation for intubation was induced by vecuronium bromide. Anesthesia was maintained by vecuronium, fentanyl, propofol, and flunitrazepam. Animals were continuously monitored by electrocardiography and pulse-oximetry. Sufficient analgesics, acetyl salicylic acid, and antibiotics were given after the first and second operation.
A model of chronic myocardial ischemia was employed to mimic clinical one-vessel coronary artery disease. In the first operation, an operative stenosis of the left anterior descending artery (LAD) was created in all pigs. One week later (second operation), the animals were examined by analyzing different parameters (see below). Afterwards, pigs were designated to one of five different experimental groups. Twelve weeks after the second operation, the animals were re-examined (same parameters as before) and killed (third operation).
2.3.1.2. First operation
The heart was exposed via an anterolateral minithoracotomy. An ultrasonic transit time (UTT) flow probe (Transonic Systems, Inc., Ithaca, NY) was positioned at the LAD distal to the first diagonal branch continuously registering the downstream blood flow. To establish a severe stenosis, a non-elastic Dacron stripe (5 mm) was placed U-like around the LAD below the first diagonal branch upstream of the UTT probe. The open edge was closed manually by successive sutures until a blood flow reduction of 50% was achieved. The stenosis resulted in an area at risk of about 25% of the LV anterior free wall. The wound was closed in layers, and the animals were allowed to recover.
2.3.1.3. Second operation
By means of a re-minithoracotomy, the heart was 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), baseline measurements of segmental myocardial shortening, left ventricular stroke work index, and time derivative of maximal left ventricular pressure were assessed as described below. Animals were designated to one of five groups and received intramyocardial injections of plasmid DNA in the ischemic area (see experimental groups). A control group was left untreated. The thorax was closed, and the pigs were allowed to recover.
2.3.1.4. Third operation
After 3 months, a sternotomy was performed. The animals were re-examined similarly to the second operation and killed. Samples of treated ischemic myocardium were snap-frozen in liquid nitrogen.
2.4. Experimental groups
Animals of the three therapy groups received either human VEGF121 (VEGF), human FGF-2 (FGF-2), or porcine MCP-1 (MCP-1). The cytokines were applied as 2 mg naked plasmid DNA (see above) supplemented with 400 IE heparin and diluted to a total volume of 2.8 ml. This amount was divided into 15 units and injected intramyocardially distributed across the ischemic area. Control animals were injected with vector DNA only (Vector) or were left untreated (Ischemia).
One pig died in tabula 1 h after VEGF plasmid injection. Samples of treated ischemic myocardium were snap-frozen in liquid nitrogen for molecular biologic analysis.
2.5. Parameters
2.5.1. Presence of plasmid DNA and mRNA
Myocardial samples were ground in a mortar under liquid nitrogen, transferred to a 2-ml tube, and homogenized by ultrasound (5x5 s). Control tissue was taken from untreated non-ischemic myocardium. One milliliter of Trireagent (Sigma) was added to the samples, and RNA and DNA were extracted following the standard protocol supplied. Five micrograms of RNA were subjected to RT-PCR using random primers and M-MLV reverse transcriptase (both from Invitrogen, Karlsruhe, Germany) according to the manufacturers’ instructions. Controls were done without M-MLV reverse transcriptase to rule out DNA contamination.
RT-PCR products and DNA were amplified by PCR using plasmid-specific primers (forward: TCT TAC TGA CAT CCA CTT, reverse: (ATT AAC CCT CAC TAA AGG GAA) at 90 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min for 25 cycles. Plasmid without an insert served as positive control.
2.5.2. Segmental myocardial shortening (SMS)
Segmental myocardial shortening (SMS) was assessed 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. SMS was assessed after 1 week chronic ischemia (2nd operation, baseline), and after 3 months ischemia (3rd operation) and calculated as follows:
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2.5.3. Hemodynamic measurements
The following assessments were taken after 1 week (2nd operation, baseline) and after 3 months (3rd operation) of chronic ischemia: left ventricular pressure, maximum of the first derivative of left ventricular pressure at a defined left atrial pressure (dp/dtmax), measured using a Millar catheter (Millar, Houston, TX), as well as maximal left ventricular stroke work index (LVSWI). Stress was induced by IV application of dobutamine for 10 min at a dose of 5 µg/kg per min.
Left ventricular stroke work was calculated as:
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2.6. Statistical analysis
The absolute values (mean±S.D.) are presented in Tables 1–3. Differences were analyzed based on the Mann–Whitney U-test for non-normally distributed data. Changes in the area at risk were calculated within the groups using the Wilcoxon's signed rank test. A P-value of asymptotic significance of less than 0.05 was considered statistically significant. The respective figures show the 3-month data as mean percentage±S.D. of the 1-week values.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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After 3 months ischemia, regional contractility in untreated and MCP-1-treated myocardium remained unchanged compared to the 1-week baseline (P=0.345, P=0.484), whereas vector, VEGF, and FGF-2 therapies resulted in improvement (P=0.046, P=0.028, P=0.028) at rest. Vector, VEGF and FGF-2 yielded better results than untreated ischemia, and were superior even to MCP-1 (Fig. 3 a).
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Some animals did not tolerate examination under dobutamine, therefore those experiments had to be cut short and the corresponding data were excluded.
3.4. Systemic hemodynamics
3.4.1. Left ventricular stroke work index (LVSWI)
LVSWI was assessed after 1 week ischemia and again after 3 months at rest and under stress conditions, except for untreated control animals, where stress data were not acquired. After 3 months there was a decline in LVSWI in the untreated and the MCP-1 groups at rest compared to 1 week ischemia (P=0.018, P=0.012). In contrast, VEGF and FGF-2, as well as vector only were able to prevent this loss of function (P=0.128, P=0.091, P=0.686), the final value not differing from 1 week-baseline (Fig. 4)
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3.4.2. Left ventricular contractility (dp/dtmax)
Left ventricular contractility was recorded after 1 week ischemia at baseline and 3 months later at rest. No significant change between values after 1 week and after 3 months ischemia appeared in any group (ischemia, P=0.08, vector, P=0.5, VEGF, P=0.128, MCP-1, P=0.345, FGF-2, P=1.0). However, VEGF and FGF-2 achieved better results than untreated controls (P=0.022, P=0.012), whereas vector and MCP-1 did not (P=0.055, P=0.475). There were no differences between any of the groups at baseline (Fig. 5)
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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After 3 months, VEGF, FGF-2, and vector only rendered functional improvements in regional contractility at rest and in left ventricular stroke work index. However, only VEGF and FGF-2 were effective in improving regional contractility under dobutamine stress and left ventricular contractility (dp/dtmax). In contrast, MCP-1 showed no positive effect.
The benefits of vector injection support the observation that mere mechanical needling of ischemic myocardium can induce vessel growth and maintain function as shown by Chu et al. [7]. In addition, remnants of lipopolysaccharides in the plasmid preparations may contribute to an inflammation with subsequent vessel development [8]. However, growth factors VEGF and FGF-2 rendered better results in at least some of the experiments and appear therefore to be superior compared to vector only.
Vascular endothelial growth factor (VEGF) is a strong mitogenic and migration-promoting factor for vascular endothelial cells and therefore a major initiator of angiogenesis, its expression being determined by hypoxia [9]. In addition, it has several vasoprotective properties [10] and mobilizes vascular endothelial progenitor cells from bone marrow [11]. In cardiomyocytes, VEGF promotes cell-extracellular matrix interaction [12] and intercellular signaling [13] thereby supporting coordinated contraction. A recent phase I/II double-blind randomized clinical study demonstrated the efficiency of intramyocardial VEGF gene therapy [14].
Fibroblast growth factor-2 (FGF-2, basic fibroblast growth factor) is involved in the regulation of various cellular functions of different myocardial cell types. It stimulates angiogenesis and, by affecting inflammatory reactions and stimulating artery-building cells, arterial vessel development as well. A number of direct and indirect protective effects of FGF-2 on cardiomyocytes has been shown in vivo (reviewed by Detillieux [15]). It was proven useful in a phase I randomized clinical study in combination with coronary artery bypass grafting [16].
So far, our results are in line with previous studies on VEGF and FGF-2. There was no major difference between the effects of either growth factor.
However, the devastating results following MCP-1 therapy came as a surprise.
One might ask whether the therapy was successful with regard to intramyocardial MCP-1 synthesis, since we do not show the in vivo presence of any of plasmid-dependent growth factors by immunohistochemistry. Taking an additional biopsy would have meant another thoracotomy and interference with the processes in the target area, since plasmid-driven protein synthesis can be expected several hours after gene injection at the earliest. In deference to the animals, we decided against this additional intervention. However, CMV promotor-driven gene expression from DNA plasmids had already been shown in non-ischemic myocardium [17] and in our model [18], and we proved MCP-1 synthesis in vitro in cardiac cells. Furthermore, VEGF and FGF-2 were efficient, and even vector DNA rendered some functional benefits. Thus, there is no evidence for unsuccessful gene transfer and expression in the case of MCP-1.
MCP-1 is expressed by activated vascular endothelial cells, cardiomyocytes, smooth muscle cells, and monocytes/macrophages. It has been shown to successfully promote the development of collateral circulation and to increase blood conductance in rabbit and porcine models of femoral arterial occlusion [4,19]. In these studies, the protein was applied via an intraarterial pump beginning at the time-point of occlusion. It has also been suggested that MCP-1 expression by bone marrow cells participates in augmentation of collateral perfusion and myocardial function, when bone marrow cells are injected into ischemic myocardium [20]. MCP-1 can also mediate angiogenesis directly [21]. It is assumed to protect cardiomyocytes from cell death caused by hypoxia and to play an important role in regulating their response to ischemia in vivo [22]. On the other hand, MCP-1 has been accused of accelerating reperfusion injury following brief ischemia [23].
In chronic ischemic myocardium, MCP-1 causing inflammation with edema might not be as beneficial as proposed from different tissue models or in vitro experiments, given the sophisticated structure of myocardial stroma and of the conducting system. Another explanation for the failure of our MCP-1 therapy might be an incorrect time-point in the course of adaptation to ischemia.
Three limitations of our study have to be mentioned: first, animals of the untreated ischemic control group were not analyzed under the indicated stress conditions except for regional contractility after 3 months ischemia, so that the spontaneous development is not known. Second, the focus of evaluation of the different therapies was on myocardial function. Additional information might have been obtained by vessel counting or assessment of perfusion. Third, we were not able to assess the actual local inflammatory response to the injected plasmids and the expressed cytokines histologically, since we decided against a biopsy (as discussed above) and analyzed the myocardium only 3 months after application of the therapies. After such a long while, the state of the myocardium no longer reflects the immediate effect of the injected agents.
In conclusion, injection of naked plasmid DNA encoding VEGF121 or FGF-2 improved myocardial function in chronic ischemia in more aspects than vector only and was superior to untreated ischemia or MCP-1. This strategy can be considered a successful tool for growth-factor stimulated preservation of function in chronic myocardial ischemia.
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Acknowledgments |
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Footnotes |
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Appendix A. Conference discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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Dr Heilmann: The plasmid has a CMV promoter, it is a commercial plasmid. As for the duration of our expression, further investigations are underway. There are studies on nonischemic myocardium where the authors were able to prove an expression over 8 days.
Dr Schmid: So you express for maybe 5 to 8 days and you measure your vascularization after 3 months. Do you think that the expression for 5 days has an effect on vascularization after 3 months?
Dr Heilmann: Yes, we do think so, because the early period determines the effect of the therapy. The vascularization, the angiogenesis takes place, according to some authors, starting at about 12 hours for the first 3 to 5 days. Arteriogenesis takes a bit longer. But we aim for the long-term effect, the therapeutical effect, and so we are not so interested in what has happened after one week but rather in the long-term effect.
Dr Schmid: Maybe expression was better after one week.
Dr Heilmann: Well, perhaps, but the patient doesn't need one week, he or she wants to live longer.
Dr Schmid: Would it be better to repeat the treatment?
Dr Heilmann: Well, that is difficult, because we use intramyocardial injections, so we would like to spare the patient repeated injections.
Dr C. Maurus (Zurich, Switzerland): In line with the first question, did you try to couple VEGF and the plasmid with GFP to monitor the VEGF expression directly? First question.
And my second question: you stated that your control animals were left untouched. Did you do sham injections or could the increase in capillary formation be solely due to the mechanical injury by injecting the plasmid?
Dr Heilmann: We considered using a fusion protein of VEGF and GFP, but we didn't want to do that because we were wary of a mixed effect. The GFP gets stuck in the cell and there is no certain knowlegde about the effects. But for a therapy you wouldn't use a fusion protein.
Dr Maurus: Is there any direct monitoring of the therapy?
Dr Heilmann: Yes, we are aware of that. But we decided to stick with a pure growth factor. To your second question: Further investigations are underway. I hope to show the results next year with a sham injection of plasmid without an insert. But in any case, the investigations on angiogenesis following needling are contradictory. I'm aware of at least two papers where one group finds angiogenesis following needling and another group doesn't. So it's unresolved. And VEGF has other effects on cardiomyocytes, too, that protects the cells from apoptosis and loss of signaling.
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Conference...References |
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