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Ann Thorac Surg 2004;77:864-868
© 2004 The Society of Thoracic Surgeons
a Department of Thoracic and Vascular Surgery, Klinikum Hannover, Heidehaus, Hannover, Germany
b Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany
c Leibniz Institute For Bioartificial Organs (LEBAO), Hannover, Germany
Accepted for publication August 15, 2003.
* Address reprint requests to Dr Walles, Thoracic and Vascular Surgery, Klinikum Hannover, Heidehaus, Am Leineufer 70, 30419 Hannover, Germany
e-mail: twalles{at}yahoo.com
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Abstract |
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METHODS: We obtained reseeded inflow and outflow cannulas of 2 patients undergoing orthotopic heart transplantation after left ventricular assist device implantation 9 and 10 months before. After cell isolation we examined cellular function of reseeded cells and their capability to form a functional endothelial layer applying immunohistologic markers and quantitative Western blot for endothelial nitric oxide synthase activity.
RESULTS: Neointima formation in inflow and outflow cannulas differs macroscopically and by histologic appearance. The neointima formation on the surface of the polyethylene terephthalate fiber (Dacron) grafts differs substantially from native aortic vessel wall with respect to cellular and extracellular matrix composition and cellular function.
CONCLUSIONS: The neointima of Dacron prostheses is composed of cells with rudimentary physiologic endothelial function. We conclude that synthetic matrices are not suitable scaffolds for generating functional cardiovascular implants.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Human study material
Dacron prostheses of implanted HeartMate LVAD systems (Thermo Cardiosystems Inc, Woburn, MA) were obtained from 2 patients (1 male, 1 female) 9 and 10 months after implantation at the time of orthotopic heart transplantation and system removal. Samples for further examinations were obtained 3 cm proximal and distal of the LVAD. The distance to the cardiac and aortal suture lines was more than 3 and 8 cm, respectively. Patients were administered intravenous heparin during the first 2 weeks. After that they received a daily dose of 100 mg of aspirin. Patients' duration on LVAD support until time of system removal was uneventful. Especially, there was no record of systemic infection.
Surgical technique for explantation
The LVAD with the IC and OC were excised during cardiopulmonary bypass. The conduits were carefully retrieved to avoid any structural deformation such as tearing or folds. The conduits were rinsed with sterile 0.9% saline solution and immersed in sterile Dulbecco's Modified Essential Medium supplemented with penicillin and streptomycin.
Tissue preparation
One half of the explanted tissue samples was used for cell isolation, the other half was fixed using Schaffer solution. The prefixed tissue blocks were polymer-fixed (methylmethacrylate resin, Technovit 9100; Kulzer, Hanau, Germany) and cut into 10-µm slides with a tempered steel blade (minimum distance from segment borders, 3 mm) for further histologic and immunohistologic assessment.
Cell isolation
The luminal surfaces of the explanted grafts were decellularized by incubation with 0.02% collagenase for 20 minutes at 37°C. The reaction was stopped by adding fetal calf serum solution. Isolated cells were cultured in endothelial-specific medium up to third passages.
Pentachrome staining
Semithin 6-µm-thick sections were fixated in acetone at -20°C and stained for proteoglycans in Alcian-blue solution for 10 minutes. Stabilization was done in ethanol for 60 minutes. Weigert's iron-hematoxylin solution was used for nuclear staining. Elastic fibers were colored with crocein acid-fuchsin for 15 minutes, rinsed in 0.5% acetic acid, and differentiated in 5% phosphorous-wolfram acid for 20 minutes. Rinsing was done in 0.5% acetic acid and followed by three washing steps with 100% ethanol. Collagen was stained in Saffron du Gatinais (saffron) for 60 minutes. Xylol and 100% ethanol were used for dehydration. Samples were washed in rinsing water after every staining step. The specimens were embedded in Eukitt. Semithin sections were stained with hemalaun.
Immunohistochemistry
To detect reendothelialization, 10-µm-thick scaffold sections were incubated for 60 minutes in monoclonal mouse antihuman CD31 integrin at 1:600 dilution. Biotin-SP-conjugated polyclonal goat antimouse was used as secondary antibody at a 1:200 dilution. For signal amplification, an immunoperoxidase avidin-biotin complex (ABC) kit (Victor stain, Burlingame, UK) was used. Diaminobenzidine was used as a chromogen. Positive control consisted of untreated human aortic tissue. Negative control was performed with mouse serum. Analog explanted prostheses were examined using the following antibodies: CD31, 
-actin, ASOII, collagen I, collagen IV, elastin, laminin, factor VIII-related antigen, and CD11b.
Acetylated low-density lipoprotein uptake test
We incubated the cells with DiI-acetylated low-density lipoproteins (DiI-Ac-LDL, 10 µg/500 mL; Molecular Probes, Eugene, OR) to characterize endothelial function. Cells were incubated for 1 hour, washed twice with phosphate-buffered solution, and overlayed with 2% formaldehyde for 10 minutes. After washing with phosphate-buffered solution the cell cultures were incubated for 1 hour in 1 mg/mL low-density lipoproteins. Fluorescence microscopy was performed at 546/590 nm. Smooth muscle cells were used as negative controls [10].
Western blotting
Proteins were isolated and separated following the NuPAGE Bis-Tris Gel instructions (Invitrogen Life Technologies, Karlsruhe, Germany). Quantitative signal detection was performed according to the instructions of the enhanced chemiluminescence solution Western blotting detection and analysis system of Amersham Biosciences. Native human aortic tissue, human arterial and venous EC, and a purified eNOS preparation (BD Biosciences, Heidelberg, Germany) served as positive controls for the Western blotting.
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Results |
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Histologic findings
The hematoxylin and eosin staining (Fig 1) showed a luminal cellularization of the Dacron prostheses and an absence of thrombus formation in IC and OC. The OC were lined with multiple irregular cell layers. The IC showed a luminal monolayer. Cell nests were scattered among the individual Dacron fibers. The outer surface was covered with a thick, heavy, vascularized connective tissue of irregular cellular composition.
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Cell isolation
Reseeding cells were isolated from IC and OC samples as described previously [11]. Isolated cells were cultured and grown up to the third passage [12]. No differences in growth properties were detected between cells of IC or OC origin. However, many isolated luminal cells had an uncommon microscopic appearance with multiple cellular pedicles.
Cellular function
The uptake and metabolism of aLDL is a specific property of EC. Viable and functional EC take up aLDL from their culture medium and can be detected by fluorescence microscopy (Fig 3A). Approximately 2% to 5% of all isolated cells of both IC and OC show a positive reaction with aLDL (Fig 3B). This indicates that more than 95% of all cells repopulating the synthetic vascular prosthesis are dedifferentiated EC or are not EC at all. Grossly, more aLDL uptake–positive cells are found in the high-pressure OC cannulas.
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–actin are two specific makers for smooth muscle cells. We were able to show the presence of migrated smooth muscle cells in both IC and OC. The determination of eNOS activity served as a second functional marker for EC function. Endothelial nitric oxide synthase activity in the isolated IC and OC cells was a fraction of native venous and arterial EC. Therefore, we put on a 10-fold cell amount in IC and OC compared with controls to obtain a sufficient signal in Western blotting (Fig 4D). However, only in the OC cell fraction were we able to detect a faint eNOS activity (Fig 4C). The Western blotting data in conjunction with the glyceraldehyde-3-phosphate dehydrogenase determination allow us to compare the eNOS activity of the isolated OC cells with native controls (Fig 5). In our studies, EC of OC origin have 20% of eNOS activity of venous EC and 10% that of arterial EC. These unphysiologic findings indicate a lack of endothelial function of the reseeded cells such as one would expect in EC dedifferentiation.
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Comment |
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The pseudointima morphology was different in the IC and OC. Disorganized collagen, intermingled with fibrin, was a characteristic feature of the loosely adherent neointima in IC, whereas regular collagen layers were found in OC. The pseudointima was composed of numerous fibroblasts and a few smooth muscle cells and covered with a nonconfluent endothelial lining. Less than 20% of the isolated luminal cells expressed endothelial function. Compared with EC of native vessels the endothelial function was less than 10% of physiologic levels.
Previously, Houel and colleagues [4, 5] characterized the pseudointima of IC as nonadherent and loosely composed of an irregular type I collagen matrix with plasma and macrophage infiltration, including smooth muscle –actin–positive cells with random orientation. In contrast to this, OC was composed of thin collagen types I and IV layers, and smooth muscle –actin–positive cells were anchored symmetrically to the Dacron surface. These results correspond with our histologic findings (Figs 1 and 2). Houel and colleagues [4] hypothesized that the difference in blood flow characteristics between the IC and OC might be responsible for the different evolutions of pseudointima observed in these conduits, influencing wall adherence and thereby defining thrombogenicity. We were able to reproduce their immunocytologic findings regarding the presence of smooth muscle –actin–positive cells (Fig 3). However, we also were able to detect CD31-positive EC in the pseudointima of both vascular conduits representing up to 10% of the isolated cell types.
Various authors reported that environmental factors influence cellular differentiation, resulting in an altered tissue morphology and function [10]. In the current study we were interested in the influence of the different IC and OC pressure environments on cell differentiation. Applying a quantitative eNOS assay, we were able to show that the cells reseeding the luminal surface of the explanted Dacron conduits have less than 10% of the physiologic endothelial eNOS activity. This finding is strongly suggestive of an advanced dedifferentiation of the reseeding EC entailing conduit thrombogenicity. Moreover, we detected a difference in the amount of eNOS activity in the high-pressure OC and the low-pressure IC. Whether this difference can be attributed to the different pressure environments alone is difficult to prove with our small number of studied grafts.
In the quest to optimize existing vascular prostheses and to develop improved vascular grafts for clinical applications, multiple attempts were undertaken to seed a thin endothelial layer in vitro on the surface of synthetic vascular prostheses—a concept referred to as tissue engineering [3, 9, 13]. Our findings indicate that human EC seeded on a synthetic vascular conduit dedifferentiate and lose their antithrombotic shielding function. This could mean that tissue engineered vascular conduits applying synthetic carrier structures would offer no advantage to already existing conduits. Whether biologic carrier matrices would perform better remains to be proved [14].
We conclude that the pseudointima of synthetic vascular conduits is composed of dedifferentiated vascular cells with impaired cell function that could, in part, be responsible for vascular graft thrombogenicity.
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Acknowledgments |
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