Potent anti-metastatic activity of combretastatin-A4.

The requirement for tumour vascularisation to permit the expansion of solid tumours beyond a threshold size of approximately 1 mm diameter has focussed attention on anti-vascular and anti-angiogenic agents for cancer therapy. Combretastatin-A4 (cis CA-4P) is a tubulin-binding agent that is cytotoxic for proliferating endothelial cells in vitro and causes anti-vascular effects in the established tumour vessels of some primary tumours. Preliminary data from Phase I clinical trials indicate that cis CA-4 may also be effective in targeting the vasculature of human tumours. As metastatic disease is the principal cause of mortality in cancer, we have investigated the effects of cis CA-4 on metastatic development using an in vivo model. We show that bolus or continuous administration of cis CA-4P results in potent inhibition of metastases derived from ectopic primary Lewis lung carcinomas in mice whereas the trans CA-4 isomer is without effect. These data further characterise the activity of CA-4 in vivo and suggest that the drug should be evaluated clinically as an anti-metastatic agent.

A common phenotype associated with atherogenesis in diverse mouse models of vascular lipid lesions.

The introduction of a range of different genetic modifications in mice results in altered lipoprotein metabolism and the development of vascular lipid lesions. At present, however, it is unclear to what extent the molecular events underlying lipid lesion formation are similar in these different mouse models of atherosclerosis. The aim of this study was to compare the protein expression pattern of lipid lesions from seven different mouse lines with varying susceptibility to vascular lipid lesion development, to determine to what extent lesions induced by different genetic interventions have a similar composition. The proteins we have measured, using quantitative immunofluorescence, are proteins whose expression is known to be modulated during atherogenesis in humans, including plasminogen activator inhibitor (PAI)-1, transforming growth factor (TGF)-beta 1, osteopontin and the macrophage marker CD11b. In all the mice lines we have investigated, PAI-1 was elevated wherever lesions developed. Active TGF-beta was depressed in the vessel wall of mice which developed lipid lesions, particularly in the intima. In contrast, TGF-beta 1 antigen (active plus latent TGF-beta 1) was increased at lesion sites. Accumulation of osteopontin and, with the marked exception of apolipoprotein(a) transgenic mice, tissue macrophages occurred at sites of lipid deposition in the vessel wall. Each lesion, irrespective of its size and the mouse strain in which it developed, had similar amounts of PAI-1, active TGF-beta and osteopontin per unit area of lesion. These data are consistent with a common phenotype accompanying atherogenesis, irrespective of the genetic basis of susceptibility.

Expression of Klf9 and Klf13 in mouse development.

Klf9 and Klf13 are members of the C(2)H(2) zinc finger family of transcription factors that are thought to be involved in regulating basal transcription. The mRNA localization of Klf9 and Klf13 during development was determined by in situ hybridization of mouse E8, E11, E13 and E16 embryo sections. The data showed that Klf9 and Klf13 are widely expressed at all the mouse embryo stages examined. Whilst the expression patterns of the two genes largely overlap there are differences in the localization or level of expression in some tissues. At E11, both genes are expressed in high levels in the cephalic mesenchyme whilst Klf13 and not Klf9 is expressed at high levels in the developing heart at E8 and E11. In the gut and bladder at E16, Klf13 is expressed in the epithelial cell layer whereas Klf9 is expressed in both the muscle and epithelial layers. Both Klf9 and Klf13 are expressed at high levels in the epidermis at E11, E13 and E16.

Microsatellite mutation of type II transforming growth factor-beta receptor is rare in atherosclerotic plaques.

A somatic mutation within a microsatellite polyA tract in the coding region of the type II transforming growth factor (TGF)-beta receptor gene was reported to occur in human atherosclerotic and restenotic lesions. This mutation occurs frequently in colorectal cancer with the replication error repair phenotype and results in loss of sensitivity to the growth inhibitory effects of TGF-beta in cells from the tumors. The mutation was proposed to account for the clonal expansion of vascular smooth muscle cells observed in atherosclerotic plaques, through loss of the growth inhibitory effect of TGF-beta. The frequency of the mutation and the extent of clonal expansion of the mutated cells have major implications for the mechanism of atherogenesis and therapeutic strategies. We analyzed a set of 22 coronary arterial and 9 aortic samples containing early to advanced atherosclerotic lesions for the mutation in the type II TGF-beta receptor polyA tract. Only 1 coronary arterial sample from an advanced lesion showed detectable amounts of the mutation, present at a low level (8% of the DNA sample). The data imply that the mutation occurs only at low frequency and is not a major mechanistic contributor to the development of atherosclerosis.

Tamoxifen effects on endothelial function and cardiovascular risk factors in men with advanced atherosclerosis.

BACKGROUND: Tamoxifen and its analogues act as selective estrogen receptor modulators (SERMs) in women, with estrogen-like activities on some plasma cardiovascular risk factors (eg, lipoproteins). Effects of SERMs on men with coronary artery disease (CAD) have not been reported. METHODS AND RESULTS: Thirty-one men with angiographically proven CAD were recruited; 16 were treated with tamoxifen (40 mg/d) for 56 days, and 15 were untreated. All the CAD patients were medicated with aspirin and an HMG-CoA reductase inhibitor for >/=6 weeks before entering the study. Ten men with angina-like symptoms but normal coronary arteries by angiography (NCA group) were also treated with tamoxifen. Blood samples were collected at days -7, 0, 7, 14, 21, 28, and 56 of treatment. Endothelium-dependent flow-mediated dilatation (ED-FMD) of the brachial artery was measured by high-resolution ultrasound at 5 visits. Tamoxifen caused an increase in %ED-FMD maximal at 28 days in the CAD group (2.1+/-0.3% to 7.5+/-0.7%; P<0.0001) and the NCA group (3.8+/-0.4% to 7.9+/-1.0%; P<0.0001), with no significant change in the untreated group. Tamoxifen also caused decreases in several plasma cardiovascular risk factors, including total cholesterol, triglycerides, lipoprotein(a), and fibrinogen. Except for the triglyceride response, these effects were similar to those reported for postmenopausal women treated with tamoxifen. CONCLUSIONS: Tamoxifen substantially increased ED-FMD in men with CAD who were taking conventional medication. Together with the effects on risk factors, the data strongly support clinical evaluation of SERMs for the treatment of men with CAD.

Combretastatin-A4 disrupts neovascular development in non-neoplastic tissue.

Combretastatin-A4 phosphate (cis-CA-4) is a tubulin-binding agent currently undergoing clinical trials as an anti-tumour drug. We have investigated whether CA-4 functions as a tumour-specific anti-vascular agent using the hyperplastic thyroid as a novel in vivo model of neovascularization. CA-4 elicited pathological changes in normal tissue, manifested as the induction of multiple, discrete intravascular thrombi. These vascular-damaging effects indicate that CA-4P does not function as a tumour-specific agent but targets neovasculature irrespective of the primary angiogenic stimulus.

Inhibiting mutations in the transforming growth factor beta type 2 receptor in recurrent human breast cancer.

Members of the transforming growth factor beta (TGF-beta) family are potent inhibitors of the growth of many epithelial cell types. Transmembrane signaling by TGF-beta occurs via a complex of the serine/threonine kinases TGF-beta type 1 receptor and TGF-beta type 2 receptor (TGFBR2), and inactivating mutations in the latter have recently been detected in some primary tumors and in several types of tumor-derived cell lines. The most common mutations that have been identified in TGFBR2 are frameshifts in a repetitive polyadenine region in replication error-positive colorectal carcinomas that result in a truncated protein and absence of receptor expression at the cell surface. A number of point mutations in the highly conserved serine/threonine kinase domain of TGFBR2 have also been reported, some of which have been correlated with either loss of trans-phosphorylation of TGF-beta type 1 receptor or constitutive activation of trans-phosphorylation. No TGFBR2 mutations have been reported in human breast tumors, but anomalous expression of TGF-beta in breast carcinomas suggests that TGF-beta signaling may be defective. We have therefore systematically examined unmatched sets of 17 primary and 17 recurrent breast tumor samples for mutations in TGFBR2, restricted to those regions of the gene in which mutations have previously been reported. None of the previously reported mutations was detected, but four novel mutations (V387M, N435S, V447A, and L452M) were found in the kinase domain in recurrent tumors. No mutations were detected in primary tumors. TGF-beta signaling was significantly inhibited by each of the N435S, V447A, and L452M mutations.

Targeting tumour vasculature: the development of combretastatin A4.

The requirement for neovascularisation to permit the development of solid tumours beyond a threshold size, has focused attention on the therapeutic potential of agents that prevent angiogenesis. The multistep nature of angiogenesis presents several targets for intervention, including the inhibition of the endothelial-cell migration or proliferation normally associated with developing vessels. Compounds that damage established tumour vasculature are also of potential clinical use. We review the development of one such antivascular drug, combretastatin A4. This tubulin-binding agent was originally isolated from an African shrub, Combretum caffrum. The disodium combretastatin A4 phosphate prodrug is currently undergoing phase I clinical trials in the UK and USA. This review assesses the in vitro and in vivo data for combretastatin and the prodrug, and the preliminary data that have emerged from the phase I clinical trials.

Nov gene encodes adhesion factor for vascular smooth muscle cells and is dynamically regulated in response to vascular injury.

Nephroblastoma overexpressed (NOV) is a member of the CCN family (connective tissue growth factor, CYR61, and NOV) of proteins that are involved in regulating the proliferation, differentiation, and adhesion of a variety of cell types. We have examined the expression of the NOV: gene and NOV protein by vascular smooth muscle cells (VSMCs), in vitro and in vivo, and the effects of recombinant NOV on VSMCs. Rat aortic VSMCs were found to express NOV: mRNA and NOV protein in vitro and in vivo. NOV: expression in adult rat tissues was very high in the aorta and was detected only weakly in the brain and lung by Northern analysis (relative levels 33:3:1). During postnatal development (3 days to 12 weeks), the expression of NOV: was correlated with markers of the differentiated smooth muscle cell phenotype (smooth muscle myosin heavy chain and SM22 alpha). In the rat carotid artery balloon injury model, NOV: was detectable by in situ hybridization and was downregulated in the media of the injured artery compared with the uninjured artery at 7 and 14 days after injury. Expression in the developing intima was barely detectable at 7 days after injury except for strong expression at the luminal surface. At 14 days after injury, NOV: expression was substantially increased throughout the intima. In vitro studies of the function of NOV protein showed that it promoted VSMC adhesion via a mechanism that was divalent cation and Arg-Gly-Asp independent but that it did not modulate VSMC proliferation or phenotype. The strong expression and dynamic regulation of NOV: in the arterial wall, together with its ability to promote VSMC adhesion, suggest that it may be involved in homeostasis and repair.

Dietary fat and reduced levels of TGFbeta1 act synergistically to promote activation of the vascular endothelium and formation of lipid lesions.

Transforming growth factor-(beta) (TGF(beta)) has a wide range of activities on vascular cells and inflammatory cells, suggesting it may have different functions during various stages of atherogenesis. We report that mice heterozygous for the deletion of the tgfb1 gene (tgfb1(+/-) mice) have reduced levels of TGF(beta)1 in the artery wall until at least 8 weeks of age. On a normal mouse chow diet, the vascular endothelium of tgfb1(+/-) mice is indistinguishable from wild-type littermates, assessed by morphology and intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. In contrast, levels of the smooth muscle isoforms of actin and myosin in medial smooth muscle cells of tgfb1(+/-) mice are significantly reduced. Following feeding a cholesterol-enriched diet for 12 weeks, high levels of ICAM-1 and VCAM-1 were detected in the vascular endothelial cells of tgfb1(+/-) mice, but not wild-type mice. Furthermore, marked deposition of lipid into the artery wall was only observed in the tgfb1(+/-) mice on the cholesterol-enriched diet. These vascular lipid lesions were accompanied by local invasion of macrophages. We conclude that deletion of a single allele of the tgfb1 gene results in a reduced level of TGFbeta1 antigen in the aorta together with reduced smooth muscle cell differentiation, whereas the addition of a high fat dietary challenge is required to activate the vascular endothelium and to promote the formation of fatty streaks resembling early atherosclerosis in humans.

Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart.

Spectroscopic Ca(2+)-indicators are thought to report values of free intracellular Ca(2+) concentration ([Ca(2+)](i)) that may differ from unperturbed values because they add to the buffering capacity of the tissue. To check this for the heart we have synthesized a new (19)F-labelled NMR Ca(2+) indicator, 1, 2-bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy]ethane ('4, 5FBAPTA'), with a low affinity (K(d) 2950 nM). The new indicator and four previously described (19)F-NMR Ca(2+) indicators 1,2-bis-[2-(1 - carboxyethyl)(carboxymethyl)amino - 5 - fluorophenoxy]ethane ('DiMe-5FBAPTA'), 1, 2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-4-fluorophenoxy]ethane ('DiMe-4FBAPTA'), 1, 2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane ('5FBAPTA') and 1, 2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane ('MFBAPTA'), with dissociation constants for Ca(2+) ranging from 46 to 537 nM, have been used to measure [Ca(2+)](i), over the range from less than 100 nM to more than 3 microM, in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4, paced at 1.0 Hz) by (19)F-NMR spectroscopy. Loading hearts with indicators resulted in buffering of the Ca(2+) transient. The measured end-diastolic and peak-systolic [Ca(2+)](i) were both positively correlated with indicator K(d). The positive correlations between indicator K(d) and the measured end-diastolic and peak-systolic [Ca(2+)](i) were used to estimate the unperturbed end-diastolic and peak-systolic [Ca(2+)](i) by extrapolation to K(d)=0 (diastolic) and to K(d)=infinity (systolic) respectively. The extrapolated values in the intact beating heart were 161 nM for end-diastolic [Ca(2+)](i) and 2650 nM for peak-systolic [Ca(2+)](i), which agree well with values determined from single cells and muscle strips.

Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myofibrils.

The measurement of cardiac Ca(2+) transients using spectroscopic Ca(2+) indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca(2+) buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca(2+) dissociation rate constants and which would account for the observed effects of (19)F-NMR indicators on the cardiac Ca(2+) transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca(2+) dependency of myofibril activation in cardiac myocytes is mediated by a single Ca(2+)-binding site on troponin C. A model based on 1:1 Ca(2+) binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca(2+) binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca(2+) binding, giving 58 and 2.0 microM respectively. In addition to reproducing our experimental data on the concentration of free Ca(2+) ions in the cytoplasm ([Ca(2+)](i)), the resulting Ca(2+) and ATP affinities given by fitting of the model also provided good predictions of the Ca(2+) dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca(2+) mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca(2+) indicators. The new model was used to estimate the extent of perturbation of the Ca(2+) transient caused by different concentrations of indicators. As little as 10 microM of a Ca(2+) indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca(2+)](i) and 30 microM will cause approx. 50% reduction in the peak-systolic [Ca(2+)](i) in a heart paced at 1.0 Hz.

TGF-beta in blood: a complex problem.

The cytokine transforming growth factor-beta (TGF-beta) was initially purified from human platelets, a rich source of this protein. In addition to platelets, TGF-beta1 is also found in other blood fractions, including plasma and the circulating leukocytes. However, more than 15 years after the initial isolation of TGF-beta1, there remains no consensus on how much TGF-beta1 is present in normal human plasma. Here we review the difficulties associated with measuring TGF-beta concentrations in complex biological fluids, and discuss the current state of knowledge on the distribution of TGF-beta isoforms in various blood fractions as well as the nature of the TGF-beta-containing protein complexes.

Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity.

Serum response factor (SRF) is a key transcriptional activator of the c-fos gene and of muscle-specific gene expression. We have identified four forms of the SRF coding sequence, SRF-L (the previously identified form), SRF-M, SRF-S and SRF-I, that are produced by alternative splicing. The new forms of SRF lack regions of the C-terminal transactivation domain by splicing out of exon 5 (SRF-M), exons 4 and 5 (SRF-S) and exons 3, 4 and 5 (SRF-I). SRF-M is expressed at similar levels to SRF-L in differentiated vascular smooth-muscle cells and skeletal-muscle cells, whereas SRF-L is the predominant form in many other tissues. SRF-S expression is restricted to vascular smooth muscle and SRF-I expression is restricted to the embryo. Transfection of SRF-L and SRF-M into C(2)C(12) cells showed that both forms are transactivators of the promoter of the smooth-muscle-specific gene SM22alpha, whereas SRF-I acted as a dominant negative form of SRF.

Mouse BTEB3, a new member of the basic transcription element binding protein (BTEB) family, activates expression from GC-rich minimal promoter regions.

Members of the three-zinc-finger family of transcription factors play an important role in determining basal transcription. We have cloned mouse BTEB3 (mBTEB3), a new member of the basic transcription element binding protein (BTEB) family, which is expressed in a wide variety of tissues. mBTEB3 activates transcription of the simian virus 40 early promoter (4-fold) and of the tissue-specific SM22alpha promoter (100-fold), suggesting that, like BTEB1 and Sp1, mBTEB3 is a basal transcription factor.

Monocyte chemoattractant protein-1 but not tumor necrosis factor-alpha is correlated with monocyte infiltration in mouse lipid lesions.

BACKGROUND: Apolipoprotein (apo)(a) transgenic mice and C57BL/6 mice fed a high fat diet develop similar-sized lipid lesions, but lesions in apo(a) mice are devoid of macrophages. We used this observation to identify which proinflammatory proteins might be involved in mediating monocyte recruitment during atherogenesis. METHODS AND RESULTS: Macrophage-deficient apo(a) transgenic mouse lesions contained similar levels of several different proinflammatory proteins, both adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and cytokines (tumor necrosis factor-alpha [TNF-alpha] and macrophage inflammatory protein-1alpha [MIP-1alpha]), similar to the macrophage-rich lesions of C57BL/6 mice. CONCLUSIONS: From this we conclude that ICAM-1, VCAM-1, TNF-alpha, and MIP-1alpha may all be necessary for vascular monocyte recruitment in vivo, but they cannot be sufficient. Monocyte chemoattractant protein-1 (MCP-1) protein was undetectable in the vessel wall taken from apo(a) transgenic mice fed a high fat diet compared with high expression in mice with lipid lesions (C57BL/6 and apoE knockout mice). Therefore elevated expression of MCP-1 but not TNF-alpha, MIP-1alpha, ICAM-1, or VCAM-1 is correlated with vascular macrophage accumulation. To test the hypothesis that monocyte infiltration during atherogenesis is MCP-1 dependent, it will be necessary to develop specific pharmacological inhibitors of MCP-1 activity.

Genetic control of the circulating concentration of transforming growth factor type beta1.

The concentration of transforming growth factor beta (TGF-beta) in plasma has been correlated with the development of several diseases, including atherosclerosis and certain forms of cancer. However, the mechanisms that control the concentration of TGF-beta in plasma are poorly understood. In a study of 170 pairs of female twins (average age 57.7 years) we show that the concentration of active plus acid-activatable latent TGF-beta1 [(a+l) TGF-beta therefore is predominantly under genetic control (heritability estimate 0.54). Single strand conformation polymorphism (SSCP) mapping of the TGF-beta1 gene promoter has identified two single base substitution polymorphisms. The two polymorphisms (G-->A at position -800 bp and C-->T at position -509 bp) are in linkage disequilibrium (correlation coefficient Delta = 0.215, P < 0.01). The C-509T polymorphism is significantly associated with the plasma concentration of (a+l) TGF-beta1, explaining 8.2% of the additive genetic variance of (a+l) TGF-beta1 concentration. It is therefore possible that predisposition to atherosclerosis, bone diseases or various forms of cancer may be correlated with the presence of particular alleles at the TGFB1 locus.

Transforming growth factor-beta1 gene polymorphisms and coronary artery disease.

1. Transforming growth factor-beta1 is a cytokine with a very wide spectrum of biological activities. Previous studies have shown that it is involved in a number of physiological and pathological processes including heart disease. In our study we aimed to scan the transforming growth factor-beta1 locus for polymorphisms and to identify haplotypes significantly associated with a predisposition to coronary atherosclerosis.2. Two patient groups comprising 244 angiographically normal individuals and 655 patients with coronary artery disease were recruited from London and Sheffield. DNA samples from these subjects were screened for mutations in the transforming growth factor-beta1 locus and all subjects were genotyped by a coupled polymerase chain reaction-restriction enzyme digestion method.3. Five polymorphisms have been identified in the transforming growth factor-beta1 gene at positions G-800A, C-509T in the promoter region, Leu10-->Pro, Arg25-->Pro in exon 1 and Thr263-->Ile in exon 5. No significant difference in frequencies for any of the five polymorphisms was found between controls and patients with coronary artery disease. Similarly, there was no correlation between these polymorphisms and hypertension.4. The genotypes of all the individuals participating in the study were assigned to seven main haplotypes of the transforming growth factor-beta1 locus. Based on species comparison data we propose that GCCGC is the ancestral haplotype in humans.5. Our data suggest that these transforming growth factor-beta1 polymorphisms are not associated with coronary artery disease and therefore their presence alone would not be a genetic risk factor for predisposition to coronary artery disease.

Expression of alternatively spliced human latent transforming growth factor beta binding protein-1.

Latent transforming growth factor beta binding protein-1 (LTBP1) is important in regulating the localisation and activation of transforming growth factor beta(TGFbeta). Three forms of LTBP1 mRNA have previously been described, LTBP1L, LTBP1S and LTBPdelta53. Here, we have analysed the LTBP1 coding sequence and identified two other spliced forms, LTBP1delta55 and LTBP1delta41. LTBP1delta55 is a short form of LTBPIL which lacks 55 amino acids including two consensus N-glycosylation sites and LTBP1delta41 is a form of LTBP1 which lacks the 12th EGF-like repeat. Furthermore, sequencing of genomic clones showed that splicing to generate LTBP1L occurs using an intra-exonic 3' splice acceptor site in the first coding exon of LTBP1S and that LTBP1delta55 arises from the alternative use of an exonic 3' splice acceptor site at the end of the following intron. LTBP1delta41 arises from skipping the exon which encodes the 12th EGF-like repeat. LTBP1delta55 and LTBP1delta41 mRNA are expressed in a wide variety of human tissues but the proportions of each splice form vary in the tissues.

Increased PAI activity and PAI-1 antigen occurring with an oral fat load: associations with PAI-1 genotype and plasma active TGF-beta levels.

Whether the post-prandial lipemic response is linked to potentially pro-atherogenic and/or prothrombotic changes in plasminogen activator inhibitor (PAI) and transforming growth factor-beta (TGF-beta) is uncertain. The aim of our study was to determine whether PAI-1 antigen and PAI activity were elevated during post-prandial lipemia following a standard fat tolerance test. We also investigated changes in TGF-beta1 antigen and TGF-beta activity, to determine whether changes in TGF-beta activity were associated with changes in PAI measurements. Lastly, the influence of genotype at a common insertion/deletion polymorphism in the PAI-1 promoter on changes in PAI activity and PAI-1 antigen was examined. Fat tolerance tests were undertaken in 57 healthy middle-aged men to investigate associations between plasma concentrations of lipoproteins, PAI (antigen and activity) and TGF-beta. PAI-1 concentration increased by 76% after 8 h (P < 0.0001). PAI activity also increased by 64% (P = 0.0054) and TGF-beta activity decreased by 10% (P < 0.0001). Increases in PAI-I antigen and PAI activity varied markedly between individuals. To investigate these heterogeneous responses we examined whether genotype at the common insertion/deletion polymorphism of the PAI-1 promoter accounted for these differences. Individuals with at least one 4G (deletion) allele showed potentially pro-atherogenic changes in both PAI-1 and TGF-beta, compared to individuals who were homozygous for the 5G (insertion) allele. In conclusion, increased PAI and decreased TGF-beta activity occur during a fat tolerance test and this effect may be modulated by a common insertion/deletion polymorphism in the PAI-1 promoter.