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Eur J Cardiothorac Surg 2002;21:1002-1008
© 2002 Elsevier Science NL

Insulin and local growth factor PDGF induce intimal hyperplasia in bypass graft culture models of saphenous vein and internal mammary artery

Bei Huanga*, Thomas Dreyerb, Martin Heidtc, Julie C.M. Yud, Monika Philippa, Friedrich W. Hehrleinc, Norbert Katza, Nadia Al-Fakhria

a Institute of Clinical Chemistry, Justus Liebig University, Giessen, Germany
b Institute of Pathology, Justus Liebig University, Giessen, Germany
c Clinic of Cardiovascular Surgery, Justus Liebig University, Giessen, Germany
d Oxford Molecular Inc., Campbell, CA, USA

Received 13 July 2001; received in revised form 20 January 2002; accepted 18 February 2002.

* Corresponding author. Institute of Pathology, Julius Maximilians University, Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany. Tel.: +49-931-20147786; fax: +49-931-20147440
e-mail: bei.huang{at}mail.uni-wuerzburg.de


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: In arteriosclerosis and bypass graft stenosis, intimal proliferation is controlled by local and systemic growth factors, such as platelet derived growth factor (PDGF) or insulin. Intimal hyperplasia can be produced in organ culture models. Our aim was to compare neointima formation in two organ culture models of internal mammary artery (IMA) and saphenous vein (SV), with special reference to the influence of systemic and local growth stimuli. Methods: Rings of freshly isolated human SV and IMA were cultured over a 3-, 6- or 8-day period. They were distributed into five groups of incubation protocols: incubation with 10% serum; insulin 50 ng/ml and 100 ng/ml; PDGF–BB 5 ng/ml and 10 ng/ml. Frozen sections of cultured rings and pre-culture segments were subjected to elastic stain and immunohistochemistry. Antibodies directed against beta-actin and smooth muscle alpha-actin were used to characterize smooth muscle cell phenotype and against proliferating cell nuclear antigen (PCNA) to demonstrate proliferating cells. Results: Growth factor incubation caused massive intimal hyperplasia with increased elastic fibers in SV and intimal smooth muscle cell as well as matrix accumulation in IMA. Intimal thickening, PCNA and beta-actin expression reached their maximum on day 6 of culture. In both culture models, serum, insulin and PDGF caused increasing intimal thickening, with more pronounced effects in SV. Conclusions: These organ culture models demonstrate the effects of insulin and PDGF on intimal hyperplasia in IMA and SV representing models for arteriosclerosis and bypass graft stenosis and stressing the role of insulin and growth factors for neointima development.

Key Words: Arteries • Cell culture/isolation • Growth factors • Smooth muscle • Veins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The proliferation of intimal smooth muscle cells (SMC), with the accumulation of proteoglycans, collagens, elastin and fibronectin in the extracellular matrix, is an important feature of several vascular pathologies including arteriosclerosis and the response of human saphenous vein (SV) to implantation as an arterial bypass graft [1,2]. It has been proposed that neointima formation results from the interaction of extrinsic factors, such as lipoproteins or increased arterial pressure leading to the production of mitogens [1], and the intrinsic growth regulatory properties of vascular endothelium and SMC [2]. Culture of cells isolated from human and animal vessels has identified multiple factors including platelet derived growth factor (PDGF), which promote proliferation in arteriosclerosis [3]. It has also been proposed that insulin plays a causative role in the formation of the arteriosclerotic lesion in diabetes through stimulation of SMC proliferation [4].

Different organ culture models of SV and internal mammary artery (IMA) have been described [5,6], but the mechanisms of intimal proliferation have not been fully understood. In our study, we used an arterial and a venous ex-vivo culture model, in which the structural integrity of the vessel wall is maintained and the different cell types are located within their native extracellular matrix. The aim of this study was to compare neointima formation in two organ culture models of IMA and SV. Additionally, the reaction of the two culture models to growth stimuli, i.e. PDGF and insulin, was demonstrated. SMC proliferation, migration and actin expression, as well as vessel wall architecture and elastin distribution were investigated.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patient samples and surgical procedure
Samples were obtained from five patients with late-stage coronary heart disease (CHD) (four men and one woman, mean age 64 years, range 54–71 years), who underwent coronary artery bypass surgery. Ethical permission was obtained from the ethical committee of Justus Liebig University. Segments of IMA used for the experiment were angiographically and macroscopically normal. An angiogram of IMA was obtained during pre-operative coronary angiography. Patient profiles were as follows: arteriosclerosis risk factors: hypercholesterolemia in 3/5 patients, hypertension in 3/5, diabetes in 2/5, hyperuricemia in 2/5, nicotine in 1/5. Severity of CHD: two-vessel disease in 1/5 patients, three-vessel and/or diffuse coronary artery disease in 4/5. The pre-operative medication included beta-receptor blockers in 4/5 patients, nitrates in 4/5, amino-salicylic acid in 4/5, anti-cholesteremics in 3/5, ACE inhibitors in 2/5, and cortisone in 1/5. Immediately before and during anaesthesia, the following drugs were administered to each patient: flunitrazepam 2 mg and morphin sulfate 30 mg as pre-medication, anaesthesia was performed with i.v.-narcolept analgesia/intubation using sufentanil 375 µg (and remifentanil 7.5 mg in some cases) and nitrous oxide gas mixture. Additionally, arterenol 20 µg and the antibiotic cefuroxim 1.5 g were administered intravenously as well as blood transfusions, if necessary. Our investigation conforms with the principles outlined in the Declaration of Helsinki [7]. Segments of SV consisted of surgically prepared excess vein using a ‘no touch’ technique [8,9], that was left over after the completion of coronary anastomoses. Segments of IMA consisted of excess surgically prepared artery [10], dissected on a pedicle of retrosternal fat tissue with side branches ligated by metal clips. Both vessel segments were initially perfused with heparin, then maintained in cooled Ringer's solution (4°C) and immediately transferred to the tissue culture laboratory for preparation.

2.2. Tissue culture
Intimal hyperplasia was provoked in established organ culture models of SV and IMA [5,6]. Preperation of vessel segments was carried out as follows: excess fat and adventitial tissue was carefully dissected from the freshly isolated vessels. Rings of SV and IMA were gently cut at about 2 mm intervals with a scalpel blade, and endothelial cells remained undenuded. We took the vessel rings to Dulbecco's modified Eagle medium (DMEM) culture medium (Life Technologies, Germany) containing 10% fetal calf serum (FCS) (Biochrom, Germany) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), amphotericin B (0.025 µg/ml) (all from Sigma, Germany) and L-ascorbic acid (Merck, Germany). Cultures were maintained at 37°C in a humidified atmosphere with 5% (v/v) CO2 in air. The culture medium was replaced every 2–3 days. When examining a 14-day culture period, the vessels were maintained without difficulty until the 8th day of culture and then tended to show signs of degeneration. Therefore, rings were taken out of culture on day 3, 6 and 8 in all the following experiments.

Vessel rings were distributed into five groups: (a) serum (culture medium containing 10% FCS without growth factors); (b) addition of insulin 50 ng/ml medium; (c) addition of insulin 100 ng/ml; (d) addition of PDGF–BB 5 ng/ml; (e) addition of PDGF–BB 10 ng/ml (Fig. 1 ).



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  		Fig. 1. The presented data were derived from morphometric, computer-assisted measurements of intimal thickness of saphenous vein (SV) and internal mammary artery (IMA) segments analyzed ‘pre-culture’ (baseline) and after incubation with serum (10% FCS), insulin 50 or 100 ng/ml, and PDGF 5 or 10 ng/ml (incubation groups). (A): SV. The bars represent the median (µm) of differences between SV intimal thickness measurements of the different incubation groups and pre-culture segments. Intimal thickness measurements of pre-culture segments are defined as baseline (zero). The lines demonstrate the range from 10 to 90% percentile (µm). Data were tested for significance of difference using from Mann–Whitney test for non-parametric analysis; differences were considered significant for P<0.05. The increase in intimal thickness in the incubation groups versus pre-culture shows high statistical significance (P<0.001). Additionally, the different dosages of insulin and PDGF show significant differences in SV intimal thickness (P<0.001 for insulin 50 versus 100 ng/ml, P<0.05 for PDGF 5 versus 10 ng/ml and versus insulin 50 ng/ml). Differences between SV intimal thickness of incubation groups PDGF 10 ng/ml and insulin 100 ng/ml were not statistically significant (P>0.05) (P values not shown in the plot). (B): IMA. The bars demonstrate the median (µm) of differences between IMA intimal thickness measurements of the different incubation groups and pre-culture segments, whereby pre-culture intimal thickness measurements are defined as baseline. Lines represent the range from 10 to 90% percentile (µm). The increase in intimal thickness in the incubation groups versus pre-culture shows high statistical significance (P<0.001, Mann–Whitney test). In addition, different dosages of insulin and PDGF show significant differences in IMA intimal thickness (P<0.001 for PDGF 5 versus 10 ng/ml and versus insulin 50 ng/ml and for PDGF 10 ng/ml versus insulin 100 ng/ml). The differences between IMA intimal thickness of the incubation groups insulin 50 versus 100 ng/ml show no statistical significance (P>0.05) (P values not shown in the plot).

 
2.3. Conventional staining and measurement procedures
Transverse cryostate sections (6 µm) were mounted on gelatin-coated slides and acetone-fixed. Conventional stain (haematoxylin–eosin) as well as elastic stain were applied. Measurements of intimal thickness were carried out with the help of a calibrated Merz-grating at equidistant points along the section length of each vessel using an image analyser (IBAS-2 Kontron, IBAS-Standard 2.0, 3CCD Sony). The intima of SV was defined as the layer of connective tissue between endothelium and media. The development of an intimal hyperplasia in SV was defined as the newly formed layer of cells and matrix substances adjacent to the endothelium above the pre-existing intima after culture, distinguished by loosely packed cells within an abundance of extracellular matrix (Fig. 2 ). In IMA, intima and neointima were defined as the area between endothelium and internal elastic lamina (Fig. 2). Intimal thickness was compared in pre-culture sections of each vessel with corresponding sections of incubation groups (a)–(e) as depicted in Fig. 1. Results were obtained from five identical experiments and expressed as the median of intimal thickness measurements of the respective incubation groups versus pre-culture segments as well as 10 and 90% percentile. Data were tested for significance of difference using Mann–Whitney test for non-parametric analysis, as the data showed no standard distribution in Kolmogorov–Smirnov analysis. Differences were considered significant, if P value was less than 0.05. Statistic tests were carried out with the aid of SPSS software, version 9.0.



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  		Fig. 2. Corresponding sectors of transverse sections of saphenous vein (SV) or internal mammary artery (IMA) vessel rings on day 8 of organ culture. Sections of SV (A) and IMA (B) organ culture after insulin (100 ng/ml) incubation subjected to Von-Willebrand-factor immunostaining (endothelial cells). The endothelium (red arrows) of SV and IMA was not overgrown on the luminal side (A, B). The intimal hyperplasia zone is marked by white double-headed arrows, the media is marked by asterisks (A, B). In IMA, the lamina elastica interna is highlighted by a black arrow (B). Note the different architecture of intimal hyperplasia in SV (A) and IMA (B) organ culture. 100x (A) and 400x (B) magnification.

 
2.4. Immunohistochemistry
SMC distribution was investigated by immunohistochemistry with monoclonal antibodies against smooth muscle alpha-actin (Dako, Germany), which characterizes contractile SMC, and beta-actin (Sigma, Germany), and which is expressed predominantly in synthetic SMC [11]. Additionally, we demonstrated proliferating cells with a monoclonal antibody against proliferating cell nuclear antigen (PCNA) (Sigma, Germany) and endothelial cells through von Willebrand factor antibody (TaKaRa, Japan). Following initial incubation with normal goat serum to inhibit non-specific binding, sections were incubated with each antibody in the appropriate dilution (smooth muscle alpha-actin: 1:25; beta-actin: 1:200; PCNA: 1:3000; von Willebrand factor: 1:20) for 12 h at 4°C.

The reactions were visualized by a biotin–streptavidin–alkaline phosphatase conjugate of secondary antibody (incubation 30–min each) and Fast Red stain (both from Sigma, Germany). The sections were counterstained with Mayer's hamalum. In negative control sections, the primary antibody was substituted by phosphate-buffered saline (PBS). We compared pre-culture sections against groups (a)–(e) of each vessel for smooth muscle alpha-actin and beta-actin expression. PCNA positive-staining cells were counted per microscopic field at 400-fold magnification, analyzing 10 fields per section.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Transverse sections of segments of SV before culture showed a typical histologic appearance and consisted of an endothelial cell layer, a relatively thin medial layer (Fig. 3 ), separated from the connective tissue-rich adventitial layer by the external elastic lamina. Histological examination of IMA showed elastic fibers between axially and longitudinally arranged SMC layers in the media and a well-defined, highly convoluted internal elastic lamina as a typical histologic feature of muscular type arteries, as well as an endothelial and adventitial layer (Fig. 4 ). Microscopy of both vessels showed that the endothelial layer was undamaged. Following 8 days in culture, the endothelial monolayer was still intact and continuous in venous and arterial segments, and arterial and venous wall architecture were also intact.



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  		Fig. 3. Sections of SV organ culture subjected to elastic stain (demonstrating elastic fibers) of pre-culture (A), 10% serum (B), insulin 100 ng/ml (C) and PDGF 10 ng/ml (D) incubation showing increasing intimal hyperplasia through growth factors and an increase in elastic fibers. Sections of SV organ culture subjected to immunohistochemical staining against beta-actin demonstrate the increase in beta-actin positive, synthetic smooth muscle cells in media and hyperplasia zone and the development of intimal hyperplasia from pre-culture (E) to incubation with growth factors, such as insulin 100 ng/ml (F). Double-headed arrows indicate measurement lines of intimal thickness from endothelium to the edge of intimal hyperplasia. 100x magnification.

 


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  		Fig. 4. Sections of IMA organ culture subjected to elastic stain, demonstrating elastic fibers, of pre-culture (A), 10% serum (B), insulin 100 ng/ml (C) and PDGF 10 ng/ml (D) incubation, showing increasing intimal thickness through growth factors. The strongest increase in elastic fibers was demonstrated in PDGF–BB incubation (D), just as in SV (Fig. 3D). Note the reduced effect of serum (B) and insulin incubation on IMA intimal hyperplasia (C) compared to SV (Fig. 3B,C). Sections of IMA organ culture subjected to beta-actin immunostaining show the development of IMA intimal hyperplasia and an increase in beta-actin positive, synthetic smooth muscle cells before (E) and after growth factor incubation with PDGF 10 ng/ml (F). Double-headed arrows indicate measurement lines of intimal thickness from the endothelium to the lamina elastica interna. 400x magnification.

 
After culture, SV and IMA segments all exhibited intimal hyperplasia consisting of several layers of cells embedded in an extracellular matrix of differing extent, that may contain different mucopolysaccharide substances, proteins and glycoproteins [12]. In this study, only elastin distribution and immunostaining for cellular and proliferation markers was specifically investigated (Figs. 2–4).

In SVs, intimal thickening was produced easily, moderate intimal thickening developed even when incubated with 10% FCS only (Figs. 1 and 3). This intimal thickening showed different morphological features than the arterial neointima (therefore termed ‘intimal hyperplasia’). The intimal hyperplasia of SV developed above the pre-existing intima, consisted of densely packed extracellular matrix and was overall more cellular than arterial neointima. There was no strict demarcation to the media, whereas in IMA the lamina elastica interna separated the neointima from the media (Fig. 2). The growth of intimal hyperplasia in SV was caused by an increase in elastic fibers, demonstrated by elastic stain. Especially the intimal layer showed accumulation of short, probably degraded, elastic fibers (Fig. 3). Between these fibers, we demonstrated alpha-actin (not shown) and beta-actin positive SMC (Fig. 3), which were PCNA-negative indicating a lack of current proliferation. In the media, some beta-actin positive, longitudinally arranged SMC and individual proliferating cells (PCNA) were demonstrated. We used insulin as growth factor because it is thought to play a causative role in the formation of arteriosclerotic lesions in diabetes [13,14]. The successful dosage in vitro was 10-fold above the values found in plasma of healthy individuals, but was comparable to high plasma levels of patients with severe type II diabetes [15]. Additionally, we also used PDGF, a local growth factor, which yields the greatest degree of growth stimulation for SMC in vitro [16]. With insulin (Figs. 1 and 3), intimal hyperplasia of SV increased up to 621% average (100 ng insulin/ml) against pre-culture, caused by an increase in elastic fibers and (lesser) SMC. With PDGF (Figs. 1 and 3), there was even a greater increase in intimal hyperplasia up to 764% average (10 ng PDGF/ml). In media and hyperplasia zone, there was a corresponding increase in cell count of PCNA-positive cells (not shown): approximately two-fold with insulin 100 ng/ml and approximately six-fold with PDGF 10 ng/ml as compared to the serum group (analyzed in 400-fold magnification); pre-culture segments showed no PCNA reactivity. Intimal thickening of SV increased mainly from day 3 to day 6 and only minimally from day 6 to day 8. After incubation of SV with PDGF, we found a greater increase in alpha-actin and beta-actin positive cells compared to insulin.

In IMA, we found varying results. It seemed to depend upon the preservation of the vessel, whether the artery segment could be maintained over an 8-day culture period. On the other hand, individual factors seemed to determine, whether or not neointima formation occurred, with some vessels being resistant even to growth stimuli in increased dosage up to 1000 ng/ml of insulin and 20 ng/ml of PDGF, as determined in preceding tests. Generally, the cultured IMA showed slight to moderate intimal thickening in the serum group (Figs. 1 and 4). The intimal thickness correlated with the thickness of pre-existing intima. Histological examination of cultured IMA showed the development of a neointima containing SMC identified by immunohistochemistry for alpha-actin and beta-actin (Fig. 4). The growth of intimal hyperplasia with insulin, respectively, PDGF (Fig. 4) was caused by accumulation of SMC and by an increase in extracellular matrix, resembling early neointima formation in arteriosclerosis [2] though without lipidic deposits and macrophage accumulation, as these were not contained in the organ culture model. However, we did not find the distinctly different effects of growth factors in the cell count of PCNA positive cells in IMA, as demonstrated in SV. PDGF also had a stronger effect than insulin on intimal thickening in IMA (Figs. 1 and 4).

When comparing the two models, beta-actin (Figs. 3 and 4) and PCNA (data not shown) positive cells showed a corresponding increase within the individual models, but a less extensive increase in IMA than in SV.

Endothelial cells, demonstrated by immunostaining with von Willebrand factor, were constantly situated on the luminal border of either SV or IMA (Fig. 2) with a moderate increase in adventitial vasa vasorum under growth stimuli. We did not find overgrowth of the endothelial layer by SMC from the cut edges.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The two organ culture models were used to investigate the basis of intimal proliferation in a setting which preserved the anatomical relationship of endothelium, smooth muscle and extracellular matrix. Morphological examination of the intimal surface showed that the endothelium was maintained over a large proportion of the surface during preparation for culturing of the vessels. The morphological appearance of endothelial cells was largely unaltered after the culture period. Therefore, the physiological interaction of endothelium and SMC was not interrupted, as in many other cell culture models (cell monolayers). It can be assumed that the morphological changes observed in the culture systems were comparable to pathophysiological processes occurring in vivo.

We found short, probably degraded elastic fibers accumulating in the intimal layer, mainly in SV culture. Degradation and increase of elastin peptide was demonstrated in neointima [17] and explained by an increased turnover of elastin with consecutive activation of elastases.

Other authors [18] have reported that the SMC of neointima in cultured SV seemed to originate from the cut edges and assumed that they migrated from the adventitia of the vein segments. According to these reports, endothelial cells originally lining the luminal surface of the vessel segments became overgrown by these cells. In our study, the SV culture model showed moderate intimal thickening already without growth factor addition. However, there was no overgrowth of the endothelial layer by SMC from the cut edges (Fig. 2). The classical concept of neointima formation states that intimal SMC migrate across the internal elastic lamina into the vascular intima, proliferate and produce extracellular matrix, thereby forming an important component of the occlusive mass of fibroatheromatous plaques in arteriosclerotic arteries and in SV grafts. Referring to this concept, we suppose that in the SV culture model SMC forming the intimal hyperplasia stemmed from the media.

The cultured IMA showed slight or moderate intimal thickening in the serum group. A positive correlation was also found between the degree of existing intima and intimal thickening. Hence, a neointima may be more likely to develop, where previous migration of SMC from the medial layer had already taken place to a minor extent.

In vivo, local vasoactive peptides or systemic growth factors act on vessels of patients with f.i. diabetes, hypertension or arteriosclerosis. Quiescent SMC express the PDGF receptor on their surface [19]. The PDGF–BB homodimer yields the greatest degree of growth stimulation for SMC [3]. PDGF also plays an essential role in SMC migration and intimal hyperplasia formation in human SV organ culture [20]. In light of these results, we chose PDGF as an example for a local growth factor. Diabetic patients have a greater risk for coronary heart disease events after coronary bypass graft surgery than non-diabetic patients [21]. Epidemiological prospective and clinical studies suggest that hyperinsulinemia may be an important risk factor for ischemic heart disease [13,14]. So we examined the effect of insulin on the organ culture models.

In fact, intimal hyperplasia was amplified in our experiments through PDGF–BB and insulin in SV (Figs. 1 and 3). The effect of insulin on venous intimal hyperplasia was augmented by increasing the dosage of insulin (Fig. 1). According to investigations done in preparation of this study, a dosage of insulin higher than 100 ng/ml did not increase intimal hyperplasia in SV. But the fact that this insulin level can be reached in vivo is alarming considering the long-term effects on arterial bypass grafts. The dosage of PDGF–BB used in this study may be reached systemically in vivo in patients with acute coronary syndromes [22]. The demonstrated effects of PDGF–BB and insulin on the venous intimal hyperplasia may be a possible pathomechanism of vein graft stenosis.

In our study, intimal hyperplasia was less pronounced in IMA than in SV. The effects of both growth factors on intimal growth were also smaller in IMA (Figs. 1 and 4) than in SV (Figs. 1 and 3), with PDGF being more effective than insulin. Even when stimulated with 10% FCS supplement to the culture medium, SV showed stronger growth of intimal hyperplasia than IMA. Studies of other authors, which were carried out in vivo as well as in vitro, may give possible explanations of this phenomenon. It has previously been shown for coronary bypass grafts that IMA grafts have a higher patency rate than SV grafts [23,24]. In vitro, SMC outgrowth from explants of SV and IMA of the same patients is more pronounced in SV than in IMA [25]. Additionally, PDGF–BB stimulates 3H-thymidine incorporation and increases cell number in SV, but not in IMA. SV shows a stronger downregulation of cell cycle inhibitor p27Kip1 upon stimulation [25]. Taken together, these results indicate that arterial SMC may have an inherent inhibiting effect on cell proliferation. They may explain the relative resistance to growth stimuli of SMC in IMA, which may contribute to the longer patency of arterial versus venous coronary bypass grafts. Increasing the dosage of insulin had no significant influence on the amount of intimal growth in IMA, however, increasing the dosage of PDGF had significantly more effect (Fig. 1). We suggest that PDGF has a stronger effect on arterial SMC proliferation and intimal hyperplasia than insulin. This emphasizes the impact of PDGF on neointimal proliferation in arteriosclerosis.

In conclusion, PDGF and insulin both show dramatic effects on intimal hyperplasia of arteries and veins in vitro. These effects may also be expected to occur in vivo, as local and systemic growth factors play a crucial role in arteriosclerosis and bypass graft stenosis. The differing effects of insulin and PDGF on the venous and arterial organ culture models may contribute to the differences in patency rates between arterial and venous bypass grafts. Further studies on the effects of growth factors could undoubtedly increase our understanding of the formation of arteriosclerotic lesions and of neointima development in bypass grafts.


   Acknowledgments
 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft DFG (German Research Foundation) since April 1998.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Zwolak R.M., Adams M.C., Clowes A.W. Kinetics of vein graft hyperplasia: association of tangential stress. J Vasc Surg 1987;5:126-136.[Medline]
  2. Munro J.M., Cotran R.S. The pathogenesis of atheroclerosis: atherogenesis and inflammation. Lab Invest 1988;58:249-267.[Medline]
  3. D'Amore P.A., Smith S.R. Growth factor effects on cells of the vascular wall: a survey. Growth Factors 1993;8(1):61-75.[Medline]
  4. Forsyth E.A., Aly H.M., Najjar S.F., Neville R.F., Sidawy A.N. Transforming growth factor beta 1 inhibits the proliferative effect of insulin on human infragenicular vascular smooth muscle cells. J Vasc Surg 1997;25(3):432-436.[Medline]
  5. Soyombo A.A., Angelini G.D., Bryan A.J., Jasani B., Newby A.C. Intimal proliferation in an organ culture of human saphenous vein. Am J Pathol 1990;137(6):1401-1410.[Abstract]
  6. Holt C.M., Francis S.E., Rogers S., Gadsdon P.A., Taylor T., Clelland C., Soyombo A., Newby A.C., Angelini G.D. Intimal proliferation in an organ culture of human internal mammary artery. Cardiovasc Res 1992;26(12):1189-1194.[Abstract/Free Full Text]
  7. Declaration of Helsinki. Cardiovasc Res 1997;35:2–3.
  8. Gottlob R. The preservation of venous endothelium by dissection without touching and by an atraumatic technique of vascular anastomosis. Minerva Chir 1977;32:693-700.[Medline]
  9. Angelini G.D., Breckenridge I.M., Williams H.M., Newby A.C. A surgical preparative technique for coronary bypass grafts of human saphenous vein which preseves medial and endothelial functional integrity. J Thorac Cardiovasc Surg 1987;94:393-398.[Abstract]
  10. Nashef S.A., Angelini G.D. Preparation of the internal mammary artery. Br J Hosp Med 1990;44(5):339-342.[Medline]
  11. Owens G.K., Thompson M.M. Development changes in isoactin expression in rat aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation. J Biol Chem 1986;261:13373-13380.[Abstract/Free Full Text]
  12. In: Ayad S., Boot-Handford R.P., Humphries M.J., Kadler K.E., Shuttleworth C.A., eds. The extracellular matrix. London: Academic Press, 1994.
  13. Eschwege E., Fontbonne A. Hyperinsulinemia and macroangiopathy: the epidemiological perspective. Transplant Proc 1992;24(3):767-768.[Medline]
  14. Despres J.P., Lamarche B., Mauriege P., Cantin B., Dagenais G.R., Moorjani S., Lupien P.J. Hyperinsulinemia as an independent risk factor for ischemic heart disease. N Engl J Med 1996;334(15):952-957.[Abstract/Free Full Text]
  15. In: Tietz N.W., ed. Clinical guide to laboratory tests, 3rd ed. Philadelphia, PA: W.B. Saunders, 1995:364-368.
  16. Butler D.M., Leizer T., Hamilton J.A. Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. J Immunol 1989;142(9):3098-3103.[Abstract]
  17. Fulop T., Jr, Wei S.M., Robert L., Jacob M.P. Determination of elastin peptide in normal and arteriosclerotic human sera by ELISA. Clin Physiol Biochem 1990;8(6):273-282.[Medline]
  18. Slomp J., Gittenberger deGroot A.C., van Munsteren J.C., Huysmans H.A., van Bockel J.H., van Hinsbergh V.W., Poelmann R.E. Nature and origin of the neointima in whole vessel wall organ culture of the human saphenous vein. Virchows Arch 1996;428(1):59-67.[Medline]
  19. Fager G., Hansson G.K., Ottosson P., Dahllof B., Bondjers G. Human arterial smooth muscle cells in culture. Effects of platelet-derived growth factor and heparin on growth in vitro. Exp Cell Res 1988;176(2):319-335.[Medline]
  20. George S.J., Williams A., Newby A.C. An essential role for platelet-derived growth factor in neointima formation in human saphenous vein in vitro. Atherosclerosis 1996;120:227-240.[Medline]
  21. Hoogwerf B.J., Waness A., Cressman M., Canner J., Campeau L., Domanski M., Geller N., Herd A., Hickey A., Hunninghake D.B., Knatterud G.L., White C. Effects of aggressive cholesterol lowering and low-dose anticoagulation on clinical and angiographic outcomes in patients with diabetes – the post coronary artery bypass graft trial. Diabetes 1996;48(6):1289-1294.[Abstract]
  22. Nilsson J., Volk-Jovinge S., Svensson J., Landou C., De-Faire U., Hamsten A. Association between high levels of growth factors in plasma and progression of coronary atherosclerosis. J Intern Med 1992;232(5):397-404.[Medline]
  23. Grondin C.M., Campeau L., Lesperance J., Enjalbert M., Bourassa M.G. Comparison of late changes in internal mammary artery and saphenous vein grafts in two consecutive series of patients ten years after operation. Circulation 1984;70((Suppl 1)):1208-1212.
  24. Holt C.M., Francis S.E., Newby A.C., Rogers S., Gadsdon P.A., Taylor T., Clelland C., Soyombo A., Newby A.C., Angelini G.D. Comparison of response to injury in organ culture of human saphenous vein and internal mammary artery. Ann Thorac Surg 1993;55:1522-1528.[Abstract]
  25. Yang Z., Oemar B.S., Carrel T., Kipfer B., Julmy F., Luscher T.F. Different proliferative properties of smooth muscle cells of human arterial and venous bypass vessels: role of PDGF receptors, mitogen-activated protein kinase, and cyclin-dependent kinase inhibitors. Circulation 1998;97(2):181-187.[Abstract/Free Full Text]



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