Bimodal concentration-dependent effect of thrombin on endothelial cell proliferation and growth factor release in culture.

BACKGROUND: The role of thrombin in the stimulation of endothelial cell (EC) proliferation is controversial. The aim of this study was to investigate if thrombin regulates cell proliferation and production of platelet-derived growth factor (PDGF), bovine fibroblast growth factor (bFGF), and transforming growth factor beta(1) (TGF-beta(1)) by bovine aortic ECs. METHODS: ECs, obtained from thoracic aortas of calves, were stimulated with thrombin at various concentrations (from 0.05 to 1.0 IU/ml) in serum free culture. Mitogenic activity of thrombin on ECs was determined by tritiated thymidine uptake. The release of PDGF, bFGF, and TGF-beta(1) was assessed by ELISA. PDGF release was confirmed by Western blot and bFGF and TGF-beta(1) mRNA expression was determined by polymerase chain reaction (PCR). RESULTS: Thrombin at high concentrations did not cause any increase in EC proliferation after 72 h of culture and induced inhibition of EC proliferation after 96 h and 8 days of culture. It induced a decrease in PDGF release and an increase in TGF-beta(1) release. Thrombin at low concentrations induced a significant increase in EC proliferation at 72 h, 96 h, and 8 days of culture. It induced an increase in PDGF release and a decrease in TGF-beta(1) release. bFGF release was higher than control at all thrombin concentrations. These data were confirmed by Western blot and PCR studies. CONCLUSIONS: Thrombin regulates EC growth through the inhibition of EC proliferation at high concentrations and through the stimulation of EC proliferation at low physiological concentrations. EC proliferation is partially mediated by autocrine production of PDGF, bFGF, and TGF-beta(1).

Nicotine reorganizes cytoskeleton of vascular endothelial cell through platelet-derived growth factor BB.

BACKGROUND: Cigarette smoking has been directly linked to atherosclerosis formation and vascular graft failures but the role of nicotine in these processes is not yet completely understood. We investigated the release of platelet-derived growth factor BB (PDGF BB) by the bovine aortic endothelial cell (EC) after nicotine administration at concentrations similar to those found in plasma of active and passive smokers and the role of PDGF BB, autocrinally released, in EC cytoskeletal modification. METHODS: EC were stimulated in a serum-free medium for 72 h with (-)-nicotine (from 6 x 10(-4) to 6 x 10(-8) M). The release of PDGF BB was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on EC was also determined. The EC cytoskeleton was studied with specific antibodies anti-alpha-actin fibers and anti-vimentin and the modification induced by PDGF BB was assessed by blocking PDGF BB activity with specific antibodies. RESULTS: The greatest PDGF BB release was noted at a (-)-nicotine concentration of 6 x 10(-6) M (P < 0.001). The addition of antibody anti-PDGF BB to EC exposed to (-)-nicotine decreased tritiated thymidine uptake by 20% (P < 0.001). EC exposed to (-)-nicotine concentrations of 6 x 10(-6) and 6 x 10(-8) M had a significant alteration in the expression of alpha-actin fibers and vimentin as compared with control. Administration of the antibody anti-PDGF BB in the culture medium reversed cytoskeletal alteration. CONCLUSIONS: Nicotine enhanced the release of PDGF BB by EC which in turn caused an alteration in cytoskeletal organization.

Nicotine-induced smooth muscle cell proliferation is mediated through bFGF and TGF-beta 1.

BACKGROUND: Cigarette smoking influences and enhances the development of atherosclerosis. We investigated if nicotine, an important constituent of cigarette smoking, has a stimulatory effect on bovine smooth muscle cell proliferation in vitro through the mediation of bFGF and TGF-beta 1. METHODS: Bovine aortic smooth muscle cells (SMC) were stimulated with (-)-nicotine at various concentrations ranging from 6 x 10(-4) mol/L to 6 x 10(-8) mol/L. SMC viability and count were assessed. The presence of bFGF and TGF-beta 1 in serum-free conditioned media was determined by the inhibition antibody-binding assay, and the mitogenic activity of (-)-nicotine on SMC was analyzed by the 3H-thymidine uptake. Polymerase chain reaction was used to study the expression of bFGF and TGF-beta 1. RESULTS: The bFGF release after (-)-nicotine stimulation was greater than in the controls, whereas TGF-beta 1 release was lower. The greatest mitogenic activity was found at a (-)-nicotine concentration of 6 x 10(-6) mol/L. The addition of monoclonal antibody anti-bFGF decreased the 3H-thymidine uptake of SMC exposed to (-)-nicotine, whereas the addition of monoclonal antibody anti-TGF-beta 1 increased the 3H-thymidine uptake of stimulated SMC. bFGF mRNA expression was significantly higher in SMC exposed to (-)-nicotine than in the controls, but TGF-beta 1 mRNA expression was significantly lower in SMC exposed to 6 x 10(-6) mol/L (-)-nicotine than in SMC treated with the other concentrations of (-)-nicotine and in controls. CONCLUSIONS: Nicotine is a potent regulator of bFGF and TGF-beta 1 production and release by aortic SMC, and it seems to play an important role in the development and progression of atherosclerosis and neointimal fibrous hyperplasia.

Nicotine induces platelet-derived growth factor release and cytoskeletal alteration in aortic smooth muscle cells.

BACKGROUND: Cigarette smoking is implicated in atherosclerotic plaque formation, but the role of nicotine in this process is not completely understood. The release of platelet-derived growth factor (PDGF) by the bovine aortic smooth muscle cell (SMC) after nicotine administration at a concentration similar to that ingested by active and passive smokers and the role of PDGF in SMC cytoskeletal modification were studied. METHODS: SMC, harvested with enzymatic digestion from calf aorta, were stimulated in a serum-free medium for 72 hours with (-)-nicotine (from 6 x 10(-4) mol/L to 6 x 10(-8) mol/L). The release of PDGF was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on SMCs was also determined. The SMC cytoskeleton was studied with specific antibodies anti-alpha-actin fibers, anti-vimentin, and anti-beta-tubulin, and the modification induced by PDGF was assessed by blocking PDGF activity with specific antibodies. RESULTS: The greatest PDGF release (1.24 +/- 0.14 ng/10(4) cells vs control 0.43 +/- 0.07 ng/10(4) cells) was noted at a (-)-nicotine concentration of 6 x 10(-7) mol/L (P < .001). The addition of monoclonal antibody anti-PDGF decreased the tritiated thymidine uptake of SMCs exposed to (-)-nicotine compared with the control (29% vs 5%-P < .001). SMCs exposed to (-)-nicotine concentration of 6 x 10(-7) mol/L and 6 x 10(-8) mol/L had a significant alteration in the expression of alpha-actin fibers, vimentin, and beta-tubulin compared with control. The administration of antibody anti-PDGF in the culture medium reversed cytoskeletal alteration. CONCLUSIONS: Nicotine enhanced the release of platelet-derived growth, which in turn caused an alteration in cytoskeletal organization.

Nicotine regulates basic fibroblastic growth factor and transforming growth factor beta1 production in endothelial cells.

Nicotine, a constituent of cigarette smoking, may induce atherosclerosis through the production of growth factors. The pattern of bFGF and TGF beta1 production and release by bovine aortic endothelial cells (EC) stimulated with nicotine (from 6 x 10(-4) to 6 x 10(-8) M) was studied. EC viability and count were assessed. The presence of bFGF and TGF beta1 in serum-free conditioned media was determined by the inhibition antibody-binding assay and Western blot analysis. Mitogenic activity of nicotine on EC was also determined. Polymerase chain reaction (PCR) was used to study the expression of bFGF and TGF beta1. The bFGF release after nicotine stimulation was greater than controls, whereas TGF beta1 release was lower. At a nicotine concentration of 6 x 10(-6) M we noted the greatest mitogenic activity. The addition of monoclonal antibody anti-bFGF decreased the tritiated thymidine uptake of EC exposed to nicotine but the addition of monoclonal antibody anti-TGF beta1 had no significant effect. bFGF mRNA expression was significantly higher in EC exposed to nicotine than in controls, whereas TGF beta1 mRNA expression was not modified. From these data we concluded that nicotine regulates bFGF production and release and TGF beta1 release and may have a key role in the development and progression of atherosclerosis.

Thrombin induces production of growth factors from aortic smooth muscle cells.

BACKGROUND: Myointimal hyperplasia is a common complication of arterial recontructive surgery. The serine protease thrombin has a major role in vessel wall healing and eventual myointimal hyperplasia formation. The aim of this study was to determine the effect of thrombin on the production of PDGF AA and bFGF by arterial smooth muscle cells. MATERIALS AND METHODS: Bovine smooth muscle cells were stimulated with thrombin in a serum-free culture. The release of PDGF AA and bFGF was assessed by ELISA. The effect of thrombin on the proliferation of confluent monolayers of bovine smooth muscle cells was determined by tritiated thymidine uptake. RESULTS: Smooth muscle cells stimulated with thrombin released more PDGF AA (P < 0.001) and bFGF (P < 0.001) than the control. Addition of anti-PDGF AA and anti-bFGF antibodies to the medium of smooth muscle cell cultures neutralized the mitogenic effect of thrombin (P < 0.001). CONCLUSIONS: The findings of our study suggest that thrombin may lead to myointimal hyperplasia formation through induction of PDGF and bFGF production by smooth muscle cells.

Growth factors and myointimal hyperplasia in experimental aortic allografts.

OBJECTIVES: To analyse the role of growth factors (platelet derived growth factor, PDGF; basic fibroblast growth factor, bFGF; interleukin 1, IL-1) in the genesis of myointimal hyperplasia in arterial allografts. MATERIALS: Two groups of experiments were performed: isografts and allografts. The isograft group consisted of 15 inbred Lewis rats in which a 1 cm long segment of aorta was inserted as an abdominal aortic interposition graft. The aortic segments were obtained from syngenic Lewis rats. The allograft group consisted of 15 inbred Lewis rats, in which a 1 cm long segment of aorta was interposed at the abdominal aorta level. The aortic segments were obtained from allogenic Brown-Norway rats. CHIEF OUTCOME MEASURES: The animals were killed 4 weeks after surgery and were analysed by morphometric analysis (n = 3 for each group). In addition, production of PDGF, bFGF and IL-1 by aortic segments (n = 12 for each group) in organ culture was assessed. MAIN RESULTS: Allografts had more myointimal hyperplasia, than isografts (p < 0.05). PDGF and bFGF production, generally considered to be the cause of myointimal hyperplasia, was not increased in allografts. IL-1 production was higher in allografts (p < 0.001). MAIN CONCLUSIONS: Myointimal hyperplasia in aortic allografts is dependent on growth factors produced by the graft itself. These growth factors are different from PDGF and bFGF that generally have been implicated in the genesis of naturally occurring myointimal hyperplasia and atherosclerosis. IL-1 may have a principal role in the genesis of myointimal hyperplasia in arterial allografts.

Fluid shear stress increases the release of platelet derived growth factor BB (PDGF BB) by aortic endothelial cells.

OBJECTIVES: The aim of this study was to determine the correlation between shear stress and the release of Platelet Derived Growth Factor (PDGF BB) by aortic endothelial cells. DESIGN AND SETTING: Laboratory in vitro study. MATERIALS: Bovine aortic endothelial cells were seeded in fibronectin-coated cylinders at 1.0 x 10(6) cells/tube and allowed to reach confluence and to adhere for 48 hours. The experimental groups were subjected to nonpulsatile, laminar flow of 50, 100, 150 ml/min in polystyrene cylinders (i.d. 10 mm) of a closed circulatory loop giving a shear stress on the endothelial cells of 3, 6, 9 dyn/cm2. The control group was subjected to similar incubation conditions without flow. OUTCOME MEASURES: The release of PDGF BB by endothelial cells was measured by ELISA and Western Blot Analysis. RESULTS: Shear stress increased significantly (p < 0.01) the release of PDGF BB by endothelial cells. CONCLUSIONS: PDGF BB release by endothelial cells may be one of the mechanisms linking hemodynamic forces and adaptation of blood vessels wall.

Hemodynamic forces modulate simultaneously the release of growth factors and the organisation of cytoskeleton of aortic smooth muscle-cells.

Recurrent stenosis because of myointimal hyperplasia or atherosclerosis after carotid endarterectomy occurs in 5-15% of the cases. The key event is the abnormal proliferation of arterial Smooth Muscle Cells (SMC). After endarterectomy SMC are directly exposed to the blood flowing under pressure. The aim of the present study was to determine the changes in morphology, cytoskeleton organisation, and release of growth factors by SMC exposed to laminar flow. Subconfluent SMC were exposed to a level of shear stress of 6 dyne/cm2 (100 ml/min) for 24 hours under conditions of steady laminar flow. The changes in morphology and cytoskeleton organisation were analysed by light and scanning electron microscopy, and by fluorescence microscopy. Growth factors release was assessed by ELISA. After exposure to laminar flow, SMC assumed a spindle-like shape; they lost many of their protrusions and there was a clear reorganisation of the cytoskeleton and simultaneously their released a higher quantity of PDGF and bFGF. In this study, we found simultaneous changes in cytoskeleton organisation and release of growth factors in SMC exposed to flow. Cytoskeleton reorganisation might be the mechanism through which SMC respond to changes in blood flow. These findings may help to explain the genesis of myointimal hyperplasia following carotid endarterectomy.

Growth factor production by arterial and vein grafts: relevance to coronary artery bypass grafting.

BACKGROUND: Occlusion caused by myointimal hyperplasia, atherosclerosis, or both is the main reason for late failure of saphenous vein coronary artery bypass grafts. On the other hand, internal mammary artery grafts are usually spared from atherosclerosis. Evidence exists that platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are involved in the genesis of myointimal hyperplasia and atherosclerosis. The aim of this study was to assess the production of PDGF and bFGF by arterial and vein grafts. METHODS: In 20 inbred Lewis rats alpha 1 cm long segment of arterial graft was interposed at the level of the abdominal aorta. In a control group of 20 Lewis rats alpha 1 cm long segment of vein graft was implanted at the level of the abdominal aorta. Animals were killed 4 weeks after operation, and the grafts were studied in serum-free organ culture to assess the production of PDGF and bFGF. RESULTS. Arterial grafts produced a smaller quantity of PDGF and bFGF than vein grafts (p < 0.01) Higher mitogenic activity was present in the conditioned media from vein grafts than in the conditioned media from arterial grafts (p < 0.001). A large amount of myointimal hyperplasia was present in all vein grafts. CONCLUSIONS: This phenomenon could explain the rarity of atherosclerotic changes in internal mammary coronary bypass grafts.

Growth factor production after polytetrafluoroethylene and vein arterial grafting: an experimental study.

PURPOSE: Occlusion caused by myointimal hyperplasia appears to be the main reason of late failure of polytetrafluoroethylene (PTFE) arterial bypass grafts. Evidence exists that growth factors are involved in the genesis of myointimal hyperplasia. The aim of this study was to assess the release of platelet-derived growth factor (PDGF) and basic fibroblastic growth factor (bFGF) by PTFE arterial grafts. METHODS: In 15 inbred Lewis rats a 1 cm long segment of PTFE was interposed at the level of the abdominal aorta. In a control of another 15 Lewis rats in a vein graft was implanted at the level of the abdominal aorta. Animals were killed four weeks after implantation and the tissue was studied in organ culture for release of PDGF AA, PDGF BB, and bFGF. RESULTS: PTFE grafts released a greater quantity of PDGF AA than did control vein grafts (28 +/- 4 ng/cm2/72 hr vs 7 +/- 2 ng/cm2/72 hr). Similarly, PTFE grafts released a greater quantity of bFGF than did arterial vein grafts (308 +/- 22 ng/cm(2)/72hr vs 204 +/- 20 ng/cm2/72 hr). CONCLUSIONS: We conclude that PTFE arterial grafts released a high quantity of growth factor, which could explain, in part, the occurrence of distal anastomotic myointimal hyperplasia.

bFGF release is dependent on flow conditions in experimental vein grafts.

OBJECTIVES: Basic Fibroblastic Growth Factor (bFGF) is a powerful mitogen for smooth muscle cells and has been implicated in the genesis of Myointimal hyperplasia. The aim of this study was to determine the release of bFGF by veins in different haemodynamic conditions. DESIGN AND SETTING: Laboratory animal study. MATERIALS: In 39 Lewis rats, a 1 cm long segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngenic Lewis rats. Arterial Vein Grafts (AVG) were harvested after 4 weeks (AVG 4) and 12 weeks (AVG 12). In 16 animals the arterial vein grafts were explanted 4 weeks after the initial operation and reimplanted (Reimplanted Vein Grafts: RVG) in syngenic Lewis rats as venous-venous bypass grafts at the level of the left iliac vein and harvested after 2 weeks (RVG 2) and 8 weeks (AVG 8). OUTCOME MEASURES: The tissue was studied in organ culture in a serum-free system for (1) release of bFGF (immunoassay) and (2) mitogenic activity of the conditioned media. Scanning electron and light microscopy studies were also performed. RESULTS: bFGF release by veins increased significantly (p < 0.01) when veins were inserted in the arterial circulation, and decreased significantly (p < 0.01) when grafts were reimplanted in the venous system. bFGF release (ng/cm2): [Formula: see text] CONCLUSION: Vein inserted in the arterial circulation release a higher quantity of bFGF. This could explain in part, the formation of myointimal hyperplasia in arterial vein graft.

Shear stress induces changes in the morphology and cytoskeleton organisation of arterial endothelial cells.

OBJECTIVES: The aim of this study was to determine the changes in the morphology and cytoskeleton organisation of endothelial cells (EC) determined by exposure to a laminar flow. Cultured EC were exposed to a wall shear stress of 6 dyne/cm2 for 24 hours. CHIEF OUTCOME MEASURES: The morphology of EC was analysed by light and scanning electron microscopy. The organisation of the cytoskeleton was determined by double fluorescence labeling with antibody anti-vimentin, anti-vinculin, anti-tubulin, and with rhodamine-labeled phalloidin. RESULTS: EC exposed to laminar flow become round-shaped with decreased area of adhesion to the substrate. There was a clear reorganisation of the cytoskeleton after exposure to shear stress; the distribution of actin changed from a stress fibre pattern to a more diffuse membrane-associated distribution. These changes in shape and cytoskeleton organisation were reversible after a 48-hour resting period. CONCLUSIONS: EC respond to laminar flow in a predictable manner and these findings may be correlated to the functional changes of EC observed after exposure to shear stress.

Shear stress influences the release of platelet derived growth factor and basic fibroblast growth factor by arterial smooth muscle cells. Winner of the ESVS prize for best experimental paper 1993.

OBJECTIVES: To determine the correlation between haemodynamic forces and the release of two mitogens for smooth muscle cells (SMC): Platelet Derived Growth Factor (PDGF) and basic Fibroblast Growth Factor (bFGF). METHODOLOGY: Bovine aortic smooth muscle cells were seeded on fibronectin coated polystyrene cylinders and allowed to reach confluence. The cells were subjected to a laminar flow of 50 cc/min (3 dyne/cm2), 100 cc/min (6 dyne/cm2) and 150 cc/min (9 dyne/cm2) in an in vitro system. Control cells were subjected to similar incubation conditions without flow. PRINCIPAL RESULTS: Shear stress increased the release of mitogens by SMC. The release of mitogens was proportional to the level of shear stress and was still evident 24 hours after flow cessation. Conditioned serum-free medium from SMC subjected to shear stress increased tritiated thymidine uptake in Swiss 3T3 fibroblasts 13-fold as compared to conditioned serum-free medium from control SMC not subjected to shear stress (p < 0.01) and threefold as compared to standard control (p < 0.001). Addition of an excess of anti-PDGF antibody reduced the mitogenic activity of the conditioned medium by 30% (p < 0.01). Addition of an excess of anti-bFGF antibody reduced the mitogenic activity of the conditioned medium by 60% (p < 0.01). CONCLUSIONS: Increasing shear stress promotes the release of both PDGF and bFGF from arterial SMC in culture and is a possible explanation for atherosclerosis formation.