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Ann Thorac Surg 2001;71:1273-1279
© 2001 The Society of Thoracic Surgeons
a Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada
Accepted for publication June 26, 2000.
Address reprint requests to Dr Del Rizzo, Laboratory for Experimental Cardiovascular Surgery, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Ave, Winnipeg, MB, Canada R2H 2A6
e-mail: delrizzo{at}cc.umanitoba.ca
e-mail: ddelrizzo{at}exchange.hsc.mb.ca
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Abstract |
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Methods. End-to-side anastomosis of porcine radial artery to porcine coronary artery were constructed. The vessels were trimmed and incubated under standard tissue culture conditions for 14 days. Appropriate controls were treated similarly. The vessels were frozen, cryosectioned, and immunostained for the expression of the proliferation marker proliferating cell nuclear antigen (PCNA). A proliferative index (PCNA positive nuclei/total nuclei) was calculated for comparative purposes.
Results. Limited PCNA staining was observed in noncultured vessel segments (0.046 ± 0.045). A slight increase in this index was observed in vessels that had been placed into culture without manipulation (0.230 ± 0.141) and in vessels subjected to an arteriotomy (0.462 ± 0.249). However, the most significant increase was obtained after construction of an anastomosis (4.98 ± 6.66). No change in total cell number was evident over the course of the experiment or in relation to the treatment.
Conclusions. Culture conditions and incision slightly stimulate cell proliferation in porcine coronary artery segments when compared with basal conditions of a native artery. In contrast, construction of an anastomosis increases proliferation 108-fold. Therefore, surgical manipulation of arterial conduits during construction of an anastomotic site is the primary trigger for intimal hyperplasia, independent of dissection and incision of the vessel. Furthermore, these data indicate the organ culture model we have developed will be useful for examining the cellular and molecular mechanisms that mediate intimal hyperplasia at the site of a coronary artery bypass graft anastomosis.
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Introduction |
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Proliferation of smooth muscle cells (SMC) is regarded as an essential component of the hyperplastic process. This is a growth factor-mediated event. Cellular injury has been shown to trigger an enhanced release of a number of growth factors that induce cell proliferation in aortic smooth muscle cells [2, 3]. Serotonin (5-HT), angiotensin II (AngII), endothelin, and platelet-derived growth factor (PDGF) are primarily involved in cardiovascular remodeling [4–6]. These growth factors activate intracellular signaling pathways involved in cell proliferation via specific cell surface receptors. As a result of metabolic or mechanical induced cellular injury, mitogen-activated protein (MAP) kinase is activated and undergoes translocation from the cytosol to the nucleus. This molecular event is a key element in signal transduction. MAP kinase activation provides the intracellular signal that leads to the stress-dependent induction of proto-oncogenes (mainly c-fos and c-jun) with the resultant induction of cellular proliferation [7–11]. The products of c-fos and c-jun combine as a heterodimer to form the AP-1 transcription factor. Further downstream from this event, there is an increase in the expression of proliferation cell nuclear antigen (PCNA) [12]. PCNA is a nuclear protein that functions as an accessory for DNA polymerase delta. Expression of PCNA has been correlated with entry into S phase, and the presence of PCNA is thus considered a specific marker of cellular proliferation. PCNA is therefore ideally suited to monitor cellular proliferation in organ culture and intact animal models in response to a defined stimulus.
A major focus of our group has been the development model systems that may be used to assess the safety and efficacy of selective inhibitors of vascular neointimal hyperplasia. We describe herein a novel porcine organ culture model that we have developed and successfully employed to study the cellular events that occur in response to the creation of a CABG anastomosis. We provide evidence that cellular proliferation occurs at the anastomotic site. We anticipate that our model system will provide a mechanism by which the early cellular changes that occur in response to vascular injury can be studied.
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Material and methods |
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Imaging equipment
Tissue samples were examined on an Olympus BH-2 Series Microscope with a BH2-RFC reflected light fluorescence attachment (Olympus Corporation, Lake Success, NY). The presence of secondary antibodies was detected with the use of specific filters (Olympus U excitation filter [BH2-DMU] and G excitation filter [BH2-DMG]; Olympus Corporation). Images were captured and digitized with the aid of a DC-330 digital camera (DAGE-MTI Inc, Michigan City, IN). Digitized images were then examined on Adobe PhotoShop software (Adobe PhotoShop version 4.0; Adobe Systems Inc, San Jose, CA).
Porcine tissue
Fresh porcine hearts and forelimbs were obtained from the local abattoir within 30 minutes from the time of sacrifice, and transferred to the laboratory on ice. The hearts were kept on ice until use. Each experiment was completed within 2 hours of sacrifice. The porcine radial artery (RA) was identified, dissected, and stored in phosphate-buffered saline (PBS) at 4°C, supplemented with 10 mg verapamil/15 mL PBS, until it was used. Care was employed to avoid stretching of the artery during harvest.
Surgical technique
The porcine left anterior descending coronary artery (LAD) was identified and exposed by sharp dissection of the epicardium. An arteriotomy measuring approximately 5 to 7 mm in length was made in the LAD. An end-to-side anastomosis of the RA to the LAD was then constructed with 7-0 polypropylene suture utilizing a continuous suture technique identical to that used by one of us (D.F.D.R.) in clinical practice. Upon completion of the anastomosis, the RA was trimmed to a length of 5 to 7 mm from the surface of the LAD. A segment of the LAD containing the anastomosis and 3 to 4 mm of control vessel on either side of the anastomosis was then dissected from the surrounding tissues and placed into culture (Fig. 1).
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Cryosectioning
Vessel segments were oriented transversely, and the first 1.5 mm of the section was removed to avoid artifacts generated at the cut sites. Transverse sections (7 µm) were cut, placed on Super Frost slides, and stored at -70°C. A representative photomicrograph of a cryosectioned radial artery to LAD anastomosis is shown in Figure 2.
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An antibody dilution series was used to determine the optimal working concentration for each antibody. Slides with mounted sections were immersed in 3% BSA/TBS-T for 1 hour, in order to block nonspecific binding sites. After each period of incubation in the presence of the appropriate (primary or secondary) antibody, the slides were washed five times with TBS-T to remove any unbound antibody. All staining occurred at room temperature in a humidified chamber.
To determine the presence of PCNA, sections were initially incubated with the primary antibody (diluted in 1% BSA/TBS-T) for 1 hour. Secondary antibody, conjugated to Cy-3, was subsequently applied to the sections, which were then incubated for 1 hour in a light-proof chamber. Thereafter, the sections were stained with Hoechst 33342 for 1 minute, followed by 10 washes in TBS-T. Slides were blotted to remove excess fluid before application of Crystal Mount (Biomeda) and coverslips.
Image capturing and cell counting
Slides were examined on an Olympus BH-2 Series Microscope with a BH2-RFC reflected light fluorescence attachment. Vessels were initially examined with the use of a U excitation filter to illustrate nuclei that were stained with Hoechst 33342 nuclear stain. The presence of PCNA was determined by viewing the sections through a G excitation filter.
Images were digitally captured onto a DAGE-MTI CD-330 digital camera viewed through the D Plan Apo 40x ultraviolet objective, with the camera set to the SynchroScan integration feature. Each segment of vessel was examined for Hoechst 33342 and Cy-3 staining. In either case, integration was used to compensate for the diminished sensitivity of the camera relative to the human eye. All images were saved as Windows Bitmap files and analyzed with PhotoShop graphic software.
The number of both total and PCNA-positive cells was obtained by viewing multiple fields of the digitally captured images. For each segment of vessel, the corresponding Hoechst 33342 and Cy-3 images were overlaid, and opacity was varied to ensure that Cy-3 staining corresponded with a nucleus. Figure 3 shows an anastomotic section that has been stained with PCNA and Hoechst 33342 stains. For each section, a proliferative index is determined:
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Determination of proliferative index for various experimental conditions was performed in the following manner. For each condition (see Tables 1 to 4), a number of animals were used. Each animal was assigned a number (shown in the left-hand column of each table). The number of cells per section and the number of PCNA+ cells per section are given as absolute values. At the bottom of each table, the average number of cells ± 1 SD are presented. The total number of animal hearts used in each set of experiments corresponds to the number of animals shown in each table.
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Results |
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Proliferative index of porcine LAD coronary arteries in culture
These experiments were designed to control for any possible mitogenic effect of the culture medium and to ensure that placing the vessels in organ culture did not result in a loss of cell number, suggesting that the culture conditions may be toxic. The data were analyzed as described in the previous experiments. Sections of LAD from 6 animals were placed into 24-well tissue culture plates and cultured for 14 days. After 14 days in culture, the sections of vessel were fixed as described and subsequently processed. The results are shown in Table 2. The average number of cells per section was 2,241.5 ± 504.3. When compared with baseline levels (Table 1), 14 days in tissue culture had no apparent effect in the number of cells per section of artery (p = 0.87). There was, however, a modest but significant (p < 0.001) increase in the number of PCNA+ cells (4.8 ± 2.9), corresponding to a proliferative index of 0.230 ± 0.141. These results indicate that the culture conditions have a mitogenic effect on porcine LAD smooth muscle cells. Furthermore, because cellularity was maintained, the culture conditions do not appear to be toxic to the cells.
Cellular proliferation in injured vessels
Vessel injury is known to induce cellular proliferation. We hypothesized that creation of an anastomosis would therefore be associated with a significant increase in the number of PCNA+ cells. However, it was also necessary to determine whether or not the creation of an arteriotomy in isolation would be sufficient to cause this phenomenon. Consistent with our previous experiments, raw data for each individual animal are presented. In 9 animals a 5- to 7-mm arteriotomy was performed on the anterior surface of the LAD. A segment of LAD containing the arteriotomy was then placed into culture for 14 days. Thereafter, the segments were examined. The results of each experiment are shown in Table 3. There were 2,658.1 ± 575.0 cells per section versus 2,280.5 ± 469.0 in the controls (Table 1, p = 0.11). The number of PCNA+ cells was 12.4 ± 7.4 and the proliferative index was 0.462 ± 0.249.
In 14 animals, an anastomosis of the radial artery to the LAD was constructed as shown in Figure 1 and placed in organ culture for 14 days. There were no significant differences in the cell count in these sections when compared with day 0 controls (2,280.5 ± 469.0 vs 1,830.7 ± 777.7, p = 0.07). In addition, there were no differences in cellular concentration between day 14 control versus day 14 arteriotomy (p = 0.16) or between day 14 control and day 14 anastomosis (p = 0.18). Thus, when taken together, the data suggest there is no loss in cellularity in the various experimental groups relative to baseline fresh control vessels. There was, however, a very significant increase in the number of PCNA+ cells at the anastomotic site (68.9 ± 56.6) when compared with all other groups, and the proliferative index was 5.0 ± 6.6.
The proliferative index for each of the four conditions is summarized in Figure 4. For each condition (day 14 control, day 14 arteriotomy, day 14 anastomosis), there was a significant increase (p < 0.0001) in the proliferative index relative to control. However, the most striking results were seen with the day 14 anastomosis group, where there was a 108.2-fold increase in proliferation relative to control.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Occlusion of saphenous vein grafts after CABG surgery exhibits a quadri-modal distribution that follows specific time intervals: (1) acute (hours to days); (2) early (within 1 year); (3) intermediate (between 1 and 6 years); and (4) late (beyond 6 and usually by 10 years). By year 1, about 15% of grafts are occluded. Thereafter, graft attrition occurs at a rate of 1% to 2% during the intermediate phase, but then the rate accelerates to about 4% per year after the sixth postoperative year [15]. Late vein graft attrition is due to the development of progressive atherosclerosis in either the conduit, the native circulation, or a combination of both.
In sharp contrast, the pathophysiology of acute and early vein graft failure is distinctly different. Very early occlusion results from thrombosis, presumably due to the loss of endothelial cells and the associated increase in platelet activity [16]. Kockx and colleagues [17] examined the conduits of 11 patients who died within a few days of their surgery. In grafts that were in place for less than 12 hours, these investigators found a narrow rim of fibrin and platelets on the luminal surface. By 4 to 10 days, there was obvious loss of endothelial cells, adherent fibrin, and platelet thrombi within the conduits they examined. This knowledge became the rational basis for the routine use of aspirin and other antiplatelet agents in an effort to improve early graft patency. A meta-analysis has demonstrated that the use of these agents in the perioperative period is in fact associated with improved 1-year patency rates [18]. This fact not withstanding, early attrition remains a very significant clinical problem, as shown by patency data from the IMAGE trial [14]. Furthermore, it is even less certain whether or not these agents will have any effect at all in altering the natural history of intermediate and late graft disease.
If grafts remain patent beyond the acute prothrombotic phase, they undergo neointimal proliferation at the site of the anastomosis. This is a constitutive response to vessel injury and is a major component of coronary restenosis. In fact, intimal hyperplasia is the principle cause of graft occlusion beyond the first month and before the first year after surgery [15]. In the normal vessel, smooth muscle cells are thought to proliferate at a very low rate, perhaps less than 0.1% per day [19]. However, it is generally accepted that after vessel injury, there is a marked increase in vascular smooth muscle cell proliferation. Our experiments support this hypothesis. We have shown that baseline proliferation of porcine coronary artery smooth muscle cells is very low. These results are summarized in Table 1. When fresh segments of the porcine LAD were subjected to immunohistochemical analysis, the proliferative index (based on the number of PCNA+ cells relative to total cell count) was 0.046 ± 0.045.
The next set of experiments performed were designed to evaluate the effect of exposure to a mitogen-rich environment on baseline proliferation. Oxidative stress and diabetes are two conditions that are associated with the genesis of vascular occlusive disease. It is thought that these conditions produce a hyperreactive state that promotes proliferation [20, 21]. Consistent with this notion, we found that (Table 2, Fig 4) after 14 days in organ culture, the proliferative index increased fivefold relative to baseline (0.230 ± 0.141 vs 0.046 ± 0.045, p < 0.001). Intentional injury to the vessel by performing an arteriotomy had an additional stimulatory effect on proliferation. After 14 days in culture, the proliferative index in the vessel wall adjacent to the arteriotomy increase 10-fold relative to baseline (0.462 ± 0.249 vs 0.046 ± 0.045) and twofold relative to uninjured vessels that were cultured in identical conditions for the same period of time (0.462 ± 0.249 vs 0.230 ± 0.141, p < 0.001; Table 3, Fig 4). Finally, creation of a coronary artery bypass anastomosis had a very dramatic effect of proliferation. The number of PCNA+ cells increased from 1.0 ± 0.8 in fresh controls to 68.9 ± 56.6 at the anastomotic site (Tables 1, 4, Fig 4), representing a 108-fold increase in the proliferative index. While a comprehensive analysis of the origins of the PCNA+ cells was beyond the scope of this study, we have evidence (work in progress) that proliferation is occurring within the smooth muscle cell population.
Any organ culture model designed to examine vascular remodeling is limited by the fact that the contributions of pulsatile flow and blood borne elements (platelets, lymphocytes, leukocytes) are absent. This not withstanding, such models are useful tools for the systematic analysis of the early molecular and cellular events that are thought to be important in the genesis of vascular occlusive disease. Such models represent an evolutionary intermediate between cell culture and live animal experimentation, and allow for the testing of pharmacological and or gene therapy approaches in a relatively cost-effective system. Our data very strongly indicate that extensive proliferation occurs at the site of a CABG anastomosis and support the long-held clinical position that intimal hyperplasia is a significant factor in early restenosis. Two major endeavors currently underway in our laboratories are the characterization of the proliferative cell type by in situ hybridization, and the testing of novel compounds in an effort to modulate the proliferative response associated with vessel injury. If successful, these efforts may lead to the development of original therapeutic modalities with a very significant clinical relevance.
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Acknowledgments |
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