| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 2000;70:1145-1152
© 2000 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Department of Surgery, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, USA
Address reprint requests to Dr Mannion, Suite 607 College, 1025 Walnut St, Philadelphia, PA 19107
e-mail: john.mannion{at}mail.tju.edu
Presented at the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
Methods. Veins (six/time point) were examined at 0.5, 1, 2, 4, 8, 24, and 48 hours after grafting in crossbred pigs. Terminal transferase-mediated dUTP nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) stains were used to assess apoptosis and proliferation. Apoptosis was also corroborated with confocal and electron microscopy.
Results. Apoptosis was high in both cell populations: at 8 hours, SMC and NSMC apoptosis peaked at 14.5% ± 3.5% and 49.9% ± 7.8%, respectively. In contrast, cell proliferation was different between the two populations. SMC proliferation was low at all time points, whereas NSMC proliferation rose to 22% ± 5.4% by 48 hours.
Conclusions. Medial SMCs are removed through apoptosis and appear to be replaced by fibrous tissue (NSMCs) early after vein grafting. This reciprocal change between the medial SMC and NSMC populations may contribute to late vein graft degeneration.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
The tenth TSDA Resident Research Award was given to Evelio Rodriguez, MD, a cardiac surgery resident in the Department of Surgery, Division of Cardiothoracic Surgery, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, who is completing a thoracic surgery research Fellowship in the Department of Surgery. He received a monetary award of $2,500 and an engraved desktop award.
The TSDA, with support by Medtronic, Inc, makes this award annually, using the above selection procedure. The resident author of the selected study is recognized at the STS meeting.
The effectiveness of the saphenous vein as a coronary bypass conduit is limited by two developments: neointimal formation and "accelerated atherosclerosis." As a result of these pathologic changes, at 10 years, only 60% of saphenous veins remain patent and fewer still (30%) are free of disease [1, 2]. These disappointing results have encouraged the use of multiple arterial grafts, which have a superior short- and long-term patency. However, the saphenous vein, for a variety of technical reasons, will likely remain a commonly used conduit.
Prevention of neointima has been the yardstick that is used to measure the success or failure of interventions to improve vein graft morphology. Because the intima normally contains few cells, neointimal formation is secondary to cell proliferation and migration from the vein graft wall [3]. The cell type felt to be responsible for these changes is the contractile smooth muscle cell (SMC), which dedifferentiates to a synthetic phenotype. These cells proliferate, migrate, and produce the extracellular matrix that results in neointima. Alternatively, the proliferation of non-smooth muscle cells (NSMCs) has been noted [4]. As a result of this understanding of vein graft remodeling, therapeutic efforts have centered on limiting neointima by inhibiting cell proliferation of both SMCs and NSMCs.
Several recent observations have led us to believe that there may be additional remodeling mechanisms, which, if altered, might result in a different approach to vein graft preservation. Apoptosis of contractile SMCs occurs as a consequence of overstretch injury in coronary angioplasty models [5–7]. Because medial SMCs are reduced early after grafting in both human vein grafts [8], and in a porcine model [9], we hypothesized that a similar apoptotic response might occur in vein graft media, which is mechanically stretched by systemic pressure. The loss of these well-differentiated cells could alter the extracellular matrix of the media and contribute to the well-described tendency for accelerated atherosclerosis.
The purpose of this study is to examine the changes that occur to the contractile SMC and the NSMC within the saphenous vein media within the first 48 hours after grafting. The rate of programmed cell death and cell proliferation for the two cell types are estimated. The results suggest that the contractile SMC does not proliferate early grafting and is partially eliminated through apoptosis. Although NSMCs undergo a high rate of apoptosis, they also have a vigorous cell proliferation response. In conjunction with the recently described migratory activity of adventitial fibroblasts [10–12], these results explain how the well-differentiated SMCs of the media are replaced with fibrous tissue.
|
Material and methods |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
All animals were equilibrated for 3 days at the housing facility before surgery, and pretreated with aspirin (650 mg PO). After premedication with Telazole (3 to 5 mg/kg; Webster, PA) and Atropine (0.03 mg/kg), the animals were anesthetized with Diprivan (10 to 15 mg/kg/h; Zeneca, Wilmington, DE), and ventilated via an endotracheal tube with 100% oxygen. Under aseptic conditions, and after intravenous antibiotics (Cefazolin, 20 mg/kg), the right and left greater saphenous veins were harvested. Branches were secured with 4-0 silk ties. The veins were not distended.
A midline neck incision was made and both carotid arteries were dissected free from adjacent tissues. In animals harvested at 4 hours (n = 6), bilateral groin incisions were also made and the right and left common femoral arteries isolated. After administration of 5,000 units of heparin, 4 cm of artery was excised and reversed saphenous interposition grafting was performed, with the beveled ends of the vein anastomosed to the transected edges of the arteries using 7-0 Prolene.
Vein grafts were harvested (six per time point) at 0.5, 1, 2, 4, 8, 24, and 48 hours after surgery. When veins were harvested 4 hours or less after surgery, the animals were maintained under anesthesia until the time of harvest. For veins harvested at later time points, the animals were allowed to recover. They were reanesthetized at the appropriate time and euthanized with Euthasol (80 mg/kg; Webster). The animals were cared for in compliance with the "Principles of Laboratory Animal Care" formulated by the National Institutes of Health (NIH publication no. 96-23, revised 1996).
Tissue sampling and preservation
Porcine saphenous veins were harvested after assessing their patency, by dividing the artery distal to the vein graft and assessing blood flow. The grafts were removed, rinsed with saline solution, and divided into 5-mm segments. The anastomoses were discarded. The segments were immersed in HistoChoice fixative (Amresco, Solon, OH) overnight, and then embedded in paraffin. Sections, 5 µm thick, were then placed on Vectabond (Vector Laboratories, Burlingame, CA)-coated slides.
Terminal transferase-mediated dUTP nick end labeling (TUNEL) technique
Briefly, sections were deparaffinized in xylene, rehydrated in serial alcohol dilutions, and incubated for 5 minutes with 0.6% hydrogen peroxide in methanol. The TdT-FragEL kit (Oncogene Research Laboratories, Cambridge, MA) was used for detection of apoptotic cells. Slides were incubated for 90 minutes with TdT enzyme (1:60) at 37°C. Tissues were stained with diaminobenzidine-tetrahydrochloride substrate for 25 minutes and counterstained with hematoxylin. After dehydration, the slides were mounted with Permount (Fisher, Pittsburgh, PA) and examined under the light microscope.
Immunohistochemistry
The Vectastain Elite ABC system (Vector Laboratories) was used for single-label immunohistochemistry. Sections were deparaffinized, incubated with 0.6% hydrogen peroxide in methanol for 30 minutes, and blocked with 5% horse serum. After washing in phosphate-buffered solution (PBS), sections were incubated with primary antibodies for 2 hours at room temperature. The following primary antibodies were used: a monoclonal mouse DE-R-11 antibody recognizing intermediate filament desmin (1:50; Novocastra Laboratory, Newcastle, UK), a monoclonal mouse PC-10 antibody recognizing cell proliferation (1:50; DAKO, Carpinteria, CA), a monoclonal mouse anti-porcine macrophage IgG2b antibody (1:100; ATCC HB 142.1; American Type Culture Collection, Rockville, MD), and a monoclonal mouse anti-CD8 antibody (DAKO). Afterwards, the slides were washed and incubated with biotinylated secondary horse anti-mouse IgG or anti-goat IgG (1:2,000; Vector Laboratories) for 1 hour, and stained with diaminobenzidine-tetrahydrochloride substrate kit (Vector Laboratories) for horseradish peroxidase followed by counterstain with aqueous hematoxylin (Fisher).
Double immunohistochemistry (TUNEL/DESMIN) was performed as follows: sections were stained using the TUNEL technique as mentioned above. The slides were then incubated with blocking 5% horse serum overnight, washed in PBS, and incubated with DE-R-11 antibody for 2 hours. Slides were then washed in PBS and incubated with biotinylated secondary horse anti-mouse IgG (1:2,000; Vector Laboratories) for 1 hour. A second stain with VIP substrate kit (Vector Purple) was performed. Finally, slides were briefly counterstained with Nuclear Fast Red (Vector Laboratories). Negative controls were performed with nonimmune serum used instead of the primary antibody.
Positive controls for the monocyte/macrophage antibody were performed in: 1) porcine blood smears, 2) porcine liver sections, and 3) the THP-1 monocyte cell line (ATCC). Positive controls for the CD8 antibody were performed in sections of porcine lymph nodes.
Confocal microscopy
Four saphenous vein grafts, harvested 8 hours after surgery, were evaluated using confocal microscopy. This time point was selected because medial apoptosis peaked 8 hours after grafting as determined with TUNEL stains. Sections, 10 µm thick, were stained with the FITC-TdT kit (Oncogene Research) in order to label apoptotic cells with a fluorescent marker. Briefly, the sections were deparaffinized, rehydrated, and washed in PBS. The slides were then incubated with FITC-TdT enzyme for 1 hour and washed with PBS. The slides were mounted with Fluorescein-FragEL Mounting Media (Oncogene Research). Sections were examined under a confocal laser scanning microscope (MRC 600; Zeiss, NY; BioRad, Hercules, CA). Images were acquired and evaluated with COMOS imaging software (BioRad). Immunofluorescent cells were identified and their morphology was assessed. Cells were deemed apoptotic if they revealed one or more of the following: (1) nuclear fragmentation, (2) chromatin condensation, (3) presence of apoptotic bodies, or (4) nuclear pyknosis.
Electron microscopy
Four saphenous vein grafts, harvested 8 hours after surgery, were evaluated using electron microscopy. Samples were placed in a 2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L Na Cacodylate buffer at pH 7.4 fixative. Samples were rinsed with Na Cacodylate buffer three times, and immersed in 1% osmium tetraoxide in the same buffer for 1 hour. Then, the samples were dehydrated, embedded with Spurrs (Polysciences, Warrington, PA), and then polymerized. Ultra-thin sections (80 to 85 nm) were double-stained with 2.5% uranyl acetate/50% ethanol and bismuth. Sections were examined (transmission mode) and photographed using the electron microscope (Hitachi 7000 STEM-scanning transmission electron microscope). Electron micrographs were taken on Kodak 4489 film (Kodak, Rochester, NY). A total of 10 high-power fields (7,000x) per vein graft were examined. The micrographs were evaluated for cellular changes characteristic of apoptosis. Cells were deemed apoptotic if they revealed one of the following: (1) cell shrinkage, (2) nuclear fragmentation, (3) chromatin condensation, (4) presence of apoptotic bodies, or (5) nuclear pyknosis.
Quantitative measurements
Definition of muscle and non-muscle bundles
The media of saphenous veins is composed of two cell populations: SMC and NSMC, which are apparent in bundles. Serial desmin-stained sections were evaluated in order to differentiate medial SMC from NSMC bundles. Desmin-positive bundles separated by desmin-negative bundles are easily identified within the media of saphenous veins during the first 48 hours after grafting. Representative desmin stains at different time points after surgery are shown in Figure 1.
|
Statistical analysis
All numeric data are presented as mean ± SE; an unpaired two-sided t test was used to determine statistical significance. Observations between time points were deemed statistically significant if the p value was less than 0.05. All analysis were performed using SAS software (version 6.12; SAS Institute, Cary, NC).
|
Results |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
Apoptosis within saphenous vein media
Contractile SMC apoptosis was low at baseline, peaked at 8 hours, and declined towards baseline by 24 to 48 hours (p < 0.01) (Fig 2A).
|
|
|
|
|
|
|
|
Comment |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
Medial degeneration and fibrosis in saphenous vein grafts were described shortly after their clinical application, but the causes and prevention have remained uncertain. Medial ischemia [13], inflammation [14, 15], and mechanical trauma [16] have been proposed as causes of fibrosis. This study suggests an additional explanation: medial remodeling is associated with a prominent rate of preprogrammed cell death. The triggers for this phenomenon are uncertain, but it is clear that these changes have their origin in the early hours after grafting.
Contractile SMCs can be eliminated from the media of the saphenous vein by three processes: dedifferentiation, necrosis, or apoptosis. The most widely accepted explanation for SMC loss is dedifferentiation, in which a SMC loses expression of certain cytoskeletal markers, such as desmin. The reduction of the number of desmin-containing cells that we previously described is theoretically consistent with this concept, although it is difficult to determine if a transformed cell is derived from a SMC or a NSMC lineage. However, the results of this study suggest that dedifferentiation is not the only explanation for SMC loss within the first 48 hours. Approximately 15% of SMCs are eliminated by apoptosis at 8 hours alone. Because apoptosis is a rapid process that can be completed within 2 to 4 hours [17], additional SMCs are probably lost between 8 and 24 hours after grafting. Thus, apoptosis offers an identifiable explanation for the reduction of desmin staining within the media.
Necrosis can be identified and distinguished from apoptosis. Necrosis is characterized by cell swelling, plasma membrane rupture, and an ensuing inflammatory response [18]. Necrosis is caused by factors external to the cell, and has been called cell death "by murder." In contrast, apoptosis, or "cell suicide," is a programmed cell death, in which a cascade of cysteine proteases cause DNA fragmentation, chromatin condensation, cell shrinkage, and engulfment by neighboring cells. Apoptosis occurs without pronounced inflammation. The paucity of macrophage infiltration by 48 hours, and the occurrence of only sporadic cells with necrotic characteristics on electron microscopy, suggest that apoptosis may be more prominent than necrosis within the first 48 hours of grafting. Undoubtedly, both necrosis and apoptosis contribute to the reduction of the contractile SMCs, although the relative contribution of each process requires further study.
There are several techniques that can identify apoptosis and the type of cell in which this process is occurring. Agarose gel electrophoresis of DNA is specific, but not sensitive [19], and cannot distinguish which cells are apoptotic. TUNEL detects DNA fragmentation in situ. In this study, TUNEL-positive cells within the muscle bundles of the vein graft were assumed to be SMCs by virtue of their location. This assumption was reasonable, considering that many TUNEL-positive cells were also desmin positive, as determined with double immunostaining. Because DNA fragmentation can also occur in necrotic cell death, TUNEL-positive cells must be confirmed to be apoptotic with an additional technique [20]. Confocal microscopy determined that nearly all TUNEL-positive cells had nuclear changes consistent with apoptosis; quantification of medial cells under TEM revealed that the apoptotic rates of SMCs and NSMCs were consistent with the TUNEL findings.
Vascular remodeling involves a balance between cell proliferation and cell death. When proliferation is favored, hypertrophy occurs, as in response to hypertension [21]; when apoptosis predominates, as in aortic aneurysms [22] or in atherosclerotic plaques [23], atrophy can occur, with unfavorable results. Atrophy appears to occur early in saphenous vein grafts. In the first 48 hours, the contractile SMCs undergo apoptosis without proliferation, contributing to their partial early disappearance.
In contrast, apoptosis of the NSMCs is counterbalanced by proliferation. It may be argued that despite proliferation, a net loss of the NSMC population should occur, because the proliferative rate for the NSMCs is lower than their apoptosis rate. However, other factors act to increase the population of the NSMC in the media. First, NSMCs migrate from the adventitia, which increase cell number in the media. Second, medial proliferation is known to peak at 5 to 7 days [24], whereas apoptosis is less prominent at later time points [25]. Given that the cells that proliferate within the first 48 hours are the NSMCs, it is reasonable to conclude that these cells continue their proliferative response.
Predominantly, pharmacologic interventions intended to limit neointimal hyperplasia have focused on reducing cell proliferation [26–28]. Theoretically, the proliferation of NSMC should be inhibited and the SMC should be preserved. This study suggests that preservation of the well-differentiated medial SMC is possible by preventing apoptosis. The loss of the contractile SMC is not an inevitable response to grafting; medial hypertrophy with contractile SMCs is possible (data not shown).
To our knowledge, this is the first study that examines cell proliferation and cell death within the media within the first several hours after saphenous vein grafting. Within this early time frame, the two major cell populations of the media react differently. The nonmuscle bundles are most sensitive, with apoptosis and proliferation even induced with a gentle surgical technique that avoids distension. Arterialization further increases the activity within the nonmuscle bundles. In contrast, the contractile SMCs appear undisturbed until apoptosis is present 8 hours after surgery. Within the first 48 hours, there is no significant proliferation within these bundles. The different kinetics of cell proliferation and apoptosis may contribute to the replacement of the media with fibrous tissue.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
|---|
Related Article
This article has been cited by other articles:
|
|
 |
|
  T. Schachner, G. Laufer, and J. Bonatti In vivo (animal) models of vein graft disease. Eur. J. Cardiothorac. Surg., September 1, 2006; 30(3): 451 - 463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Schachner Pharmacologic inhibition of vein graft neointimal hyperplasia J. Thorac. Cardiovasc. Surg., May 1, 2006; 131(5): 1065 - 1072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Fogelstrand, K. Osterberg, and E. Mattsson Reduced neointima in vein grafts following a blockage of cell recruitment from the vein and the surrounding tissue Cardiovasc Res, August 1, 2005; 67(2): 326 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Schachner, A. Oberhuber, Y. Zou, A. Tzankov, H. Ott, G. Laufer, and J. Bonatti Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts Eur. J. Cardiothorac. Surg., February 1, 2005; 27(2): 302 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Saunders, G. Pintucci, C. S. Bizekis, R. Sharony, K. M. Hyman, F. Saponara, F. G. Baumann, E. A. Grossi, S. B. Colvin, P. Mignatti, et al. Vein graft arterialization causes differential activation of mitogen-activated protein kinases J. Thorac. Cardiovasc. Surg., May 1, 2004; 127(5): 1276 - 1284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Krejca, J. Skarysz, P. Szmagala, D. Plewka, G. Nowaczyk, A. Plewka, and A. Bochenek A new outside stent - does it prevent vein graft intimal proliferation? Eur. J. Cardiothorac. Surg., December 1, 2002; 22(6): 898 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sartore, A. Chiavegato, E. Faggin, R. Franch, M. Puato, S. Ausoni, and P. Pauletto Contribution of Adventitial Fibroblasts to Neointima Formation and Vascular Remodeling: From Innocent Bystander to Active Participant Circ. Res., December 7, 2001; 89(12): 1111 - 1121. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
