Karen E. Porter

The development of an in vitro flow model of human saphenous vein graft intimal hyperplasia

Objective : Although the role of blood flow has been investigated in animal models of intimal hyperplasia, there have been no detailed studies in intact human vein owing to the difficulties in designing a suitable laboratory model. The aim of this study was to develop a flow model of human vein graft intimal hyperplasia. Methods : Organ cultures of human saphenous vein were exposed to laminar flow by culturing in a closed circulatory system under predetermined conditions of venous and arterial shear stress for 14 days. Following fixation and processing, paraffin sections were immunostained and neointimal thicknesses measured. Results : It was found that arterial flow completely inhibited neointima formation, but venous flow only partly suppressed the response when compared with vein cultured under static conditions. These results are in agreement with previous in vivo studies in a primate graft model, where increased shear stress inhibited intimal proliferation. Conclusion : The endothelial cell is believed to be the key mediator of haemodynamic effects which influence smooth muscle cell proliferation, and the flow rig developed in this study offers the potential to study inter-cellular interactions within the intact vessel. Furthermore, this method provides the facility to study the effects of different flow conditions on segments of vein from the same patient. This model has scope for further development and sophistication which may ultimately lead to increasing our understanding of the aetiology of vein graft stenoses, and hence formulation of preventative strategies.

Osteopontin Is Not a Marker for Proliferating Human Vascular Smooth Muscle Cells

Osteopontin (OP) is a secreted glycoprotein that contains the Arg-Gly-Asp (RGD) cell-binding sequence that binds calcium and is chemotactic and adhesive for rat vascular smooth muscle cells (VSMCs). OP gene expression is upregulated in cultured rat VSMCs in vitro and after balloon carotid injury in vivo, suggesting that OP may be a marker for proliferating VSMCs in vivo and in vitro. Our in situ hybridization studies of human atherosclerotic coronary vessels, however, have shown OP mRNA expression in plaque macrophages but not VSMCs. The current study investigated OP mRNA expression in cultured human VSMCs and macrophages and in an organ culture model of neointima formation in human saphenous vein. OP mRNA expression was not detected by Northern blot analysis of total RNA from subconfluent or confluent cultures of human VSMCs of any passage maintained in normal growth medium or after stimulation with TGF beta 1 (20 ng/mL), angiotensin II (1 mumol/L), or basic fibroblast growth factor (10 mg/mL) but was just detectable after stimulation with activation vitamin D3 (1 mumol/L). In contrast, cultured human macrophages expressed high levels of OP mRNA that were not dependent on lipid loading. OP mRNA was detected in isolated foci in all layers of saphenous veins maintained in organ culture for 14 days, including <2% of neointimal cells, a distribution that paralleled that of tissue macrophages. These results suggest that OP gene expression is not a marker for proliferation of human VSMCs in vitro and highlight a fundamental difference in the biology of human and rodent VSMCs.

A Quantitative Study of Long Saphenous Vein Morphology in Patients Undergoing Arterial Surgery

Objective: To quantify the incidence and extent of structural changes present in the long saphenous vein of patients with arterial disease. Design: Observational study of saphenous vein morphology. Setting: Departments of Surgery and Pathology, Leicester Royal Infirmary. Patients: Sixty vein biopsies from patients undergoing arterial surgery. Main outcome measures: Intimal and medial thickness and morphology. Results: Smooth muscle cell hyperplasia, elastosis and fibrosis contributed to intimal thickening (> 10 μm) in 87% of veins. This was frequently associated with medial longitudinal muscle hypertrophy. Intimal thickness had a skewed distribution with a median (range) of 33 (8–381) μm, The upper limit of the normal range was 200 μm. The median (range) medial thickness was 293 (131–468) μm. Conclusions: Intimal thickening is common in the long saphenous vein of patients undergoing arterial surgery but is extensive in only a small proportion. The upper limit of the normal range was 200 μm.

Effect of recombinant growth factors on human saphenous vein smooth muscle cells

Dear Editor: Proliferation of vascular smooth muscle cells (SMC) is one of the principal features of intiinal hyperplasia (IH), the pathology unde> lying vein graft stenoses after arterial bypass surgex 7. Numerous growth factors have been shown in animal or in cell culture experiments to have a proliferative effect on vascular SMC. However, there are variations between species and between SMC derived from the arterial and the venous systems. As saphenous vein is the conduit most frequently used in arterial bypass surgery, the most appropriate cell to study is the human saphenous vein smooth muscle cell. The purpose of this study was to investigate the effects of various putative mitogens on isolated SMC derived from human saphenous veiq. Previous work from our department has shown that a paraerine mediator of SMC proliferation is produced in the human saphenous vein organ culture model (1). This mediator is able to promote intimal hyperplasia in a cocuhure model in a segment of vein denuded of its endothelium. The mediator is produced by an intact segment of vein and is likely to be a product of the intact endothelium. The mediator is able to stimulate proliferation of human saphenous vein SMC in the denuded segment of vein when culture medium supplemented with 30% fetal calf serum (FCS) has no effect. The mediator or mediators of this proliferation are likely to be one of a number of growth factors. In order to investigate which growth factors have a significant proliferative effect on human saphenous vein SMC, a variety of growth factors that are known to be present in either venous endothelium or venous SMC were added to human saphenous vein SMC in culture. Human saphenous vein SMC were obtained using an explant technique based on the method described by Chamley-Campbell et al. (2). Briefly, a segment of vein was chopped into small fragments of approximately 1 mm a within a small volmne (2 ml) of Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FCS, and the vein fragments with their medium were transferred to a 25 cm a culture flask that was maintained in a tissue culture incubator at 37 ° C. After 2 ~ wk in culture, SMC migrated out of the exptants and, when the cells had become subeonfluent, they were passaged by the use of trypsin. SMC grown in culture were characterized bv their typical hill and valley configuration, and by immunofluorescent staining for alpha-actin. Further trypsinization was used for subsequent repassaging of the cells at a 1:3 ratio each time. All cells were used for proliferation experiments between Passages 2 and 5. There has been some suggestion that further passaging leads to a loss of viability (2). SMC were rendered quiescent by replacing their medium with RPMI containing 0.4% FCS. This level of serum is sufficient to maintain viability but does not stimulate proliferation. Other investigators have used levels of 0.4% (3,8), 0.5% (5,6,9,19), or 1% (13,16) serum to growth arrest SMC. The cells were growth arrested for a period of 72 h to ensure that they were all at the Go stage in the growth cycle before the proliferation assays were begun. After the 72-h period of growth arrest, the cells were harvested, resuspended at a concentration of 10 cells/ml in either 2.5% FCS medium or 15% FCS medium, and plated out in 24-well plates. The level of 2.5% serum was found to have minimal growth-promoting activity while not completely arresting the cells. At lower levels of serum, the cells could not be stimulated to proliferate following the addition of various growth factors. Other im estigators have shm~n incorporation of thyinidine into SMC at lower serum levels (7,17) but cell replication is inhibited without a minimum amount of serum present. Plasma-derived serum (2%) (11) or between 0.5% and 3% FCS have been used to provide nlinimal stimulation by other workers (10,12,16). A separate plate was used for the study of each growth factor. Each plate contained l04 ceils in 21 of the 24 wells. The growth factors investigated in this experiment ~ele those considered by Ross (14) to have an effect on SMC proliferation in atherosclerosis in humans and are known to be produced by vascular endothelial or smooth muscle cells, namely platelet-derived growth factor (PDGF), basic fibroblast grmvth factor (bFGF), transforming growth factor beta (TGF[3), tumor necrosis factor alpha (TNFct), interleukin-1 alpha (ILIa), and insulinlike growth factor-1 (IGF-1). Each growth factor was individually added to 21 of the 1-ml wells containing 104 SMC. In addition, one plate of SMC contained only medium with 2.5% FCS (baseline control), and a further plate contained medium supplemented with 15% FCS (positive control). The experiment was carried out over a 14-d period. The medium was completely replaced in all but three of the wells on ever 7 2nd d and new growth factor was added with the new medium. In the remaining three wells, the cells were halwested with tlTpsin and counted. The concentration of SMC in three separate wells was measured for each growth factor at each time point and a mean value was calculated. From this, a growth curve was constructed for each growth factor. A total of 10 experiments were performed using 10 different clones of SMC. Because there was considerable variation between clones, each curve within one experiment was compared to the control growth curve within the experiment containing only the 2.5% FCS with no added growth factor. Ceil counts were made on ever): 2qd d and a growth curve was calculated for each growth factor and for the 2.5% FCS control and the 15% FCS positive control. The area under the curve was taken as representing the amount of growth over the time period. The areas were calculated using Simpsons rule (A = dx*(y[0] + 4*y[1] + y[2])/6, where A = area under curve, dx = equally spaced intervals on the X axis, y = the Y values at each point). The growth areas were compared to the area fox the 2.5% FCS area for each cell clone. This gave a growth ratio for each growth factor and for the 15% FCS medium for each SMC clone. The areas were compared