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Eur J Cardiothorac Surg 2001;19:156-163
© 2001 Elsevier Science NL
Second Department of Surgery, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka-Cho, Yoshida-Gun, Fukui 9101193, Japan
Received 31 August 2000; received in revised form 20 November 2000; accepted 4 December 2000.
Corresponding author. Tel.: +81-776-61-8379; fax: 81-776-61-8114
e-mail: riweiw{at}fmsrsa.fukui-med.ac.jp
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Key Words: Transmyocardial laser revascularization • Angiogenesis • Matrix metalloproteinase • Platelet-derived endothelial cell growth factor
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The capacity of extracellular matrix (ECM) macromolecules to influence angiogenesis, and the formation of new microvessels from existing blood vessels, has been well documented [2,3]. Destruction and compositional alterations of the ECM are key events in angiogenesis [4]. Matrix metalloproteinases (MMPs) degrade the ECM; it has been shown that the secretion of MMPs by microvascular endothelial cells is an essential first step in the formation of new blood vessel [5]. Otherwise, platelet-derived endothelial cell growth factor (PD-ECGF) is initially cloned as a novel non-heparin-binding angiogenic factor present in platelets, and it is known to be chemotactic for endothelial cells in vitro and angiogenic in vivo [6].
In the present study, we measured the activity of MMPs and PD-ECGF in an acute canine myocardial infarction model treated with carbon dioxide (CO2) laser, for the purpose of examining the relationship between MMPs, PD-ECGF and angiogenesis in the early phase of TMLR.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1. Model of acute canine myocardial infarction
Fourteen adult beagle dogs without gender distinction (8.2 to 14.3 kg) were given a premedication of ketamine hydrochloride (125 to 150 mg intramuscular). The dogs were anesthetized with intravenous pentobarbital sodium (25 mg/kg), intubated, and ventilated mechanically with room air. Under sterile conditions, a left thoracotomy was performed through the fourth or fifth intercostal space and the pericardium was incised vertically to expose the heart. The left anterior descending coronary artery (LAD) was dissected just distal to the first diagonal branch and the artery was ligated permanently. To minimize arrhythmia, an intravenous drip of lidocaine (75 µg/kg per min) was administered during the whole operative procedure and an additional bolus dose (1.5 mg/kg) was given before LAD ligation in each dog. After all of the protocols were completed, the incision was closed in the routine manner and the dogs were allowed to recover from anesthesia. They were given routine postoperative care after the surgical procedure. Cefazolin sodium (25 to 30 mg/kg) was administered twice daily for 5 days and pain medication was given as needed.
The dogs were randomly divided into two groups before operation: (1) The Laser group (n=6), see laser protocol; (2) The control group (n=6), after LAD ligation the pericardium and the chest were closed in layers.
2.2. Laser protocol
After LAD ligation, transmural laser channels were created in the beating heart with a CO2 laser (Sharplan lasers, Inc, USA 1055): at 55-watts, with 0.3 s for a single pulse. The CO2 laser was aimed with helium-neon laser guidance placed vertically against the epicardium of the region supplied by the left anterior descending coronary artery distal to the first diagonal branch. Channels with an approximate 1 mm diameter were made with a distribution of approximately one per cm2, and an average of 10 channels (ranging from 8 to 11) were created in each heart. Epicardial surface bleeding from laser sites was controlled with several minutes of light, direct pressure. No epicardial purse string sutures were necessary to control the bleeding.
2.3. Harvesting tissue samples
Two weeks after the initial operation, animals were anesthetized and mechanically ventilated as before. A median sternotomy was performed and hearts were isolated by careful dissection of adhesions. Animals were sacrificed with an overdose of pentobarbital sodium (0.1 g/kg intravenous). The hearts were removed immediately and washed with cold phosphate-buffered saline. The transmural samples (each of approximately 0.5 g) from the center of the infarcted LAD territory (LAD), right ventricular wall (RV), left circumflex artery perfuse area (Lcx) and interventricular septum, except the LAD perfuse area (IS), were cut. Samples were frozen directly in liquid nitrogen and stored at -80°C for MMPs and PD-ECGF activity analysis. Tissues for histological analysis were preserved in 10% zinc formalin.
2.4. Myocardial MMPs extraction
The myocardial tissues were washed three times with cold saline, homogenized in an ice-cold extraction buffer (50 mg of tissue in 1 ml of extraction buffer) containing 10 mmol/l cacodylic acid (pH 5.0), 0.15 mol/l NaCl, 1 mmol/l ZnCl2, 20 mmol/l CaCl2, 1.5 mmol/l NaN3, and 0.01% Triton X-100, and incubated at 4°C with continuous agitation for 24 h without stirring [7]. The tissue extracts were centrifuged at 4°C, 17 500xg for 20 min. The resultant pellet was re-extracted with fresh buffer (100 mg of tissue in 1 ml of extraction buffer). The final protein concentrations of the mixture were detected with a standardized colorimetric assay: Bio-Rad protein assay [8]. Aliquots were stored at -80°C until use.
2.5. Myocardial PD-ECGF extraction
The myocardial tissue (~ 50 mg) was crushed and added to 400 µl of a lysis buffer containing 50 mmol/l Tris-HCl (pH 6.8), 1% Triton X-100, 2 mmol/l phenylmethylsulphonyl fluoride and 0.02% mercaptoethanol, homogenized with a supersonic waves homogenizer at 4°C. The extract was centrifuged at 4°C, 15 000xg for 30 min [9]. The supernatants were harvested and the protein concentration was measured with Bio-Rad protein assay. Samples were stored at -80°C until use.
2.6. Gelatin zymography
The matrix metalloproteinase activities of the myocardial tissue were measured by using a 7.5% standard sodium dodecyl sulfate (SDS)-polyacrylamide gel containing a final gelatin concentration of 1 mg/ml under non-reducing conditions. Gelatin was used as a substrate because connective tissue degrading enzymes such as gelatinase rapidly cleave it, and it was easily incorporated into polyacrylamide gels. Extracts were thawed on ice, diluted to a final protein concentration of 400 µg/ml with distilled water, mixed with a SDS sample buffer (pH 7.6, V/V, 1: 1.25) containing 50 mmol/l Tris-HCl, 2% SDS and 10% glycerol, and incubated at room temperature for 30 min. The 30 µl of complexes were loaded onto the gel and electrophoresed at 200 V for approximately 45 min at room temperature. After electrophoresis, the gel was cut into two pieces and soaked twice in 2.5% Triton X-100 in 0.05 mol/l Tris (pH 7.5) with 30 min of gentle shaking for each to exchange the SDS in the substrate gels, and rinsed in water (3x30 s). One half of the gel was incubated for 18 h at 37°C in 50 mmol/l Tris-HCl buffer (pH 7.6), containing 5 mmol/l CaCl2 and 0.02% NaN3; the other half was incubated in the same buffer added to 10 mmol/l ethylenediaminetetraacetic acid (EDTA) or 1 mmol/L 1,10-phenanthroline. The gels were stained with 0.1% amido black in 50% methanol, 5% acetic acid for 30 min, and destained twice for 30 min each in 5% methanol and 7% acetic acid. The gels were analyzed with a densitograph (ATTO Densitograph 4.0, Tokyo Japan).
2.7. Measurement of PD-ECGF activity
(A) Thirty microliters of the myocardial extract was incubated with 50 µl 0.2 mol/l Tris - 0.1 mol/l arsenate buffer (pH 7.5) and 20 µl 50 mmol/l thymidine at 37°C for 30 min. After incubation, adding 900 µl 0.2 mol/l NaOH stopped the reaction. (B) For the negative control, 50 µl 0.2 mol/l Tris - 0.1 mol/l arsenate buffer was added to the 30 µl of the myocardial extract, and 900 µl 0.2 mol/l NaOH was added to inhibit the reaction before adding 20 µl 50 mmol/l thymidine. Then the complexes were incubated under similar conditions. The thymine formed in the solutions was measured with absorbance at 300 nm using a spectrophotometric method. The difference of A minus B was defined as the PD-ECGF activity and expressed as mol thymine formed g-1 protein h-1.
2.8. Histologic evaluation
Tissues from LAD areas were fixed with 10% buffered formalin and routinely dehydrated and embedded in paraffin. Serial sections of 4–5 µm were cut and stained with hematoxylin and eosin for morphologic change. Standard double immunohistochemical techniques were used to stain the tissue with antibody against proliferating cell nuclear antigen (PCNA, PC-10, Nichirei) and von Willebrand Factor (vWF, polyclonal rabbit anti-human antibody, Dako, Japan). Bound antibody was detected with a LSAB2 kit (Dako, Japan). Complexes were visualized with diaminobenzidine (DAB) for vWF and cobalt chloride-DAB for PCNA. The counterstaining was not performed. The tissues were also stained with anti-human PD-ECGF antibody (monoclonal anti-human TdrPase antibody, IC6-203, Roche, Japan). Measurements were performed around the channel remnants (CRs), which were identified by the following criteria: (1) identifiable laser puncture scar under a low-power magnification (40x); (2) presence of inflammatory cells and granulation tissue. Each of the most vascular areas was counted in the 100x magnification fields for vWF positive microvessels and 200x magnification fields for vWF/PCNA positive microvessels. A vessel lumen was necessary for vessel identification when the microvessel count was performed. After myocardial infarction in dogs, the myocardial necrosis is frequently heterogeneous and formed by complex interdigitations between necrotic and viable areas, therefore, five-subepicardium and five-subendocardium counts, a total of ten fields, were analyzed in each dog heart. Three doctors performed the counting and the average values were used for statistical analysis.
2.9. Statistical analysis
The Unpaired t-test or Mann–Whitney U-test was used for the analysis between the two groups. The Friedman test was used for the analysis between LAD, IS, RV and Lcx in each group. The Spearman rank correlation test was used for the analysis of the correlation between MMPs, microvessels and PD-ECGF. Values in the tables and figures are given as the mean±1 SD or the median with interquartile range (25–75%) and minimum–maximum where applicable.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1. Gelatinase activity
The activities of latent and active MMPs were assayed by gelatin zymography under non-reducing conditions and denatured using SDS, as shown in Fig. 1A: The lytic bands were found at more than 212; 94; 86; 67 and 60 kDa, and represent different molecular weights of gelatinase. All of these gelatinolytic activities were completely blocked by the addition of 10 mmol/L EDTA or 1 mM 1,10-phenanthroline (Fig. 1B). This demonstrated that these enzymes have the characteristics of matrix metalloproteinases. The molecular weights of these lytic bands were identified with a protein marker and control MMPs (Sigma), and they represented a big molecular mass of gelatinase, proMMP-9, active MMP-9, proMMP-2 and active MMP-2, respectively. The activities of MMPs are shown in Table 1. In the two groups, the active MMP-9 expressed very low activity in only a few samples, so they were excluded from the analysis. In the same group, the proMMP-2, active MMP-2 and proMMP-9 in the LAD area increased significantly compared with IS, RV and Lcx. There were no significant differences between IS, RV and Lcx in the two groups (Table 1) or between each other in the same group (P=0.13 in laser and P=0.22 in control). In the laser group, the proMMP-2, active MMP-2 and proMMP-9 activity in the LAD area were prominently higher than in the control group (Table 1).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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In the present study, we found that TMLR greatly influenced vascular growth patterns. Compared with the control hearts, there was a significant increase in the number of vessels and proliferating endothelial vessels in the region beyond the edge of channel remnants. These findings are similar to those obtained by Kohmoto et al. with normal and ischemic canine hearts [1,12]. To attract new vessels, a tissue must release an endothelial cell chemoattractant, and the ECM must be degraded for the endothelial migration. In other words, angiogenesis can be defined as consisting of three essential steps: (1) matrix degradation, (2) endothelial cell migration, and (3) morphogenic differentiation of endothelial cells into tubes. Matrix metalloproteinases are a family of zinc-dependent endopeptidases that function in the turnover of ECM components. Type IV collagen is a major component of the blood vessel basement membrane, and its degradation is essential in tumor cell invasion of the vasculature. Type IV collagenase consists of two forms of MMPs: MMP-2 (type IV collagenase A, gelatinase A) and MMP-9 (type IV collagenase B, gelatinase B), and has been demonstrated to facilitate the invasion of tumor cells by proteolytic cleavage of the basement membrane and support endothelial cell invasion essential for tumor angiogenesis [14,15]. Using natural and synthetic MMPs’ inhibitors has established that MMP activity is required for angiogenesis [16]. Tyagi et al. reported that MMP-2 and MMP-9 were involved in migration of intimal endothelial and (or) vascular smooth muscle cells through the dense ECM following collateral vessel development in a canine model of chronic coronary occlusion [17]. This demonstrated that MMPs play a similar role in myocardial angiogenesis. In the present study, the MMPs existed almost as a zymogenic form or were not expressed in the uninfarcted areas of IS, RV and Lcx. This suggests that MMPs were not expressed in normal myocardium or had no active function in normal myocardium. We also found that the MMPs’ activity increased in the control group as previously reported [18]. This increase in activity is believed to be important in the remodeling of the uninfarcted cardiac stroma and collateral vessels [17,19]. Recently, one study reported that inhibition of MMPs’ activity reduces left ventricular remodeling and depresses cardiac function after myocardial infarction [20]. In the present study, although we did not show any index of myocardial function, which has been reported on previously, we think that the increase of MMP in the laser treated area, not only correlates with angiogenesis but also correlates with the development of myocardial function. At least during the early phase of TMLR angiogenesis correlates with the expression of MMPs. Since active MMPs are the biological relevant enzymatic forms, and active MMP-2 was only expressed in the LAD area, and was not expressed in normal myocardium (IS, RV, Lcx), this suggests that active MMP-2 plays a pivotal role in angiogenesis induced by TMLR.
The platelet-derived endothelial cell growth factor, also known as thymidine phosphorylase (dThdPase), catalyses the reversible phosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate; 2-deoxyribose-1-phosphate has recently been identified as an endothelial cell chemoattractant and angiogenesis-inducing factor [21]. It has many roles, such as angiogenesis, wound healing (blood vessel repair), neuronal survival and growth inhibition of astrocytes amongst others. As the largest source of PD-ECGF/dThdPase in the body is found in platelets, this strongly suggests that it has a role in maintaining the integrity of blood vessels and promoting the repair of the endothelium. The net increase in vasculature is seen when PD-ECGF/dThdPase is overexpressed and may be caused largely by stabilizing and maintaining the existing vasculature. A recent study reported that PD-ECGF/dThdPase inhibits arterial smooth muscle cell proliferation and accelerates endothelial cell replication, favorably modulating arterial wall response to injury.
In oncogenous research, it has been identified that the level of PD-ECGF/dThdPase expression correlated with the density of microvessels [22,23]. Kurizaki et al. [24] reported that the expression of MMP-2 activity was correlated significantly with the expression of vascular endothelial growth factor; and MMP-9 expression correlated significantly with thymidine phosphorylase expression in human breast cancer tissue. These findings suggest that MMPs and growth regulators may have a cooperative function in angiogenesis. We also found a cooperation in the present study upon analyzing the correlation between microvessel count, MMPs’ activity, and PD-ECGF/dThdPase activity. Immunohistochemical staining of laser treated areas further demonstrated that the higher the density of microvessels, the higher the expression of PD-ECGF/dThdPase. These findings further suggest that TMLR induced angiogenesis correlates with MMPs and PD-ECGF/dThdPase during the early phase.
Some limitations reside in the present study: (A) TMLR was performed in a canine model, in which many collaterals exist between the coronary arteries, and they perhaps perfuse the infarcted or ischemic myocardium. (B) In clinic, TMLR is used in chronic ischemia, either as an isolated treatment or in conjunction with coronary artery by-pass grafting. Therefore, the results may not be directly applicable to the clinical situation. Further study that more closely mimics the clinical situation in which TMLR is applied should be done in the future.
In summary, our conclusions coincide with the angiogenesis theory in TMLR research reported previously. We presented new evidence of TMLR induced angiogenesis from the angiogenic growth factor-PD-ECGF/dThdPase, which stimulates endothelial mitogenesis and chemotaxis, and angiogenic promotion diathesis-MMPs, which contribute to the microenvironment alteration for angiogenesis with TMLR during the early phase.
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Acknowledgments |
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References |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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