A comparative study of carotid atherosclerotic plaque microvessel density and angiogenic growth factor expression in symptomatic versus asymptomatic patients.

OBJECTIVE: A challenge facing clinicians is identifying patients with asymptomatic carotid disease at risk of plaque instability. We hypothesise that locally released angiogenic growth factors contribute to plaque instability. METHODS: Carotid endarterectomy specimens from eight symptomatic and eight asymptomatic patients were interrogated for microvessel density and angiogenic growth factor expression histologically using immunofluorescence, and biochemically using quantitative real-time polymerase chain reaction (q-RT-PCR). Bio-Plex suspension array was used to assess circulating biomarkers in venous blood from the same patients and six healthy age-matched controls. RESULTS: Immunofluorescence demonstrated significantly greater neovessel density in symptomatic plaques (P=0.010) with elevated expression of hepatocyte growth factor (HGF) (P=0.001) and its receptor MET (P=0.011) than in asymptomatic plaques. The q-RT-PCR demonstrated up-regulation of Endoglin (CD105), HGF (P=0.001) and MET (P=0.011) in the plaques of symptomatic versus asymptomatic patients. Bio-Plex suspension array demonstrated elevated HGF (P=0.002) serum levels in symptomatic versus asymptomatic patients and healthy controls, and decreased platelet-derived growth factor (PDGF) (P=0.036) serum levels in symptomatic versus asymptomatic patients. CONCLUSION: Plaque instability may be mediated by HGF-induced formation of new microvessels, and decreased vessel stability resulting from decreased PDGF. Suspension array technology has the potential to identify circulating biomarkers that correlate with plaque rupture risk.

Synchronous oesophagectomy and hepatic resection for metastatic oesophageal cancer: report of a case.

BACKGROUND: Oesophageal cancer with liver metastasis is rare and when diagnosed is usually advanced and surgical management is contraindicated.Method-Results: We report the case of a patient who presented with oesophageal cancer and liver metastasis. The patient received chemotherapy combined with RFA to liver tumour. Subsequently she was subjected to oesophagectomy and liver resection of segment 5 extended into segment 8. Patient underwent adjuvant chemotherapy post-operatively and remains disease-free until now, 29 months after operation. CONCLUSION: Oesophageal cancer with concomitant liver metastasis is a rare and lethal disease. Multimodal management including surgery may offer prolonged survival in highly selected patients.

Identification of a 'snapshot' of co-expressed angiogenic markers in laser-dissected vessels from unstable carotid plaques with targeted arrays.

BACKGROUND/AIMS: Angiogenesis is a feature of the atherogenic process, with intimal neovascularisation arising from vessels in the adventitia, adjacent to a plaque. Immature, leaky blood vessels from unstable plaques proliferate abnormally and, being poorly invested with smooth muscle cells, may contribute to instability of the plaque by facilitation of inflammatory cell infiltration and haemorrhagic complications. METHODS: We used laser-capture microdissection to isolate angiogenic areas of the extracellular matrix (containing CD105/flt-1-positive, fragile thin-walled vessels) and non-angiogenic vascular areas (CD105-negative, with smooth muscle cell covering) of complicated endarterectomy plaques, and specifically designed angiogenesis-TaqMan real-time PCR microarrays to identify gene expression. RESULTS: Important pro-angiogenic components, including Notch-3, delta-like-4 (DLL4), Tie-2, angiopoietin-1 (Angio-1) and receptor for advanced glycation end products (RAGE), and one anti-angiogenic factor, endostatin, were up-regulated in these regions. Immunohistochemistry demonstrated localisation within intimal, active (CD105-positive) microvessels and co-localisation of Notch-3 and DLL4/Tie-2 and Angio-1 in the same vessels indicating multiple/synergistic signalling mechanisms associated with vessel development. CONCLUSION: These data, although providing only a snapshot of information, demonstrate that plaque vascularisation occurs in the presence of multiple angiogenically active factors. Knowledge of their combined effects could help in the formulation of novel therapeutics designed to stabilise or prevent their formation in the treatment of atherosclerosis.

Oligosaccharides of hyaluronan induce angiogenesis through distinct CD44 and RHAMM-mediated signalling pathways involving Cdc2 and gamma-adducin.

We used short-interfering RNA (siRNA) to knockdown the hyaluronan (HA) receptors CD44 and the receptor for hyaluronan-mediated motility (RHAMM) in vascular endothelial cells to investigate their role in angiogenesis. We showed that CD44 and RHAMM single knockdown inhibited low molecular weight hyaluronan (o-HA)-induced endothelial cell tube formation in Matrigel, but no change in the control, epidermal growth factor-induced tube formation was observed. Using a Kinexus phosphoprotein array and confirmational Western blotting we were able to show a differential effect on HA-induced protein expression after CD44 and RHAMM knockdown. CD44 knockdown abolished o-HA-induced membrane phospho-protein kinase C-alpha (PKC-alpha) and down-stream phospho-gamma-adducin expression. Using the PKC inhibitor Go6976, we demonstrated the involvement of PKC-alpha and gamma-adducin in o-HA-induced tube formation, whilst o-HA-induced enzymatic activity of MMP9 was also reduced. This suggests that endothelial tube formation involves activation of MMP9 via PKC-alpha. Furthermore, the involvement of gamma-adducin in o-HA-induced F-actin cytoskeleton rearrangement was CD44-dependent and the reduction of CD44 expression lead to a change in endothelial cell morphology. Both RHAMM and CD44 knockdown completely inhibited o-HA-induced Cdc2 (Cdk1) phosphorylation suggesting a possible involvement in cell cycle control. Although CD44 and RHAMM are both involved in o-HA-induced endothelial tube formation in Matrigel, they mediate distinct angiogenic signalling pathway and for the first time we demonstrated the specific involvement of gamma-adducin in CD44/o-HA-induced endothelial tube formation. The data presented here extend our understanding of key stages of the processes of o-HA-induced angiogenesis which may have relevance to tumour progression.

B-cell translocation gene 2 is over-expressed in peri-infarct neurons after ischaemic stroke.

OBJECTIVES: Recovery from stroke is dependent on the survival of neurons in the dynamic peri-infarcted region. Although several markers of neuronal injury and apoptotic cell death have been described, administration of neuroprotective drugs directed at specific molecules has had limited success. A complete understanding of deregulated genes associated with neuronal death would be beneficial. Our previous microarray studies identified increased expression of a novel protein, the B-cell translocation gene 2 (BTG2), in infarcted regions. METHODS: We have used immunohistochemistry and Western blotting to examine the expression and localization of BTG2 in stroked brain tissue and immunofluorescent staining of human fetal brain neurons to determine if oxygen-glucose deprivation affected its expression. RESULTS: We show that BTG2 is strongly expressed in peri-infarcted and infarcted regions of brain tissue, localizing in neuronal nuclei and cytoplasm, whilst being absent or very weakly expressed in normal looking contralateral tissue. Exposure of human fetal brain neurons to oxygen-glucose deprivation also induced BTG2 expression in the cytoplasm and perinuclear regions of cells staining positive for propidium iodide (a marker of nuclear damage). CONCLUSIONS: BTG2 may be a modulator of cell survival and differentiation and could help to protect against cell death by inhibition of necrosis and/or apoptotic signalling pathways.

An investigation of the kinetic and anti-angiogenic properties of plant glycoside inhibitors of thymidine phosphorylase.

We investigated the potential of symplocomoside (1) and symponoside (2), glycosides isolated from the bark of Symplocos racemosa to inhibit thymidine phosphorylase (TP) activity and associated angiogenesis. Compound 1 was a reversible, noncompetitive inhibitor of deoxythymidine binding to TP (IC(50) = 65.45 +/- 5.08 microM; K(i) = 62.83 +/- 2.10 microM) and 2 was a reversible, uncompetitive inhibitor (IC(50) = 94.17 +/- 4.05 microM; K(i) = 101.95 +/- 1.65 microM). Molecular modeling analysis indicated that both compounds bound at the active site of the enzyme but not solely to amino acid residues involved in catalysis. Both compounds were active in in vitro angiogenic assays inhibiting endothelial cell migration and invasion in Matrigel, but did not inhibit growth factor-induced proliferation and were not cytotoxic. Compound 1 may have potential as an anti-angiogenic and anti-tumor agent.

New VEGF antagonists as possible therapeutic agents in vascular disease.

BACKGROUND: In this review we provide the reader with an analysis of the importance of VEGF in modulating the angiogenic process in vascular diseases. OBJECTIVES: We have described the key role of VEGF in the development of the major angiogenic diseases including ocular retinopathies, solid tumour growth and atherosclerotic plaque development. METHODS: Following a brief description of the disease, a detailed literature review of the mechanisms through which VEGF induces promotion of neovascularisation and current anti-VEGF therapies is provided for the reader. RESULTS/CONCLUSIONS: Current and future potential clinical therapies are discussed in particular concerning our thoughts on future directives involving adenoviral-mediated gene targeting, nanotechnology and combinational therapies.

Anti-angiogenic activity of sesterterpenes; natural product inhibitors of FGF-2-induced angiogenesis.

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. We have investigated the anti-angiogenic potential of two naturally occurring sesterterpenes, leucosesterterpenone (compound 1) and leucosterlactone (compound 2) isolated from the Himalayan plant Leucosceptrum canum and identified as having biological activity in preliminary screening. Compound 1 inhibited fibroblast growth factor-2-induced proliferation, migration in a wounding assay, chemotaxis and tube formation with small vessel (human dermal) and large vessel (bovine aortic) endothelial cells while compound 2 was largely inactive. Both compounds were also active in an in vivo angiogenic model using the chick chorioallantoic membrane. Neither compounds showed inhibitory activity in the absence of fibroblast growth factor-2. We were able to demonstrate in a binding assay that compounds 1 and 2 bound to the fibroblast growth factor-2 receptor-1 with IC(50) values of 1.4 +/- 0.956 and 132.47 +/- 7.90 muM, respectively, with a concomitant down regulation of phosphorylated ERK1/2 but did not bind to receptor-2. Compound 1 was less hydrophobic than compound 2 and this may contribute to its increased activity. Compound 1 is a new addition to the small number of inhibitors of fibroblast growth factor-2-induced angiogenesis. The compound was a specific inhibitor in that it had no effect on vascular endothelial growth factor or epithelial growth factor-induced angiogenesis. Since angiogenesis is essential for tumour development we conclude that these compounds may have potential as anti-tumour agents.

A feasibility, pharmacokinetic and frequency-escalation trial of intraperitoneal chemotherapy in high risk gastrointestinal tract cancer.

AIMS: To assess the feasibility, pharmacokinetics and maximum tolerable frequency (MTF) of intraperitoneal (IP) 5-fluorouracil and leucovorin (FU/LV) added, as a regional boost, to intravenous chemotherapy after resection of gastrointestinal cancer. METHODS: Fifty-three patients were recruited following gastrointestinal cancer resection (43 colon; 10 stomach/small bowel) with serosal involvement. Peritoneal ports were implanted and IP fluid distribution evaluated ultrasonically. Twelve patients were studied for pharmacokinetics; 44 (41 evaluable) for MTF. Treatment was weekly intravenous bolus FU/LV for 6 months; to this was added IP FU/LV (400/20 mg/m(2) in 1500 ml 4% icodextrin) with increasing frequency from 4 weekly to 1 weekly in four successive cohorts. RESULTS: Peritoneal fluid distribution was excellent. Intraperitoneal FU exposure (AUC) after IP treatment was >1000-fold plasma AUC after IP treatment (regional pharmacokinetic advantage), and >100-fold plasma AUC after intravenous treatment (regional therapeutic advantage). IP therapy was well tolerated if given every 4, 3 or 2 weeks, but not weekly: 11/13, 7/8, 10/13 and 0/7 patients respectively completed treatment without IP modification in these cohorts. Problems with peritoneal access occurred in 20% of patients. CONCLUSION: Adding fortnightly IP FU/LV to a standard intravenous regimen is safe, tolerable and provides high peritoneal FU exposure. More reliable peritoneal access is needed to improve the feasibility of this otherwise promising therapeutic approach.

Leukaemia inhibitory factor is over-expressed by ischaemic brain tissue concomitant with reduced plasma expression following acute stroke.

Leukaemia inhibitory factor (LIF) is a glycoprotein of the interleukin-6 family, which has potent pro-inflammatory properties and is involved in regulation of neuronal differentiation. We have previously identified its upregulation in gene microarrays following acute ischaemic stroke in man. LIF expression and localization was measured in human ischaemic stroke autopsy specimens, in a rat model of middle cerebral artery occlusion (MCAO) and in human foetal neural cell cultures following oxygen-glucose deprivation (OGD) by Western blotting and immunohistochemistry. Circulating LIF was determined in the plasma of patients in the hyper-acute stroke phase using a multiplex enzyme-linked-immunosorbent serologic assay system. Patients demonstrated an increase in LIF expression in peri-infarcted regions with localization in neurons and endothelial cells of microvessels surrounding the infarcted core. The rat MCAO model showed similar upregulation in neurons with a peak increase at 90 min. Circulating serum LIF expression was significantly decreased in the hyper-acute phase of stroke. Brain-derived neurons and glia cultured in vitro demonstrated an increase in gene/protein and protein expression respectively following exposure to OGD. Increased LIF expression in peri-infarcted regions and sequestration from the peripheral circulation in acute stroke patients are characteristic of the pathobiological response to ischaemia and tissue damage.

Pathophysiology of acute ischaemic stroke: an analysis of common signalling mechanisms and identification of new molecular targets.

Stroke continues to be a major cause of death and disability. The currently available therapies have proven to be highly unsatisfactory (except thrombolysis) and attempts are being made to identify and characterize signalling proteins which could be exploited to design novel therapeutic modalities. The pathophysiology of stroke is a complex process. Delaying interventions from the first hours to days or even weeks following blood vessel occlusion may lead to worsening or impairment of recovery in later stages. The objective of this review is to critically evaluate the major mechanisms underlying stroke pathophysiology, especially the role of cell signalling in excitotoxicity, inflammation, apoptosis, neuroprotection and angiogenesis, and highlight potential novel targets for drug discovery.

Indications for and anticipated difficulty of third molar surgery: a comparison between a dental hospital and a specialist high street practice.

OBJECTIVE: To investigate the indications for and anticipated difficulty of third molar surgery between two different referral settings. DESIGN: A prospective study involving completion of a proforma pre- and post-operatively. SETTING: A dental teaching hospital and a specialist surgical dentistry practice in 2003. SUBJECTS AND METHODS: Patients referred for the assessment of their third molars were recruited. Details of the clinical and radiographical assessment for each patient were recorded pre-operatively and the extent of surgery required post-operatively. RESULTS: The main indication for referral for third molar extraction was pericoronitis in both centres. A larger number of patients were assessed and treated in a shorter period of time at the surgical dentist compared with the dental hospital. The surgical dentist was accurate in his assessment of the difficulty of surgery 96% of the time compared with 66% for the dental hospital staff. CONCLUSIONS: This study highlights the benefits for patients in being treated by a surgical dentist. As dental students require exposure to surgical dentistry in order to attain a level of competence, a reduction in the number of patients being referred to dental hospitals may impact upon students' ability to both assess and perform surgical procedures. This may mean that undergraduates will be less able to fulfil the recommendations of the General Dental Council. An outreach programme for final year dental students to surgical dentistry practices would benefit all concerned.

The cooperative transforming effects of PAX3-FKHR and IGF-II on mouse myoblasts.

Alveolar rhabdomyosarcoma (ARMS) cells express high levels of PAX3-FKHR and IGF-II. In this study, we have investigated the effects of PAX3-FKHR and IGF-II on the expression of muscle regulatory factors (myf5, MyoD and myogenin), and platelet derived growth factor-B (PDGF-B) and vascular endothelial growth factor (VEGF) in mouse C2C12 myoblasts in vitro. PAX3-FKHR induced cell cycling of C2C12 cells and promoted proliferation whilst blocking myogenesis. IGF-II inhibited their differentiation without influencing proliferation. Western blotting showed that PAX3-FKHR and IGF-II blocked the expression of myogenin and MyoD respectively. Since MyoD affects early myogenesis and myogenin controls terminal differentiation, a combination of PAX3-FKHR and IGF-II synergistically blocks myogenesis at several different stages in differentiation. We have also shown that the major survival and angiogenic cytokines, PDGF-B and VEGF, were induced by IGF-II and PAX3-FKHR respectively. A combination of PAX3-FKHR and IGF-II could synergistically up regulate the expression of PDGF-B and VEGF and stabilize their high expression levels. Our results suggest that high expression of PAX3-FKHR and IGF-II in ARMS synergistically play a key role in oncogenesis and tumour progression of ARMS.

Irinotecan, cisplatin and mitomycin in inoperable gastro-oesophageal and pancreatic cancers - a new active regimen.

Irinotecan, mitomycin and cisplatin all demonstrate activity in gastro-oesophageal cancers. This novel combination was administered to outpatients with previously untreated inoperable gastro-oesophageal or pancreatic cancer, in a 28-day cycle. A total of 26 out of 31 patients with gastro-oesophageal cancer and 12 out of 14 patients with pancreatic cancer have been treated with this combination, and were evaluable for response. The overall response rates for patients with gastro-oesophageal cancer was 42%, with a median survival of 9.5 months. In patients with pancreatic cancer, the overall response rate was 42% with a median survival of 8 months. There was a statistically significant increase in survival between those patients who achieved a stable disease response and those who achieved either a partial response or complete response. The toxicity profiles for both cancers were virtually identical. There were five treatment-related deaths, and a high admission rate (42%). Thus irinotecan, mitomycin and cisplatin is a new combination with activity in inoperable upper gastro-oesophageal cancers, but with a high toxicity profile. Future developments include reducing the dose of irinotecan and number of cycles of therapy to four.

Effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: possible relevance to wound healing in diabetes.

Ineffectual wound healing in hyperglycaemic patients suffering from diabetes mellitus is characterised by a reduction in capillary reformation (angiogenesis). Basic fibroblast growth factor (FGF-2) is secreted by fibroblasts, macrophages and in particular endothelial cells (EC) in response to tissue injury and is important in promotion of neovascularisation. Recently, glycation of FGF-2 has been shown to significantly reduce its activity in vitro. We have examined the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary formation and associated signal transduction in bovine aortic EC (BAEC). FGF-2 was exposed to 0.25 M glucose-6-phosphate (G-6-P) for 24-72 h and the degree of glycation determined by matrix assisted laser desorption ionisation mass spectrometry. Native FGF-2 was heterogeneous with Mw in the range 15,153.6-17,903 Da. After 24 h incubation with G-6-P there was evidence of glycation, and the mass increase corresponded to addition of 2.7 mol of G-6-P residues; after 48 h, 4 mol sugar was added and this increased to 8.7 after 72 h. Dimerisation of FGF-2 was observed after 72 h of treatment. Induction of mitogenesis in BAEC was significantly reduced by 25%-40% after treatment for 48-96 h with glycated (24 h) FGF-2 (gFGF-2; 100 pg/ml-5 ng/ml; P < 0.05), whilst capillary tubule formation was significantly reduced by between 60% and 90% (100 pg/ml-1 ng/ml; P < 0.05) after 5 days compared to native FGF-2. Subsequent investigation of the signal transduction molecules associated with mitogenesis showed a reduction in FGF-2 induced tyrosine phosphorylated proteins of approximate Mw 20-150 kDa between 10 min and 24 h, in particular, mitogen activated protein kinase (MAPK)/early response kinase (ERK-1, ERK-2), after glycation. To determine the reason for reduced angiogenic activity of gFGF-2, we compared its binding characteristics to that of native FGF-2. Total binding of gFGF-2 to the cell surface was significantly reduced in BAEC analysed by FACS compared to native FGF-2 (P < 0.05). Further investigation using 125I-labelled differentially washed samples, demonstrated a significant reduction in gFGF-2 binding to the high affinity tyrosine kinase receptor (46%) compared to native FGF-2. In summary, glycation of FGF-2 in vitro occurs rapidly within 24 h in the presence of elevated levels of G-6-P. Glycation caused a significant reduction in the ability of FGF-2 to bind to the tyrosine kinase receptor and activate signal transduction pathways responsible for both mitogenesis and capillary formation in BAEC. These results could help to explain the mechanism behind impaired wound healing in patients with diabetes mellitus.

Activation of MAP kinase (ERK-1/ERK-2), tyrosine kinase and VEGF in the human brain following acute ischaemic stroke.

We examined expression of vascular endothelial growth factor (VEGF), phosphorylation of mitogen activated protein kinase (MAP) kinase (ERK1 and ERK2) and tyrosine phosphorylation in 19 patients (aged 58-90 years; mean 75) who died 1-44 days after acute ischaemic stroke. In the grey matter penumbra, 13 of 19 patients showed an increase in MAP kinase tyrosine phosphorylation (ERK1; 2.0- to 8-fold, ERK2; 2.2- to 11-fold) compared with normal contralateral tissue. In almost all cases, ERK-2 phosphorylation was higher than ERK1. Of these 13 patients, 11 also showed a general increase in tyrosine kinase phosphorylation, and eight expressed increased levels of VEGF protein (2.5- to 5-fold). In tissue examined directly from the infarct core, activation of the above proteins was not observed in the, majority of patients. In the white matter, seven of 19 patients (penumbra), and nine of 19 patients (stroke) had an increase in MAP kinase tyrosine phosphorylation (ERK1; 2.0- to 4.6-fold and ERK-2; 2.3- to 5.4-fold respectively) compared with normal contralateral tissue. There was no relationship between activation of MAP kinase and expression of VEGF. Examination of phosphorylated MAP kinase by immunohistochemistry revealed an increase in immunoreactivity in neurones, astroglial cells, reactive microglia and endothelial cells in areas surrounding infarcts, especially in areas with the highest density of microvessels. In conclusion, chronic activation of tyrosine phosphorylated events, in particular redistribution and phosphorylation of MAP kinase (ERK1/ERK2) occurs consistently in the grey matter penumbra of brain tissue following ischaemic stroke, and may be associated with increase in expression of VEGF. These signal transduction events could be important determinants of the extent of neuronal survival and/or angiogenic activity in the recovering brain tissue.

Serial measurement of vascular endothelial growth factor and transforming growth factor-beta1 in serum of patients with acute ischemic stroke.

BACKGROUND AND PURPOSE: Both vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-beta1) are expressed in higher than normal concentrations in the penumbra of patients after ischemic stroke. Because both cytokines are central to the processes of angiogenesis, tissue inflammation, and fibrosis, we performed serial measurements of these cytokines in patients with cerebral infarction and determined their relationship to stroke etiology and volume. METHODS: We serially (at days 0, 1, 3, 7, and 14) measured the serum levels of VEGF and active TGF-beta1 in 29 patients with acute ischemic stroke. Age-matched healthy subjects (n=26) were used as controls. RESULTS: Expression of VEGF was significantly increased in the majority of patients after acute stroke at each of the time points compared with normal controls. Highest expression occurred at day 7 (588+/-121 pg/mL; P=0.005), and it remained significantly elevated at 14 days after stroke. Expression of VEGF correlated with infarct volume, clinical disability (Scandinavian Stroke Scale), and peripheral leukocytosis and was significantly higher in patients with atherothrombotic large-vessel disease and ischemic heart disease (P<0.05 in all cases). In contrast, expression of active TGF-beta1 was not significantly different from control patients at any of the measured time points. When the mean concentration of TGF-beta1 from each patient (pooled time points) was compared with the control mean, a significant increase was found in only 2 patients, whereas levels decreased in 12 patients (P<0.05). There was no correlation between circulating active TGF-beta1 and VEGF expression, leukocytosis, stroke subtype, or patient disability as assessed by Scandinavian Stroke Scale score. CONCLUSIONS: VEGF but not TGF-beta1 showed a dramatic increase in serum of stroke patients. Correlation between stroke severity and VEGF concentration suggests it could be involved in the subsequent repair processes resulting in partial recovery after stroke. Correlation between VEGF expression and peripheral leukocytosis suggests that these changes may also reflect the immunologic status of the patient. VEGF may play an important role in the pathophysiology of acute ischemic stroke and could be of value in future treatment strategies.

Physiological concentrations of gangliosides GM1, GM2 and GM3 differentially modify basic-fibroblast-growth-factor-induced mitogenesis and the associated signalling pathway in endothelial cells.

It has been suggested that gangliosides can influence the growth of cells by modulation of growth-factor-receptor signalling. The activation of endothelial cells (EC) during angiogenesis is crucial for tumour growth and for metastasis, also for numerous other physiological and pathological situations. Pre-treatment of bovine aortic endothelial cells (BAEC) with GM1 or GM2 (5-20 microM) inhibited basic-fibroblast-growth-factor (bFGF)-induced mitogenesis, but GM3 (0.1-20 microM) acted synergistically, increasing proliferation above that of bFGF alone (p < 0.05). The mitogenic effect of all 3 gangliosides was markedly reduced if the cells were washed to remove excess gangliosides from the medium before addition of bFGF. We further show that GM1 and to a lesser extent GM2 modify bFGF binding to its receptor and inhibit the associated mitogenic signal-transduction pathway of protein-tyrosine phosphorylation of 40 to 120 kDa, PLCgamma1, MAP kinase and protein-kinase-C activation. In contrast, GM3 increased tyrosine phosphorylation and MAP kinase activity, as compared with bFGF alone. The observed differential modulation of bFGF-induced mitogenesis by GM1, GM2 and GM3 was at concentrations routinely occurring in the serum of cancer patients. The results suggest that circulating gangliosides may have a role in regulating solid-tumour growth by modulating angiogenesis.